HK1118082A - Methods of amplifying and sequencing nucleic acids - Google Patents

Description

Methods for nucleic acid amplification and sequencing

Technical Field

The present invention relates to a method and an apparatus for determining a base sequence of DNA. More particularly, the present invention relates to a method and apparatus that can automatically or semi-automatically amplify and determine the base sequence of a genome.

Background

The development of rapid and sensitive nucleic acid sequencing methods using automated DNA sequencers has revolutionized modern molecular biology. With a series of machines and a consistent effort by a panel of technicians, it is now possible to analyze the complete genome of plants, fungi, animals, bacteria and viruses. However, the goal of rapid and automated or semi-automated sequencing of genomes in a short time has not been possible. There continues to be technical problems with accurate sample preparation, amplification and sequencing.

One technical problem that has hampered genomic sequence analysis is the inability of researchers to rapidly prepare numerous nucleic acid samples containing a complete genome in a short period of time.

Another technical problem is the inability to representatively amplify a genome at a level consistent with the sensitivity of current sequencing methods. Modern economically viable sequencing machines, while sensitive, still require more than one million copies of the DNA fragment to be sequenced. Current methods that provide high copies of DNA sequencing involve various cloning or in vitro amplification that cannot amplify the number of individual clones (600,000 or more, tens of millions for the human genome) necessary to economically sequence a full genome.

Yet another technical problem is the limitation of current sequencing methods, which are capable of performing at most one sequencing reaction per hybridization of oligonucleotide primers. Hybridization of sequencing primers is generally the rate limiting step in limiting the throughput of a DNA sequencer.

In most cases, the polymerase chain reaction (PCR; Saiki, R.K. et al, Science1985, 230, 1350-. However, it is neither economical nor efficient to utilize current PCR technology to meet the increasing demands of modern genetics, especially when the need for whole genome sequencing is considered.

Efforts to maximize time and cost effectiveness have generally focused on two areas: reducing the reaction volume required for amplification and increasing the number of simultaneous amplifications performed. Miniaturization confers benefits of reduced sample and reagent use, reduced amplification time, and increased throughput scale.

Although conventional thermocyclers require relatively long cycle times due to the limitations of the thermal block (Woolley, A.T. et al, anal. chem.1996, 68, 4081-. Constant-current PCR devices utilize etched microchannels connected to a fixed temperature zone to reduce the reaction volume to sample levels below microliters (Lagaly, E.T. et al, Analytical Chemistry 2001, 73, 565-570; Schnegas, I. et al, Labon aChip-Tlae Royal Society of southern 2001, 1, 42-49).

Glass capillaries heated with air (Kalinina, O. et al, Nucleic Acids Res.1997, 25, 1999-. Similar reaction volumes have been achieved with microfabricated silicon thermal cyclers (Burns, M.A. et al, Proc. Natl. Acad. Sci. USA 1996, 93, 5556-.

In many cases, these miniaturisation have reduced the total PCR reaction time to less than 30 minutes for modified electrical heating elements (Kopp, M.U. et al, Science1998, 280, 1046-.

Some technologies employ both increased throughput and miniaturization; it keeps the reaction volume below 1. mu.l, as in the 1536-well system designed by Sasaki et al (Sasaki, N. et al, DNA Res.1997, 4, 387-391). As another example, Nagai et al (Nagai, H. et al, biosens. BioElectron.2001, 16, 1015-. Unfortunately, the recovery of amplicons from these methods has proven problematic for use, requiring evaporation through permselective membranes.

Despite these significant increases in reaction volume and cycle time, none of the previous strategies provided massively parallel amplification, which was necessary to significantly increase throughput to the levels required for complete genome analysis. DNA sequencers are still slower and more expensive than desired. In a pure research environment, it may be acceptable if one sequencer is slow and expensive. However, this inefficient sequencing method is not satisfactory even for a well-priced research institute when it is desirable to utilize a DNA sequencer in a clinical diagnostic setting. Massively parallel sequencing of thousands of clonally amplified target targets would greatly facilitate large-scale whole genome library analysis without the time-consuming sample preparation process and expensive error-prone cloning process. Successful high-volume, solid-phase, clonal DNA amplification can be used for a variety of purposes. Thus, it is apparent that there is a need for preparing genomic or large template nucleic acids for sequencing, amplifying the nucleic acid templates, and sequencing the amplified template nucleic acids without the limitation of one sequencing reaction per hybridization. Furthermore, there is a need for a system to interface these techniques into a viable automated or semi-automated sequencing instrument.

Brief description of the invention

The present invention describes an integrated system comprising a system for (1) nucleic acid sample preparation,

(2) nucleic acid amplification, and (3) DNA sequencing.

The present invention provides a novel method for preparing libraries of various DNA sequences, particularly sequences derived from large template DNA or whole (or partial) genomic DNA. Single-stranded DNA sequences are prepared from large template DNA or whole or partial DNA genomes by fragmentation, filling-in, adaptor ligation, nick repair, and isolation of single-stranded DNA. The method provides for generating a ssDNA library attached to a solid support comprising: (a) generating a ssDNA template library; (b) attaching a ssDNA template to a solid support; and (c) isolating the solid support to which the ssDNA template is attached.

The present invention also provides a method of amplifying each member of a DNA library in a single reaction tube, for example, by separately packaging a plurality of DNA samples in microcapsules of an emulsion, simultaneously performing amplification of a plurality of packaged nucleic acid samples, and releasing the amplified plurality of DNAs from the microcapsules for subsequent reactions. In one embodiment, a single copy of the nucleic acid template is hybridized to the DNA capture beads, resuspended and emulsified in a complete amplification solution into a microreactor (typically 100-200 microns in diameter), after which amplification (e.g., PCR) is used to increase the copy number clone of the starting template to more than 1,000,000 copies of a single nucleic acid sequence, preferably 2,000,000-20,000,000 copies of a single nucleic acid sequence. For example, amplification reactions can be performed simultaneously with at least 3,000 microreactors per microliter of reaction mixture, and can be performed simultaneously with over 300,000 microreactors in a single 100 μ l volume of a test tube (e.g., a PCR reaction tube). The invention also provides methods for enriching those beads that comprise a successful DNA amplification event (i.e., by removing beads to which no DNA has been attached).

The invention also provides methods for sequencing nucleic acids from multiple primers in a single primer hybridization step. Two or more primers are hybridized to the template DNA to be sequenced. All but one of the sequencing primers were then protected. Sequencing is performed again by extending the unprotected primer (e.g., pyrosequencing). The extension can either be allowed to complete (with additional polymerase and dNTPs if necessary) or terminated (by polymerase and ddNTPs). Removing chain completion and/or termination reagents. One of the protected primers is then deprotected and sequenced by extension of the newly deprotected primer. This process is continued until all sequencing primers have been deprotected and sequenced. In a preferred embodiment, both ends of the double stranded nucleic acid are sequenced using two primers (one protected and one deprotected).

The invention also provides an apparatus and method for sequencing nucleic acids using a pyrophosphate-based sequencing method. The device has a Charge Coupled Device (CCD) camera, a microfluidic cavity, a cartridge (cartridge) holder, a pump, and a flow valve. The device utilizes chemiluminescence as a detection method, which has an inherently low background for pyrosequencing. In a preferred embodiment, the sequencing sample cartridge (cartridge), referred to as a 'PicoTiter plate', is formed from a commercial fiber optic panel that is acid etched to produce hundreds of thousands of very small wells, each 75pL in volume. The device includes a novel reagent delivery container suitable for use in the arrays described herein to provide fluidic reagents to picotiter plates, and reagent delivery methods incorporating the reagent delivery container. Photons from each well on the picotiter plate are transmitted to a specific pixel on the CCD camera to detect the sequencing reaction.

Brief Description of Drawings

FIG. 1 depicts a schematic representation of the complete process of library preparation, including template DNA fragmentation (FIG. 1A), end-filling (FIG. 1B), adaptor ligation (FIG. 1C), nick repair, strand extension and gel separation (FIG. 1D). FIG. 1E depicts a schematic representation of the amplification and sequencing stages of template DNA (FIG. 1E). FIG. 1F depicts a representative gel comprising a sample preparation of a 180-350 base pair adenovirus DNA library according to the methods of the present invention. FIG. 1G depicts a detailed schematic representation of library preparation, amplification and sequencing.

FIG. 2A depicts a schematic representation of a universal adaptor design according to the present invention. Each universal adaptor is generated from two complementary ssDNA oligonucleotides designed to contain a 20bp nucleotide sequence for PCR primers, a 20bp nucleotide sequence for sequence primers, and a unique 4bp recognition sequence consisting of a non-repetitive nucleotide sequence (i.e., ACGT, CAGT, etc.). FIG. 2B depicts a representative universal adaptor sequence pair for use in the present invention. Adaptor a sense strand: SEQ ID NO: 1; adaptor a antisense strand: SEQ ID NO: 2; adaptor B sense strand: SEQ ID NO: 3; adaptor B antisense strand: SEQ ID NO: 4. FIG. 2C depicts a schematic representation of a universal adaptor design for use in the present invention.

FIG. 3 depicts strand displacement and extension of nicked double stranded DNA fragments according to the present invention. Following ligation of the universal adaptors generated from the synthetic oligonucleotides, T4 DNA ligase treatment will generate double stranded DNA fragments containing two nicked regions (FIG. 3A). Addition of the strand displacing enzyme (i.e., Bst DNA polymerase I) will bind to the nick (fig. 3B), strand displace the nicked strand and complete nucleotide extension of the strand (fig. 3C) to produce a non-nicked double-stranded DNA fragment (fig. 3D).

FIG. 4 depicts the isolation of directionally ligated single stranded DNA according to the present invention using streptavidin coated beads. Following ligation of universal adaptors a and B (the two different adaptors are sometimes referred to as "first" and "second" universal adaptors), the double stranded DNA will comprise adaptors in four possible compositions: AA. BB, AB and BA. When universal adaptor B contains 5' biotin, the AB, BA and BB populations are captured and separated using a magnetic streptavidin coated solid support (population AA is eluted). The BB population remains on the beads and is not released when each end of the double stranded DNA is attached to the beads. However, only groups AB and BA will release a single-stranded DNA fragment complementary to the binding strand after washing in the presence of a low salt buffer. Single-stranded DNA fragments were isolated from the supernatant and used as templates for subsequent amplification and sequencing. The method is described in more detail below.

FIG. 5 depicts a schematic representation of the structure of a DNA capture bead.

FIG. 6 depicts a schematic representation of one embodiment of a bead emulsion amplification method.

Figure 7 depicts a schematic representation of an enrichment process that removes beads with no DNA attached thereto.

FIGS. 8A-B depict a schematic representation of a double-ended sequencing reaction according to the present invention.

Figure 9 depicts a double-ended sequencing demonstration of a pyrosequencing (pyroqencing) device according to the present invention.

FIGS. 10A-F depict an exemplary double-ended sequencing process.

FIGS. 11A-D depict diagrammatic representations of rolling circle based amplification using anchor primers.

FIG. 12 depicts a diagram of a sequencing device according to the present invention.

FIG. 13 depicts a diagram of an agent delivery/perfusion chamber according to the present invention.

FIG. 14 depicts a so-called PicoTiter board of the present invention^TMMicrographs of the cavitated (cavitated) fiber optic bundle of (a).

FIG. 15 depicts a micrograph of a picotiter plate covered with beads having DNA templates immobilized thereon and sulfurylase and luciferase immobilized thereon.

FIG. 16 depicts a reagent flow chamber and FORA (PicoTiter plate)^TM) Shown schematically.

Fig. 17 depicts a diagram of an analytical instrument of the present invention.

FIG. 18 depicts a graphical representation of a microscopic parallel sequencing reaction in a PicoTiter plate.

Figure 19 depicts a micrograph of a single well reaction.

FIG. 20 depicts a PicoTiter board^TMAnd (4) adding a sample box. "A" refers to a PicoTiter plate with wells facing the cassette^TMBetween PicoTiter plate^TMThe distance between the open side of the hole and the filling chuck wall is 0.3 mm; "B" refers to a silicon sealing gasket; "C" means an inlet; "D" refers to an inlet sample addition tube; "E" refers to an outlet and "F" refers to an outlet tube. PicoTiter plate with plastic clip^TMIs fixed in the box. Liquid is filled through filling tube D and enters the PicoTiter plate through inlet C^TMThe open sides of the aperture are in spaced relation to the cartridge wall. Filling the area defined by the silicone sealing gasket B and the excess liquid leaves the cartridge through the outlet E and the outlet tube F.

FIG. 21 depicts PicoTiter plate amplification at dissociation perspectiveA chamber. "A" refers to an amplification chamber cover with 6 retaining pegs; "B" refers to a closed cell foam insulating mat; "C" refers to a 25mm by 75mm standard glass microscope slide; "D" refers to a 0.25mm thick silicon wafer; "E" refers to PicoTiter plate^TM(ii) a "F" refers to the amplification chamber base; "G" refers to a second 0.25mm thick silicon wafer.

FIG. 22 depicts a solid-phase PicoTiter plate^TMSchematic representation of PCR. Cylindrical structure symbolizing a single PicoTiter board ^TMAnd (4) a hole. The grey spheres symbolize the beads with immobilized primers. As indicated by the arrows, the forward "F" (red) and reverse "R" (blue) primers are shown in the 5 'to 3' direction. The synthetic sequences complementary to the forward and reverse primers are shown as dark red (F-complement) and dark blue (R-complement) bars. The single-stranded template DNA is shown as a solid gray line and the newly synthesized DNA strand is a broken gray line. The fluorescently labeled hybridization probes are shown as green bars.

FIGS. 23A-C depict hybridization of fluorescent probes to bead-immobilized test DNA fragments. FIGS. 23A (top left) and 23B (top right) show the specificity of mixed probe populations hybridized to fragment A and fragment B immobilized on control beads, respectively. Fragment B beads showed Alexa Fluor 647 signal (red), while fragment B beads showed Alexa Fluor 488 signal (green). Fig. 23C (lower panel) depicts probe fluorescence from DNA capture beads after PTPCR. The beads showed homologous fragment a and fragment B signals, as well as a mixture of templates, showing varying degrees of yellow.

Figure 24 depicts representative BioAnalyzer output values from single stranded DNA library analysis.

FIG. 25 depicts the insert flanked by PCR primers and sequencing primers.

FIG. 26 depicts truncated products resulting from mismatches of PCR primers in the cross-hybridization region (CHR).

Fig. 27 depicts melting temperature based calculations for primer candidates.

Fig. 28A-D depict the assembly of a nebulizer for use in the method of the invention. A cap was placed on top of the sprayer (fig. 7A) and the cap was secured with a sprayer clip fitting (fig. 7B). The bottom of the nebulizer was connected to a nitrogen source (FIG. 7C) and the entire device was coated with parafilm (FIG. 7D).

FIG. 29A depicts representative results of a LabChip analysis of a single stranded DNA library after spraying and filling.

FIG. 29B depicts representative size distribution results for adaptor-ligated single-stranded DNA libraries after spraying, filling-up, and gel purification.

FIG. 30 is a depiction of a fixture used to place a tube on an oscillating plate beneath a vertical syringe pump. The fixture was modified to control the three sets of bead emulsion amplification reaction mixtures. The syringe was filled with PCR reaction mixture and beads.

Fig. 31 is a depiction of the optimal placement of the syringe in a vertical syringe pump and the positioning of the emulsion tube under the syringe outlet.

Fig. 32 is a depiction of the optimal placement of the syringe pump push module relative to the syringe plunger, and the optimal placement of the clamp on the oscillation plate. With this arrangement, the syringe components are discharged into the agitated emulsion oil.

FIG. 33 is a depiction of a bead (see arrow) suspended in a single microreactor according to a method of the present invention.

FIG. 34 is a depiction of the results of double-ended sequencing showing that the sequence of both ends of the DNA template was determined. SEQ ID NO: 44: atgcacatggttgacacagtggt, respectively; SEQ ID NO: 45: atgcacatggttgacacagtgg, respectively; SEQ ID NO: 46: atgccaccgacctagtctcaaactt are provided.

FIG. 35 illustrates a coating of beads comprising two oligonucleotide sequences for double-stranded sequencing.

FIG. 36 illustrates the process of solution phase PCR and bead targeting-a step in a preferred embodiment of double-ended sequencing.

FIG. 37 illustrates emulsion breaking and recovery of amplified template DNA on beads-a step in a preferred embodiment of double-ended sequencing.

FIG. 38 depicts a schematic representation of a preferred method of double-stranded sequencing.

FIG. 39 is a graph showing the results of sequencing the genome of Staphylococcus aureus (Staphylococcus aureus).

Figure 40 illustrates the average read length in experiments involving double-ended sequencing.

Figure 41 illustrates the number of wells per genome span in a double-ended sequencing experiment.

Figure 42 illustrates typical output values and alignment strings from double-ended sequencing methods. The sequence is displayed sequentially, from top to bottom: SEQ ID NO: 47-SEQ ID NO: 60.

Detailed Description

A new platform is presented herein that allows simultaneous amplification of thirty thousand isolated PCR reactions in a volume as low as 39.5 picoliters. The pooled PTPCR products from the entire reaction can be recovered by one washing step and analyzed for the presence and abundance of specific templates by real-time PCR. More interestingly, it is shown herein that these PTPCR products can be driven to a solid support and detected by hybridization of two colored fluorescent probes, allowing high-volume, solid-phase, clonal DNA amplification and massively parallel sequencing.

The present invention relates to a method and apparatus for performing genomic sequencing, which meets the following objectives: (1) preparing nucleic acids for sequencing in a rapid and efficient manner, (2) amplifying the nucleic acids in a representative manner, and (3) performing multiple sequencing reactions with only one primer hybridization. The present invention is particularly suited for genotyping, detection and diagnosis from small samples of nucleic acids in a cost-effective manner.

Defining:

unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, with exemplary suitable methods and materials being described below. For example, a method comprising more than two steps may be described. In such a method, not all steps may be required to achieve certain goals and the present invention claims separate steps to achieve these separate goals. All publications, patent applications, patents, and other references are incorporated herein by reference. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

As used herein, the term "universal adaptor" refers to two complementary and annealed oligonucleotides designed to comprise one PCR primer nucleotide sequence and one sequencing primer nucleotide sequence. Optionally, the universal adaptor may further include a unique discriminating key sequence, which consists of non-repeating nucleotide sequences (i.e., ACGT, CAGT, etc.). A set of universal adaptors comprises two unique and distinct double stranded sequences that can be ligated to the ends of double stranded DNA. Thus, the same universal adaptor or different universal adaptors can be ligated to either end of the DNA molecule. When included in a single-stranded larger DNA molecule or when present as an oligonucleotide, the universal adaptor may be referred to as a single-stranded universal adaptor.

"target DNA" may mean a DNA whose sequence is determined by the method and apparatus of the present invention.

A binding pair may mean a pair of molecules that interact by specific non-covalent interactions that depend on the three-dimensional structure of the molecules involved. Typical pairings of specific binding partners include antigen-antibody, hapten-antibody, hormone-receptor, nucleic acid strand-complementary nucleic acid strand, substrate-enzyme, substrate analog-enzyme, inhibitor-enzyme, sugar-plant lectin, biotin-avidin, and virus-cell receptor.

As used herein, the term "discriminating key sequence" refers to a sequence consisting of at least one (i.e., a, C, G, T) of each of the four deoxyribonucleotides. The same discriminating sequence can be used for a complete library of DNA fragments. Alternatively, a library of DNA fragments derived from different organisms may be traced using different discriminative key sequences.

As used herein, the term "plurality of molecules" refers to DNA isolated from the same source, whereby different organisms can be prepared by the same method. In one embodiment, the plurality of DNA samples are derived from DNA, whole genomic DNA, cDNA, large fragments of viral DNA, or reverse transcripts derived from viral RNA. Such DNA may be derived from any source, including mammalian (i.e., human, non-human primate, rodent, or canine), plant, avian, reptile, fish, fungus, bacteria, or virus.

As used herein, the term "library" refers to a subset of smaller DNA produced from a single DNA template, either fragmented or whole genome.

As used herein, the term "unique" in "unique PCR primer region" refers to a sequence that is not present or is present at very low copy levels within the DNA molecule to be amplified or sequenced.

As used herein, the term "compatible" refers to one end of double-stranded DNA to which an adaptor molecule can be attached (i.e., blunt end or sticky end).

As used herein, the term "fragmentation" refers to the process of converting larger molecules of DNA into smaller fragments of DNA.

As used herein, the term "large template DNA" is DNA of greater than 25kb, preferably greater than 500kb, more preferably greater than 11MB, and most preferably 5MB or greater.

As used herein, the term "stringent hybridization conditions" refers to those conditions under which only complementary sequences will hybridize to each other.

The invention described herein is generally a system and method for processing nucleic acids. The systems and methods can be used to process nucleic acids in a variety of ways that utilize nucleic acid sequencing. Such sequencing can be performed to determine the identity of nucleic acid sequences, single nucleotide polymorphism detection in nucleic acid fragments, nucleic acid expression profiling (comparing nucleic acid expression profiles between two or more states-e.g., comparing between diseased and normal tissue or comparing between untreated tissue and tissue treated with drugs, enzymes, radiation, or chemotherapy), haplotyping (comparing variations in genes or genes on each of two alleles present in a human subject), karyotyping (diagnostically comparing one or more genes in a test tissue with the same genes from a "normal" karyotyped subject prior to pregnancy to detecting congenital defects-the tissue is typically from embryo/fetus), and genotyping (comparing one or more genes from a first individual of a species with the same genes from other individuals of the same species).

The system has multiple components. These include (1) a nucleic acid template to be processed, (2) a picotiter plate comprising the nucleic acid template, (3) a flow chamber and fluid delivery means that allows a nucleic acid processing reagent to flow over the nucleic acid template, wherein the processing reagent generates light when processing the nucleic acid, (4) a light capture means that detects light emitted when processing the nucleic acid and converts the captured light into data, and (5) data processing means that processes the data to generate useful information about the processed nucleic acid. Each of these components of the system will be discussed in detail below.

1. Nucleic acid templates and preparation thereof

Nucleic acid template

Nucleic acid templates, e.g., nucleic acid libraries, capable of being sequenced according to the present invention may generally comprise open circular or closed circular nucleic acid molecules. A "closed loop" is a covalently closed circular nucleic acid molecule, e.g., a circular DNA or RNA molecule. An "open loop" is a linear single-stranded nucleic acid molecule having a 5 'phosphate group and a 3' hydroxyl group.

In one embodiment, the single stranded nucleic acid comprises at least 100 copies of a particular nucleic acid sequence, each copy being covalently end-to-end linked. In certain embodiments, the open loop is formed in situ from a linear double stranded nucleic acid molecule. The ends of a given open loop nucleic acid molecule may be ligated by a DNA ligase. The sequences at the 5 'and 3' ends of the open circular molecule are complementary to two regions of contiguous nucleotides in the second nucleic acid molecule, e.g., an adapter region (sometimes referred to as an adapter) of an anchor primer, or two adjacent regions in the second DNA molecule. Thus, the ends of the open loop molecules can be ligated using DNA ligase or extended by DNA polymerase in a gap-filling reaction. Open loops are described in detail in Lizardi, U.S. Pat. No. 5,854,033, which is incorporated herein by reference in its entirety. After annealing of the open loops to the anchor primer, for example, one open loop can be converted to a closed loop in the presence of a DNA ligase (for DNA) or an RNA ligase.

If desired, the nucleic acid template may be provided as a padlock probe. The Padlock probe is a linear oligonucleotide that includes a target-complementary sequence at each end, and is separated by a linker sequence. The linker may be ligated to the ends of members of a library of nucleic acid sequences that have been, for example, physically sheared or digested with restriction enzymes. After hybridization to the target sequence, the 5 '-and 3' -terminal regions of these linear oligonucleotides are juxtaposed. This juxtaposition allows for covalent binding of two probe fragments (if properly hybridized) by enzymatic ligation (e.g., with T4 DNA ligase), thus converting the probes into a circularly closed molecule that is ligated to a specific target sequence (see, e.g., Nilsson et al, 1994.Science 265: 2085-. Due to their specificity and selectivity for gene sequence variants (see, e.g., Lizardi et al, 1998.nat. Genet.19: 225- "232; Nilsson et al, 1997.nat. Genet.16: 252-" 255) and due to the fact that the resulting reaction products remain localized to specific target sequences, the resulting probes are suitable for simultaneous analysis of many gene sequences. Furthermore, intramolecular ligation of many different probes is expected to be less susceptible to non-specific cross-reactivity than multiplex PCR-based methodologies in which non-homologous pairing of primers can result in unrelated amplification products (see, e.g., Landegren and Nilsson, 1997.Ann. Med.29: 585-.

Libraries of starting nucleic acid templates comprising single-stranded or double-stranded nucleic acid molecules can be constructed, provided that the nucleic acid sequences include regions that can anneal, if present in the library, or can be made to anneal to an anchor primer sequence. For example, when used as a template for rolling circle amplification, a region of a double-stranded template needs to be at least transiently single-stranded to function as a template for extension of an anchor primer.

The library template may include a variety of components, including but not limited to one or more regions complementary to the anchor primer. For example, the template library may include a region complementary to a sequencing primer, a region of control nucleotides, and an insert consisting of a sequencing template that is subsequently characterized. As explained in more detail below, the control nucleotide region is used to calibrate the relationship between the amount of by-product and the number of integrated nucleotides. As used herein, the term "complement" refers to a nucleotide sequence that is capable of hybridizing to a specific nucleotide sequence to form a matched duplex.

In one embodiment, the library template comprises (i) two different regions complementary to the anchor primer, (ii) a region homologous to the sequencing primer, (iii) an optional control nucleotide region, (iv) an intervening sequence of, for example, 30-500, 50-200, or 60-100 nucleotides to be sequenced. The template can of course include two, three or all four of these features.

The template nucleic acid may be constructed from any source of nucleic acid, e.g., any cell, tissue, or organism, and can be generated by any art-recognized method. Suitable methods include, for example, ultrasonication of genomic DNA and digestion with one or more Restriction Endonucleases (REs) to produce fragments of the desired length range from the starting population of nucleic acid molecules. Preferably, the one or more restriction enzymes have different four base recognition sequences. Examples of such enzymes include, for example, Sau3Al, MspI, and TaqI. Preferably, the enzyme is used in combination with an anchor primer having a region comprising the recognition sequence of the corresponding restriction enzyme. In certain embodiments, one or both of the anchor primer adapter regions comprises other sequences that border known restriction enzyme recognition sites, thereby allowing capture or annealing of the anchor primer of the target-specific restriction fragment to the anchor primer. In other embodiments, the restriction enzyme is a type IIS restriction enzyme.

Alternatively, a template library may be prepared by generating a complementary dna (cdna) library from RNA, e.g., messenger RNA (mrna). If desired, the cDNA library can be further treated with restriction endonucleases to obtain 3 'ends of specific RNAs, internal fragments, or fragments comprising the 3' ends of isolated RNAs. The adaptor region in the anchor primer may be complementary to the target sequence, which is believed to occur in the template library, for example, in the generation of known or suspected sequence polymorphisms within the fragment by endonuclease digestion.

In one embodiment, an indicator oligonucleotide may be attached to a member of a library of templates to allow for subsequent association of a template nucleic acid with a population of nucleic acids from which the template is derived. For example, one or more samples of the starting DNA population can be separately fragmented using any of the previously disclosed methods (e.g., restriction digestion, ultrasonication). An indicator oligonucleotide sequence specific for each sample is attached, e.g., ligated, to the ends of the fragmented members of the class group. The indicator oligonucleotide may function as a circularized, amplified and optionally sequenced region which allows it to be used to indicate or encode a nucleic acid to identify the starting sample from which it originates.

Libraries of different templates made with multiple distinguishable indicator primers can be mixed together for subsequent reactions. Determining the sequence of the library members makes it possible to identify sequences corresponding to the indicator oligonucleotides. Based on this information, the origin of any given segment can be inferred.

The invention includes a sample preparation process that results in a solid or moving solid matrix array comprising a plurality of anchor primers or adapters covalently attached to a template nucleic acid.

When the template nucleic acid is circular, the covalently linked anchor primer and one or more copies of the target nucleic acid are preferably formed by annealing the anchor primer to a complementary region of the circular nucleic acid, and subsequently extending the annealed anchor primer with a polymerase to cause formation of a nucleic acid comprising one or more copies of a sequence complementary to the circular nucleic acid.

The attachment of the anchor primer to the solid or mobile solid substrate may occur before, during or after the extension of the annealed anchor primer. Thus, in one embodiment, one or more anchor primers are attached to a solid or moving solid substrate, followed by annealing of the anchor primer to the target nucleic acid and extension in the presence of a polymerase. Alternatively, in a second embodiment, the anchor primer is annealed to the target nucleic acid first, and the 3' OH terminus of the annealed anchor primer is extended with a polymerase. The extended anchor primer is then attached to a solid or mobile solid substrate. By varying the sequence of the anchor primer, it is possible to specifically amplify different target nucleic acids present in a population of nucleic acids.

Listed below is a preferred embodiment for preparing template nucleic acids for amplification and sequencing reactions. The present invention includes a method of preparing sample DNA comprising seven general steps: (a) fragmenting a large template DNA or whole genome DNA sample to produce a plurality of digested DNA fragments; (b) generating compatible ends on the plurality of digested DNA samples; (c) ligating a set of universal adaptor sequences to the ends of the fragmented DNA molecules to produce a plurality of adaptor-ligated DNA molecules, wherein each universal adaptor sequence has a known and unique base sequence comprising a common PCR primer sequence, a common sequencing primer sequence and a discriminating four base key sequence, and wherein an adaptor is attached to biotin; (d) isolating the plurality of ligated DNA fragments; (e) removing any portion of the plurality of ligated DNA fragments; (f) nick repair and strand extension of the plurality of ligated DNA fragments; (g) attaching each ligated DNA fragment to a solid support; and (h) isolating a population of single-stranded adaptor-ligated DNA fragments with unique adaptors at each of their ends (i.e., to provide directionality).

The following discussion summarizes the basic steps involved in the method of the present invention. The steps are described in a particular order, however, as known to those skilled in the art, the steps may be modified to achieve the same results. The inventors claim protection of this modification. In addition, some steps may be minimized, as also known to those skilled in the art.

Fragmentation

In the practice of the methods of the present invention, fragmentation of a DNA sample can be performed by any method known to those of ordinary skill in the art. Preferably, the fragmentation is performed by enzymatic or mechanical means. The mechanical means may be ultrasonic or physical shearing. The enzymatic method may be performed by digestion with a nuclease (e.g., deoxyribonuclease (dnase I)) or one or more restriction endonucleases. In a preferred embodiment, the fragmentation results in an end of unknown sequence.

In a preferred embodiment, the enzymatic method is dnase I. DNase I is a general purpose enzyme that nonspecifically cleaves double-stranded DNA (dsDNA) to release 5' phosphorylated di-, tri-, and oligonucleotide products. DNase I in the presence of Mn²⁺、Mg²⁺And Ca²⁺But the buffer without other salts had the best activity. The purpose of the DNase I digestion step is to fragment one large DNA genome into smaller species comprising the library. The cleavage properties of DNase I will result in random digestion of the template DNA (i.e.no sequence bias) and will lead when used in the presence of manganese-based buffers Blunt-ended dsDNA fragments predominate (Melgar, E. and D.A.Goldtwait.1968. Deoxygenic acidic nucleotides.II. the effects of metals on the mechanism of action of deoxyribonuclease I.J.biol.chem.243: 4409). The range of digestion products produced after dnase I treatment of a genomic template depends on three factors: i) the amount (units) of enzyme used; ii) digestion temperature (. degree.C.); and iii) incubation time (minutes). The DNase I digestion conditions listed below have been optimized to produce genomic libraries having a size of 50-700 base pairs (bp).

In a preferred embodiment, DNase I digests large template DNA or whole genomic DNA1-2 minutes to produce a set of polynucleotides. In another preferred embodiment, the DNase I digestion is performed between 10 ℃ and 37 ℃. In yet another preferred embodiment, the digested DNA fragment is between 50bp to 700bp in length.

Make up well

In Mn²⁺Digestion of genomic DNA (gDNA) with DNase I when present will produce DNA fragments that are blunt-ended or have a bulge end that is one or two nucleotides long. In a preferred embodiment, Pfu DNA polymerase is used to generate an increased number of blunt ends. In another embodiment, blunt ends may be generated with a less potent DNA polymerase such as T4 DNA polymerase or Klenow DNA polymerase. Pfu "filling up" or blunt end was used to increase the number of blunt ends generated after digestion of genomic templates with DNase I. The use of Pfu DNA polymerase for fragment filling will result in filling of the protruding portion. Furthermore, Pfu DNA polymerase does not exhibit DNA elongase activity, but has 3 '→ 5' exonuclease activity, which will result in removal of single and double nucleotide extensions further increasing the number of blunt-ended DNA fragments available for adaptor ligation (Costa, G.L. and M.P.Weiner.1994a. protocols for cloning and analysis of blank-ended PCR-generated DNA fragments. PCRmethods Appl3 (5): S95; Costa, G.L., A.Grafsky and M.P.Weiner.1994b. cloning and analysis of PCR-generated DNA fragments. PCR Methods Appl3 (6): 338; Costa, G.L. and M.P.Weiner.1994c. Polishing wit. PCR-generating DNA fragments. PCR Methods Appl3 (6): 338) h T4 or Pfupolymerase increases the efficiency of cloning of PCR products.NucleicAcids Res.22(12)：2423)。

Adapter ligation

If the nucleic acid library is attached to a solid substrate, the nucleic acid template is then preferably annealed to the anchor primer sequence using well-established techniques (see, e.g., Hatch et al, 1999, Genet. anal. Bionaol. Engineer.15: 35-40; Kool, U.S. Pat. No. 5,714,320 and Lizardi, U.S. Pat. No. 5,854,033). In general, any method of annealing the anchor primer to the template nucleic acid sequence is suitable so long as it results in complete or near complete specific complementarity between the adapter region or anchor primer sequence region and the sequences present in the template library.

In a preferred embodiment, a universal adaptor sequence is added to each DNA fragment after fragmentation and blunt-ending of the DNA library. The universal adaptor is designed to include a set of unique PCR priming regions, typically 20bp in length, located adjacent to a set of unique sequencing priming regions, typically 20bp in length, optionally followed by a discriminating key sequence consisting of at least one of each of the four deoxyribonucleotides (i.e., A, C, G, T). In a preferred embodiment, the discriminating key sequence is 4 bases in length. In another embodiment, the discriminating key sequence may be a combination of 1-4 bases. In yet another embodiment, each unique universal adaptor is forty-four bp (44bp) in length. In a preferred embodiment, universal adaptors are ligated to each end of the DNA fragments using T4 DNA ligase to add a total of 88bp nucleotides to each DNA fragment. Different universal adaptors are designed specifically for each DNA library preparation and will therefore provide a unique identifier for each organism. The size and sequence of the universal adaptors can be modified as will be apparent to those skilled in the art.

For example, to prepare two different universal adaptors (i.e., "first" and "second"), single stranded oligonucleotides can be ordered from a supplier (i.e., Integrated DNA Technologies, IA or operon Technologies, CA). In one embodiment, the universal adaptor oligonucleotide sequence is modified during synthesis with two or three phosphothioester linkages at the 5 'and 3' ends in place of the phosphodiester linkage. Unmodified oligonucleotides are rapidly degraded by nucleases and are therefore of limited utility. Nucleases are enzymes that catalyze the hydrolytic cleavage of a polynucleotide chain by cleaving the phosphodiester bond between nucleotide bases. Thus, one simple and widely used nuclease-resistant chemistry available for oligonucleotide use is phosphothioester bond modification. In a thioester phosphate, a sulfur atom replaces a non-bridging oxygen on the oligonucleotide backbone, making it resistant to all forms of nuclease digestion (i.e., resistant to digestion by both endonucleases and exonucleases). Each oligonucleotide was HPLC purified to ensure that there were no contaminating or spurious oligonucleotide sequences in the synthesized oligonucleotide preparation. Universal adaptors are designed to allow for directed ligation to blunt-ended fragmented DNA. Each set of double-stranded universal adaptors is designed to have a PCR priming region that contains non-complementary 5' four base overhangs that cannot ligate to blunt-ended DNA fragments and prevent ligation to each other at these ends. Thus, binding can only occur between the 3 'end of the adapter and the 5' end of the DNA fragment and between the 3 'end of the DNA fragment and the 5' end of the adapter. Double-stranded universal adaptor sequences are generated by using single-stranded oligonucleotides designed with sequences that allow annealing of substantially complementary oligonucleotide sequences and prevent cross-hybridization between two non-complementary oligonucleotides. In one embodiment, 95% of the universal adaptors are formed by annealing of complementary oligonucleotides. In a preferred embodiment, 97% of the universal adaptors are formed by annealing of complementary oligonucleotides. In a more preferred embodiment, 99% of the universal adaptors are formed by annealing of complementary oligonucleotides. In a most preferred embodiment, 100% of the universal adaptors are formed by annealing of complementary oligonucleotides.

One of the two adapters may be attached to the support binding moiety. In a preferred embodiment, 5' biotin is added to the first universal adaptor to allow subsequent separation of the ssDNA template and non-covalent coupling of the universal adaptor to the surface of a solid support saturated with one biotin-binding protein (i.e., streptavidin, neutravidin, or avidin). Other linkages are well known in the art and may be used in place of biotin-streptavidin (e.g., antibody/epitope, receptor/ligand, and oligonucleotide pairing or complementarity). In one embodiment, the solid support is a bead, preferably a polystyrene bead. In a preferred embodiment, the beads have a diameter of about 2.8 μm. As used herein, such beads are referred to as "sample preparation beads".

Each universal adaptor can be prepared by binding and annealing two ssDNA oligonucleotides, one comprising a sense sequence and the second comprising an antisense (complementary) sequence. A schematic representation of the universal adaptors is presented in FIG. 2.

Isolation of ligation products

Ligation of the universal adaptors results in fragmented DNA formed from adaptors on each end, unbound single adaptors, and adaptor dimers. In a preferred embodiment, agarose gel electrophoresis is used as a method to separate a population of adapter DNA libraries from unligated single adapters and adapter dimers. In other embodiments, the fragments may be isolated by size exclusion chromatography or sucrose sedimentation. The process of DNA digestion by DNase I generally results in a library population of 50-700 bp. In a preferred embodiment, after agarose gel electrophoresis in the presence of a DNA tag, the addition of a set of 88bp universal adaptors will convert the DNA library population to a larger size and will result in a migration pattern of approximately 130-800bp in size; adaptor dimers will migrate to 88 bp; while adapters that are not ligated will migrate to 44 bp. Therefore, many 200-800bp sized double stranded DNA libraries can be physically separated from agarose gels and purified using standard gel extraction techniques. In one embodiment, gel separation of the adapted ligated DNA library will result in recovery of a library population of 200-400bp in size. Other methods of discriminating adaptor-ligated fragments are known to those skilled in the art.

Incision repair

Because the DNA oligonucleotide used for the universal adaptor is not 5 'phosphorylated, there will be gaps in the 3' junctions of the fragmented DNA after ligase treatment (see FIG. 3A). Both "gaps" or "nicks" can be filled in by using a DNA polymerase that can bind, strand displace, and extend the gapped DNA fragments. DNA polymerases that lack 3 '→ 5' exonuclease activity but exhibit 5 '→ 3' exonuclease activity have the ability to recognize nicks, replace nicked strands, and perform strand extension in a manner that results in repair of nicks and formation of non-nicked double-stranded DNA (see fig. 3B and 3C) (Hamilton, s.c., j.w.farchas and m.c.davis.2001.dnapolymerases as enzymes for biotechnology.biotechnology 31: 370).

Several modifying enzymes are used for the nick repair step, including but not limited to polymerases, ligases, and kinases. DNA polymerases that can be used for such use include, for example, escherichia coli (e.coli) DNA polI, thermoanaerobacterium hydrosulfuricus (e.coli) polI, and bacteriophage phi 29. In a preferred embodiment, the strand displacing enzyme Bacillus stearothermophilus polI (BstDNA polymerase I) is used to repair nicked dsDNA and form unnotched dsDNA (see FIG. 3D). In another preferred embodiment, the ligase is T4 and the kinase is a polynucleotide kinase.

Isolation of Single-stranded DNA

After generating unnotched dsDNA, ssDNAs comprising both the first and second adaptor molecules are isolated (the desired population is indicated below with an asterisk; "A" and "B" correspond to the first and second adaptors). A double stranded DNA library will have adapters bound in the following configuration:

universal adaptor A-DNA fragment-Universal adaptor A

Universal adaptor B-DNA fragment-Universal adaptor A^*

Universal adaptor A-DNA fragment-Universal adaptor B^*

Universal adaptor B-DNA fragment-Universal adaptor B

The universal adaptors are designed so that only one universal adaptor has a 5' biotin moiety. For example, if the universal adaptor B has a 5' biotin moiety, streptavidin-coated sample preparation beads can be used to bind all double-stranded DNA library species with adaptor B. A genomic library containing two universal adaptor a species will not contain a 5' biotin moiety and will not bind to streptavidin-containing sample preparation beads and thus can be eluted. The only species that can remain attached to the beads are those with universal adaptor a and B and those with two universal adaptor B sequences. Having two universal adaptor B sequences (i.e., biotin moieties on each 5' end) will bind to the streptavidin-coated sample preparation beads on each end, since each strand contained in the double strand will be bound. Double stranded DNA species having one universal adaptor A and one universal adaptor B will contain one single 5' biotin moiety and will thus only bind to the streptavidin coated beads at one end. The sample preparation beads are magnetic, so when magnetized the sample preparation beads will always be coupled to the solid support. Thus, in the presence of a low salt ("melt" or denatured) solution, only those DNA fragments containing a single universal adaptor A and a single universal adaptor B sequence will release complementary unbound strands. This single-stranded DNA population can be collected and quantified by, for example, pyrophosphate sequencing, real-time quantitative PCR, agarose gel electrophoresis, or capillary gel electrophoresis.

Templates attached to the beads

In one embodiment, ssDNA libraries generated according to the methods of the invention are quantified to calculate the number of molecules per unit volume. These molecules are annealed to a solid support (bead) that contains oligonucleotide capture primers that are complementary to the PCR primer regions at the universal adaptor ends of the ssDNA species. The beads are then transferred to an amplification procedure. The clonal populations of a single species captured on the DNA beads can then be sequenced. In one embodiment, the solid support is a bead, preferably an agarose gel bead. As used herein, such beads are referred to as "DNA capture beads".

Beads as used herein may be of any convenient size and made of any known material. Examples of such materials include: minerals, natural polymers and synthetic polymers. Specific examples of these materials include: cellulose, cellulose derivatives, acrylic resins, glass; silica gel, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, polystyrene crosslinked with divinylbenzene and the like (see, Merrifield Biochemistry 1964, 3, 1385-. In one embodiment, the DNA capture beads are 20-70 μm in diameter. In a preferred embodiment, the DNA capture beads have a diameter of 20-50 μm. In a more preferred embodiment, the DNA capture beads are about 30 μm in diameter.

In one aspect, the invention includes a method of generating a library of solid supports comprising: (a) preparing a population of ssDNA templates according to the methods disclosed herein; (b) attaching each DNA template to a solid support, such that each solid support has a molecule of DNA; (c) amplifying the population of single-stranded templates such that amplification produces a clonal population of each DNA fragment on each solid support; (d) clonal populations of beads were sequenced.

In one embodiment, the solid support is a DNA capture bead. In another embodiment, the DNA is a reverse transcript of genomic DNA, cDNA or viral RNA. The DNA may be attached to a solid support, for example, by biotin-streptavidin linkage, covalent linkage, or by complementary oligonucleotide hybridization. In one embodiment, each DNA template is ligated to a set of universal adaptors. In another embodiment, the universal adaptor pair comprises a common PCR primer sequence, a common sequencing primer sequence and a discriminating key sequence. Isolating single-stranded DNA providing a unique end; the single stranded molecules are then attached to a solid support and amplification techniques are applied to clone the amplified population. The DNA may be amplified by PCR.

In another aspect, the invention provides a library of solid supports made by the methods described herein.

Nucleic acid templates (e.g., DNA templates) prepared by this method can be used for a number of molecular biological assays, such as linear extension, rolling circle amplification, PCR, and sequencing. This method can be implemented in a ligation reaction, for example, by using a high bead to DNA molar ratio. The capture of single-stranded DNA molecules will follow a pulson distribution and will form a small set of beads with no DNA attached and a small set of beads with two molecules of DNA attached. In a preferred embodiment, it will be a bead-on-molecule DNA. Furthermore, it is possible to include other components in the adaptors, which can be used for other operations in the isolation of the library.

2. Nucleic acid template amplification

In order to sequence a nucleic acid template according to the methods of the invention, the copy number must be amplified to generate a sufficient copy number of the template to produce a signal detectable by the light detection method. Any suitable nucleic acid amplification method may be used.

A number of in vitro nucleic acid amplification techniques have been described. These amplification methodologies can be divided into those methods: (i) require temperature cycling Polymerase Chain Reaction (PCR) (see, e.g., Saiki et al, 1995.Science 230: 1350-; q.beta.replicase systems (see, e.g., Lizardi et al, 1988.Biotechnology 6: 1197-; strand displacement amplification Nucleic Acids Res.1992 Apr 11; 20(7): 1691-6; and in PNAS 1992 Jan 1; 89(1): 392-6; and NASBA J Virol methods.1991 Dec; 35(3): 273-86.

In one embodiment, isothermal amplification is used. Isothermal amplification also includes rolling circle-based amplification (RCA). RCA is described, for example, in Kool, U.S. patent No. 5,714,320 and Lizardi, U.S. patent No. 5,854,033; hatch et al, 1999, genet.anal.biomol.enginer.15: 35-40. The result of the RCA is a single DNA strand extended from the 3' end of the anchor primer (and thus attached to the solid support matrix) and comprising a concatemer containing multiple copies of the circular template annealed to the primer sequence. Generally, 1,000 to 10,000 or more copies of circular templates, each having, for example, a size of about 30-500, 50-200, or 60-100 nucleotides, can be obtained with RCA.

The RCA amplification product after annealing of the circular nucleic acid molecule to the anchor primer is illustrated in fig. 11A. The circular template nucleic acid 102 is annealed to an anchor primer 104, which anchor primer 104 has been attached to a surface 106 at its 5 'end and has a free 3' OH for extension. The circular template nucleic acid 102 includes two adapter regions 108 and 110 that are complementary to the sequence region of the anchor primer 104. Also included in the circular template nucleic acid 102 are insert 102 and 114 regions, the 114 region being homologous to a sequencing primer used in the sequencing reaction discussed below.

After annealing, the free 3' -OH on the anchor primer 104 can be extended using sequences in the template nucleic acid 102. The anchor primer 102 may be extended along the template multiple times, each repetition adding to the sequence extending from the anchor primer a sequence complementary to the circular template nucleic acid. Four repeats, or four cycles of rolling circle replication, are shown in FIG. 11A as extended anchored primer amplification products 114. Extension of the anchor primer forms an amplification product that is either covalently or physically attached to the substrate 106. A number of in vitro nucleic acid amplification techniques can be used to extend the anchor primer sequence. The amplification is typically carried out in the presence of a polymerase, e.g., a DNA or RNA directed DNA polymerase, and one, two, three or four types of nucleotide triphosphates, and, optionally, auxiliary binding proteins. In general, any polymerase capable of extending the 3 ' -OH group of a primer can be used as long as it lacks 3 ' to 5 ' exonuclease activity. Suitable polymerases include, for example, those from Bacillus stearothermophilus (Bacillus stearothermophilus), Thermus aquaticus (Thermus aquaticus), Pyrococcus furiosus (Pyrococcus furiosus), Thermococcus litoralis and Thermus thermophilus (Thermus thermophilus), bacteriophages T4 and T7, and the E.coli DNA polymerase IKOW fragment. Suitable RNA-directed DNA polymerases include, for example, reverse transcriptase from avian myeloblastosis virus, reverse transcriptase from Moloney murine leukemia virus, and reverse transcriptase from human immunodeficiency virus-I.

Other embodiments of the circular template and anchor primer are shown in more detail in FIGS. 11B-11D. FIG. 11B is a schematic representation of an annealed open loop linear substrate that can function as an anchor primer extension template after ligation. A template molecule having the sequence 5 ' -tcg tgt gag gtc tca gca tct tat gtatat tta ctt cta ttc tca gtt gcc taa getgca gcca-3 ' (SEQ ID NO: 5) was annealed to an anchor primer having a 5 ' terminal biotin linker and the sequence 5'-gac ctc aca cga tgg ctg cagctt-3' (SEQ ID NO: 6). Annealing of the template results in juxtaposition of the 5 'and 3' ends of the template molecule. The 3' OH of the anchor primer can be extended using a circular template.

The use of the circular template and anchor primer for identifying single nucleotide polymorphisms is shown in FIG. 11C. Shown is a generic anchor primer having the sequence 5'-gac ctc aca cga tgg ctg cag ctt-3' (SEQ ID NO: 7). The anchor primer anneals to a SNP probe having the sequence 5'-ttt ata tgtatt cta cga ctc tgg agt gtg cta ccg acg tcg aat ccg ttg act ctt atc ttc a-3' (SEQ ID NO: 8). The SNP probe is then hybridized to a gene containing the SNP region, which has the sequence 5'-cta gct cgt aca tat aaa tga aga taa gatcct g-3' (SEQ ID NO: 9). Hybridization of the nucleic acid sequence comprising the polymorphism to the SNP probe complex allows for subsequent ligation and circularization of the SNP probe. The SNP probe is designed such that its 5 'and 3' ends anneal to genomic regions, bordering the region of the polymorphic site, as shown in FIG. 11C. The circularized SNP probes can then be extended and sequenced using the methods described herein. Nucleic acids lacking the polymorphism do not hybridize, resulting in juxtaposition of the 5 'and 3' ends of the SNP probe. In this case, the SNP probe cannot be ligated to form a circular substrate required for subsequent extension.

FIG. 11D illustrates the use of a gapped oligonucleotide with a circular template molecule. An anchor primer having the sequence 5'-gac ctc aca cga gta gca tgg ctg cag ctt-3' (SEQ ID NO: 10) was attached to the surface through a biotin linker. A template molecule having sequence 5'-tcg tgt gaggtc tca gcatct tat gta tat tta ctt cta ttc tca gtt gcc taa gct gca gcc a-3' (SEQ ID NO: 11) is annealed to the anchor primer to form a partially single-stranded or gapped region in the anchor primer flanked by double-stranded regions. A nicked molecule having the sequence 5 '-tgc tac-3' is then annealed to the anchor primer. Ligation of both ends of the gapped oligonucleotide to a template molecule results in the formation of a circular nucleic acid molecule that can function as a template for rolling circle amplification.

RCA can occur when replication of the duplex molecule begins at the point of initiation. Subsequently, a nick opens one of the strands and the free 3' -terminal hydroxyl moiety generated by the nick is extended by the action of DNA polymerase. The newly synthesized strand eventually replaces the original parent DNA strand. This type of replication described above is called Rolling Circle Replication (RCR) because the replication point can be imagined as rolling a circle on a circular template strand and, in theory, it can proceed indefinitely. In addition, since the newly synthesized DNA strand is covalently bound to the starting template, the replacement strand has a starting genomic sequence (e.g., a gene or other target sequence) at its 5' -end. In RCR, the starting genomic sequence is followed by any number of "copy units" that are complementary to the starting template sequence, where each copy unit is synthesized by continued rotation of the starting template sequence. Thus, each subsequent rotation replaces the DNA synthesized in the previous replication cycle.

By using RCA reactions, strands representing tandem copies of many circularized molecular complements can be generated. For example, recently, in vitro isothermal cascade amplification reactions using RCA to obtain a circularized padlock probe have been used to detect single copies of genes in human genomic DNA samples (see Lizardi et al, 1998.nat. Genet. 19: 225-. In addition, RCA has also been used to detect single DNA molecules in solid phase-based assays, although difficulties arise when applying this technique to in situ hybridization (see Lizardi et al, 1998.nat. Genet.19: 225-.

If desired, RCA can be performed at elevated temperatures, e.g., at temperatures greater than 37 deg.C, 42 deg.C, 45 deg.C, 50 deg.C, 60 deg.C, or 70 deg.C. In addition, RCA can be initially performed at a lower temperature, e.g., room temperature, followed by a shift to an elevated temperature. The elevated temperature RCA is preferably performed with a thermostable nucleic acid polymerase and primers capable of annealing stably and specifically at elevated temperatures.

RCA may also be performed with non-naturally occurring oligonucleotides, such as peptide nucleic acids. In addition, RCA may be performed in the presence of an accessory protein such as a single-strand binding protein.

The development of a method for amplifying short DNA fragments which have been immobilized on a solid support has recently been described in the literature (see, for example, Hatch et al, 1999, Genet. anal.Biomol. Engineer.15: 35-40; Zhang et al, 1998, Gene 211: 277-85; Baner et al, 1998, Nucl. acids Res.26: 5073-5078; Liu et al, 1995, J.am. chem. Soc.118: 1587-1594; Fire and Xu, 1995, Proc. Natl.Acad. Sci.USA 92: 4641-4645; Nilsson et al, 1994, Science 265: 2085-2088). RCA targets specific DNA sequences by hybridization and a DNA ligase reaction. The cyclic product is then used as a template in a rolling circle replication reaction.

Other examples of isothermal amplification systems include, for example, (i) self-sustained sequence replication (see, e.g., Guatelli et al, 1990.Proc. Natl. Acad. Sci. USA 87: 1874-.

PCR amplification of nucleic acid templates

In a preferred embodiment, polymerase chain reaction ("PCR") is used to generate additional copies of the template nucleic acid. The PCR amplification step may be performed before or after dispensing the nucleic acid template into the picotiter plate.

Bead emulsion PCR amplification

In a preferred embodiment, the PCR amplification step is performed prior to dispensing the nucleic acid template into the picotiter plate.

In a particularly preferred embodiment, the novel amplification system, referred to herein as "bead emulsion amplification", is carried out by attaching the template nucleic acid (e.g., DNA) to be amplified to a solid support, preferably in the form of generally spherical beads. A single-stranded template DNA library prepared according to the sample preparation method of the present invention is a suitable source of a starting nucleic acid template library attached to beads for use in such an amplification method.

The beads are attached to a large number of single primers (i.e., primer B in FIG. 6) that are complementary to a region of the template DNA. Template DNA anneals to the bead-bound primers. The beads are suspended in the reaction mixture in the aqueous phase and subsequently coated in a water-in-oil emulsion. The emulsion consists of droplets of a separate aqueous phase surrounded by a thermostable oil phase, which is about 60 to 200 μm in diameter. Each droplet preferably contains an amplification reaction solution (i.e., reagents necessary for nucleic acid amplification). An example of amplification is a PCR reaction mixture (polymerase, salts, dNTPs) and a pair of PCR primers (primer A and primer B). See, fig. 6A. A subset of the droplet populations also comprise DNA beads containing DNA templates. This subset of droplets is the basis for amplification. Microdroplets not within this subset do not have template DNA and do not participate in amplification. In one embodiment, the amplification technique is PCR and the PCR primers are present in a ratio of 8: 1 or 16: 1 (8 or 16 of one primer to 1 of the second primer) to perform asymmetric PCR.

In general, the DNA was annealed to an oligonucleotide (primer B) immobilized to the bead. During thermocycling (FIG. 6B), the bond between the single-stranded DNA template and the immobilized B primer on the bead is broken, releasing the template into the surrounding micro-coating solution. The amplification solution, in this case a PCR solution, comprises additional solution phase primers a and B. Solution phase B primers bind readily to the complementary B' region of the template because the binding kinetics of the solution phase primers are faster than the immobilized primers. In early PCR, both A and B strands amplified equally well (FIG. 6C).

In the middle PCR (i.e., between cycles 10 and 30) the B primer was depleted, stopping exponential amplification. The reaction then enters asymmetric amplification and the amplicon population becomes dominated by a strands (fig. 6D). In late PCR (FIG. 6E), asymmetric amplification increased the concentration of A strands in solution after 30 to 40 cycles. Excess A strands begin to anneal to the bead immobilized B primers. The thermostable polymerase then synthesizes immobilized, amplicon bead-bound B strands using the a strand as a template.

In the final PCR (FIG. 6F), continued thermal cycling forces further annealing to the bead-bound primers. Solution phase amplification may be minimal at this time but the immobilized B chain concentration increases. Subsequently, the emulsion is broken and the immobilized product is rendered single stranded by denaturation (by heat, pH, etc.) which removes the complementary a strand. The A primer is annealed to the A' region of the immobilized strand and added to the immobilized strand with a sequencing enzyme and any necessary accessory proteins. The beads are then sequenced using accepted pyrophosphate techniques (e.g., as described in U.S. Pat. Nos. 6,274,320, 6,258,568 and 6,210,891, which are incorporated herein by reference in their entirety).

Template design

In a preferred embodiment, the DNA template amplified by the bead emulsion amplification method may be a population of DNAs such as, for example, a genomic DNA library or a cDNA library. Preferably each member of the population has a common nucleic acid sequence at a first end and a common nucleic acid sequence at a second end. This can be achieved, for example, by ligating a first adaptor DNA sequence to one end and a second adaptor DNA sequence to a second end of the population of DNAs. Many DNA and cDNA libraries, by the nature of the cloning vector (e.g., Bluescript, Stratagene, La Jolla, CA), conform to this description by having one common sequence at the first end of each member DNA and a second common sequence at the second end. The DNA template may be any size that enables in vitro amplification, including the preferred amplification techniques PCR and asymmetric PCR. In a preferred embodiment, the DNA template is between about 150 and 750bp in size, such as, for example, about 250bp in size.

Binding nucleic acid template to capture beads

In the first step, a single stranded nucleic acid template to be amplified is attached to a capture bead. The nucleic acid template may be attached to the solid support capture bead in any manner known in the art. There are many methods in the art for attaching DNA to a solid support, such as the preferred beads. According to the present invention, covalent chemical attachment of DNA to the amine-coated capture beads is achieved by linking the 5' -phosphate on the DNA to the bead via a phosphoramide linkage using a standard coupling agent, such as a water-soluble carbodiimide. Another option is to first couple specific oligonucleotide linkers to the beads using similar chemistry, and then attach DNA to the linkers on the beads using DNA ligase. Other attachment chemistries for attaching oligonucleotides to beads include the use of N-hydroxysuccinamide (NHS) and its derivatives. In this method, one end of the oligonucleotide may comprise a reactive group (e.g., an amide group) that forms a covalent bond with the solid support, while the other end of the linker comprises a second reactive group that is capable of binding to the oligonucleotide to be immobilized. In a preferred embodiment, the oligonucleotide is bound to the DNA capture bead by a covalent bond. However, non-covalent linkages, such as chelation or antigen-antibody complexes, may also be used to attach the oligonucleotide to the bead.

Oligonucleotide linkers may be employed which specifically hybridize to sequences at the ends of the DNA fragments, such as overlapping ends from restriction enzyme sites or sticky ends of phage lambda-based cloning vectors, but blunt-ended ligation may also be advantageously employed. These methods are described in detail in us 5,674,743. Preferably any method used to immobilize the beads will continue to bind the immobilized oligonucleotides throughout the steps of the method of the invention.

In one embodiment, each capture bead is designed with a plurality of nucleic acid primers that recognize (i.e., are complementary to) a portion of the nucleic acid template, which thus hybridizes to the capture bead. In the methods described herein, it is desirable to perform clonal amplification of the template species, so that preferably only one unique nucleic acid template is attached to any one capture bead.

Beads as used herein may be of any convenient size and made of any number of known materials. Examples of such materials include: minerals, natural polymers and synthetic polymers. Specific examples of these materials include: cellulose, cellulose derivatives, acrylic resins, glass, silica gel, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, polystyrene crosslinked with divinylbenzene and the like (see Merrifield Biochemistry1964, 3, 1385- Cotton, silica gel, controlled pore (conjugate) glass, metal, cross-linked dextran (e.g., Sephadex)^TM) And agarose gel (Sepharose)^TM) And solid supports known to those skilled in the art. In a preferred embodiment, the capture beads are agarose gel beads having a diameter of about 25-40 μm.

Emulsification action

The capture beads with attached single stranded template nucleic acid are emulsified into a heat stable water-in-oil emulsion. The emulsion may be formed according to any suitable method known in the art. A method of producing an emulsion is described below, but any method of producing an emulsion may be utilized. Such methods are known in the art and include adjuvant, counter-current, cross-flow, rotary drum, and membrane methods. Furthermore, the size of the microcapsules can be adjusted by varying the flow rate and flow rate of the components. For example, in drop-wise addition, the size of the drops and the total delivery time may be varied. Preferably, the emulsion comprises bead "microreactors" having a density of about 3,000 beads/microliter.

The emulsion is preferably generated by suspending the template-attached beads in an amplification solution. As used herein, the term "reaction solution" means a sufficient mixture of reagents necessary to perform amplification of template DNA. An example of an amplification solution is provided in the following example, it being understood that various modifications can be made to the PCR solution.

In one embodiment, the bead/amplification solution mixture is added dropwise to a spinning biocompatible oil (e.g., light mineral oil) mixture and emulsified. The oil used may be supplemented with one or more biocompatible emulsion stabilizers. These emulsion stabilizers may include Atlox 4912, Span 80 and other recognized and commercially available suitable stabilizers. Preferably, the size of the droplets formed is from 5 to 500 microns, more preferably from about 50 to 300 microns, and most preferably from 100 to 150 microns.

The size of the microreactor is not limited. The microreactors should be large enough to contain sufficient amplification reagents for the desired degree of amplification. However, the microreactors should be small enough to enable amplification of a population of microreactors, each microreactor containing a member of a DNA library, by conventional laboratory equipment (e.g., PCR thermal cycling equipment, test tubes, incubators, etc.).

Due to the above limitations, the optimal size of the microreactor may be between 100 and 200 microns in diameter. Microreactors of this size allow amplification of a DNA library comprising approximately 600,000 members in a volume of microreactor suspension of less than 10 ml. For example, if PCR is the amplification method of choice, 10ml fits 96 tubes of a conventional thermal cycler with 96 tube capacity. In a preferred embodiment, the suspension of 600,000 microreactors has a volume of less than 1 ml. Less than 1ml of suspension can be amplified in approximately 10 tubes of a conventional PCR thermal cycler. In a most preferred embodiment, the suspension of 600,000 microreactors has a volume of less than 0.5 ml.

Amplification of

After coating, the template nucleic acid may be amplified by any suitable DNA amplification method including transcription based amplification systems (Kwoh D. et al, Proc. Natl. Acad. Sci. (U.S.A.). 86: 1173 (1989); Gingeras T.R. et al, PCT Application WO 88/10315; Davey, C. et al, European Patent Application publication No.329,822; Miller, H.I. et al, PCT Application WO 89/06700, and "race" (Frohman, M.A., In: PCR Protocols: A Guide Methods and applications, Acadamic Press, NY (1990)) and "one-sided PCR" (Oa, O. et al, Proc. Natl. Acad. Sci. A. 198392, USA) (19883. 1989), as well as other Methods discussed In U.S. Ser. No. 5, Natl. Acad. No. 5, USA et al, (1989) and "Loop PCR amplification Methods (see also U.S. Ser. 3, No. 5, 1989).

In a preferred embodiment, the DNA amplification is performed by PCR. PCR according to the present invention can be performed by coating the target nucleic acid bound to the beads with a PCR solution containing all reagents necessary for PCR. PCR can be achieved by exposing the emulsion to any suitable thermal cycling protocol known in the art. In a preferred embodiment, amplification is performed for 30-50 cycles, preferably about 40 cycles. Preferably, but not necessarily, the amplification process is followed by one or more cycles of hybridization and extension following the amplification cycle. In a preferred embodiment, hybridization and extension is performed for 10-30 cycles, preferably about 25 cycles (e.g., as described in the examples). Conventionally, the template DNA is amplified until at least two million to fifty million copies of template DNA per bead, preferably about one million to thirty million copies per bead, are immobilized.

Recovery of broken emulsions and beads

After template amplification, the emulsion is broken (also known in the art as "demulsification"). There are many ways to break emulsions (see, e.g., U.S. Pat. No. 5,989,892 and references cited therein) and one skilled in the art will be able to select the appropriate method. In the present invention, a preferred method of breaking the emulsion is to add additional oil to cause the emulsion to separate into two phases. The oil phase is then removed and a suitable organic solvent (e.g., hexane) is added. After mixing, the oil/organic solvent phase is removed. This step may be repeated several times. Finally, the aqueous layer above the beads was removed. The beads are then washed with an organic solvent/annealing buffer (e.g., one of the suitable annealing buffers described in the examples) mixture, followed by washing in the annealing buffer. Suitable organic solvents include alcohols such as methanol, ethanol, and the like.

The beads containing the amplified template can then be resuspended in an aqueous solution for use, for example, in a sequencing reaction according to known techniques. (see Sanger, F. et al, Proc. Natl. Acad. Sci. U.S. A.75, 5463-5467 (1977); Maxam, A.M. & Gilbert, W. Proc Natl Acad Sci USA 74, 560-564 (1977); Ronaghi, M. et al, Science281, 363, 365 (1998); Lysov, I. et al, Dokl Akad Nauk SSSR 303, 1508-1511 (1988); Bains W. & Smith G.C.J.TheorBiol 135, 303-307 (1988); Drnanc, R. et al, Genomics 4, 114-128 (1989); Khrapko, K. R. et al, FEBS Lett 256.118-122 (1989); sequencing J. TM., Klotp. A. 1984, 114-128 (1989); Khrapkp. K. R. et al, FEBS Lett 256.118-122; sequencing 1989; VzP. J. Strin PCR) and, if the PCR products were to be removed as described in Ser. 5, PCR primers, PCR, see, PCR, if the primers were used for example, PCR, for example, for the primers, for the PCR, for the template for the.

Briefly, the second chain is melted away using any well-known method, such as NaOH, low ionic (e.g., salt) forces, or heat treatment. After this melting step, the beads were deposited and the supernatant was discarded. The beads were resuspended in annealing buffer, sequencing primers were added, and annealed to single stranded template attached to the beads using standard annealing cycles.

Purification beads

In this regard, the amplified DNA on the beads can be sequenced either directly on the beads or in different reaction vessels. In one embodiment of the invention, the DNA is sequenced directly on the beads by transferring the beads to a reaction vessel and subjecting the DNA to a sequencing reaction (e.g., pyrophosphate or Sanger sequencing). Alternatively, the beads may be isolated and the DNA may be removed from each bead and sequenced. In another case, the sequencing step can be performed on each individual bead. However, this method, although commercially successful and technically feasible, may not be the most efficient, as many beads will be negative beads (beads with no attached amplified DNA). Thus, beads without nucleic acid template can be removed prior to dispensing to picotiter plates using the following optional method.

If the goal of initial DNA attachment is to minimize beads with two different copies of DNA, then a high percentage of beads may be "negative" (i.e., no amplified nucleic acid template attached thereto). For useful pyrosequencing, each bead should contain multiple copies of a single type of DNA. This need is approached by maximizing the total number of beads with a single DNA-binding fragment (prior to amplification). This goal can be achieved by observation of a mathematical model.

For the general case where "N" number of DNAs are randomly distributed among M number of beads, the relative bead population containing any number of DNAs depends on the ratio of N/M. The fraction of beads containing N DNAsR (N) can be calculated using the pussong distribution:

R(N)＝exp-(N/M)×(N/M)^Na/N! (wherein X is a multiplier)

The following table shows some calculated values for various N/M (average DNA fragment to bead ratio) and N (number of fragments actually bound to beads).

N/M	0.1	0.5	1	2
					R(0)	0.9	0.61	0.37	0.13
R(1)	0.09	0.3	0.37	0.27
					R(N＞1)	0.005	0.09	0.26	0.59

The top row in the table represents the various ratios of N/M. R (0) represents the fraction of beads without DNA, R (1) represents the fraction of beads with one attached DNA (before amplification) and R (N > 1) represents the fraction of beads with more than one attached DNA (before amplification).

The table shows that the maximum fraction of beads containing a single DNA fragment is 0.37 (37%) and occurs at a fragment/bead ratio of 1. In this mixture, about 63% of the beads are not useful for sequencing because they have no DNA or more than one species of DNA. Furthermore, controlling the fragment/bead ratio requires complex calculations and variations can produce batches of beads with significantly smaller fractions of available beads.

This inefficiency can be significantly improved if amplicon-containing beads (resulting from binding of at least one fragment) can be separated from those without amplicons (resulting from beads without bound fragments). An amplicon is defined as any nucleic acid molecule produced by an in vitro nucleic acid amplification technique. Binding can be performed at a low average fragment/bead ratio (N/M < 1) to minimize the ratio of beads with more than one DNA bound. The separation step will remove most or all of the beads without DNA, leaving an enriched population of beads with one amplified DNA. These beads can be applied to any sequencing method, such as, for example, pyrosequencing. Since the fraction with one amplicon (N ═ 1) has been enriched, any sequencing method will be more efficient.

As an example, when the average fragment/bead ratio is 0.1, 90% of the beads will have no amplicon, 9% of the beads will have one amplicon and be useful, and 0.5% of the beads will have more than one amplicon. The enrichment process of the present invention will remove 90% of the zero amplicon beads, leaving a population of beads with a sequencable fraction (N ═ 1) of:

1-(0.005/0.09)＝94％.

dilution of the fragments into a bead mixture while isolating the amplicon-containing beads can result in a 2.5-fold higher enrichment than the preferred unenriched method. 94%/37% (see table N/M ═ 1) 2.5. An additional benefit of the enrichment method of the invention is that the final fraction of the sequenceable beads is relatively insensitive to changes in N/M. Thus, the complex calculation to derive the optimized N/M ratio is neither necessary nor can it be done at a lower level of accuracy. This will eventually make the method more suitable for operation by less trained personnel or automation. An additional benefit of the method is that the zero amplicon beads can be recycled and reused. Although recycling is not necessary, it can reduce the cost or overall volume of reagents, making the methods of the invention more suitable for certain purposes, such as, for example, portable sampling, remote robotic sampling, and the like. Furthermore, all the benefits of the method (i.e., less trained personnel, automation, recycling of reagents) will reduce the cost of the method. The method is described in more detail below.

The enrichment method can be used to treat beads that have been amplified in the bead emulsion method above. The amplification is designed such that each amplified molecule contains the same DNA sequence at its 3' end. The nucleotide sequence may be 20 mu but may be any sequence from 15 bases or more, such as 25 bases, 30 bases, 35 bases or 40 bases or more. Naturally, although the longer oligonucleotide ends are functional, they are not required. One skilled in the art can introduce such a DNA sequence into the end of the amplified DNA. For example, if PCR is used for amplification of DNA, the sequence may be part of one member of a PCR primer pair.

A schematic representation of the enrichment process is shown in fig. 7. Here, amplicon bound beads were mixed with 4 blank beads to represent a fragment diluted amplification bead mixture. In step 1, a biotin-labeled primer complementary to the 3' end of the amplicon is annealed to the amplicon. In step 2, a DNA polymerase and four natural deoxyribonucleoside triphosphates (dNTPs) are added to the bead mixture and the biotin-labeled primer is extended. This extension is to enhance the linkage between the biotin-labeled primer and the bead-bound DNA. This step can be omitted if the biotin-labeled primer-DNA linkage is strong (e.g., in a high ionic environment). In step 3, streptavidin-coated beads that are easily attracted by a magnetic field (referred to herein as "magnetic streptavidin beads") are introduced into the bead mixture. Magnetic beads are commercially available from, for example, Dynal (M290). The streptavidin capture moiety binds to biotin of the hybridized amplicon, which then specifically localizes the amplicon bound beads to magnetic streptavidin beads.

In step 5, a magnetic field (given by a magnet) is applied near the reaction mixture, which results in all "magnetic streptavidin beads/amplicon bound bead mixtures" being located along the side of the tube closest to the magnetic field. It is expected that magnetic beads without amplicon binding beads attached will also be located along the same side. Beads without amplicons remain in solution. The bead mixture was washed and beads that were not immobilized by a magnet (i.e., blank beads) were removed and discarded. In step 6, the extended biotin-labeled primer strand is separated from the amplicon strand by "melting" -a step that can be accomplished, for example, by heat or by changing pH. The heat may be 60 ℃ under low salt conditions (e.g., in a low ion environment, such as 0.1 XSSC). The change in pH can be achieved by adding NaOH. The mixture is then washed and the supernatant containing the amplicon bound beads is recovered, while the unbound magnetic beads are retained by the magnetic field. The resulting enrichment beads can be used for DNA sequencing. Note that the primers on the DNA capture beads can be the same as the primers of step 2 above. In this case, annealing of the amplicon-primer complementary strand (with or without extension) is the source of target-capture affinity.

The biotin-streptavidin pairing can be replaced by a variety of capture-target pairings. Two classes are those whose binding pair can then be cleaved and reversibly bound under practically achievable conditions. If cleavage of the target-capture complex is desired, cleavable pairs include thiol-thiol, digoxin/digoxin-resistant, -Captavidin^TM。

Step 2 is optional, as described above. If step 2 is omitted, it may not be necessary to separate the magnetic beads from the amplicon binding beads. Amplicon binding beads with attached magnetic beads can be used directly for sequencing. If sequencing is performed in a single microwell, separation would not be necessary if the amplicon bound bead-magnetic bead complexes could be placed in the microwell.

Although magnetic capture beads are convenient to use, the capture moiety can be bound to other surfaces. For example, streptavidin can be chemically bound to a surface, such as the inner surface of a test tube. In this case, the amplification bead mixture may be flowed through. Amplicon bound beads will tend to remain until "melted" while white beads will flow through. This arrangement may be particularly advantageous for automating the bead preparation process.

Although the above embodiments are particularly useful, other methods for separating beads are also contemplated. For example, the capture beads can be labeled with a fluorescent moiety that will cause the target-capture bead complexes to fluoresce. Target-capture bead complexes can be isolated by flow cytometry or fluorescent cell sorting. The use of large capture beads will allow separation by filtration or other particle size separation techniques. Since both the capture and target beads are capable of forming complexes with a variety of other beads, it is possible to aggregate into a mass of cross-linked capture-target beads. Large clumps will make separation possible by washing away only non-clumped blank beads. The methods are described, for example, in Bauer, j; chromatology B, 722(1999)55-69 and Brody et al, Applied Physics Lett.74(1999) 144-146.

The DNA capture beads, each containing multiple copies of a single species of nucleic acid template prepared according to the above method, are then suitable for distribution onto picotiter plates.

Nucleic acid amplification on picotiter plates

In an alternative embodiment, the nucleic acid template is distributed onto the picotiter plate prior to amplification and subsequently amplified in situ on the picotiter plate. This method is described in detail in the examples.

3. Sequencing a nucleic acid template

The method according to the invention utilizes pyrosequencing to sequence nucleic acid templates. This technique is based on the detection of pyrophosphate released during DNA synthesis. See, for example, Hyman, 1988.A new method of sequencing DNA. anal biochem.174: 423-36; ronaghi, 2001.Pyrosequencing shades light on DNA sequencing. genome res.11: 3-11.

In the cascade of enzymatic reactions, visible light is produced in proportion to the number of incorporated nucleotides. The cascade is initiated with a nucleic acid polymerization reaction in which the inorganic Ppi is released following incorporation of nucleotides by the polymerase. The released Ppi is converted to ATP by ATP sulfurylase, which provides energy to the luciferase to oxidize luciferin and generate light. Since the added nucleotides are known, the sequence of the template can be determined. Solid phase pyrophosphate sequencing utilizes immobilized DNA in a three-enzyme system (see figure). To enhance signal to noise ratio, dATP α S was substituted for native dATP. Typically dATP α S is a mixture of two isomers (Sp and Rp); the use of pure 2 ' -deoxyadenylate-5 ' -O ' - (1-thiotriphosphate) Sp-isomer in pyrophosphate sequencing allows for longer reads up to twice the read length.

4. Device for sequencing nucleic acids

The present invention provides an apparatus for sequencing nucleic acids, which generally comprises one or more reaction chambers for performing a sequencing reaction, means for delivering reactants into and out of the reaction chambers, and means for detecting sequencing reaction events. In another embodiment, the device includes a reagent delivery container comprising a plurality of chambers on a planar surface. In a preferred embodiment, the device is connected to at least one computer to control the individual components of the device and to store and/or analyze information obtained from the detection of sequencing reaction events.

The invention also provides one or more reaction chambers arranged on an inert matrix material, also referred to herein as a "solid support", which allows discrete localization of nucleic acid templates and reactants in a sequencing reaction in a defined space and allows detection of sequencing reaction events. Thus, as used herein, the term "reaction chamber" or "analyte reaction chamber" refers to a localized region on a matrix material that facilitates, for example, the interaction of reactants in a nucleic acid sequencing reaction. As discussed in more detail below, the sequencing reactions claimed herein preferably occur on multiple, serially-connected, individual nucleic acid samples, particularly simultaneously sequencing multiple nucleic acid samples derived from genomic and chromosomal nucleic acid templates (e.g., DNA).

The device of the invention preferably comprises a sufficient number of reaction chambers to perform such a plurality of individual sequencing reactions. In one embodiment, there are at least 10,000 reaction chambers, preferably at least 50,000 reaction chambers, more preferably more than 100,000 reaction chambers, even more preferably more than 200,000 reaction chambers.

Since the number of simultaneous sequencing reactions is limited by the number of reaction chambers, throughput can be increased by assembling plates containing wells of increased density. The following table shows this process for 14X 43mm and 30X 60mm active areas derived from 25X 75mm and 40X 75mm arrays, respectively.

Table: development of higher hole number arrays

Distance between each other	Diameter of hole	Number of holes	Number of holes
				(um)	(um)	(14×43mm)	(30×60mm)
50	44	275K	800K
				43	38	375K	1.2M
35	31	575K	1.6M
				25	22	1.1M	3.2M

The reaction chambers on the array are typically in the form of cavities or wells in the matrix material, having a width and depth that enables the reactants to be placed. Nucleic acid templates are typically dispensed into reaction chambers on one or more solid supports or beads; the reactants are in a medium that facilitates the reaction and flow through the reaction chamber. When in the form of a cavity or well, it is preferred that the chambers be of sufficient size and order so that the necessary reactants can be (i) introduced into the chamber, (ii) reacted in the chamber and (iii) prevented from mixing between the chambers. The shape of the aperture or cavity is preferably circular or cylindrical, but may be a shape having multiple faces so as to approximate a circle or cylinder. In a preferred embodiment, the shape of the aperture or cavity is substantially hexagonal. The cavity can have a smooth wall surface. In a further embodiment, the cavity can have at least one irregular wall surface. The cavity can have a flat bottom or a concave bottom.

The reaction chambers may be spaced apart by a distance of between 5 μm and 200 μm. The separation distance is determined by measuring the center-to-center distance of two adjacent reaction chambers. In general, the reaction chambers may be spaced apart by a distance of between 10 μm and 150 μm, preferably between 20 μm and 100 μm, most preferably between 40 μm and 60 μm. In one embodiment, the reaction chamber has a width (diameter) between 0.3 μm and 100 μm, more preferably between 20 μm and 70 μm, most preferably between 30 μm and 50 μm. The depth of the reaction chamber is preferably between 10 μm and 100 μm, preferably between 20 μm and 60 μm. Alternatively, the reaction chamber may have a depth of between 0.25 and 5 times the width of a reaction chamber, or, in another embodiment, between 0.3 and 1 times the width of a reaction chamber.

In a preferred embodiment, the array is formed from a cut fiber bundle (i.e., a fused bundle of fibers) and the reaction chambers are formed by etching one surface of the fiber reactor array. Cavities may also be formed in the matrix by etching, molding or micromolding.

Each cavity or reaction chamber typically has a depth of between 10 μm and 100 μm; alternatively, the depth is between 0.25 and 5 times the cavity width dimension, preferably between 0.3 and 1 times the cavity width dimension.

In one embodiment, the array described herein generally includes a flat top surface and a flat bottom surface, which is light conductive, so that light signals from the reaction chamber can be detected through the bottom flat surface. In these arrays, the distance between the top and bottom surfaces is typically no more than 10cm, preferably no more than 2cm, typically between 0.5mm and 5mm, most preferably about 2 mm.

In a particularly preferred embodiment, the solid support is referred to as a picotiter plate, the reaction chambers have a center-to-center spacing of about 43 μm to 50 μm, a pore diameter of between 38 μm to 44 μm, and a pore volume of between 10 to 150pL, preferably between 20 to 90pL, more preferably between 40 to 85pL, and most preferably about 75 pL.

In one embodiment, each chamber or reaction chamber of the array contains reagents for analyzing nucleic acids or proteins. Typically, those reaction chambers that contain nucleic acids (not all reaction chambers in the array need be) contain only a single species of nucleic acid (i.e., a single sequence of interest). There may be a single copy of such a nucleic acid in any particular reaction chamber, or there may be multiple copies. It is generally preferred that the reaction chamber contains at least 100,000 copies of the nucleic acid template sequence, preferably at least 1,000,000 copies, more preferably between 2,000,000 and 20,000,000 copies, and most preferably between 5,000,000 and 15,000,000 copies of the nucleic acid. The ordinarily skilled artisan will appreciate that variations in the copy number of nucleic acids in any one reaction chamber will affect the number of photons generated in a pyrosequencing reaction and can be routinely adjusted to provide more or less photon signals than are needed. In one embodiment, the nucleic acid is amplified to provide the desired number of copies using PCR, RCA, ligase chain reaction, other isothermal amplification, or other conventional methods of nucleic acid amplification. In one embodiment, the nucleic acid is single stranded.

Solid support material

Any material can be used as the solid support material as long as the surface allows for stable attachment of primers and detection of nucleic acid sequences. The solid support material may be flat or may be cavitated, e.g., at the end of a cavitated fiber or micro-wells etched, patterned or otherwise micro-formed to a flat surface, e.g., using techniques commonly used in the construction of microelectromechanical systems. See, for example, Rai-Choudhury, HANDBOOK OF MICROLITHOGRAPHY, MICROMACHINING, AND MICROFABRICATION, VOLUME 1: MICROLITHOGRAPHY, Volume PM39, SPIE Press (1997); madou, CRC Press (1997), Aoki, biotech. 98-9 (1992); kane et al, biomaterials.20: 2363-76 (1999); deng et al, anal. clam.72: 3176-80 (2000); zhu et al, nat. genet.26: 283-9(2000). In some embodiments, the solid support is optically transparent, e.g., glass.

Lithographic techniques commonly used in electronic integrated circuit construction can be utilized, as described in, for example, U.S. Pat. nos. 5,143,854, 5,445,934, 5,744,305, and 5,800,992; chee et al, Science 274: 610-614 (1996); fodor et al, Nature 364: 555-; fodor et al, Science 251: 767 773 (1991); gushin et al, anal. biocerrz.250: 203-211 (1997); kinosita et al, Cell 93: 21-24 (1998); Kato-Yamada et al, J.biol.chem.273: 19375-19377 (1998); and Yasuda et al, Cell 93: 1117-1124(1998) to construct an array of attachment sites on an optically transparent solid support. Photolithography and electron beam lithography render solid supports or substrates with linking groups that allow attachment of modified biomolecules (e.g., proteins or nucleic acids) susceptible to light. See, for example, Service, Science 283: 27-28 (1999); Rai-Choudhury, HANDBOOK OF MICROLITHOGRAPHY, MICROMACHINING, ANDMICROFABRICATION, VOLUME 1: MICROLITHOGRAPHY, Volume PM39, SPIE Press (1997) or, alternatively, can utilize a method such as that described in Zaradzinski et al, Science 263: 1726 and 1733(1994) to produce an array of photosites.

The matrix material is preferably made of a material that facilitates detection of a reaction event. For example, in a typical sequencing reaction, binding of dntps to sample nucleic acids to be sequenced can be monitored by the detection of photons generated by the action of enzymes on phosphate released in the sequencing reaction. Thus, having a matrix material made of a transparent or light-conducting material facilitates the detection of photons.

In certain embodiments, the solid support may be coupled to a bundle of optical fibers that are used to detect and transmit the photoproducts. The total number of fibers within the bundle can be varied to match the number of individual reaction chambers in the array for the sequencing reaction. The number of fibers incorporated into the bundle is designed to match the resolution of the detection device, allowing 1: 1 imaging. The overall size of the bundle is selected so as to optimize the usable area of the detection device while maintaining the desired reagent (flow) characteristics in the reaction chamber. Thus, for a 4096 × 4096 pixel CCD (electrically coupled device) array having 15 μm pixels, the fiber bundle is selected to be about 60mm × 60mm or to have a diameter of about 90 mm. The desired number of fibers are initially fused into a bundle or array of fibers, the ends of which may then be cut and polished to form a "slice" of the desired thickness (e.g., 1.5 mm). The resulting optical fiber sheet has handling characteristics similar to the glass plane. The individual fibers may be of any size diameter (e.g., 3 μm to 100 μm).

In some embodiments, two fiber bundles are used: the first bundle is attached directly to the detection device (also referred to herein as a fiber bundle or linker) and the second bundle is used as a reaction chamber matrix (sheet or substrate). In this case, the two are optionally placed in direct contact with a light coupling fluid, thereby imaging the reaction centers onto the detection device. If a CCD is used as the detection device, the sheet may be slightly larger to maximize the use of the CCD area, or slightly smaller to match the format of a typical microscope slide-25 mm by 75 mm. Within the limits of the state of the art, the diameter of the individual fibers within the bundle is chosen to maximize the likelihood that a single reaction will be imaged onto a single pixel in the detection device. An exemplary diameter of the fiber bundle is 6-8 μm and the flake is 6-50 μm, although any diameter of 3-100 μm can be used. Fiber bundles are commercially available from CCD camera manufacturers. For example, the sheet can be obtained from Incom, inc. (Charlton, MA) and cut and polished from a large fusion of optical fibers, typically 2mm thick, although perhaps 0.5 to 5mm thick. The resulting optical fiber chip has similar processing characteristics to glass flat or glass microscope slides.

The reaction chamber can be formed in a matrix made of fiber optic material. The cavity is formed in the surface of the fiber by, for example, treating the end of the fiber bundle with an acid to form a depression in the fiber material. Thus, in one embodiment, the cavity is formed by a fiber optic bundle, preferably by etching one end of the fiber optic bundle. Each cavitated surface is capable of forming a reaction chamber. Such an array is referred to herein as a fiber optic reactor array or FORA. The depth of the recess is from about half the diameter of a single optical fiber to two to three times the diameter of the fiber. The lumen can be introduced into the end of the fiber by placing one side of the optical fiber sheet in an acid bath for varying periods of time. The length of time may vary depending on the overall depth of the reaction chamber desired (see, e.g., Walt et al, 1996.arzal. clz. em. 70: 1888). The wide slot chamber can have a uniform flow rate dimension of about 14mm x 43 mm. Thus, with this approximate flow rate scale and about 4.82 × 10^-4Cavity/. mu.m²Density, the device can have about 290,000 chambers accessible to the fluid. Several methods are known in the art for attaching molecules to cavities (or detecting attached molecules) etched into the ends of fiber optic strands. See, for example, Michael et al, anal. chem.70: 1242-1248 (1998); ferguson et al, Nature Biotechnology 14: 1681-; healey and Walt, anal. chem.69: 2213-2216(1997). A pattern of reactive sites can also be created in the microwells using photolithographic techniques similar to those used to create a reaction patch on a flat support. See, Healey et al, Science 269: 1078 1080 (1995); munkholm and Walt, anal.cher7n.58: 1427-1430(1986), and Bronk et al, anal. chem.67: 2750-27 57(1995)。

The opposite side (i.e., the unetched side) of the optical fiber wafer is typically highly polished to allow optical coupling (e.g., by immersion in oil or other optical joining fluid) to the second optical fiber bundle. This second fiber bundle exactly matches the diameter of the optical wafer containing the reaction chamber and serves as a conduit to deliver the photo-product to an attached detection device, such as a CCD imaging system or camera.

In a preferred embodiment, for example, by 15% H in aqueous solution₂O₂/15％NH₄OH volume: volume, then six deionized water rinses, then 0.5M EDTA, then six deionized water rinses, then 15% H₂O₂/15％NH₄OH followed by six successive washes of deionized water (incubation for one and a half hours in each wash) cleaned the optical fiber sheet thoroughly.

The surface of the optical fiber sheet is preferably coated to facilitate its use in sequencing reactions. The coated surface is preferably optically clear, allowing easy attachment of proteins and nucleic acids, and does not negatively affect the activity of immobilized proteins. In addition, the surface preferably minimizes non-specific adsorption of macromolecules and enhances the stability of attached macromolecules (e.g., attached nucleic acids and proteins).

Suitable materials for coating the array include, for example, plastics (e.g., polystyrene). The plastic may preferably be spin coated or sprayed (0.1 μm thick). Other materials for coating the array include gold layers, such as 24K gold, 0.1 μm thick, with adsorbed self-assembled monolayers of long chain mercaptoalkanes. Biotin is then covalently coupled to the surface and saturated with biotin-binding proteins (e.g., streptavidin or avidin).

Coating materials may additionally include those systems used to attach anchor primers to substrates. It is also possible to coat the array with organosilanes that allow direct covalent coupling through amino, thiol or carboxyl groups. Other coating substances include photoreactive linkers such as photobiotin, (Amos et al, "biomedical Surface Modification Using macromolecular coupling Technology," encyclopedic Handbook of Biomaterials and bioinformation reering, Part A: Materials, Wise et al (ed.), New York, Marcel Dekker, p. 895926, 1995).

Other coating materials include hydrophilic polymer gels (polyacrylamides, polysaccharides), preferably polymerized directly on the surface, or polymer chains covalently attached after polymerization (Hjerten, J.chromatogr.347, 191 (1985); Novotny, 4naL Cliem.62, 2478(1990), and polymers (triblock copolymers, such as PPO-PEO-PPO, also known as F-108) that specifically adsorb to polystyrene or silanized glass surfaces (Ho et al, Lang7nuir 14: 3889-94, 1998), and passive adsorption layers of biotin-binding proteins.

In addition, the above materials can also be derivatized with one or more functional groups that are well known in the art for enzyme and nucleotide immobilization, for example, metal chelating groups (e.g., nitrilotriacetate, iminodiacetic acid, pentadentate chelators) that bind 6 × His-tagged proteins and nucleic acids.

Surface coatings that increase the available binding sites for subsequent processing, such as attachment of enzymes (discussed later), can be used beyond the theoretical binding capacity of 2D surfaces.

In a preferred embodiment, the individual fiber diameters (i.e., 6 μm to 12 μm) used to create the fused fiber bundle/sheet are larger than those used in optical imaging systems (i.e., 3 μm). Thus, a single reaction site can be imaged using several optical imaging fibers.

In a particularly preferred embodiment, a plate known as the PicoTiter plate^TMThe sample cartridge for nucleic acid template sequencing of (a) was formed from a commercial fiber optic faceplate, acid etched to produce the well structure. Each optical fiber core is about 44 microns in diameter, has a cladding of 2-3 microns, and is etched by acidEach well was formed to form a reaction well volume of about 65pL-85pL, most preferably about 75 pL. The use of etched holes on the fiber optic faceplate surface has three purposes: i) delaying the diffusion of the emitted light from different areas of the array; ii) separation of the reaction chamber ("tube") containing the amplified template molecules, and iii) a very efficient high power aperture coupled to the CCD. Finally, the larger the number of sequencing templates fixed within the well, the more optical signals can be obtained.

Delivery tool

One example of a means for delivering reactants into a reaction chamber is a perfusion chamber of the present invention, shown in FIG. 13. The perfusion chamber comprises a closed cell having transparent upper and lower sides. It is designed to allow the flow of solutions on the substrate surface and to allow rapid exchange of reagents. Thus, it is suitable for performing, for example, a pyrosequencing reaction. The chamber shape and size can be adjusted to optimize reagent exchange to include mass flow exchange, diffusive exchange, or in laminar or turbulent flow regimes.

The perfusion chamber is preferably detached from the imaging system when it is being prepared and placed on the imaging system only when sequencing analysis is being performed. In one embodiment, the solid support (i.e., a DNA chip or glass slide) is placed with a metal or plastic housing that can be assembled and disassembled to allow replacement of the solid support. The lower side of the solid support of the perfusion chamber carries an array of reaction chambers and the image of the array of reaction centers is focused onto a CCD imaging system using a conventional optical-based focusing system using a high-power aperture objective.

Thereby enabling analysis of many samples in parallel. Using the method of the invention, a number of nucleic acid templates can be analysed in this way by allowing a solution comprising an enzyme and one nucleotide to flow over the surface and subsequently detecting the signal generated by each sample. This process may then be repeated. Alternatively, several different oligonucleotides complementary to the template may be distributed on the surface, followed by hybridization of the template. The incorporation of deoxynucleotides or dideoxynucleotides can be monitored for each oligonucleotide by using the signals generated by the various oligonucleotides as primers. By combining signals from different regions of the surface, sequence-based analysis can be performed by four cycles of polymerase reactions using various dideoxynucleotides.

When the support is present in the form of a cavitated array, for example, at the end of a picotiter plate or other microwell array, suitable delivery means include flow and wash and also, for example, flow, spray, electrospray, inkjet delivery, stamping, ultrasonic atomization (Sonotek corp., Milton, NY) and rolling. When Spraying is used, the reagents can be delivered in a uniform thin layer onto the picotiter plate by either industrial-type nozzles (Spraying Systems, co., Wheaton, IL) or nebulizers for Thin Layer Chromatography (TLC), such as a CAMAG TLC nebulizer (CAMAG Scientific inc., Wilmington, NC). These sprayers spray the reagents into aerosol particles of 0.3-10 μm in size.

The successive reagent delivery steps are preferably separated by washing steps using techniques well known in the art. These washes can be performed, for example, using the methods described above, including high velocity flow nebulizers, or by liquid flow over picotiter plates or microwell arrays. Washing can occur at any time after the starting material reacts with the reagent to form a product in each reaction chamber, but before the reagent delivered to any one reaction chamber diffuses out of that reaction chamber into any other reaction chamber. In one embodiment, any one reaction chamber is free of products formed in any other reaction chamber, but the products are made using one or more co-reagents.

One embodiment of the complete device is illustrated in fig. 12. The device includes an inlet conduit 200 connected to a detachable perfusion chamber 226. The inlet conduit 200 allows for the entry of sequencing reagents through a plurality of tubes 202 and 212, each of which is connected to a plurality of sequencing formulation reagent containers 214 and 224.

Reagent is introduced into perfusion chamber 226 through conduit 200 using a pressurizing system or pump driven forward flow. Typically, the reagent flow rate is 0.05-50 ml/min (e.g., 1-50 ml/min), and the volume is 0.100ml to constant flow (washing). The valves are under computer control to allow circulation of the nucleotides and wash reagents. Sequencing reagents, such as polymerases, can be either pre-mixed with nucleotides or added to the aqueous stream. The manifold combines all six tubes 202 and 212 into one for feeding the perfusion chamber. Thus allowing several reagent delivery ports into the perfusion chamber. For example, one of the ports may be used to allow the input of aqueous sequencing reagents while the other port allows these reagents (and any reaction products) to be aspirated from the perfusion chamber.

In a preferred embodiment, one or more reagents are delivered to a fixed array or an array attached to a population of mobile solid supports, such as beads or microspheres. The beads or microspheres need not be spherical and irregularly shaped beads may be used. They are generally constructed of a variety of substances, such as plastics, glass or ceramics, and the size of the beads varies from nanometers to millimeters depending on the width of the reaction chamber. A variety of bead compounds may be used, for example, toluylene, polystyrene, acrylic polymers, latex, paramagnetic, thoria sol, carbon graphite and titanium dioxide. The construction or chemistry of the beads can be selected to facilitate attachment of the desired reagents.

In another embodiment, the bioactive agent is first synthesized and subsequently covalently attached to the bead. As will be appreciated by those skilled in the art, this will be done depending on the composition of the bioactive agent and the bead. The functionalization of solid support surfaces, such as certain polymers having chemically reactive groups such as sulfhydryl, amine, carbonyl, and the like, is generally known in the art.

In a preferred embodiment, the nucleic acid template is delivered to a picotiter plate on a bead. Luciferase and sulfurylase were delivered to each well on the beads as was DNA polymerase (see figure). It is noted that one or more of the nucleic acid template, luciferase and sulfurylase may be delivered separately on separate beads, or together on the same bead. To allow sequencing at elevated temperatures, we cloned and modified thermostable sulfurylase from Bacillus stearothermophilus. We also cloned and modified several immobilized enzyme active luciferases, including both fluorescent electrons and fireflies. In a preferred embodiment firefly luciferase is used.

"blank" beads can be used that have a surface chemistry that promotes the attachment of functional groups desired by the user. Other examples of such blank bead surface chemistries include, but are not limited to, amino groups including aliphatic and aromatic amines, carboxylic acids, aldehydes, amides, chloromethyl groups, hydrazides, hydroxyl groups, sulfonates, and sulfates.

These functional groups can be used to add any number of different candidate agents to the bead, typically using known chemical methods. For example, a candidate agent comprising a saccharide can be attached to an amino-functionalized support; the furfural is made using standard techniques and subsequently reacted with amino groups on the surface. In an alternative embodiment, a sulfhydryl linker may be used. There are many thiol-reactive linkers in the art, such as SPDP, maleimide, alpha-haloacetyl and pyridyl disulfide (see, e.g., 1994Pierce Chemical Company catalog, technical section on cross-linkers, page 155-200, incorporated herein by reference), which can be used to attach cysteines containing protein preparations to a support. Alternatively, the amino groups on the candidate agent can be used to attach to amino groups on the surface. For example, a number of stable bifunctional groups are known in the art, including homobifunctional and heterobifunctional linkers (see Pierce Catalog and Handbook, p. 155-). In another embodiment, carboxyl groups (from the surface or from candidate agents) may be derivatized using well-known linkers (see Pierce catalog). For example, carbodiimide activated carboxyl groups are attacked by good nucleophiles such as amines (see Torchilin et al, clinical Rev. Therepapeutic Drug Carrier Systems, 7 (4): 275-308 (1991)). Protein candidates can also be attached using other techniques known in the art, e.g., antibodies attached to polymers, see Slinkin et al, biocoyy. cliem.2: 342-; torchilin et al, supra; trubetskoy et al, Bioconjugate chem.5: 220-235(1994). It should be understood that the candidate agents may be attached in a variety of ways, including those listed above. Preferably, the manner of attachment does not significantly alter the functionality of the candidate agent; i.e. it should be attached in such a flexible way as to allow it to interact with the target.

Specific techniques for immobilizing enzymes on beads are known in the art. In one case, NH may be utilized₂The surface is chemically beaded. Surface activation was achieved with 2.5% glutaraldehyde in phosphate buffered saline (10mM) which provided a pH of 6.9 (138mM NaCl, 2.7mM KC 1). The mixture was stirred on a shaker at room temperature for about 2 hours. The beads were then washed with ultrapure water plus 0.01% -0.02% Tween 20 (surfactant) and washed again with pH7.7 PBS plus 0.01% Tween 20. Finally, the enzyme is added to the solution, preferably after prefiltration using a 0.45 μm amicon micro-pure filter.

The moving population of solid supports is placed in a reaction chamber. In certain embodiments, 5% to 20% of the reaction chambers may have mobile solid supports, 20% to 60% of the reaction chambers may have mobile solid supports or 50% to 100% of the reaction chambers may have mobile solid supports with at least one reagent immobilized thereon. Preferably, at least one reaction chamber has a mobile solid support having at least one reagent immobilized thereon and adapted for use in a nucleic acid sequencing reaction.

In certain embodiments, the reagent immobilized on the mobile solid support may be a polypeptide having sulfurylase activity, a polypeptide having luciferase activity, or a chimeric polypeptide having both sulfurylase and luciferase activities. In one embodiment, it may be a fusion protein of ATP sulfurylase and luciferase. Since the sulfurylase reaction product is consumed by luciferase, access to the two enzymes can be achieved by covalently linking the two enzymes in the form of a fusion protein. The invention can be used not only to open up substrate channels, but also to reduce product cost and possibly double the binding sites on streptavidin-coated beads.

In another embodiment, the sulfurylase is a thermostable ATP sulfurylase. In a preferred embodiment, the thermostable sulfurylase is active at temperatures above ambient (at least 50 ℃). In one embodiment, the ATP sulfurylase is from a thermophilic organism (thermophile). In another embodiment, the mobile solid support may have immobilized thereon a first reagent which is a polypeptide having sulfurylase activity and a second reagent which is a polypeptide having luciferase activity.

In another embodiment, the reagent immobilized on the mobile solid support may be a nucleic acid; preferably the nucleic acid is a single stranded concatemer. In a preferred embodiment, the nucleic acid may be used to sequence a nucleic acid, for example, a pyrosequencing reaction.

The invention also provides a method for detecting or quantifying ATP activity using a moving solid support; ATP may preferably be detected or quantified as part of a nucleic acid sequencing reaction.

A picotiter plate that has been "covered" with a moving solid support having nucleic acids or reagent enzymes attached thereto is shown in FIG. 15.

5. Method for sequencing nucleic acids

Followed by pyrophosphate-based sequencing. The sample DNA sequence and extension primer are then subjected to a polymerase reaction in the presence of a nucleotide triphosphate, which is added to a separate aliquot of the sample-primer mixture or continued to the same sample-primer mixture, whereby if the nucleotide triphosphate is complementary to the base of the target position, it will be incorporated and release pyrophosphate (PPi). The release of PPi is then detected to indicate which nucleotide was incorporated.

In one embodiment, a region of the sequence product is determined by annealing a sequencing primer to a region of the template nucleic acid, and subsequently contacting the sequencing primer with a DNA polymerase and a known nucleotide triphosphate, i.e., dATP, dCTP, dGTP, dTTP, or an analog of one of these nucleotides. The sequence can be determined by detecting the by-products of the sequence reaction as described below.

The sequencing primer can be of any length or base composition so long as it is capable of specifically annealing to a region of the amplified nucleic acid template. No particular structure is required for the sequencing primer, so long as it is capable of specifically directing amplification of a region on the template nucleic acid. Preferably, the sequencing primer is complementary to a region of the template that is intermediate between the sequence to be tested and the sequence that can hybridize to the anchor primer. The sequencing primer is extended with a DNA polymerase to form a sequence product. Extension is carried out in the presence of one or more types of nucleotide triphosphates and, if desired, auxiliary binding proteins.

Incorporation of dNTPs is preferably determined by testing for the presence of sequencing by-products. In a preferred embodiment, the nucleotide sequence of the sequencing product is determined by measuring inorganic pyrophosphate (PPi) that is released by nucleotide triphosphates (dNTPs) when the dNMP is incorporated into the extended sequence primer. This sequencing method, known as PyroSequencing AB (Stockholm, Sweden), can be carried out in solution (liquid phase) or as a solid phase technique. In, for example, W09813523a1, Ronaghi et al, 1996, anal. biochem.242: 84-89, Ronaghi et al, 1998.Science 281: PPi-based sequencing methods are generally described in 363-365(1998) and USSN 2001/0024790. The entire disclosure of these PPi sequencing disclosures is incorporated herein by reference. See also, for example, U.S. Pat. Nos. 6,210,891 and 6,258,568, each of which is incorporated herein by reference in its entirety.

The pyrophosphate released under these conditions can be measured enzymatically (e.g., by light generation in the luciferase-luciferin reaction). These methods allow nucleotides to be identified at a given target site and allow for simple and rapid sequencing of DNA without the need for electrophoresis and the use of potentially dangerous radiolabels.

PPi can be detected by a variety of different methodologies, and a variety of enzymatic methods have been previously described (see, e.g., Reeves et al, 1969.anal. biochem. 28: 282-287; Guillory et al, 1971.anal. biochem. 39: 170-180; Johnson et al, 1968.anal. biochem. 15: 273; Cook et al, 1978.anal. biochem. 91: 557-565; and Drake et al, 1979.anal. biochem. 94: 117-120).

PPi released as a result of incorporation of dNTPs by a polymerase can be converted to ATP using, for example, ATP sulfurylase. This enzyme has been identified as involved in sulfur metabolism. Both reduced and oxidized forms of sulfur are mineral nutrients essential for plant and animal growth (see, e.g., Schmidt and Jager, 1992, Ann. Rev. plant Physiol. plant mol. biol. 43: 325-349). In both plants and microorganisms, the active uptake of sulfate is followed by reduction to sulfide. Since sulfide has a very low oxidation/reduction potential relative to existing cellular reductants, the initial step of assimilation requires its activation by ATP-dependent reactions (see, e.g., Leyh, 1993, Crit. Rev. biochem. mol. biol. 28: 515-542). ATP sulfurylase (ATP: sulfate adenylyltransferase; EC 2.7.7.4) catalyzes the initiation reaction in inorganic sulfate metabolism (SO 4) ^-2) (ii) a See, e.g., Robbins and Lipmann, 1958. j.biol.chem.233: 686 + 690; hawes and Nicholas, 1973, biochem.j.133: 541-550). SO4 in this reaction^-2Is activated to adenosine 5' -phosphate sulfate (APS).

ATP sulfurylase has been highly purified from several sources, such as Saccharomyces cerevisiae (Saccharomyces cerevisiae) (see, e.g., Hawes and Nicholas, 1973, Brochett. J.133: 541 550); penicillinba chrysogenum (see, e.g., Renoso et al, 1990.J biol. chem.265: 10300-; rat liver (see, e.g., Yu et al, 1989.Arch. biochem. Biophys.269: 165-174); and plants (see, e.g., Shaw and Anderson, 1972.biochem. J.127: 237-. Furthermore, it has been described from prokaryotes (see, e.g., Leyh et al, 1992, J.biol.Cllem.267: 10405-; eukaryotes (see, e.g., Cherest et al, 1987. mol.Gen.Genet.210: 307-; plants (see, e.g., Leustek et al, 1994. plantaphysiol.105: 897-; and animals (see, e.g., Li et al, 1995. J.biol.chem.270: 29453-29459). Depending on the particular source, the enzyme is a homo-oligomer or a heterodimer (see, e.g., Leyh and Suo, 1992, J.biol.chem.267: 542-.

In certain embodiments, a thermostable sulfurylase is used. Thermostable sulfurylases can be obtained, for example, from the genera Archaeoglobus (Archaeoglobus) or Pyrococcus (Pyrococcus). There are sequences of thermostable sulfurylases in the databases acc.no.028606, acc.no. q9ycr4, and acc.no. p56863.

ATP sulfurylase has been used for many different purposes, for example, bioluminescent detection of ADP at high ATP concentrations (see, e.g., Schultz et al, 1993, anal. biochem. 215: 302-304); continuous monitoring of DNA polymerase activity (see, e.g., Nyrbn, 1987, anal. biochem. 167: 235-238); and DNA sequencing (see, e.g., Ronaghi et al, 1996.Ahal. biochem.242: 84-89; Ronaghi et al, 1998.Science 281: 363-.

Several tests have been developed to detect the positive ATP sulfurylase reaction. The colorimetric molybdolysis test is based on the phosphate test (see, e.g., Wilson and Bandwirski, 1958.J.biol. chem.233: 975-981), while the continuous spectrophotometric molybdolysis test is based on the detection of NADH oxidation (see, e.g., Seubert et al, 1983.Arch. biochem. Biophys.225: 679-691; Seubert et al, 1985.Arch. biochin. Bioplys.240: 509-523). The latter test requires the presence of several detection enzymes. In addition, several radioactivity tests have been described in the literature (see, e.g., Daley et al, 198) AnaLBiochem.157: 385-395). For example, one test is based on the results of³²Of p-labelled ATP release³²Detection of ppi (see, e.g., Seubert et al, 1985.Arch. biochem. Biophys.240: 509-523), while the other is based on³⁵S incorporation [ 2 ]³⁵S]Labeled APS (this assay also requires pure APS kinase as a conjugated enzyme; see, e.g., Seubert et al, 1983.Arch. biochem. Bioplay.225: 679-691); the third reaction depends on³⁵SO4^-2Composed of³⁵S]Labeled APS release (see, e.g., Daley et al, 1986.anal. biochem. 157: 385- > 395).

For the detection of the reverse ATP sulfurylase reaction, a continuous spectrophotometric test has been previously described (see, e.g., Segel et al, 1987.Methods enzymol.143: 334-349); a bioluminescence test (see, e.g., Balharry and Nicholas, 1971, anal. biochem. 40: 1-17); a kind of³⁵SO4^-2Release testing (see, e.g., Seubert et al, 1985.Arch. biochem. Bioplys.240: 509-523); and a³²ppi incorporation test (see, e.g., Osslund et al, 1982. plantaphysiol.70: 39-45).

ATP produced by ATP sulfurylase can be hydrolyzed using an enzymatic reaction to generate light. Luminescent chemical reactions (i.e., chemiluminescence) and biological reactions (i.e., bioluminescence) are widely used in analytical biochemistry to sensitively measure various metabolites. In bioluminescent reactions, the chemical reaction that results in light emission is enzyme catalyzed. For example, the luciferin-luciferase system allows for specific testing of ATP while the bacterial luciferase-oxidoreductase system can be used for monitoring of nad (p) H. Both systems have been expanded to the analysis of a variety of substances by coupling reactions involving the production or utilization of ATP or NAD (P) H (see, e.g., Kricka, 1991. Cheminessence and biolinessence technologies. Clin. chem.37: 1472-1281).

The development of New reagents has made it possible to obtain stable light emission proportional to the concentration of ATP (see, for example, Lundin, 1982.Applications of fluorescent luciferase In; luminescennt Assays (Raven Press, New York) or NAD (P) H (see, for example, Lovgren et al, continuously monitoring of NADH-converting reactions by background luminescence. J.appl.biochem.4: 103-111). with these stable light emitting reagents, it is possible to perform end-point tests and calibrate each test by adding known amounts of ATP or NAD (P) H.

Suitable enzymes for converting ATP to light include luciferases, for example, insect luciferases. Luciferase, an end product of catalysis, produces light. The most well understood light-emitting enzyme is that of the firefly Photinus pyralis (Coleoptera spp.). The corresponding genes have been cloned and expressed in bacteria (see, for example, de Wet et al, 1985.Proc. Natl. Acad. Sci. USA 80: 7870-7873) and plants (see, for example, Ow et al, 1986.Science 234: 856-859), as well as in insects (see, for example, Jha et al, 1990. FEBSLett.274: 24-26) and mammalian cells (see, for example, de Wet et al, 1987.mol. cell. biol.7: 725-7373; Keller et al, 1987.Proc. Natl. Acad. Sci. USA 82: 3264-3268). In addition, a variety of luciferase genes have recently been cloned and partially identified from the Jamaica click beetle, Pyroplorus plagiophihalamus (Coleoptera) species (see, e.g., Wood et al, 1989. J.biolun. Chemimum.4: 289-301; Wood et al, 1989.Science 244: 700-702). Different luciferases are sometimes capable of producing light at different wavelengths, which may enable simultaneous monitoring of light emission at different wavelengths. These aforementioned characteristics are therefore unique and add new space for the utilization of current reporting systems.

Firefly luciferase catalyzes bioluminescence in the presence of luciferin, adenosine 5' -triphosphate, magnesium ion and oxygen, yielding a quantum yield of 0.88 (see, e.g., McElroy and Selinger, 1960.Arclz. biochem. Biophys. 88: 136-145). The firefly luciferase bioluminescent reaction can be utilized as an assay for ATP detection with approximately 1X 10^{- 13}Restriction of detection of M (see, e.g., Leach, 1981.J.Appl biochem.3: 473-517.) furthermore, of luciferase-mediated detection systemsThe overall degree of sensitivity and convenience has generated significant interest in the development of biosensors based on firefly luciferases (see, e.g., Green and Kricka, 1984.Talanta 31: 173-.

Using the enzymes described above, the sequencing primer is exposed to a polymerase and known dNTPs. If the dNTP is incorporated at the 3' end of the primer sequence, the dNTP is cleaved and a PPi molecule is released. Subsequently PPi is converted to ATP using ATP sulfurylase. Preferably, the ATP sulfurylase is present in a sufficiently high concentration such that the conversion of PPi proceeds with first order kinetics associated with PPi. In the presence of luciferase, ATP is hydrolysed to generate one photon. The reaction preferably has a sufficient concentration of luciferase present in the reaction mixture to allow the reaction, ATP → ADP + PO ₄ ^3-The + photon (light) follows first order kinetics associated with ATP. Photons can be measured using the methods and apparatus described below. In one embodiment, the detection is performed using PPi and a coupled sulfurylase/luciferase reaction to generate light. In certain embodiments, one or both of the sulfurylase and luciferase are immobilized on one or more mobile solid supports that are assigned to each reaction site.

The present invention thus allows the detection of the release of PPi during a polymerase reaction giving a real-time signal. The sequencing reaction can be continuously monitored in real time. The present invention thus makes possible a method for rapid detection of PPi release. The reaction has been estimated to occur in less than 2 seconds (Nyren and Lundin, supra). The rate limiting step is the conversion of PPi to ATP by ATP sulfurylase, whereas the luciferase reaction is fast and takes an estimated less than 0.2 seconds. The incorporation rate of polymerases has been evaluated by a variety of methods and it has been found, for example, that for Klenow polymerase, complete incorporation of one base can take less than 0.5 seconds. Thus, the estimated total time for incorporation of one base and detection by this enzymatic test is approximately 3 seconds. It will be seen that extremely fast reaction times are possible which enable real-time detection. Reaction time can be further reduced by using a more thermostable luciferase.

For most applications, it is desirable to use reagents that are free of contaminants like ATP and PPi. These contaminants can be removed by flowing the reagents through a pre-column containing apyrase and/or pyrophosphatase bound to a resin. Alternatively, apyrase or pyrophosphatase may be bound to magnetic beads and used to remove contaminating ATP and PPi present in the reagents. In addition, it is desirable to wash away diffusible sequencing reagents, e.g., unincorporated dNTPs, with a wash buffer. Any wash buffer used for pyrophosphate sequencing may be used.

In certain embodiments, the concentration of the reactants in the sequencing reaction comprises 1pmol DNA, 3pmol polymerase, 40pmol dNTP in 0.2ml buffer. See Ronaghi et al, anal. bioche7 n.242: 84-89(1996).

If desired, a sequencing reaction can be performed with each of the four predetermined nucleotides. A "complete" cycle typically involves the sequential administration of sequencing reagents for each of the nucleotides dATP, dGTP, dCTP and dTTP (or dUTP) in a predetermined sequence. Unincorporated dNTPs were washed away between the addition of each nucleotide. Alternatively, unincorporated dNTPs are degraded by apyrase (see below). The cycle is repeated as necessary until the desired amount of sequence of the sequence product is obtained. In certain embodiments, about 10-1000, 10-100, 10-75, 20-50, or about 30 nucleotides of sequence information are obtained from extension of one annealed sequencing primer.

In certain embodiments, the nucleotide is modified to include a disulfide derivative of a hapten, such as biotin. The analysis is performed by adding the modified nucleotides to the nascent primer annealed to the anchoring substrate by a post-polymerization step that includes i) sequentially binding avidin or streptavidin conjugated moieties linked to the enzyme molecule in embodiments where the modification is biotin, ii) washing away excess avidin or streptavidin linked enzyme, iii) flow of a suitable enzyme substrate under conditions suitable for enzyme activity, and iv) detection of the enzyme substrate reaction product. In this embodiment the hapten is removed by addition of a reducing agent. This method allows nucleotides to be identified at a given target site and allows for simple and rapid sequencing of DNA without the need for electrophoresis and the use of potentially dangerous radiolabels.

One preferred enzyme for detecting haptens is horseradish peroxidase. If desired, a wash buffer may be used between the additions of the various reactants herein. Unreacted dNTPs used to extend the sequencing primer can be removed using apyrase. The wash buffer can optionally include apyrase.

Exemplary haptens, e.g., biotin, digoxigenin, the fluorescent dye molecules cy3 and cy5, and luciferin are incorporated into extended DNA molecules with various efficiencies. Attachment of the hapten can occur through the linkage of the sugar, base, and through the phosphate portion of the nucleotide. Exemplary methods of signal amplification include fluorescence, electrochemical, and enzymatic. In a preferred embodiment using enzymatic amplification, the enzymes, e.g., Alkaline Phosphatase (AP), horseradish peroxidase (HRP), β -galactosidase, luciferase can include those known to have luminescent substrates, and the means for detecting these luminescent (chemiluminescent) substrates can include a CCD camera.

Modified bases are added in a preferred manner and detection is carried out by removing or inactivating the hapten conjugate moiety by use of a cleavage or inactivating agent. For example, if the cleavable linker is a disulfide, the cleavage agent can be a reducing agent, such as Dithiothreitol (DTT), β -mercaptoethanol, and the like. Other ways of inactivation include heat, cold, chemical denaturants, surfactants, hydrophobic agents, and suicide inhibitors.

Luciferase is capable of directly hydrolyzing dATP with the release of photons. This results in a false positive signal because hydrolysis occurs independently of incorporation of dATP into the extended sequencing primer. To avoid this problem, a dATP analogue can be used which is incorporated into DNA, i.e.which is a substrate for DNA polymerase but not luciferase. One such analog is α -thio-dATP. Thus, the use of a-thio-dATP avoids the generation of spurious photons that can occur upon hydrolysis of dATP that is not incorporated into the growing nucleic acid strand.

In general, PPi-based detection can be calibrated by measuring light that is released upon addition of a control nucleotide to the sequencing reaction mixture immediately after addition of the sequencing primer. This allowed normalization of the reaction conditions. The sequential incorporation of two or more identical nucleotides is indicated by a corresponding increase in the amount of light released. Thus, a two-fold increase in the released light relative to the control nucleotide indicates that two consecutive dNTPs are incorporated into the extended primer.

If desired, apyrase may be "washed" or "flowed" over the surface of the solid support to facilitate degradation of any remaining unincorporated dNTPs in the sequencing reaction mixture. Apyrase also degrades the ATP produced and thus "turns off" the light produced by the reaction. After treatment with apyrase, any remaining reaction is washed away in preparation for subsequent dNTP incubation and photon detection steps. Alternatively, apyrase may be bound to a solid or mobile solid support.

Double-ended sequencing

In a preferred embodiment, we provide a method of sequencing from both ends of a nucleic acid template. Traditionally, sequencing of both ends of a double-stranded DNA molecule requires at least hybridization of a primer, sequencing of one end, hybridization of a second primer, and sequencing of the other end. An alternative approach is to isolate the individual strands of the double stranded nucleic acid and sequence each strand separately. The present invention provides a third option that is faster and less labor intensive than the first two methods.

The invention provides a method for sequencing nucleic acid by a plurality of primers in sequence. DNA sequencing in this application refers to sequencing with a polymerase, in which the sequence is determined as Nucleotide Triphosphates (NTPs) are incorporated into the growing strand of the sequencing primer. An example of this type of sequencing is the pyrosequencing detection method (see, e.g., U.S. Pat. Nos. 6,274,320, 6,258,568 and 6,210,891, each of which is incorporated herein by reference in its entirety).

In one embodiment, the invention provides a method for sequencing both ends of a template double stranded nucleic acid. The double-stranded DNA comprises two single-stranded DNAs; referred to herein as a first single-stranded DNA and a second single-stranded DNA. The first primer is hybridized to the first single-stranded DNA and the second primer is hybridized to the second single-stranded DNA. The first primer is unprotected and the second primer is protected. "protection" and "protected" are defined herein as the addition of a chemical group to the reaction site of a primer that prevents the primer from polymerization by a DNA polymerase. In addition, the addition of such chemical protecting groups should be reversible so that upon reconstitution, the currently deprotected primer can once again function as a sequencing primer. The nucleic acid sequence is determined in one direction (e.g., from one end of the template) by extending the first primer with a DNA polymerase using conventional methods such as pyrophosphate sequencing. The second primer is then deprotected and the sequence is determined by extending the second primer in the other direction (e.g., from the other end of the template) using DNA polymerase and conventional methods such as pyrophosphate sequencing. The sequences of the first and second primers are specifically designed to hybridize to both ends of the double-stranded DNA or anywhere along the template in the present method.

In another embodiment, the invention provides a method of sequencing a nucleic acid by a plurality of primers. In this method, a plurality of sequencing primers are hybridized to a template nucleic acid to be sequenced. All but one sequencing primer was reversibly protected. The protected primers are oligonucleotide primers that cannot be extended with the polymerases and dNTPs commonly used in DNA sequencing reactions. A reversibly protected primer is a protected primer that can be deprotected. In the present invention all protected primers are reversibly protected. After deprotection, the reversibly protected primer functions as a normal sequencing primer and is able to participate in a normal sequencing reaction.

The invention provides a method for sequencing nucleic acid by a plurality of primers in sequence. The method comprises the following steps: first, one or more template nucleic acids to be sequenced are provided. Second, a plurality of sequencing primers are hybridized to the template nucleic acid. The number of sequencing primers can be represented by n, where n can be any positive number greater than 1. The number may be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. The n-1 number of primers may be protected by a protecting group. Thus, for example, if n is 2, 3, 4, 5, 6, 7, 8, 9 or 10, then n-1 will be 1, 2, 3, 4, 5, 6, 7, 8, 9 respectively-the remaining primers (e.g., the n number of primers- (n-1) number of protected primers equals 1 remaining primer) are unprotected. Third, the unprotected primer is extended and the template DNA sequence is determined by conventional methods such as, for example, pyrosequencing. Fourth, after sequencing of the first primer, one of the remaining protected primers is deprotected. Fifth, the unprotected primer is extended and the template DNA sequence is determined by conventional methods such as, for example, pyrosequencing. Optionally, the method can be repeated until all protected primers are sequenced.

In another aspect, the invention includes a method of sequentially sequencing nucleic acids comprising the steps of: (a) hybridizing 2 or more sequencing primers to the nucleic acid, wherein all but one of the primers are reversibly protected; (b) determining the sequence of one strand of the nucleic acid by polymerase extension from an unprotected primer; (c) deprotecting one of the reversibly protected primers to an unprotected primer; (d) repeating steps (b) and (c) until all reversibly protected primers are deprotected and used as determined sequences. In one embodiment, the method comprises a further step between steps (b) and (c) of terminating the extension of the unprotected primer by contacting the unprotected primer with a DNA polymerase and one or more nucleotide triphosphates or dideoxynucleotides. In yet another embodiment, the method further comprises between said steps (b) and (c) another step of terminating the extension of the unprotected primer by contacting the unprotected primer with a DNA polymerase and a dideoxy triphosphate nucleotide from ddATP, ddTTP, ddCTP, ddGTP or a combination thereof.

In another aspect, the invention includes a method of sequencing a nucleic acid, comprising: (a) hybridizing a first unprotected primer to a first strand of said nucleic acid; (b) hybridizing a second protected primer to the second strand; (c) contacting the first and second strands with a polymerase such that the first unprotected primer extends along the first strand; (d) terminating extension of the first sequencing primer; (e) deprotecting the second sequencing primer; and (f) contacting the first and second strands with a polymerase, thereby extending the second sequencing primer along the second strand. In a preferred embodiment, the ending comprises capping or terminating the elongation.

In another embodiment, the invention provides a method of sequencing both ends of a template double stranded nucleic acid comprising a first and a second single stranded DNA. In this embodiment, the first primer is hybridized to the first single-stranded DNA and the second primer is hybridized to the second single-stranded DNA in the same step. The first primer is unprotected and the second primer is protected.

After hybridization, the nucleic acid sequence is determined in one direction (e.g., from one end of the template) by extending the first primer with a DNA polymerase using conventional methods such as pyrophosphate sequencing. In a preferred embodiment, the polymerase lacks 3 '-5' exonuclease activity. The second primer is then deprotected and its sequence determined by extension of the second primer in the other direction (e.g., from the other end of the template) with a DNA polymerase using conventional methods such as pyrophosphate sequencing. As previously described, the sequences of the first and second primers are designed to hybridize to both ends of the double-stranded DNA or anywhere along the template. This technique is particularly useful for sequencing many template DNAs that have unique sequencing primer hybridization sites at both ends. For example, many cloning vectors provide a unique sequencing primer hybridization site flanking the insertion site to facilitate subsequent sequencing of any cloned sequence (e.g., Bluescript, Stratagene, La Jolla, Calif.).

One advantage of this method of the invention is that both primers can be hybridized in a single step. The benefits of this and other methods are particularly useful in parallel sequencing systems where hybridization is more involved than normal systems. An example of a parallel sequencing system is disclosed in pending U.S. patent application Ser. No. 10/104,280, which is incorporated herein by reference in its entirety.

The oligonucleotide primers of the present invention can be synthesized by conventional techniques, for example, using a commercial oligonucleotide synthesizer and/or by ligating together subfragments so synthesized.

In another embodiment of the invention, the length of a double stranded target nucleic acid can be determined. Methods for determining the length of a double-stranded nucleic acid are known in the art. The length determination may be performed before or after the nucleic acid is sequenced. Known methods for measuring the length of nucleic acid molecules include gel electrophoresis, pulsed-field gel electrophoresis, mass spectrometry, and the like. Since a blunt-ended double-stranded nucleic acid is composed of two single strands of the same length, a length measurement of one strand of the nucleic acid is sufficient to determine the length of the corresponding double strand.

The sequencing reaction according to the invention also allows the length of the template nucleic acid to be determined. First, the complete sequence from one end of the nucleic acid to the other will allow the length to be determined. Second, the sequencing of both ends can overlap in the middle, allowing the two sequences to be joined. The complete sequence can be determined and the length revealed. For example, if the template is 100bps long, bases 1-75 can be determined by sequencing from one end; bases 25-100 can be determined by sequencing from the other end; thus there is an overlap of 51 bases in the middle from base 25 to base 75; from this information, the complete sequence from 1 to 100 can be determined and the length of 100 bases can be revealed from the complete sequence.

Another method of the present invention is directed to a method comprising the following steps. First, a plurality of sequencing primers, each having a different sequence, are hybridized to the DNA to be sequenced. The number of sequencing primers can be any value greater than 1, such as, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. All but one of these primers are reversibly protected. Unprotected primers were extended and sequenced in a sequencing reaction. Typically, when one primer is fully extended, it cannot be extended and will not affect subsequent sequencing by the other primer. If desired, the sequencing primer can be terminated using an excess of polymerase and dNTPs or using ddNTPs. If a termination step is taken, the termination reagents (dNTPs and ddNTPs) should be removed after this step. Subsequently, one of the reversibly protected primers is deprotected and sequencing is continued from the second primer. The steps of deprotecting the primers, sequencing from the deprotected primers, and optionally, terminating sequencing from the primers are repeated until all protected primers are deprotected and used for sequencing.

The reversibly protected primers should be protected with different chemical groups. By selecting an appropriate deprotection method, one primer can be deprotected without affecting the protecting groups of the other primers. In a preferred embodiment, the protecting group is PO ₄. I.e., the second primer is PO₄Deprotection is achieved and is accomplished by T4 polynucleotide kinase (using its 3' phosphatase activity). In another preferred embodiment, the protection is a thio or phosphothiol group.

The template nucleic acid may be DNA, RNA or Peptide Nucleic Acid (PNA). Although DNA is the preferred template, RNA and PNA can be converted to DNA by known techniques such as random primer PCR, reverse transcription, RT-PCR or a combination of these techniques. In addition, the methods of the invention can be used to sequence nucleic acids of known or unknown sequence. Sequencing of nucleic acids of known sequence can be used, for example, to confirm the sequence of synthetic DNA or to confirm the identity of a suspected pathogen with a known nucleic acid sequence. The nucleic acid may be a mixture of more than one nucleic acid group. It is known that a subset of sequences can be sequenced in a long nucleic acid or in a population of unrelated nucleic acids using a sequencing primer of sufficient specificity (e.g., 20 bases, 25 bases, 30 bases, 35 bases, 40 bases, 45 bases, or 50 bases). Thus, for example, the template may be a 10Kb sequence or ten sequences of 1Kb each. In a preferred embodiment, the template DNA is between 50bp to 700bp in length. The DNA may be single-stranded or double-stranded.

In the case where the template nucleic acid is single-stranded, a plurality of primers may be hybridized to the template nucleic acid as follows:

5 '-primer 4- -3' 5 '-primer 3- -3' 5 '-primer 2-3' 5 '-primer 1-3'

3 '- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -'

In this case, it is preferred that the initial unprotected primer is the primer that hybridizes at the most 5' end of the template. See primer 1 in the figure above at this position extension of primer 1 does not replace (by strand replacement) primers 2, 3 or 4 when sequencing from primer 1 is complete, primer 2 can be deprotected and nucleic acid sequencing can begin. Sequencing from primer 2 can replace primer 1 or the extension of primer 1, but has no effect on the remaining protected primers (primers 3 and 4). By using this sequence, each primer can be used in turn and sequencing reactions of one primer do not affect sequencing of the next primer.

One feature of the invention is the ability to utilize multiple sequencing primers on one or more nucleic acids and the ability to sequence from multiple primers using only one hybridization step. In the hybridization step, all sequencing primers (e.g., n number of sequencing primers) can be hybridized to the template nucleic acid simultaneously. In conventional sequencing, sequencing of a primer typically requires a hybridization step. It is a feature of the present invention that sequencing of n primers (as defined above) can be performed by a single hybridization step. This effectively eliminates n-1 hybridization steps.

In a preferred embodiment, the sequences of the n number of primers are sufficiently different such that the primers do not cross-hybridize or auto-hybridize. Reciprocal hybridization refers to the hybridization of one primer to another primer due to the complementarity of the sequences. One form of reciprocal hybridization is commonly referred to as "primer dimer". For primer dimers, the 3' ends of the two primers are complementary and form a structure that, when extended, is approximately the sum of the lengths of the two primers. Auto-hybridization refers to the condition where the 5 'end of a primer is complementary to the 3' end of the primer. In this case, the primers have a tendency to self-hybridize to form hairpin-like structures.

The primer may interact or be specifically associated with the template molecule. By the term "interact" or "associate" is meant herein that two substances or compounds (e.g., a primer and a template; a chemical moiety and a nucleotide) are sufficiently bound (e.g., attached, bound, hybridized, linked, annealed, covalently linked, or associated) to one another to enable a desired test to be performed. By the term "specific" or "specifically" is meant herein that two components selectively bind to each other. The parameters required to achieve a specific interaction can be routinely determined, for example, using methods conventional in the art.

To achieve greater sensitivity or to facilitate analysis of complex mixtures, protected primers can be modified (e.g., derivatized) with chemical moieties designed to give distinct signals. For example, each protected primer can be derivatized with a different natural or synthetic amino acid attached to the oligonucleotide strand at one or more positions along the hybridizing portion of the strand through an amide bond. Of course, the chemical modification can be detected either after it has been cleaved from the target nucleic acid or when it is associated with the target nucleic acid. By allowing each protected primer to be identified in a distinguishable manner, it is possible to analyze (e.g., screen) a large number of different target nucleic acids in a single assay. Many such tests can be performed quickly and easily. Such assays or sets of assays can therefore be performed with high throughput efficiency as defined herein.

In the method of the present invention, after the first primer is extended and the sequence of the template DNA is determined, the second primer is deprotected and sequenced. There is no interference between the sequencing reaction of the first primer and the sequencing reaction of the second primer, which is currently unprotected, because the first primer is fully extended or terminated. Because the first primer is fully extended, sequencing of the second primer using conventional methods such as pyrosequencing will not be affected by the presence of the extended first primer. The invention also provides a method of reducing any possible contamination of the signal from the first primer. Signal contamination refers to the incidence of incomplete extension of the first primer. In this case, the first primer will continue to elongate as the latter primer is deprotected and extended. Extension of both the first and second primers can interfere with determination of the DNA sequence.

In a preferred embodiment, the sequencing reaction (e.g., a chain extension reaction) of one primer is first terminated or terminated before the sequencing reaction of the second primer is initiated. The chain extension reaction of DNA can be terminated by contacting the template DNA with DNA polymerase and dideoxynucleotide triphosphates such as ddATP, ddTTP, ddCTP and ddGTP. After termination, the dideoxynucleotide triphosphates can be removed by washing the reaction with a solution without ddNTPs. A second method to avoid further extension of the primer is to add nucleotide triphosphates (dNTPs such as dATP, dTTP, dGTP and dCTP) and DNA polymerase to the reaction to fully extend any incompletely extended primer. After complete extension, the dNTPs and polymerase are removed before the next primer is deprotected. By terminating or terminating one primer before the other primer is deprotected, the signal-to-noise ratio of a sequencing reaction (e.g., pyrosequencing) can be improved.

Steps (a) optionally terminating or ending the sequencing, (b) deprotecting a new primer, and (c) sequencing from the deprotected primers may be repeated until the sequence is determined by extension of each primer. In this method, the hybridization step comprises "n" primers and one unprotected primer. The unprotected primer is first sequenced and steps (a), (b) and (c) above may be repeated.

In a preferred embodiment, pyrophosphate sequencing is used for all sequencing performed according to the method of the invention.

In another preferred embodiment, double-ended sequencing is performed according to the procedure outlined in FIG. 10. This process can be divided into six steps: (1) generating capture beads (fig. 10A); (2) driver bead (DTB) PCR amplification (fig. 10B); (3) SL reporter preparation (fig. 10C); (4) sequencing of the first strand (fig. 10D); (5) preparation of second strand (fig. 10E and 10F); and (6) analysis of each strand (FIG. 10G). This exemplary process is outlined below.

In step 1, an N-hydroxysuccinimide (NHS) -activated capture bead (e.g., Amersham Biosciences, Piscataway, NJ) is coupled with a forward primer and a reverse primer. NHS coupling forms chemically stable amide bonds with ligands containing primary amino groups. The capture beads were also coupled to biotin (fig. 10A). Beads (i.e., solid nucleic acid capture supports) used herein can be of any convenient size and made of any number of known materials. Examples of such materials include: minerals, natural polymers and synthetic polymers. Specific examples of these materials include: cellulose, cellulose derivatives, acrylic resins, glass; silica gel, polystyrene, gelatin, polyvinylpyrrolidone, a copolymer of vinyl and acrylamide, polystyrene crosslinked with divinylbenzene or the like (see Merrifield Biochemistry1964, 3, 1385- ^TM) And agarose gel (Sepharose)^TM) And solid supports known to those skilled in the art. In a preferred embodiment, the capture beads are agarose gel beads having a diameter of about 25-40 μm.

In step 2, template DNA that has been hybridized to the forward and reverse primers is added and the DNA is amplified by a PCR amplification strategy (fig. 10B). In one embodiment, the DNA is amplified by emulsion polymerase chain reaction, driver bead polymerase chain reaction, rolling circle amplification, or loop-mediated isothermal amplification. In step 3, streptavidin is added after the addition of sulfurylase and luciferase, which are coupled to streptavidin (fig. 10C). The addition of auxiliary enzymes during the sequencing process has been described in U.S. patent application Ser. No. 10/104,280 and U.S. patent application Ser. No. 10/127,906, which are incorporated herein by reference in their entirety. In one embodiment, the template DNA has DNA adaptors ligated to both the 5 'and 3' ends. In a preferred embodiment, the DNA is coupled to the DNA capture beads by hybridization of a DNA adaptor to a complementary sequence on the DNA capture beads.

In the first step, the single stranded nucleic acid template to be amplified is attached to a capture bead. The nucleic acid template may be attached to the capture bead in any manner known in the art. There are a variety of methods in the art for attaching DNA to microbeads. Covalent chemical attachment of DNA to beads can be achieved by using standard coupling agents, such as water-soluble carbodiimides, to attach the 5' -phosphate on DNA to amine-coated microspheres via phosphoramidate linkages. Another option is to first couple specific oligonucleotide linkers to the beads using similar chemistry and then attach DNA to the linkers on the beads using DNA ligase. Other ligation chemistries include the use of N-hydroxysuccinamide (NHS) and its derivatives to attach oligonucleotides to beads. In this method, one end of the oligonucleotide may comprise a reactive group (e.g., an amide group) that forms a covalent bond with the solid support, while the other end of the linker comprises another reactive group that is capable of binding to the oligonucleotide to be immobilized. In a preferred embodiment, the oligonucleotide is bound to the DNA capture bead by a covalent bond. However, non-covalent linkages, such as chelation or antigen-antibody complexes, may be used to attach the oligonucleotides to the beads.

Oligonucleotide linkers that specifically hybridize to unique sequences at the ends of the DNA fragment, such as overlapping ends from restriction enzyme sites or "sticky ends" of phage lambda-based cloning vectors, may be employed, but blunt-end ligation may also be advantageously used. These methods are described in detail in US 5,674,743, the specification of which is incorporated herein by reference. Preferably any method for immobilising beads will continue to bind the immobilised oligonucleotides throughout the steps of the method of the invention. In a preferred embodiment, the oligonucleotide is bound to the DNA capture bead by a covalent bond. However, non-covalent linkages, such as chelation or antigen-antibody complexes, may be used to attach the oligonucleotides to the beads.

In step 4, the first strand of DNA is sequenced by dispensing the capture beads onto a PicoTiter plate (PTP) and sequencing in a manner known to those of ordinary skill in the art (e.g., pyrosequencing) (fig. 10D). After sequencing, a mixture of dNTPs and ddNTPs is added to "cap" or terminate the sequencing process (FIG. 10E). In step 5, the second strand of the nucleic acid was prepared from the blocked primer strand by removing ddNTPs by adding apyrase and removing the 3' phosphate group by polynucleotide kinase (PNK) (FIG. 10F). Polymerase was then added to prime the second strand, followed by sequencing according to standard methods known to those of ordinary skill in the art (fig. 10G). In step 7, the sequences of both the first and second strands are analyzed to determine adjacent DNA sequences.

Detection means

The solid support is optionally connected to an imaging system 230, which includes a CCD system associated with a conventional light beam or fiber optic bundle. In one embodiment, the perfusion chamber matrix includes a thin layer of fiber array such that light generated near the aqueous interface is transmitted directly through the fibers to the exterior of the matrix or chamber. When the CCD system includes a fiber optic connector, imaging can be achieved by placing the perfusion chamber substrate in direct contact with the connection. Alternatively, conventional optics may be used to image the light directly onto the CCD sensor from outside the fiber matrix, for example, by using a 1-1 magnification high power aperture lens system. When the matrix does not provide optical fibre coupling, a lens system may also be used as described above, in which case either the matrix or the perfusion chamber cover is optically transparent. An exemplary CCD imaging system is described above.

Light is collected from the reactor on the substrate surface using an imaging system 230. Light can be imaged onto, for example, a CCD using a high sensitivity, low noise device known in the art. For fiber-based imaging, it is preferable to integrate the fiber directly onto the cover glass, or for a FORA, so that the fiber that forms the microwells is also the fiber that transmits light to the detector.

The imaging system is connected to a computer control and data collection system 240. In general, any conventional hardware and software packages may be utilized. A computer control and data collection system is also connected to catheter 200 to control the delivery of the agent.

Photons generated by the pyrophosphate sequencing reaction are captured by the CCD only if they pass through a focusing device (e.g., optical lens or fiber) and are focused on a CCD element. However, the emitted photons will escape equally in all directions. When using a planar array (e.g., a DNA chip), in order to maximize its subsequent "capture" and quantification, it is preferred to collect photons as close as possible to the photon generation site, e.g., next to a planar solid support. This is achieved by: (i) the use of optical immersion oil between the cover glass and the conventional optical lens or fiber bundle, or preferably, (ii) the integration of the optical fibers directly onto the cover glass itself. Similarly, when a thin optically transparent flat surface is used, the optical fiber can also be placed against its back surface so that it is not necessary to "image" through the depth of the entire reaction/perfusion chamber.

Reaction events, e.g., photons generated by luciferase enzymes, can be detected and quantified using a variety of detection devices, e.g., photomultiplier tubes, CCD, CMOS, absorptiometers, luminometers, Charge Injection Devices (CID), or other solid state detectors, as well as the devices described herein. In a preferred embodiment, the quantification of the emitted photons is achieved by using a CCD camera equipped with a fused fiber bundle. In another preferred embodiment, the quantification of the emitted photons is achieved by using a CCD camera equipped with a microchannel plate intensifier. A thin back CCD can be used to increase sensitivity. CCD detectors are described, for example, in Bronks et al, 1995.kraal. chem.65: 2750, 2757.

An exemplary CCD system is a SpectraL instruments, Inc. (Tucson, AZ) Series 6004-port camera with a Lockheed-Martin LM485CCD chip and a single 1-1 fiber connector with a fiber diameter of 6-8 μm. This system has 4096 x 4096, or more than 16,000,000 pixels and has a quantum efficiency from 10% to > 40%. Thus, up to 40% of the photons imaged onto the CCD sensor are converted into detectable electrons depending on the wavelength.

In other embodiments, fluorescent moieties may be used as a label and detection of reaction events may be performed with a confocal scanning microscope to laser scan the surface of the array or other techniques such as existing scanning near-field optical microscopy (SNOM) with less optical resolution, thereby allowing the use of "denser" arrays. For example, with SNOM, individual polynucleotides can be distinguished when separated by a distance of less than 100nm, e.g., 10nm by 10 nm. Furthermore, scanning tunneling microscopy (Binning et al, Helvetica P/iysica Acta, 55: 726-.

Haplotype application

Virtually any sequencing application can be achieved using the methods and apparatus of the present invention. In one embodiment we claim a haplotype map. Human genetic diversity is an important factor in the variability of patient response to drugs. The most accurate measure of this diversity is the haplotype, which is the organizational structure of the polymorphic variation, as it is found on chromosomes. Recently, major government and academic genome researchers in the united states, canada and europe have recognized that haplotypes are a powerful tool that can reduce the complexity of genetic information to practical forms. Haplotypes can be used for drug discovery to improve target effectiveness and the outcome of drug screening studies, and for drug development to improve the design and reliability of clinical trials. Haplotype markers can be used to predict the efficacy and safety of new and approved drugs and will serve as the basis for a new paradigm of personalized medicine that matches patients with the appropriate dose of the appropriate drug through guidance from a database of clinical marker associations.

Multiple experimental studies have shown that adjacent SNP alleles are often in Linkage Disequilibrium (LD) with each other, such that the status of one SNP allele is often correlated with the allele of another adjacent SNP. These associations exist and will be co-delivered generation by generation due to the common history of immediately linked SNPs. The pattern of human sequence variation (haplotype) thus represents an ancestral DNA fragment. Historically meiosis has slowly dissociated alleles from neighboring alleles on ancestral chromosomes, except for immediately adjacent variations. The degree of linkage disequilibrium in the coloniser population with recent bottlenecks has been the target of many studies-particularly in the cloning of simple mendelian diseases such as cystic fibrosis (16), huntington's disease (11), bone dysplasia (DTD) (8). Although these clonal studies benefit from large chromosomal fragments that show large distance (often in the megabase range) LD spans, there is very little experimental data until recently LD spanning the human genome was noted in the world population.

We note three recent examples of large-scale investigations LD: (see, e.g., Reich, D.E., Cargill, M., Bolk, S., Ireland, J., Sabeti, P.C., Richter, D.J., Lavery, T., Kouyouumjian, R., Farhadian, S.F., Ward, R. & Lander, E.S.2001.Linkage disequilibrium in the human genome.Nature 411, 199-204.26). We extracted 19 chromosomal regions to investigate their SNP content. Genotyping was first performed on high frequency SNP span intervals of 2-160kb in Caucasian samples. LD is detectable at a distance of approximately 60kb over all regions, with significant differences between regions with distances as short as 6kb at one site and as long as 155kb at another site. Not surprisingly, LD is significantly correlated with estimated local recombination rates. Further analysis in nigeria human samples provided evidence of shorter LD in this population, although the allelic combinations were similar to caucasian samples over short distances. Overall, this work provides evidence that large block LDs are prevalent in the human genome and that genome-wide LD maps of disease genes will be feasible.

Reagent kit

The invention also includes kits for use in the methods of the invention, which may include one or more of the following components: (a) a test specific primer that hybridizes to the sample DNA such that the target site is directly adjacent to the 3' end of the primer; (b) a polymerase enzyme; (c) identifying a detection enzyme means for PPi release; (d) deoxynucleotides include, in place of dATP, a dATP analog that is capable of functioning as a substrate for a polymerase but is incapable of functioning as a substrate for the PPi detection enzyme; and (e) optionally dideoxynucleotides, optionally ddATP being replaced by a ddATP analogue capable of acting as a substrate for a polymerase but not for the PPi detection enzyme. If the kit is used to initiate PCR amplification, it may further comprise the following components: (i) a pair of PCR primers, at least one of which has means for immobilizing the primer; (ii) one is preferably a thermostable polymerase, such as Taql polymerase; (iii) PCR reaction buffer solution; and (iv) deoxynucleotides. When an enzyme label is used to assess PCR, the kit will advantageously contain a substrate for the enzyme and other components of the detection system.

One embodiment of the invention relates to a method of sequencing a nucleic acid. The method includes fragmenting a large template nucleic acid molecule to produce a plurality of fragmented nucleic acids. The fragmented nucleic acids are then delivered to aqueous microreactors in a water-in-oil emulsion such that a plurality of aqueous microreactors comprise a single copy of a fragmented nucleic acid, a single bead capable of binding to the fragmented nucleic acid, and an amplification reaction solution containing reagents necessary to perform nucleic acid amplification. In a next step, the nucleic acid is amplified in the microreactors to form amplified copies of said nucleic acid and the amplified copies are bound to beads in the microreactors. Next, the beads are delivered to an array of at least 10,000 reaction chambers on a flat surface, wherein a plurality of reaction chambers comprises no more than a single bead. Finally, sequencing reactions are performed simultaneously on multiple reaction chambers.

Another embodiment of the invention is directed to an array comprising a planar surface having a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center-to-center spacing of between 20-100 μm and each cavity has a width on a scale between 20 μm and 70 μm. In addition, in the array at least 10,000 reaction chamber. Each reaction chamber may contain at least 100,000 copies of a single stranded nucleic acid template.

Another embodiment of the invention relates to an array comprising a flat top surface and a flat bottom surface, wherein the flat top surface has at least 10,000 cavities thereon, each cavity forming an analyte reaction chamber, and the flat bottom surface is optionally conductive, thereby enabling detection of optical signals from the reaction chambers through the flat bottom surface, wherein the distance between the flat top surface and the flat bottom surface is no more than 5mm, wherein the reaction chambers have a center-to-center spacing between 20-100 μm and each chamber has a width of at least one dimension between 20 μm and 70 μm. In one embodiment, the distance between the flat top surface and the flat bottom surface does not exceed 2 mm.

Another embodiment of the invention relates to an array tool for performing independent parallel co-reactions in an aqueous environment. The array tool may comprise a substrate comprising at least 10,000 separate reaction chambers containing a starting material capable of reacting with a reagent, each reaction chamber being dimensioned such that when one or more fluids containing at least one reagent are delivered into each reaction chamber, the diffusion time of the reagent out of the well exceeds the time required for the starting material to react with the reagent to form a product.

Another embodiment of the invention is directed to a method of delivering a bioactive agent to an array. The method comprises dispersing a plurality of moving solid supports, each having at least one reagent immobilized thereon, on the array, wherein the reagents are suitable for use in a nucleic acid sequencing reaction, the array comprising a planar surface having a plurality of reaction chambers disposed thereon. The reaction chambers can have a center-to-center spacing of between 20-100 μm and each chamber has a width of at least one dimension between 20 μm and 70 μm.

Another embodiment of the invention relates to a device that simultaneously monitors the reaction chamber for light that indicates that a reaction is occurring at a particular site. The device comprises (a) a reaction chamber array formed from a planar substrate comprising a plurality of cavitated surfaces, each cavitated surface forming a reaction chamber for containing an analyte, wherein the reaction chambers have a center-to-center spacing of between 20-100 μm, each reaction chamber has a volume of between 10-150pL, the array comprising more than 10,000 separate reaction chambers; (b) an optically sensitive device arranged such that light from a particular reaction chamber is directed to a particular predetermined area of the optically sensitive device; (c) means for determining the level of light striking each predetermined area; and (d) means for recording the light level changes of each reaction chamber over time.

Another embodiment of the invention is directed to an analytical sensor comprising (a) an array formed from a first bundle of optical fibers having a plurality of cavitated surfaces at one end thereof, each cavitated surface forming a reaction chamber for containing an analyte, wherein the reaction chambers have a center-to-center spacing of between 20-100 μm and a width of between 20-70 μm, the array comprising more than 10,000 separate reaction chambers; (b) an enzymatic or fluorescent means for generating light in the reaction chamber; and (c) light detection means comprising a light capture means and a second fiber bundle for delivering light to the light detection means, said second fiber bundle being optically connected to said array such that light generated in a single reaction chamber is captured by a single fiber or a cluster of single fibers of said second fiber bundle for delivery to the light capture means.

Another embodiment of the invention relates to a method of conducting separate parallel co-reactions in an aqueous environment. The first step comprises delivering a fluid comprising at least one reagent to an array, wherein the array comprises a matrix comprising at least 10,000 separate reaction chambers, each reaction chamber being adapted to contain an analyte, wherein the reaction chambers have a volume between 10-150pL and comprise a starting material capable of reacting with a reagent, each reaction chamber being measured such that when the fluid is delivered into each reaction chamber, the diffusion time for the reagent to diffuse out of the well exceeds the time required for the starting material to react with the reagent to form a product. The second step comprises washing the fluid from the array after (i) the starting material reacts with the reagents to form a product in each reaction chamber, but before (ii) the reagents delivered to any one reaction chamber diffuse out of that reaction chamber into the other reaction chambers.

Another embodiment of the invention relates to a method of delivering a nucleic acid sequencing enzyme to an array. The array has a planar surface with a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center-to-center spacing of between 20-100 μm. The method comprises dispersing a plurality of moving solid supports having one or more nucleic acid sequencing enzymes immobilized thereon over the array, such that a plurality of reaction chambers comprise at least one moving solid support.

Another embodiment of the invention relates to a method of delivering a plurality of nucleic acid templates to an array. The array has a planar surface with a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center-to-center spacing of between 20-100 μm, the array having at least 10,000 reaction chambers. The method comprises the step of dispersing a plurality of mobile solid supports, each having no more than one species of nucleic acid template immobilized thereon, over an array, said dispersing resulting in the deployment of no more than one mobile solid support in any one reaction chamber.

Another embodiment of the invention relates to a method of sequencing a nucleic acid. The method comprises the step of providing a plurality of single-stranded nucleic acid templates disposed in a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein said reaction chambers have a center-to-center spacing of between 20-100 μm and said planar surface has at least 10,000 reaction chambers. The next step includes performing a pyrophosphate-based sequencing reaction simultaneously in all reaction chambers by annealing an effective amount of a sequencing primer to a nucleic acid template and extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to produce a sequencing product, a sequencing reaction byproduct being produced if the predetermined nucleotide triphosphate is incorporated onto the 3' end of the sequencing primer. The third step includes identifying the sequencing reaction by-products, thereby determining the nucleic acid sequence in each reaction chamber.

Another embodiment of the present invention relates to a method of determining the base sequence of a plurality of nucleotides on an array. The first step comprises providing at least 10,000 DNA templates, each individually disposed in a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center-to-center spacing of between 20-100 μm and a volume of between 10-150 pL. The second step comprises adding an activated nucleoside 5 ' -triphosphate precursor of a known nitrogenous base to the reaction mixture in each reaction chamber, each reaction mixture comprising a template-directed nucleotide polymerase and a single-stranded polynucleotide template hybridized to a complementary oligonucleotide primer at least one nucleotide residue shorter than the template to form at least one unpaired nucleotide residue on each template at the 3 ' -end of the primer strand, under reaction conditions permitting incorporation of the activated nucleoside 5 ' -triphosphate precursor into the 3 ' -end of the primer strand, provided that the nitrogenous base of the activated nucleoside 5 ' -triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the template. The third step comprises detecting whether the nucleoside 5 ' triphosphate precursor is incorporated into the primer strand, wherein incorporation of the nucleoside 5 ' triphosphate precursor indicates that the unpaired nucleotide residue of the template has a nitrogenous base component complementary to the incorporated nucleoside 5 ' triphosphate precursor. The fourth step comprises sequentially repeating steps (b) and (c), wherein each successive repetition adds and detects one type of activated nucleoside 5' triphosphate precursor of a known nitrogenous base composition. The fifth step involves determining the base sequence of the unpaired nucleotide residues of the template in each reaction chamber from the sequence in which the nucleoside precursors are incorporated.

Another embodiment of the present invention relates to a method for identifying a base at a target position in a DNA sequence of a template DNA. The first step comprises providing at least 10,000 separate DNA templates, each individually disposed in a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center-to-center spacing of between 20-100 μm, and the DNA is made single stranded before or after distribution in the reaction chambers. The second step involves providing an extension primer that hybridizes to the immobilized single-stranded DNA at a location immediately adjacent to the target location. Subjecting the immobilized single-stranded DNA to a polymerase reaction in the presence of a predetermined deoxynucleotide or dideoxynucleotide, wherein a sequencing reaction byproduct is produced if the predetermined deoxynucleotide or dideoxynucleotide is incorporated at the 3' end of the sequencing primer. The fourth step includes identifying the sequencing reaction by-product, thereby determining the nucleotide complementary to the base at the target position of each of the 10,000 DNA templates.

Another embodiment of the invention relates to an apparatus for analyzing nucleic acid sequences. The device includes: (a) a reagent delivery cuvette (cuvette), wherein the cuvette comprises an array comprising a planar surface having a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center-to-center spacing of between 20-100 μm and there are more than 10,000 reaction chambers, and wherein the delivery cuvette contains reagents for a sequencing reaction; (b) a delivery tool coupled to the delivery vessel; (c) an imaging system in communication with the delivery chamber; and (d) a data collection system in communication with the imaging system.

Another embodiment of the present invention relates to an apparatus for determining the base sequence of a plurality of nucleotides on an array. The device includes: (a) a reagent cuvette comprising a plurality of cavities on a flat surface, each cavity forming an analyte reaction chamber, wherein there are more than 10,000 reaction chambers, each reaction chamber having a center-to-center spacing of between 20-100 μm and a volume of between 10-150 pL; (b) simultaneously adding to the reaction mixture in each reaction chamber a reagent delivery means for an activated nucleoside 5 ' triphosphate precursor of a known nitrogenous base, each reaction mixture comprising a template-directed nucleotide polymerase and a single-stranded polynucleotide template hybridized to a complementary oligonucleotide primer at least one nucleotide residue shorter than the template to form at least one unpaired nucleotide residue on each template at the 3 ' end of the primer strand, under reaction conditions permitting incorporation of the activated nucleoside 5 ' triphosphate precursor into the 3 ' end of the primer strand, provided that the nitrogenous base of the activated nucleoside 5 ' triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the template; (c) detection means for detecting in each reaction chamber whether said nucleoside 5 ' triphosphate precursor is incorporated into the primer strand, wherein incorporation of said nucleoside 5 ' triphosphate precursor indicates that the unpaired nucleotide residue of the template has a nitrogenous base component complementary to said incorporated nucleoside 5 ' triphosphate precursor; and (d) means for sequentially repeating steps (b) and (c), wherein each successive repetition adds and detects one type of activated nucleoside 5' triphosphate precursor of a known nitrogenous base component; and (e) data processing means for simultaneously determining the base sequence of the unpaired nucleotide residues of the template in each reaction chamber from the incorporation sequence of the nucleoside precursor.

Another embodiment of the invention is directed to a device for processing a plurality of analytes. The device includes: (a) a flow cell configured with a matrix comprising at least 50,000 cavitated surfaces on a fiber optic bundle, each cavitated surface forming a reaction chamber for containing an analyte, wherein the reaction chamber has a center-to-center spacing of between 20-100 μm and a diameter of between 20-70 μm; (b) a fluidic means for delivering a processing reagent from one or more reservoirs to the flow chamber, thereby bringing the analyte dispensed in the reaction chamber into contact with the reagent, and (c) a detection means for simultaneously detecting a sequence of optical signals from each reaction chamber, each optical signal of the sequence being indicative of an interaction between the processing reagent and the analyte dispensed in the reaction chamber, wherein the detection means is in communication with the cavitated surface.

Another embodiment of the invention relates to a method of sequencing a nucleic acid. The first step comprises providing a plurality of single-stranded nucleic acid templates in an array having at least 50,000 isolated reaction sites. The second step involves contacting the nucleic acid template with reagents necessary to perform a pyrophosphate-based sequencing reaction coupled with light emission. The third step includes detecting light emitted by the plurality of reaction sites on portions of the optically sensitive device. The fourth step involves converting light striking each portion of the optically sensitive device into an electrical signal that can be distinguished from signals from all other reaction sites. The fifth step involves determining the sequence of the nucleic acid template for each isolated reaction site from the corresponding electrical signal based on light emission.

Another embodiment of the invention relates to a method of sequencing a nucleic acid. The first step involves fragmenting a large template nucleic acid molecule to produce a plurality of fragmented nucleic acids. The second step comprises attaching one strand of a plurality of the fragmented nucleic acids to the beads, respectively, to produce single-stranded nucleic acids attached to the beads, respectively. The third step comprises delivering a population of single stranded fragmented nucleic acids individually attached to beads to an array of at least 10,000 reaction chambers on a planar surface, respectively, wherein a plurality of wells comprises no more than one bead having single stranded fragmented nucleic acids. The fourth step includes performing sequencing reactions on multiple reaction chambers simultaneously. The sequencing reaction may have the following steps: (a) annealing an effective amount of a sequencing primer to a single-stranded fragmented nucleic acid template and extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to produce a sequencing product, producing a sequencing reaction byproduct if the predetermined nucleotide triphosphate is incorporated onto the 3' end of the sequencing primer, and (b) identifying the sequencing reaction byproduct, thereby determining the nucleic acid sequence in the plurality of reaction chambers. Alternatively, the sequencing reaction may comprise the steps of: (a) hybridizing two or more sequencing primers to a single strand of one or more nucleic acid molecules, wherein all but one of the primers are reversibly blocked primers; (b) incorporating at least one base into a nucleic acid molecule by polymerase extension from an unblocked primer; (c) preventing further elongation of the unblocked primer; (d) deblocking one of said reversibly blocked primers into an unblocked primer; and (e) repeating steps (b) to (d) until at least one of said reversibly blocked primers is deblocked and used to determine sequence.

In the following pending U.S. patent applications: additional materials and methods may be found in USSN60/443,471, presented at 29/1/2003 and USSN 60/465.071, 23/4/2003. All patents, patent applications, and references cited in this specification are herein incorporated by reference.

Examples

Example 1: sample preparation

DNA sample:

the DNA should be of high quality and free of contaminants such as proteins, nucleases, lipids and other chemicals (e.g. residual EDTA from the preparation) and salts. Preferably, the genomic DNA should have an 260/280 ratio of 1.8 or higher. If it is desired to sequence the genome of only one organism, then the DNA should be quality checked to ensure that there is no contaminating DNA, for example, a preparation of human DNA may be checked by PCR to ensure that it is not contaminated with bacterial DNA molecules. Another method of checking for contamination is by restriction digestion patterns and in particular restriction digestion followed by southern blotting using a suitable probe known to be specific for one organism (e.g., human or mouse) and a second probe known to be specific for a possible contaminating organism (e.g., e. If desired, the DNA should originate from a single clone of an organism (e.g., a colony if from a bacterium).

Step 1: DNase I digestion

The purpose of the DNase I digestion step is to fragment large pieces of DNA, such as a whole genome or large pieces of a genome, into smaller parts. This population of smaller size DNA portions produced from a single DNA template is referred to as a "library". Deoxyribonuclease I (DNase I) is an endonuclease that cleaves double-stranded template DNA. The cleavage properties of DNase I allow random digestion of template DNA (i.e.minimal sequence bias) and when used in the presence of manganese-based buffers will result in a preponderance of blunt-ended double-stranded DNA fragments (Melgar and Goldthwait 1968). Dnase I digestion of genomic templates depends on three factors: i) the amount (units) of enzyme used; ii) digestion temperature (. degree.C.); and iii) incubation time (minutes). The DNase I digestion conditions listed below were optimized to generate a DNA library of 50-700 base pairs (bp) in size.

1. The DNA was obtained and prepared in Tris-HCl (10mM, Ph 7-8) at a concentration of 0.3 mg/ml. A total of 134. mu.l of DNA (15. mu.g) was required for this preparation. It is recommended not to use DNA preparations diluted with buffers containing EDTA (i.e., TE, Tris/EDTA). The presence of EDTA is inhibitory to the enzymatic digestion of dnase I. If the DNA preparation contains EDTA, it is important that the DNA is "salted" out of solution and reconstituted with a suitable Tris-HCl buffer (10mM, pH 7-8) or nanopure water (pH 7-8).

2. In a 0.2ml tube, 10. mu.l of MnCl containing 50. mu.l of Tris pH 7.5(1M) was prepared₂DNase I buffer (1M), 1. mu.l BSA (100mg/ml), and 39. mu.l waterAnd (6) flushing liquid.

3. In a separate 0.2ml tube, 15. mu.l DNase I buffer and 1.5. mu.l DNase I (IU/ml) were added. The reaction tube was placed in a thermal cycler set at 15 ℃.

4. Mu.l (0.3mg/ml) was added to a DNase I reaction tube placed in a thermal cycler set at 15 ℃. The lid was closed and the sample was incubated for exactly 1 minute. After incubation, 50. mu.l of 50mM EDTA was added to stop the enzymatic digestion.

5. The digested DNA was purified by using QiaQuick PCR purification kit. The digestion reaction was then divided into four aliquots, each aliquot was purified using four spin columns ((37.5. mu.l/spin column.) each column was eluted with 30. mu.l Elution Buffer (EB) according to the manufacturer's instructions.

6. A3. mu.l aliquot of the digest was saved for analysis using a BioAnalzyer DNA 1000 LabChip.

Step 2: pfu complement

Digestion of the DNA template with dnase I produces DNA fragments that are predominantly blunt-ended, some fragments will have ends that comprise a one-two nucleotide long overhang. Pfu filling is used to increase the amount of blunt end species by filling (i.e., "blunting") the 5' protrusion. Furthermore, Pfu DNA polymerase has 3 '→ 5' exonuclease activity, which will result in removal of mono-and di-nucleotide extensions. Pfu supplementation increases the amount of blunt-ended DNA fragments available for adaptor ligation (Costa1994a, 1994b, 1994 c). The following Pfu replenishment method was used.

1. In a 0.2ml tube, 115. mu.l of purified DNase I digested DNA fragment, 15. mu.l of 10 Xclone Pfu buffer, 5. mu.l of dNTPs (10mM) and 15. mu.l of clone Pfu DNA polymerase (2.5U/. mu.l) were added in that order.

2. The filled reaction components were mixed well and incubated at 72 ℃ for 30 minutes.

3. After incubation, the reaction tube was removed and placed on ice for 2 minutes.

4. The priming reaction mixture was then divided into four portions and purified using QiaQuick PCR purification columns (37.5. mu.l per column). Each column was eluted with 30. mu.l of Elution Buffer (EB) according to the manufacturer's instructions. The eluates were then combined to generate a final reaction volume of 120. mu.l.

5. A3. mu.l aliquot of the digest was saved for analysis using a BioAnalzyer DNA 1000 LabChip.

And step 3: ligation of Universal adaptors to fragmented DNA libraries

After fragmentation and filling-up of the genomic DNA library, primers were added to the ends of each DNA fragment. These primer sequences are referred to as "universal adaptors" and consist of double-stranded oligonucleotides that contain specific guide regions that provide both PCR amplification and nucleotide sequencing. The universal adaptor is designed to include a unique set of 20 base pair long PCR guide regions located adjacent to a unique set of 20 base pair long unique sequencing guide regions followed by a unique 4 base "key" consisting of at least one of each deoxyribonucleotide (i.e., a, C, G, T). Each unique universal adaptor (referred to as "Universal adaptor A" and "Universal adaptor B") is forty-four base pairs (44bp) long. The universal adaptors were ligated to each end of the DNA fragments using T4 ligase to generate a total nucleotide addition of 88bp to each DNA fragment. Different designed universal adaptors are prepared for each genomic DNA library and will thus provide a unique identification for each organism.

To prepare a pair of universal adaptors, single stranded oligonucleotides can be designed on their own and produced by commercial vendors. Universal adaptor DNA oligonucleotides were designed with two phosphothio linkages at each oligonucleotide end that act to protect against nuclease activity (Samini, T.D., B.Jolles and A.Laige.2001.best minor modifides oligonucleotides encoding to cell nucleic acid activity. antisense nucleic acid Drug Dev.11 (3): 129., the contents of which are incorporated herein by reference). Each oligonucleotide was HPLC purified to ensure that there were no contaminating or spurious DNA oligonucleotides in the final preparation.

The universal adaptors are designed to allow for directed ligation to blunt-ended fragmented genomic DNA. For each universal adaptor pair, the PCR guide region comprises a 5 'four base overhang and a blunt-ended 3' key region. Orientation is achieved when the blunt-ended side of the universal adaptor ligates to blunt-ended DNA fragments and the 5' overhang of the adaptor does not ligate to blunt-ended DNA fragments. In addition, a 5' biotin is added to the universal adaptor B to allow for subsequent isolation of the ssDNA template (step 8). Each universal adaptor is prepared by annealing two single-stranded complementary DNA oligonucleotides (i.e., an oligonucleotide comprising a sense sequence and a second oligonucleotide comprising an antisense sequence) in a single tube. The following attachment method was used.

1. In a 0.2ml tube, 39. mu.l H2O (molecular biology grade water), 25. mu.l of the digested filled-up DNA library, 100. mu.l of 2 Xquick ligase reaction buffer, 20. mu.l of MMP1(10 pm/. mu.l) adaptor set, 100: 1 ratio, and 16. mu.l Quick ligase were added in that order. The ligation reaction was mixed well and incubated at room temperature for 20 minutes.

2. The ligation reaction was then removed and 10. mu.l of the ligation reaction was purified for use on a BioAnalyzer. A single spin column from Qiagen Min-Elute kit was used. Each column was eluted with 10. mu.l EB according to the manufacturer's instructions. A1. mu.l aliquot of the purified ligation reaction was loaded using a BioAnalyzer DNA 1000 LabChip. This purification step is recommended because the unpurified ligation reaction contains high amounts of salt and PEG, which would inhibit the sample from working properly on the BioAnalyzer.

3. The remaining ligation reaction (190. mu.l) was used for gel separation in step 4.

Step 3 a: microcon filtration and adaptor construction. The total preparation time was approximately 25 minutes.

Universal adaptor ligation requires a 100-fold excess of adaptors. To aid in the removal of these excess adaptors, the double stranded gDNA library was filtered through a Microcon YM-100 filter device. Microcon YM-100 membranes can be used to remove double stranded DNA smaller than 125 bp. Therefore, unbound adaptors (44bp), as well as adaptor dimers (88bp) can be removed from the ligated gDNA library population. The following filtration method was used:

1. 190. mu.l of ligation reaction from step 4 was applied to the assembled MicroconYM-100 device.

2. The device was placed in a centrifuge and centrifuged at 5000 × g for approximately 6 minutes, or until the membrane was almost dry.

3. For washing, 200. mu.l of 1 XTE were added.

4. The sample was centrifuged at 5000 Xg for about 9 minutes or until the membrane was almost dry.

5. For recovery, the reservoir (reservoir) was inserted into a new vial and centrifuged at 3000 xg for about 3 minutes. The reservoir is discarded. The recovery volume was approximately 10. mu.l. Next, 80. mu.l of TE was added.

Adapters (a and B) were HPLC purified and modified with a phosphothioester linkage prior to use. For adapter "A" (10. mu.M), 10. mu.l of 100. mu.M adapter A (44bp, sense) was mixed with 10. mu.l of 100. mu.M adapter A (40bp, antisense) and mixed with 30. mu.l of 1 × annealing buffer (V)_f50 μ l). Primers were annealed on a Sample prepLabthermal cycler (see below) using the ANNEAL program. For adaptor "B" (10. mu.M), 10. mu.l of 100. mu.M adaptor B (44bp, sense) was combined with 10. mu.l of 100. mu.M adaptor B (40bp, antisense) and 30. mu.l of 1 × annealing buffer (V)_f50 μ l) were mixed. Primers were annealed on a Sample prepLabthermal cycler (see below) using the ANNEAL program. The set of adaptors can be stored at-20 ℃ until use.

ANNEAL-A procedure for primer annealing:

1. incubating at 95 ℃ for 1 min;

2. the temperature was reduced to 15 ℃ at 0.1 ℃/sec; and

3. the temperature was maintained at 15 ℃.

There is no orientation required for genomic inserts and adaptors. Fragments may be ligated at either end. Four single-stranded DNA oligonucleotides are included in the universal adaptor set. Each single stranded oligonucleotide was synthesized at a level of 1 millimolar and subjected to HPLC purification. Each single stranded oligonucleotide includes four phosphothioester linkages at each end.

And 4, step 4: gel electrophoresis and extraction of adapted DNA libraries

The universal adaptor ligation method yields the following: 1) fragmented DNAs with adaptors on either end; 2) unbound single adaptors; or 3) formation of adaptor dimers. Agarose gel electrophoresis was used as a method to separate the adaptor DNA library population from unligated single adaptor and adaptor dimer populations. The process of DNase I digestion of genomic DNA results in a library population of 50-700bp (step 1). Adding an 88bp set of universal adaptors will shift the population to a larger size and will result in a migration pattern of approximately 130 and 800bp in size. Adaptor dimers will migrate to 88 bp; while adapters that are not ligated will migrate to 44 bp. Therefore, genomic DNA libraries of > 200bp in size can be physically separated from agarose gels and purified using standard gel extraction techniques. Gel separation of the adapted DNA library will result in recovery of a library population of ≧ 200bp in size.

The following electrophoresis and extraction methods were used.

1. A 2% agarose gel was prepared.

2. Mu.l of 10 × Ready-Load Dye was added to the remaining 90. mu.l of DNA ligation mixture.

3. The dye/ligation reaction mixture was loaded onto the gel using four adjacent lanes (25. mu.l/lane).

4. Mu.l of a 100bp gradient marker (0.1. mu.g/. mu.l) was loaded into the two lanes distant from the ligation reaction lane.

5. Run the gel at 100V for 3 hours.

6. When the running of the gel is finished, the gel is removed from the gel box and transferred to a flat surface covered with a plastic cover. The DNA bands were examined using a hand-held long-wave UV lamp. The 200-400bp fragment size was excised from the agarose gel using a sterile, single-use scalpel. Using this method, libraries of any size range can be isolated. It is also possible to separate more than one size range. When the library size range is 200-900bp, it is possible to separate several size ranges from a single well (i.e., 200-400bp and 500-700 bp).

7. The DNA embedded in the agarose gel was isolated using the Qiagen MinElute gel extraction kit according to the manufacturer's instructions. Briefly, buffer QG was added to cover the agarose in the tube. The agarose was allowed to dissolve completely. The color of the buffer QG was maintained by adjusting the pH according to Qiagen instructions to minimize sample loss. Purification was performed using two MinElute spin columns (Qiagen). The large volume of dissolved gel requires that each column be loaded several times. The column was eluted with 10. mu.l of buffer EB which was preheated at 55 ℃. The eluates were pooled to generate a 20. mu.l gDNA library.

8. A1. mu.l aliquot of each isolated DNA library was analyzed using a BioAnalyzer DNA 1000LabChip to assess the precise distribution of the DNA library population.

And 5: strand Displacement and extension of nicked double stranded DNA libraries

Since the DNA oligonucleotides used as universal adaptors are not phosphorylated, gaps are present at the 3' junctions of fragmented gDNAs. Both "gaps" or "nicks" can be filled in by using a strand displacing DNA polymerase. The polymerase recognizes the nick, displaces the nicked strand, and extends the strand in such a way as to result in repair of the nick and formation of non-nicked double-stranded DNA. The strand displacing enzyme used was a large fragment of Bst DNA polymerase.

1. In a 0.2ml test tube, 19. mu.l of the gel-extracted DNA library, 40. mu. lnH were added in that order₂O, 8. mu.l of 10 XThermoPol reaction buffer, 8. mu.l BSA (1mg/ml), 2. mu.l NTPs (10mM) and 3. mu.l BstI polymerase (8U/. mu.l).

2. The samples were mixed well and placed in a thermal cycler and incubated using strand displacement: "BST" was used for incubation. BST procedure for strand displacement and nicked double stranded DNA library extension:

1. incubation at 65 ℃ for 30 minutes;

2. incubation at 80 ℃ for 10 min;

3. incubation at 58 ℃ for 10 min; and

4. The temperature was maintained at 14 ℃.

3. A1. mu.l aliquot of the Bst-treated DNA library was analyzed using a BioAnalyzer DNA 1000 LabChip.

Step 6: preparation of streptavidin beads

After the nickless double-stranded genomic DNA is generated, single-stranded genomic DNAs containing flanking universal adaptor sequences must be isolated. This step lists the binding of biotin-labeled double stranded DNA to streptavidin beads. For the preparation of streptavidin beads, the following method was used:

1. mu.l Dynal M-270 streptavidin beads were washed with 200. mu.l of 1 × binding buffer (1M NaCl, 0.5mM EDTA, 5mM Tris, pH 7.5) by applying the magnetic beads to the MPC.

2. The beads were resuspended in 100. mu.l 2 Xbinding buffer, followed by the addition of the remaining 79. mu.l of Bst-treated DNA sample (from step 5) and 20. mu.l of water.

3. The bead solution was mixed well and placed on a tube shaker at room temperature for 20 minutes. The bead mixture was washed twice with 100. mu.l of 1 × binding buffer using MPC, followed by nH₂O wash twice. Binding and washing (B)&W) slowFlush (2 × and 1 ×): preparation of 2 XB by mixing 10mM Tris HCl (pH 7.5), 1mM EDTA and 2M NaCl&W buffer solution. The reagents were combined and mixed well as listed above. The solution can be stored at room temperature for 6 months; by mixing 2 XB &W buffer and nH₂O1: 1 mixing to make 1X B&W buffer solution. The final concentration was half of the above, i.e., 5mM TrisHCl (pH 7.5), 0.5mM EDTA, and 1M NaCl.

And 7: isolation of Single stranded DNA libraries Using streptavidin beads

After the double stranded gDNA library is bound to streptavidin beads, it is preferred to isolate only single stranded gDNAs from the ligated library, which comprise Universal adaptor A and Universal adaptor B (the desired population is indicated below with an asterisk). The library of double stranded genomic DNA fragments will have adapters bound in the following configuration:

universal adaptor A-gDNA fragment-Universal adaptor A

Universal adaptor B-gDNA fragment-Universal adaptor A^*

Universal adaptor A-gDNA fragment-Universal adaptor B^*

Universal adaptor B-gDNA fragment-Universal adaptor B

Since only the universal adaptor B has a 5' biotin moiety, it is possible to bind all gDNA library species with universal adaptor B using magnetic streptavidin containing beads. The genomic library population (or unligated species) comprising the two universal adaptor A species will not bind to streptavidin-containing beads and will be removed during the wash process. Species that remain bound to the beads after washing include those with universal adaptors a and B or those with two universal adaptor B ends.

Genomic DNA species with two universal adaptor B sequences with two biotin molecules can be bound to streptavidin-containing beads on both ends. Species with a and B adaptors having only a single biotin molecule can only bind the bead at the B "end". To isolate single-stranded populations, the bead-bound double-stranded DNA is treated with a sodium hydroxide solution that serves to disrupt hydrogen bonds between complementary DNA strands. If the DNA fragment has biotin at each end (the B-terminus of the universal adaptor), both resulting single strands will remain bound to the bead. If the fragment has only a single biotin (Universal adaptors A and B), the complementary strand is isolated from the DNA-bead complex.

The resulting single stranded genomic library is collected from the solution phase and quantified, for example, using pyrosequencing (PyroSequence) or by using an RNA Pico 6000LabChip (Agilent, Palo Alto, CA). Single-stranded genomic DNA libraries are quantified by counting the number of molecules per unit volume. The single stranded gDNA molecule (in half copies/bead to obtain a valid copy/bead) was then annealed to 25-30 μm agarose beads containing DNA capture primer (PCR primer B). The template is then amplified using emulsion polymerase chain reaction. Subsequent sequencing can be performed using known techniques. For isolation of the single stranded library, the following method was used.

1. Mu.l of the thawing solution (0.125M NaOH, 0.1M NaCl) was added to the washed beads from step 6 above.

2. The bead solution was mixed well and the bead mixture was incubated on a tube shaker at room temperature for 10 minutes.

3. Using a Dynal MPC (magnetic particle concentrator), the beads were carefully removed and the supernatant was left. 250 μ l of the supernatant included the single stranded DNA library.

4. In a separate tube, 1250. mu.l PB (from QiaQuick purification kit) was added and the solution was neutralized by adding 9. mu.l of 20% acetic acid.

5. Using a Dynal MPC, beads from 250. mu.l of supernatant comprising a single stranded gDNA library were pelleted and the supernatant carefully removed and transferred to a freshly prepared PB/acetic acid solution.

6. 1500 μ l of the solution was purified using a single QiaQuick purification spin column (load sample at 750 μ l/load twice through the same column). The single stranded DNA library was eluted with 50. mu. lEB.

Step 8 a: single-stranded gDNA was quantified using pyrosequencing. The total preparation time was about 1 hour.

1. In a 0.2ml tube, the following reagents were added in order:

25 μ l of Single-stranded gDNA

Mu.l MMP2B sequencing primer

14 μ l library annealing buffer

Total volume of 40. mu.l

2. The DNA was annealed using the ANNEAL-S program (see appendix, below).

3. Samples were run on a PSQ (pyrosequencing clamp) to measure the picomoles of template in each sample. Sequencing methods can be found in U.S. Pat. nos. 6,274,320; U.S. Pat. nos. 4,863,849; U.S. Pat. nos. 6,210,891; and U.S. Pat. No. 6,258,568, the contents of which are incorporated herein by reference in their entirety. Calculations were performed to determine the number of single stranded gDNA template molecules per microliter. The remaining 25. mu.l of the prepared single-stranded gDNA library was used for amplification and subsequent sequencing (approx. 1X 10)⁶Reaction).

And step 8 b: quantification of single stranded gDNA was performed using an RNA Pico 6000 LabChip. The total preparation time was approximately 30 minutes.

1. The mRNA Pico assay option was selected on the BioAnalyzer (Software version 2.12).

2. RNA Pico 6000LabChip was prepared on a BioAnalyzer according to the manufacturer's instructions.

3. An RNA LabChip gradient (RNA6000 gradient) was prepared according to the manufacturer's (Ambion) instructions. Briefly, the RNA LabChip gradient in solution was heated to 70 ℃ for 2 minutes. The solution was cooled on ice for 5 minutes to rapidly cool the gradient. The solution was centrifuged briefly to remove the condensate from the tube walls. RNA LabChip gradients were stored on ice and used within a day.

4. The ssDNA library to be analyzed was run in triplicate on adjacent lanes using 3 1. mu.l aliquots.

5. The concentration of each ssDNA library lane was calculated using the BioAnalyzer software (see table below and fig. 24). The average number of all three lanes was used to calculate the DNA concentration of the library using the methods listed below.

a. The peak integration lower limit line (large protuberance in fig. 24) was shifted immediately before the library peak (see below).

b. The peak integration upper limit line (large protuberance in fig. 24) was shifted immediately after the library peak (see below). In this way, the peak trim line connecting the lower and upper trim lines follows the slope of the background.

c. The base average size of the peak is determined using a mouse pointer (usually near the peak of the peak) or a defined peak is used by selection of the software.

d. The integrated value is used to calculate the amount of substance in the peak. The recovered picogram values obtained were converted into recovered molecules (see table below). The library concentration (molecules/microliter) is then determined.

Watch (A)

1	2	3	4	5	6	7	8	9
									Mean value of	Mean number	Mean number	Mean number	Mean value of
					pg/μL(1)	pg/μL(2)	pg/μL(3)	pg/μL						Size (bp)1	Size (bp)2	Size (bp)3	Size (bp)
Sample (I)	1633	1639	1645						1639	435	435	432	434

10	11	12	13	14	15
						Average molecular weight (g/mole)	Average molecular weight	Libraries
Ribonucleotides	(g/mol)	g/μL							Mol/g	Mol/. mu.L	Molecule/. mu.L
			328.2	1.42E+05	1.64E-09	7.02E-06	1.15E-14	6.93E+09

As shown in the above table, the concentration of library 1 was calculated as 1639 pg/. mu.l (column 5) and the average fragment size was 434 nucleotides (column 9). These values were obtained by the Agilent 2100 software as described in steps (a) - (d) above the average Molecular Weight (MW) of the ribonucleotides was 328.2 g/mole (column 10). The MW of the average library fragment (1.42X 10) was calculated by multiplying the average length (434) by the average ribonucleotide (328.2) ⁵G/mol, column 11). The quantified library (1639 pg/. mu.l) was converted to g/l (1.64X 10)^-9/. mu.l, column 12). By mixing grams per microliter (1.64X 10)^-9g/l, column 12) divided by the average molecular weight of the library fragments (1.42X 10)⁵Column 11) to calculate the moles/microliter (1.15X 10-¹⁴mol/. mu.l, column 14). Finally, by mixing mole/microliter (1.15X 10-¹⁴mol/. mu.l, column 14) times the Avogastron constant (6.02X 10)²³Molecule/mole) to obtain the number of molecules/microliter (6.93X 10)⁹Molecule/. mu.l, column 15).

Final library concentrations greater than 1X 10 are expected⁸Molecules/. mu.l. An even more important factor for library quality is adaptor dimer concentration. In fig. 24, the height of the library peak was determined to be about ten times greater than the adaptor dimer peak (first peak after labeling). A good quality library needs to have a peak height at least 2 times greater than the dimer peak. It should be noted that the RNA Pico 6000LabChip provided an estimate within the accuracy of 500% single stranded gDNA concentration. Thus, it is important to perform one initial sequencing run using titration of the template to determine the copy number of input gDNA per bead (cpb). The recommended input DNA is 2.5cpb, 1cpb, 0.5cpb and 0.1 cpb. This titration was easily checked using a 4slot bead loading chamber on a 14X 43 PTP.

And step 9: dilution and preservation of Single-stranded gDNA library

The single-stranded gDNA library was eluted and quantified in buffer EB. To avoid degradation, single-stranded gDNA libraries were cryopreserved in the presence of EDTA. After quantification, an equal volume of 10mM TE was added to the library stock. All subsequent dilutions were in TE. The product was as follows:

the remaining final volume of ssDNA library after PSQ analysis was 25 μ l.

The remaining final volume of ssDNA library after LabChip analysis was 47 μ l.

For dilution of the initial stock, the single-stranded gDNA library was diluted to 100,000,000 molecules/. mu.l in 1 × laboratory grade elution buffer. Single-stranded gDNA library aliquots were prepared for general use. For this purpose, 200,000 molecules/. mu.l are diluted in 1 Xlaboratory-grade elution buffer and 20. mu.l aliquots are measured. Aliquots of the library used in the single flash were stored at-20 ℃.

Step 10: emulsion polymerase chain reaction

Where increased cpb numbers are preferred, bead emulsion PCR is performed as described in U.S. patent application serial No. 06/476,504, filed 6.6.2003, which is incorporated herein by reference in its entirety.

Preparation of reagents

The stop solution (50mM EDTA) comprised 100. mu.l of 0.5M EDTA with 900. mu.l of nH₂O mixed to obtain 1.0ml of a 50mM EDTA solution. For 10mM dNTPs, 10. mu.l dCTP (100mM), 10. mu.l ATP (100mM), 10. mu.l GTP (100mM) and 10. mu.l dTTP (100mM) were mixed with 60. mu.l molecular biology grade water. All four 100mM nucleotide stocks were thawed on ice. Subsequently, 10. mu.l of each nucleotide was combined with 60. mu.l of nH₂O to reach a final volume of 100 μ l and mixed well. Next, 1ml aliquots were dispensed into 1.5ml microcentrifuge tubes. The preservation solution can be stored at-20 ℃ for one year.

The 10 × annealing buffer included 200mM Tris (pH 7.5) and 50mM magnesium acetate. For this solution, 24.23g of Tris were added to 800ml of nH₂O and the mixture was adjusted to pH 7.5. To this solution was added 10.72g of magnesium acetate and dissolved completely. The solution was brought to a final volume of 1000ml andand can be stored at 4 deg.C for one month. 10 XTE included 100mM Tris-HCl (pH 7.5) and 50mM EDTA. These reagents were added together and mixed well. The solution can be stored at room temperature for 6 months.

Example 2: primer design

As discussed above, universal adaptors are designed to include: 1) a unique set of PCR primers of generally 20bp length (located adjacent to (2)); 2) a unique set of sequencing primer regions, typically 20bp long, and 3) optionally followed by a unique discriminating key sequence consisting of at least one (i.e., a, C, G, T) of each deoxyribonucleotide. The likelihood of cross-hybridization between the primer and an undesired region of the target genome decreases with increasing genome size and increasing length of perfect match with the primer. However, this potential interaction with the cross-hybridizing region (CHR) is undesirable for reasons set forth below.

In a preferred embodiment of the invention, PCR amplification and subsequent sequencing is performed using a single stranded DNA library. The sequencing method requires digestion of a given genome into a 150-and 500-bp fragment, followed by ligation of two unique bipartite primers (consisting of both PCR and sequencing regions) to the 5 'and 3' ends of the fragment (FIG. 25). Unlike general PCR amplification, which is based on melting temperature (T.sub.t), the disclosed methods utilize synthetic primer sites, which necessitates careful primer design anew_m) The uniqueness of the primer sequence within the genome and the proximity to a specific region or target gene, the existing part of the genome is selected as the primer site.

Selecting a tetramer:

strategies for re-designing primers are found in the work on Molecular markers for hybridization experiments (see, Hensel, M. and D.W. Holden, Molecular genetic protocols for the purpose of the primer of viral in bothappic bacteria and enzymes, 1996.142(Pt 5): p.1049-58; Shoemaker, D.D. et al, quantitative analysis of yeast deletion reactions using a high throughput Molecular rod-coding sequence, 1996.14 (4): p.450-6) and PCR/LDR (polymerase chain reaction/ligation detection reaction) hybridization primers (see, Gerriry, N.P. et al, viral methods for large Molecular detection, Molecular probes for Molecular markers of hybridization experiments; see, U.S. Pat. No. 3, J.P. 32, Molecular probes for the purpose of Molecular analysis and Molecular analysis of viral in genetic analysis and Molecular analysis, P.251, Molecular analysis of Molecular analysis, p.251, Molecular analysis, p.27. Pat. 32. 3. Biocoding, Molecular analysis of Molecular analysis, Molecular analysis.

The PCR/LDR work was particularly relevant and focused on designing oligonucleotides "zip codes", composed of six with similar final T_mA specially designed tetramer-composed 24-base primer (see, Gerry, N.P. et al, Universal DNA microarray method for multiplex detection of low impedance point mutations. journal of molecular Biology, 1999.292: p.251-262; U.S. Pat. No. 6,506,594). The tetramer component was selected based on the following criteria: each tetramer is at least two bases different from others, excluding tetramers that induce self-pairing or hairpin formation, and excluding palindrome (AGCT) or repetitive tetramer (TATA). 256(4⁴) The 36 of the seed possibility permutations meet the necessary requirements and are then subjected to further definition required for acceptable PCR primer design (table 1).

TABLE 1

The table shows an indication based on the results obtained by Gerry et al 1999 j.mol.bio.292: 251-262 listed the matrix for selection of the standard tetrameric primer components. Each tetramer must be different from all others by at least two bases. The tetramer cannot be palindromic or complementary to any other tetramer. Thirty-six tetramers (bold, underlined) were selected; the sequence in italics represents an unconsidered palindromic tetramer.

Designing a primer:

PCR primers were designed to conform to the common specifications for general primer Design (see, Rubin, E. and A.A.Levy, A physical model and a computational sizing of PCR using complex templates.nucleic Acids Res, 1996.24 (18): p.3538-45; Buck, G.A. et al, Design protocols and performance of custom DNA sequencing primers.Biotechnology, 1999.27 (3): p.528-36), and actual selection was performed by computer program MMP. For efficient synthesis of a PCR/sequencing primer consisting entirely of two parts, the primer was limited to a length of 20 bases (5 tetramers). Each primer contained a two-base GC clamp at the 5 'end and a single GC clamp at the 3' end (Table 2), all sharing a similar T_m(+/-2 ℃ C.) (FIG. 27). No hairpin formation within the primer was allowed (internal hairpin stem. DELTA.G > -1.9 kcal/mol). Dimerization is also controlled; a maximum acceptable dimer of 3 bases is permissible, but it can occur in the last six 3 'bases, and the maximum allowable Δ G for the 3' dimer is-2.0 kcal/mol. In addition, penalties are applied to primers whose 3' ends are too similar to the other primers in the set, thereby avoiding cross-hybridization between the reverse complements of one primer to the other.

TABLE 2

	1-pos	2-pos	3-pos	4-pos	5-pos
						1	CCAT	TGAT	TGAT	TGAT	ATAC
2	CCTA	CTCA	CTCA	CTCA	AAAG
						3	CGAA	TACA	TACA	TACA	TTAG
4	CGTT	AGCC	AGCC	AGCC	AATC
						5	GCAA	GACC	GACC	GACC	TGTC
6	GCTT	TCCC	TCCC	TCCC	AGTG
						7	GGAC	ATCG	ATCG	ATCG	CTTG
8	GGTA	CACG	CACG	CACG	GATG
						9		TGCG	TGCG	TGCG	TCTG
10		ACCT	ACCT	ACCT
						11		GTCT	GTCT	GTCT
12		AGGA	AGGA	AGGA
						13		TTGA	TTGA	TTGA
14		CAGC	CAGC	CAGC
						15		GTGC	GTGC	GTGC
16		ACGG	ACGG	ACGG
						17		CTGT	CTGT	CTGT
18		GAGT	GAGT	GAGT
						19		TCGT	TCGT	TCGT

Table 2 shows a possible arrangement of 36 selected tetramers providing two 5 'and a single 3' G/C clip. The internal site consists of the remaining tetramer. This results in 8X 19X 9 speciesPermutation, or 493,848 possible combinations. FIG. 27 shows the first pass based on T_mThe acceptable primers of (1) were selected by reducing the number of 493,848 primers to 56,246 with a T of 64-66 ℃_mA candidate of (1).

TABLE 3 probability of complete sequence match of primers increases with decreasing match length requirement and increasing target genome size

Matching length	Complete match probability (1/4^ length)	Chance of match in adenovirus-35K bases%	Chance of matching in NCBI bacteria database-488M glass base%	Chance of base matching 3B in humans%
					20	9.1E-13	0.00％	0.04％	0.27％
19	7.3E-12	0.00％	0.65％	4.32％
					18	4.4E-11	0.00％	5.67％	34.37％
17	2.3E-10	0.00％	35.69％	99.17％
					16	1.2E-09	0.02％	97.52％	＞100％
15	5.6E-09	0.12％	＞100％	＞100％
					14	2.6E-08	0.64％	＞100％	＞100％
13	1.2E-07	3.28％	＞100％	＞100％
					12	5.4E-07	15.68％	＞100％	＞100％
11	2.4E-06	58.16％	＞100％	＞100％
					10	1.0E-05	99.35％	＞100％	＞100％
9	4.6E-05	99.77％	＞100％	＞100％
					8	2.0E-04	＞100％	＞100％	＞100％
7	8.5E-04	＞100％	＞100％	＞100％
					6	3.7E-03	＞100％	＞100％	＞100％
5	1.6E-02	＞100％	＞100％	＞100％
					4	6.4E-02	＞100％	＞100％	＞100％
3	2.5E-01	＞100％	＞100％	＞100％
					2	7.1E-01	＞100％	＞100％	＞100％
1	1.0E+00	＞100％	＞100％	＞100％

The possibility of complementary regions occurring within the target genome is not a major concern in the primer design process, although tolerance of PCR to mismatches in complex sample populations has been reported (see, e.g., Rubin, E. and A. Levy, A physical model and a formulated simulation of PCR using complex templates. nucleic acids Res, 1996.24 (18): p.3538-45). Although the possibility of finding complete pairing with 20 base primers is extremely low (4) ²⁰) (table 3), but the probability of finding fewer discontiguous pairs increases significantly with the size of the target genome. As a result, the probability of finding a perfect match of at least 10 of the 20 bases for the adenovirus genome is 99.35%. The probability of finding a perfect match of 16 bases is 97% for sequences in the NCBI database (approximately 100-fold greater than the adenovirus genome) for sequences in the human genome (30 billion genes) and 99% for 20 bases of primers.

The high probability of primer cross-hybridization to a genomic region is less problematic than predicted due to the random DNA digestion used to generate the template fragment. Thus, the efficiency of the cross-hybridizing region (CHR) is quite good. It is unlikely that CHR can successfully compete with a perfect match between PCR primers and template in solution. In addition, any primer that includes a mismatch at its 3' end is subject to significant competition penalties. Even if the CHR competes for the desired PCR primer, it will produce a truncated PCR product without a downstream site for the sequencing primer. If the truncated product can be driven to the capture bead and immobilized, one of two conditions will occur. If the CHR competes out of the solution-primer, the immobilized product will lack a sequencing primer binding site and will result in an empty PicoTiter plate (PTP) well. If the CHR competes out of the bead-bound primer, sequencing primer The object will still be present and the only effect is a shorter insertion. None of the results unduly compromise sequencing quality. Given the large amount of genomic material used in the sample preparation process (currently 25. mu.g, 5.29X 10 containing 35Kb of adenovirus genome¹⁶Single copy) oversampling can be used to provide fragments lacking the entire CHR and allow for standard PCR amplification of the region of interest.

Example 3: preparation of samples by atomization

Preparation of DNA by atomization

The purpose of the nebulization step is to fragment a large piece of DNA, such as a whole genome or a large portion of a genome, into a smaller molecule that can be subjected to DNA sequencing. This population of smaller sized DNA generated from a single DNA template is referred to as a library. The nebulization cuts the double-stranded template DNA into fragments of 50-900 base pairs. The cleaved library contained single-stranded ends that were end-repaired by a combination of T4DNA polymerase, e.coli DNA polymerase I (Klenow fragment), and T4 polynucleotide kinase. Both T4 and Klenow DNA polymerase are used to "fill in" the 3 'recessed ends (5' overhangs) of DNA by their 5 '-3' polymerase activity. The single-stranded 3 '-5' exonuclease activity of T4 and Klenow DNA polymerase will remove the 3 'overhang ends and the kinase activity of T4 polynucleotide kinase will add phosphate to the 5' hydroxyl end.

A sample was prepared by:

1. mu.g of gDNA (genomic DNA) were obtained and adjusted to a final volume of 100. mu.l in 10mM TE (10mM Tris, 0.1mM EDTA, pH 7.6; see reagents at the end of the section). By measuring an o.d. of 1.8 or higher._260/280To analyze DNA for contamination. The final gDNA concentration is expected to be about 300. mu.g/ml.

2. To the gDNA was added 1600. mu.l of ice-cold nebulization buffer (see end).

3. The reaction mixture was placed in an ice-cold nebulizer (CIS-US, Bedford, MA).

4. The cap from a 15ml snap-cap falcon tube was placed on top of the nebulizer (fig. 28A).

5. The cap was secured with a clean nebulizer clip assembly consisting of a suitable cap (falcon tube cap) and two rubber O-rings (fig. 28B).

6. The bottom of the nebulizer was attached to the nitrogen source and the entire device was wrapped with parafilm (fig. 28C and 28D).

7. While holding the nebulizer upright (as shown in fig. 28D), 50psi of nitrogen was applied for 5 minutes. The bottom of the nebulizer was tapped on a hard surface every few seconds to force the condensed liquid back to the bottom.

After 8.5 minutes the nitrogen was turned off. After the pressure was normal (30 seconds), the nitrogen source was removed from the nebulizer.

9. The parafilm was removed and the top of the nebulizer was unscrewed. The sample was removed and transferred to a 1.5ml microcentrifuge tube.

10. The nebulizer top was reloaded and the nebulizer was centrifuged at 500rpm for 5 minutes.

11. The remaining sample in the nebulizer was collected. The total recovery was approximately 700. mu.l.

12. The recovered samples were purified using QIAquick columns (Qiagen inc., Valencia, CA) according to the manufacturer's instructions. The large volume requires the column to be loaded several times. The sample was eluted with 30. mu.l of buffer EB (10mM Tris HCl, pH 8.5; provided in Qiagen kit) pre-warmed at 55 ℃.

13. The samples were quantified by UV spectroscopy (2. mu.l at 1: 100 dilution in 198. mu.l water).

Supplement with enzymes

Spraying of the DNA template produces a number of DNA fragments with ragged ends. These ends are blunted and easily ligated to the adaptor fragment by using three enzymes, T4DNA polymerase, E.coli DNA polymerase I (Klenow fragment) and T4 polynucleotide kinase.

Samples were prepared as follows:

1. the following reagents were added in order to a 0.2ml tube:

28 μ l of purified, nebulized gDNA fragment

5 mul of water

5 μ l10 XT 4DNA polymerase buffer

5μl BSA(1mg/ml)

2μldNTPs(10mM)

5μl T4DNA polymerase (3 units-μl)

50 μ l final volume

2. The solution of step 1 was mixed well and incubated in an MJ thermocycler (any precise incubator can be used) for 10 minutes at 25 ℃.

3. Mu.l of E.coli DNA polymerase (Klenow fragment) (5 units/ml) was added.

4. The reaction was mixed well and incubated at 25 ℃ for 10 minutes and 16 ℃ for a further 2 hours in an MJ thermocycler.

5. The treated DNA was purified using a QiaQuick column and eluted with 30. mu.l of buffer EB (10mM Tris HCl, pH 8.5) pre-warmed at 55 ℃.

6. The following reagents were combined in a 0.2ml test tube:

30 μ l Qiagen purified, filled, nebulized gDNA fragment

5 mul of water

5 μ l10 XT 4PNK buffer

5μl ATP(10mM)

5μl T4PNK (10 units/ml)

50 μ l final volume

7. The solutions were mixed and placed in an MJ thermocycler using the T4PNK program at 37 ℃ for 30 minutes, 65 ℃ for 20 minutes, then stored at 14 ℃.

8. The samples were purified using a QIAquick column and eluted in 30. mu.l of buffer EB which was preheated at 55 ℃.

9. A2. mu.l aliquot of the final refill was analyzed using a BioAnalyzer DNA 1000LabChip (see below).

Ligation of adapters

The process of ligating adaptors was performed as follows:

1. the following reagents were added in order to a 0.2ml tube:

20.6 μ l of molecular biology grade water

28 μ l of digested, filled gDNA library

60 ul 2 XQUICK ligase reaction buffer

Mu.l MMP (200 pmol/. mu.l) Universal adaptor set

9.6μl Rapid ligase

Total volume of 120. mu.l

The above reaction was designed for 5. mu.g and varied accordingly depending on the amount of gDNA used.

1. The reagents were mixed well and incubated at 25 ℃ for 20 minutes. The tube was kept on ice until a gel for agarose gel electrophoresis was prepared.

Gel electrophoresis and extraction of adapted gDNA libraries

Nebulization of genomic DNA produced a library population of 50-900 bp. The addition of an 88-bp universal adaptor will shift the population to a larger size and will result in a migration pattern with a larger size range (approximately 130-980 bp). Adaptor dimers will migrate to 88bp while adaptors not ligated will migrate to 44 bp. Therefore, genomic DNA libraries with a size range of 250bp or more can be physically separated from agarose gels and purified using standard gel extraction techniques. Gel separation of the adapted DNA library will result in recovery of a library population with a size range of > 250bp (the size range of the library may vary depending on the application). The size of the library after adaptor ligation ranged from 130-980 bp. It should be noted that the method can be used to perform separation of any band size range by excising different regions of the gel, such as, for example, 130-. Fragments of 250bp to 500bp were isolated using the method described below.

A150 ml agarose gel was prepared, comprising 2% agarose, 1 XTBE and 4.5. mu.l ethidium bromide (10mg/ml mother liquor). The ligated DNA was mixed with 10X Ready Load Dye and loaded onto the gel. In addition, 10. mu.l of a 100bp gradient (0.1. mu.g/. mu.l) was loaded into both lanes of the ligation reaction remote from the sample flanks. The gel was electrophoresed at 100V for 3 hours. When running the gel was completed, the gel was removed from the gel box and transferred to GelDoc, which was covered with a plastic cover. DNA bands were examined using a Prep UV lamp. The library populations of 250-and 500-bp fragment sizes were excised from the agarose gel using a sterile, single-use scalpel. This procedure is performed as quickly as possible to avoid DNA nicks. The gel strips were placed in 15ml falcon tubes. Agarose embedded gDNA was isolated using Qiagen MinElute gel extraction kit. Aliquots of each isolated gDNA library were analyzed using a BioAnalyzer DNA 1000LabChip to assess the precise distribution of the gDNA library population.

Strand replacement and extension of gDNA library and isolation of Single stranded gDNA library Using streptavidin beads

Strand replacement and extension of the nicked double stranded gDNA library was performed as described in example 1, except that the Bst treated samples were incubated in a thermal cycler at 65 ℃ for 30 minutes and placed on ice until needed. Streptavidin beads were prepared as described in example 1, except that two washes with 200. mu.l IX binding buffer and with 200. mu.l nH ₂Two washes of O were performed for the final wash. Separation of sheets Using streptavidin beads as followsChain gDNA library. The water from the washed beads was removed and 250. mu.l of the thawing solution was added (see below). The bead suspension was mixed well and incubated at room temperature for 10 minutes on a tube shaker. In a separate tube, 1250. mu.l of PB (from QiaQuick purification kit) and 9. mu.l of 20% acetic acid were mixed. The beads in 250. mu.l of the thawing solution were precipitated using DynalMPC and the supernatant was carefully removed and transferred to a freshly prepared PB/acetic acid solution. DNA from 1500. mu.l of solution was purified using a single QiaQuick purification spin column. This was done by loading the sample twice through the same column at 750. mu.l/sample. The single-stranded gDNA library was eluted with 15. mu.l of buffer EB which was pre-warmed at 55 ℃.

Quantification and preservation of Single-stranded gDNA libraries

Single-stranded gDNA was quantified using an RNA Pico 6000LabChip as described in example 1. In some cases, the single-stranded gDNA library was quantified by a second test to ensure that the initial Agilent2100 quantification was performed accurately. For this purpose, RiboGreen quantification (ssDNA quantification by fluorimetry) was performed as described to confirm Agilent2100 quantification. If the two assessments differ by more than a factor of 3, each analysis is repeated. If the quantization shows more than a factor of 3 difference between the two methods, a wider range of templates is used: beads.

Dilution and preservation of the single stranded gDNA library was performed as described in example 1. The product was as follows:

the remaining final volume of ssDNA library after LabChip analysis was 12 μ l.

The remaining final volume of ssDNA library after RiboGreen analysis was 9 μ l.

The final volume of ssDNA library remaining after TE addition was 18. mu.l.

An equal volume of TE was added to the single stranded gDNA library stock. Single-stranded gDNA library of 1X 10⁸Molecules/. mu.l buffer TE. Stocks were diluted (1/500) in TE to 200,000 molecules/. mu.l and 20. mu.l aliquots were prepared.

Post-nebulization library fragment size distribution

Typical results of Agilent 2100DNA 1000LabChip analysis from 1. mu.l of material after nebulization and priming are shown in FIG. 29A. The size distribution of most products is expected to fall around 50-900 base pairs. The expected average size (top of peak) is about 450 bp. Typical results from gel purification of adaptor-ligated library fragments are shown in fig. 29B.

Reagent

Unless otherwise specified, the reagents listed in the examples represent standard reagents that are commercially available. For example, Klenow, T4DNA polymerase Buffer, T4PNK Buffer, Quick T4DNA ligase, Quick Ligation Buffer, BstDNA polymerase (large fragment) and ThermoPol reaction Buffer are available from New England Biolabs (Beverly, MA) and dNTP mixtures are available from Pierce (Rockford, IL). Agarose, ultrapure TBE, BlueJuice gel loading buffer, and Ready-Load100bp DNA gradient were purchased from Invitrogen (Carlsbad, Calif.). Ethidium bromide and 2-propanol are available from Fisher (Hampton, NH). RNA gradients can be purchased from Ambion (Austin, TX). Other agents are known and/or listed below:

Melting solution

Component (A)	Required amount of	Manufacturer(s)	Material numbering
				NaCl(5M)	200μl	Invitrogen	24740-011
NaOH(10N)	125μl	Fisher	SS255-1
				Molecular biological grade water	9.675ml	Eppendorf	0032-006-205

The thawing solution comprised 100mM NaCl and 125mM NaOH. The listed reagents were combined and mixed well. The solution can be stored at room temperature for 6 months.

Binding and washing (B & W) buffer (2 × and 1 ×):

component (A)	Required amount of	Manufacturer(s)	Material numbering
				Ultrapure Tris-HCl (pH7.5, 1M)	250μl	Invitrogen	15567-027
EDTA(0.5M)	50μl	Invitrogen	15575-020
				NaCl(5M)	10ml	Invitrogen	24740-011
Molecular biological grade water	14.7ml	Eppendorf	0032-006-205

2×B&The W buffer included 10mM Tris-HCl (pH7.5), 1mM EDTA and 2M NaCl at final concentrations. The listed reagents were combined and mixed well. The solution can be stored at room temperature for 6 months. By mixing 2 XB&W and picopure H₂Preparation of 1 XB by O1: 1 mixing&W buffer solution. The final concentration was half that listed above, i.e., 5mM Tris-HCl (pH7.5), 0.5mM EDTA, and 1M NaCl.

Other buffers include the following 1 × T4DNA polymerase buffers: 50mM NaCl, 10mM Tris-HCl, 10mM MgCl₂1mM dithiothreitol (pH7.9 at 25 ℃ C.). TE: 10mM Tris, 1mM EDTA.

Preparation of specialty reagents

TE(10mM)：

Component (A)	Required amount of	Manufacturer(s)	Material numbering
				TE(1M)	1ml	Fisher	BP1338-1
Molecular biological grade water	99ml	Eppendorf	0032-006-205

The reagents were mixed well and the solution was able to be stored at room temperature for 6 months.

Nebulized buffer

Component (A)	Required amount of	Manufacturer(s)	Material numbering
				Glycerol	53.1ml	Sigma	G5516
Molecular biological grade water	42.1ml	Eppendorf	0032-006-205
				Ultrapure Tris-HCl (pH7.5, 1M)	3.7ml	Invitrogen	15567-027
EDTA(0.5M)	1.1ml	Sigma	M-10228

All reagents (glycerol last added) were added to Stericup and mixed well. The solution was labeled and it could be stored at room temperature for 6 months.

ATP(10mM)：

Component (A)	Required amount of	Manufacturer(s)	Material numbering
				ATP(100mM)	10μl	Roche	1140965
Molecular biological grade water	90μl	Eppendorf	0032-006-205

The reagents were mixed well and the solution was able to be stored at-20 ℃ for 6 months.

BSA(1mg/ml)：

Component (A)	Required amount of	Manufacturer(s)	Material numbering
				BSA(10mg/ml)	10μl	NEB	M0203 kit
Molecular biological grade water	90μl	Eppendorf	0032-006-205

The reagents were mixed well and the solution was able to be stored at 4 ℃ for 6 months.

Library annealing buffer, 10 backing

Component (A)	Required amount of	Manufacturer(s)	Material numbering
				Ultrapure Tris-HCl (pH7.5, 1M)	200ml	Invitrogen	15567-027
Magnesium acetate, enzyme grade (1M)	10.72g	Fisher	BP-215-500
				Molecular biological grade water	～1L	Eppendorf	0032-006-205

The 10 × annealing buffer included 200mM Tris (pH7.5) and 50mM magnesium acetate. For this buffer, 200ml of Tris was added to 500ml of picopureH₂And (4) in O. Subsequently, 10.72g of magnesium acetate was added to the solution and dissolved sufficiently. The solution was adjusted to a final volume of 1000 ml. The solution can be stored at 4 deg.C for 6 months. To avoid the possibility of laboratory contamination, the buffer was aliquoted for single or short-term use.

Adapter

Adaptor "a" (400 μ M):

component (A)	Required amount of	Manufacturer(s)	Material numbering
				Adapter A (sense; HPLC purified, phosphothioester bond, 44bp, 1000 pmol/. mu.l)	10.0μl	IDT	Customization
Adapter A (antisense; HPLC purified, phosphothioester bond, 40bp, 1000 pmol/. mu.l)	10.0μl	IDT	Customization
				Annealing buffer (10X)	2.5μl	454 Co Ltd	Upper table
Molecular biological grade water	2.5μl	Eppendorf	0032-006-205

For this solution, 10. mu.l of 1000 pmol/. mu.l adaptor A (44bp, sense) was mixed with 10. mu.l of 1000 pmol/. mu.l adaptor A (40bp, antisense), 2.5. mu.l of 10 × library annealing buffer and 2.5. mu.l of water (V)_f25 μ l). The adapters were annealed on a Sample Prep Lab thermal cycler using the ANNEAL-A program (see appendix below). More details of the adaptor design are provided in the appendix.

Adaptor "B" (400 μ M):

component (A)	Required amount of	Manufacturer(s)	Material numbering
				Adapter B (sense; HPLC purified, phosphothioester bond, 40bp, 1000 pmol/. mu.l)	10μl	IDT	Customization
Adapter B (antisense; HPLC purified, phosphothioester bond, 44bp, 1000 pmol/. mu.l)	10μl	IDT	Customization
				Annealing buffer (10X)	2.5μl	454 Co Ltd	Upper table
Molecular biological grade water	2.5μl	Eppendorf	0032-006-205

For this solution, 10. mu.l of 1000 pmol/. mu.l was added Adaptor B (40bp, sense) was mixed with 10. mu.l of 1000 pmol/. mu.l adaptor B (44bp, antisense), 2.5. mu.l of 10 × library annealing buffer and 2.5. mu.l of water (V)_f25 μ l). The adapters were annealed on a Sample Prep Lab thermal cycler using the ANNEAL-A program (see appendix below). After annealing, the adaptors "A" and "B" are combined (V)_f50 μ l). The set of adaptors can be stored at-20 ℃ until use.

20% acetic acid

Component (A)	Required amount of	Manufacturer(s)	Material numbering
				Acetic acid, ice-cold	2ml	Fisher	A35-500
Molecular biological grade water	8ml	Eppendorf	0032-006-205

For this solution, ice cold acetic acid was added to water. The solution can be stored at room temperature for 6 months.

Adaptor annealing program:

ANNEAL-A procedure for primer annealing:

1. incubating at 95 deg.C for 1 min;

2. the temperature was reduced to 15 ℃ at 0.1 ℃/sec; and

3. the temperature was maintained at 14 ℃.

End-repaired T4 polymerase/Klenow POLISH program:

1. incubating at 25 deg.C for 10 min;

2. incubation at 16 ℃ for 2 hours; and

3. the temperature was maintained at 4 ℃.

End-repair T4PNK program:

1. incubating at 37 ℃ for 30 min;

2. incubating at 65 deg.C for 20 min; and

3. the temperature was maintained at 14 ℃.

BST procedure for strand displacement and nicked double-stranded gDNA extension:

1. incubating at 65 deg.C for 30 min; and

2. The temperature was maintained at 14 ℃.

And step 9: dilution and preservation of Single-stranded DNA libraries

Single stranded DNA library in EB buffer: the final volume remaining was 25 μ l.

Initial stock dilutions were prepared as follows: using Pyrosequencing (Pyrosequencing AB, Uppsala, Sweden) results, the single-stranded DNA library was diluted to 100M molecules/. mu.L in 1 × annealing buffer (usually this is a 1: 50 dilution).

Aliquots of the commonly used single-stranded DNA library were made by diluting 200,000 molecules/. mu.L in 1 × annealing buffer and preparing 30. mu.L aliquots. Store at-20 ℃. Samples were used in emulsion PCR.

Preparation of reagents

Stop solution (50mM EDTA): 100. mu.l of 0.5M EDTA and 900. mu.l of nH₂O to make 1.0ml of a 50mM EDTA solution.

10mM dNTPs solution includes 10. mu.l dCTP (100mM), 10. mu.l dATP (100mM), 10. mu.l GTP (100mM) and 10. mu.l TTP (100mM), 60. mu.l molecular biology grade water, (nH)₂O). All four 100mM nucleotide stocks were thawed on ice. Mu.l of each nucleotide was combined with 60. mu.l of nH₂O to reach a final volume of 100 μ l and mixed well. 1ml aliquots were dispensed into 1.5ml microcentrifuge tubes and stored at-20 ℃ for no more than one year.

Annealing buffer, 10 ×: the 10 × annealing buffer included 200mM Tris (pH 7.5) and 50mM magnesium acetate. For this solution, 24.23g of Tris were added to 800ml of nH₂O and the mixture was adjusted to pH 7.5. To this solution was added 10.72g of magnesium acetate and dissolved completely. The solution was brought to a final volume of 1000 ml. The solution can be stored at 4 ℃ for one month.

10 × TE: 10 XTE comprises 100mM Tris-HCl (pH 7.5) and 50mM EDTA. These reagents were added together and mixed well. The solution can be stored at room temperature for 6 months.

Example 4: bead emulsion PCR

The following processes, including capture of template DNA, DNA amplification and recovery of beads bound to amplified template can be performed in a single tube. The emulsion format ensures physical separation of the beads into 100-and 200 μm "microreactors" within this single tube, thus allowing clonal amplification of various templates. Immobilization of the amplification product is achieved by extending the template along the oligonucleotide bound to the DNA capture bead. Typically, the copy number of the immobilized template is 10,000,000-30,000,000 copies/bead. Multiple copies of DNA capture beads with single species of nucleic acid template are readily distributed onto PTPs.

The 300,000 75 picoliter wells etched on the PTP surface provide a unique array for sequencing short DNA templates in a massively parallel, efficient and cost-effective manner. However, this requires a significant number (in millions of copies) of clonal templates in each reaction well. The methods of the invention allow the user to clonally amplify single-stranded genomic templates by performing PCR reactions in standard test tubes or microtiter plates. Single copies of the template can be mixed with capture beads, resuspended in a complete PCR amplification solution, and emulsified into microreactors (100. sup. th and 200. mu.m. diameter), after which PCR amplification produces 10 of the starting template ⁷And (5) amplifying. This method is much simpler and more cost effective than previous methods.

Binding of nucleic acid template to Capture beads

This example describes the preparation of a population of beads to which preferably only one unique nucleic acid template is attached. Successful clonal amplification relies on the delivery of a controlled number of templates (0.5 to 1) to each bead. Delivery of excess template can result in PCR amplification of the mixed template population, preventing the generation of meaningful sequence data, while a deficiency of template will result in fewer wells containing template for sequencing. This may attenuate the degree of genome coverage provided by the sequencing phase. As a result, the template concentration is preferably accurately determined by repeated quantification, and the binding method follows the following list.

Template quality control

The success of the emulsion PCR reaction relates to the quality of the template. Despite the care and care taken in the amplification period, poor quality templates will prevent successful amplification and the generation of meaningful data. To avoid unnecessary loss of time and money, it is important to check the quality of the template material before starting the emulsion PCR phase of the process. Preferably, the library should undergo two quality control steps before it is used in emulsion PCR. The concentration and distribution of the product it contains should be determined. Ideally, the library should appear as a heterogeneous population of fragments, with few or no adapter dimers (e.g., -90 bases) visible. In addition, amplification of the PCR primers should result in a fuzzy smear of one product, e.g., from 300 to 500 bp. The absence of amplification product may reflect failure to properly ligate the adapter to the template, while the presence of a single band of any size may reflect contamination of the template.

Preparation of PCR solution

The main problem at this stage is to avoid contamination of the PCR reaction mixture by stray amplicons. Contamination of the PCR reaction with residual amplicons is a serious problem that can lead to failure of the sequencing run. To reduce the possibility of contamination, appropriate laboratory techniques should be followed and the preparation of the reaction mixture should be carried out in a clean room in a laminar flow hood clean bench for UV treatment.

PCR reaction mixture

For 200. mu.l of PCR reaction mix (sufficient to amplify 600,000 beads), the following reagents were mixed in a 0.2ml PCR tube:

TABLE 4

	Mother liquor	Finally, the product is processed	Microlitre
				HIFI buffer solution	10X	1X	20
Treated nucleotides	10mM	1mM	20
				Mg	50mM	2mM	8
BSA	10％	0.1％	2
				Tween80	1％	0.01％	2
Ppase	2U	0.003U	0.333333
				Primer MMP1a	100μM	0.625μM	1.25
Primer MMP1b	10μM	0.078μM	1.56
				Polymerase enzyme	5U	0.2U	8
Water (W)			136.6
				Total up to			200

The tubes were shaken well and kept on ice until the template annealed to the beads.

DNA capture beads:

1. 600,000DNA capture beads were transferred from the storage tubes to 1.5ml microcentrifuge tubes. The exact amount used depends on the bead concentration of the normalization reagent.

2. The beads were pelleted in a bench top mini centrifuge and the supernatant removed.

3. Steps 4-11 were performed in a PCR clean room.

4. The beads were washed with 1mL of 1 × annealing buffer.

5. The capture beads were pelleted in a microcentrifuge. The tube was rotated 180 ° and centrifuged again.

6. All supernatant was removed from the tubes containing the beads except for approximately 10. mu.l. The beads were not disturbed.

7. 1mL of 1 × annealing buffer was added and the mixture was incubated for 1 min. The beads were then precipitated as in step 5.

8. All material was removed from the tube except for approximately 100. mu.l.

9. The remaining beads and solution were transferred to a PCR tube.

10. A1.5 mL tube was washed with 150. mu.l of 1 × annealing buffer by pipetting up and down several times. This was added to a PCR tube containing beads.

11. The beads were pelleted as in step 5 and all but approximately 10 μ l of supernatant was removed, taking care not to disturb the beads.

12. Remove a quantified single stranded template dna (sstdna). The final concentration was 200,000-sstDNA molecules/. mu.l.

13. Mu.l of diluted sstDNA was added to the PCR tube containing the beads. This is equivalent to 600,000 copies of sstDNA.

14. The tube was gently shaken to mix the ingredients.

15. sstDNA was annealed to the capture beads in a PCR thermocycler using the following protocol, program 80Anneal stored in the EPCR folder on the MJ thermocycler:

5 minutes at 65 ℃;

reduced to 60 ℃ at 0.1 ℃/sec;

held at 60 ℃ for 1 minute;

reduced to 50 ℃ at 0.1 ℃/sec;

Held at 50 ℃ for 1 minute;

reduced to 40 ℃ at 0.1 ℃/sec;

held at 40 ℃ for 1 minute;

reduced to 20 ℃ at 0.1 ℃/sec; and

it was kept at 10 ℃ until the next step could be carried out.

In most cases, the beads are used for amplification immediately after template binding. If the beads are not used immediately, they should be stored in the template solution at 4 ℃ until needed. After storage, the beads were treated as follows.

16. As in step 6, the beads were removed from the thermal cycler, centrifuged, and the annealing buffer removed without disturbing the beads.

17. The beads were kept in an ice bucket until emulsification was performed (example 2).

18. The capture beads include an average of 0.5-1 copies of sstDNA bound to each bead, ready for emulsification.

Example 5: emulsification action

Suitable PCR solutions for this step are described below. For 200. mu.l of PCR reaction mix (sufficient to amplify 600,000 beads), the following were added to a 0.2ml PCR tube:

	mother liquor	Finally, the product is processed	Microlitre
				HIFI buffer solution	10X	1X	20
Treated nucleotides	10mM	1mM	20
				Mg	50mM	2mM	8
BSA	10％	0.1％	2
				Tween80	1％	0.01％	2
Ppase	2U	0.003U	0.333333
				Primer MMP1a	100μM	0.625μM	1.25
Primer MMP1b	10μM	0.078μM	1.56
				Taq	5U	0.2U	8
Water (W)			136.6
				Total up to			200

This example describes how to generate a heat stable water-in-oil emulsion containing approximately 3,000PCR microreactors per microliter. Listed below are methods of preparing emulsions.

1. Add 200. mu.l of PCR solution to 600,000 beads (both fractions from example 1).

2. Resuspend the beads by blowing up and down several times.

3. The PCR-bead mixture was incubated at room temperature for 2 minutes to equilibrate the beads with the PCR solution.

4. Add 400. mu.l of the emulsion oil to a UV-irradiated 2ml microcentrifuge tube.

5. Add the "amplicon free 1/4" stirring magnetic stir bar to the emulsion oil tube. An amplicon-free stir bar was prepared as follows. The 1/4 "stir bar was made using a large stir bar. The stir bar was then:

washing (dropping or spraying) with DNA-Off;

washing with picopure water;

drying with a Kimwipe edge; and

ultraviolet irradiation for 5 minutes.

6. Remove the magnetic slips of the Dynal MPC-S tube holder. Tubes of emulsion oil were placed in a test tube rack. The test tube was placed in the center of a stirring plate set at 600 rpm.

7. The tube was shaken vigorously to resuspend the beads. This ensures that there is minimal bead clumping.

8. Using a P-200 pipette, the PCR-bead mixture was added drop-wise to the rotating oil at a rate of approximately one drop per 2 seconds, each drop being allowed to sink to the level of the magnetic stir bar and to emulsify before the next drop was added. The solution turned into a uniform milky white liquid with a similar viscosity to mayonnaise.

9. Once the entire PCR-bead mixture was added, the microcentrifuge tube was flicked several times to mix any oil on the surface with the milky emulsion.

10. Stirring was continued for an additional 5 minutes.

11. Steps 9 and 10 are repeated.

12. The stir bar is removed from the emulsified material by pulling it out of the tube with a larger stir bar.

13. Remove 10. mu.l of the emulsion and place on a microscope slide. The emulsion was covered with a cover slip and examined at 50 x magnification (10 x eyepiece and 5 x objective). A "good" emulsion is expected to consist mainly of individual beads in separate droplets (microreactors) of PCR solution in oil.

14. Suitable emulsion oil mixtures with emulsion stabilizers were prepared as follows. The components of the emulsion mixture are shown in table 5.

TABLE 5

Composition (I)	Required amount of	Origin of origin	Reference numerals
				Sigma light mineral oil	94.5g	Sigma	M-5904
Atlox 4912	1g	Uniqema	NA
				Span 80	4.5g	Uniqema	NA

The emulsion oil mixture was prepared by preheating Atlox 4912 to 60 ℃ in a water bath. Subsequently, 4.5 grams of Span 80 was added to 94.5 grams of mineral oil to form a mixture. Subsequently, 1 gram of preheated Atlox 4912 was added to the mixture. The solution was placed in a sealed container and mixed by shaking and rotation. Any signs of Atlox fixation and solidification were remedied by heating the mixture to 60 ℃, followed by shaking.

Example 6: amplification of

This example describes the amplification of template DNA in a bead-emulsion mixture. According to this method of the invention, the DNA amplification phase of the process takes 3 to 4 hours. After amplification is complete, the emulsion may be left on the thermal cycler for up to 12 hours before beginning the process of separating the beads. PCR thermal cycling was performed by placing 50-100. mu.l of the emulsified reaction mixture into a single PCR reaction chamber (i.e., PCR reaction tube). PCR was performed as follows:

1. a single pipette tip was used to transfer 50-100. mu.l amounts of the emulsion into approximately 10 separate PCR tubes or 96-well plates. For this step, the water-in-oil emulsion is highly viscous.

2. The plate is closed, or capped, with the PCR tube, and the container is placed in an MJ thermocycler with or without a 96-well plate adapter.

3. The PCR thermal cycler was programmed to run the following program:

1 cycle (4 min at 94 ℃) -hot start;

40 cycles (30 seconds at 94 ℃, 30 seconds at 58 ℃, 90 seconds at 68 ℃);

25 cycles (30 seconds at 94 ℃ C., 6 minutes at 58 ℃ C.); and

the mixture was stored at 4 ℃.

4. After the PCR reaction is complete, the amplification material is removed to continue the emulsion breaking and bead recovery.

Example 7: burst emulsion and bead recovery

This example describes how to break the emulsion and recover the beads with the amplification template thereon. Preferably, the emulsion after PCR should remain intact. The lower level of the emulsion, by visual inspection, corresponds to maintaining a milky white suspension. If the solution is clear, the emulsion may have partially dissolved into its water and oil phases, and it is likely that many beads will have a mixture of templates. If the emulsion has broken in one or two tubes, these samples should not be mixed with the other. If the emulsion has broken in all tubes, the process should not be continued.

1. All PCR reactions from the initial 600 μ Ι sample were combined into a single 1.5ml microcentrifuge tube using a single pipette tip. As mentioned above, the emulsion is very viscous. In some cases, the aspiration is repeated several times per tube. Transfer as much material as possible into a 1.5ml tube.

2. The remaining emulsified material was recovered from each PCR tube by adding 50. mu.l of Sigma mineral oil to each sample. Using a single pipette tip, each tube was pipetted up and down several times to resuspend the remaining material.

3. This material was added to a 1.5ml tube containing a large block of emulsified material.

4. The sample was vortexed for 30 seconds.

5. The samples were centrifuged in a bench top microfuge tube at 13.2K rpm in an Eppendorf microcentrifuge for 20 minutes.

6. The emulsion separates into two phases with a large white interface. The clear oil phase at the top was removed as much as possible. Leaving the cloud in the tube. The oil and aqueous layers are typically separated by a white layer. Beads were often observed to settle at the bottom of the tube.

7. The aqueous layer above the beads was removed and kept for analysis (gel analysis, Agilent 2100, and Taqman). If an interface of white material remained above the aqueous layer, 20 microliters of the lower aqueous layer was removed. This is carried out by penetrating the interface substance with a pipette tip and aspirating the solution from below.

8. To the remaining emulsion was added 1ml of hexane in a PTP Fabrication and Surface Chemistry from Fume Fume hood.

9. The sample was vortexed for 1 minute and centrifuged at full speed for 1 minute.

10. In the PTP Fabrication and Surface Chemistry from Fume Fume hood, the top oil/hexane phase was removed and placed in an organic waste container.

11. 1ml of 1 × annealing buffer in 80% ethanol was added to the remaining aqueous phase, interface and beads.

12. The sample was vortexed for 1 minute or until the white substance dissolved.

13. The samples were centrifuged at high speed for 1 minute. The tube was rotated 180 ° and centrifuged for an additional 1 min. The supernatant was removed without disturbing the bead pellet.

14. The beads were washed with 1ml of 1 × annealing buffer containing 0.1% Tween 20 and this step was repeated.

Example 8: single strand removal and primer annealing

If the beads are used in a pyrophosphate-bead sequencing reaction, then the second strand of the PCR product must be removed and the sequencing primer annealed to the single stranded template bound to the beads. This embodiment describes a method of accomplishing the same.

1. The beads were washed with 1ml of water and centrifuged twice for 1 minute. The tube was rotated 180 ° in between two centrifugations. After centrifugation, the aqueous phase was removed.

2. The beads were washed with 1ml of 1mM EDTA. The tube was centrifuged as in step 1 and the aqueous phase was removed.

3. 1ml of 0.125M NaOH was added and the sample was incubated for 8 minutes.

4. The sample was vortexed briefly and placed in a microcentrifuge tube.

After 5.6 minutes, the beads were pelleted and as much solution as possible was removed as in step 1.

6. At the completion of the 8 min NaOH incubation, 1ml of 1 × annealing buffer was added.

7. The sample was briefly vortexed and the beads were pelleted as in step 1. As much solution as possible was removed and 1ml of 1 × annealing buffer was added again.

8. The sample was briefly vortexed, the beads were pelleted as in step 1, and 800. mu.l of 1 × annealing buffer was removed.

9. The beads were transferred to a 0.2ml PCR tube.

10. The beads were transferred and as much annealing buffer as possible was removed without disturbing the beads.

11. Add 100. mu.l of 1 × annealing buffer.

12. Mu.l of 100. mu.M sequencing primer was added. The sample was shaken just before annealing.

13. Annealing was performed in an MJ thermocycler using the "80 Anneal" program.

14. The beads were washed three times with 200. mu.l of 1 × annealing buffer and resuspended with 100. mu.l of 1 × annealing buffer.

15. The beads were counted in a Hausser Hemacytometer. Typically, 300,000 to 500,000 beads are recovered (3,000-5,000 beads/. mu.l).

16. The beads were stored at 4 ℃ and could be used for sequencing for one week.

Example 9: optional enrichment step

The beads containing amplicons were enriched using the following method. Enrichment is not necessary but it can be used to perform subsequent molecular biology techniques such as more efficient DNA sequencing.

50 microliters of 10 μ M (500 picomoles total) biotin-sequencing primer was added to agarose beads containing the amplicons from example 5. The beads were placed in a thermal cycler. Primers were annealed to DNA on the beads by the thermal cycler annealing procedure of example 2.

After annealing, agarose beads were washed three times with annealing buffer containing 0.1% Tween 20. The ssDNA fragments now containing annealed biotin-sequencing primers were concentrated by centrifugation and resuspended in 200. mu.l of BST binding buffer. 10 microliter of 50,000 units/ml Bst-polymerase was added to the resuspended beads and the container holding the beads was placed on the rotator for 5 minutes. Two microliters of 10mM dNTP mix (i.e., 2.5. mu.l each of 10mM dATP, dGTP, dCTP and dTTP) were added and the mixture was incubated at room temperature for an additional 10 minutes. The beads were washed three times with annealing buffer containing 0.1% Tween20 and resuspended in the starting volume of annealing buffer.

Mu.l of Dynal streptavidin beads (Dynal Biotech Inc., Lake Success, NY; 10mg/ml of M270 or MyOne) were annealed in an annealing buffer containing 0.1% Tween20^TMBeads) were washed three times and resuspended in the starting volume of annealing buffer containing 0.1% Tween 20. The Dynal bead mixture was then added to the resuspended agarose beads. The mixture was shaken and placed in a rotator at room temperature for 10 minutes.

The beads were collected in the bottom of the tube by centrifugation at 2300g (500 rpm for Eppendorf centrifuge 5415D). The beads were resuspended in the starting volume of annealing buffer containing 0.1% Tween 20. The mixture in the tube was placed in a magnetic separator (Dynal). The beads were washed three times with annealing buffer containing 0.1% Tween20 and resuspended in the same buffer of the starting volume. Beads without amplicons were removed by a washing step as described previously. Only the agarose beads containing the appropriate DNA fragments were retained.

The magnetic beads were separated from the agarose beads by adding 500. mu.l of 0.125M NaOH. The mixture was shaken and the magnetic beads were removed by magnetic separation. The agarose beads remaining in solution were transferred to another tube and washed with 400. mu.l of 50mM Tris acetate until the pH stabilized at 7.6.

Example 10: nucleic acid sequencing Using bead emulsion PCR

The following experiments were performed to test the efficiency of the bead emulsion PCR. For this method, 600,000 agarose beads with an average diameter of 25-35 μm (as provided by the manufacturer) were covalently attached to the capture primers at a ratio of 30,000,000 and 50,000,000 copies/bead. Beads with covalently attached capture primers were mixed with 1,200,000 copies of a single-stranded adenovirus library. The library construct includes a sequence complementary to the capture primer on the bead.

The adenovirus library was annealed to the beads using the method described in example 1. Subsequently, the beads were resuspended in a complete PCR solution. The PCR solution and beads were emulsified in 2 volumes of a rotating emulsifying oil using the same method as described in example 2. The emulsified (coated) beads were subjected to PCR amplification as outlined in example 3. The emulsion was broken as outlined in example 4. The DNA on the beads was made single stranded using the method of example 5 and the sequencing primer was annealed.

Subsequently, 70,000 beads were sequenced simultaneously by pyrosequencing using a pyrosequencer from 454Life Sciences (New Haven, CT) (see pending application to Lohman et al filed concurrently with the present application entitled "method for nucleic acid amplification and sequencing", USSN60/476,592 filed 6.6.2003). Batches of 70,000 beads were sequenced and the data are listed in table 6 below.

TABLE 6

Margin of error of comparison	Comparison of				Coverage degree	Inferred read error
							Is free of	Is single	A plurality of	Is specially provided with
	0％	47916	1560		1110	54.98％							0.00％
5％							46026	3450		2357	83.16％	1.88％
	10％	43474	6001	1	3742	95.64％							4.36％

This table shows the results obtained from a BLAST analysis comparing sequences obtained from a pyrophosphate sequencer with adenovirus sequences. The first column shows the error tolerance used in the BLAST program. The last column shows the true error determined by direct comparison with known sequences.

Bead emulsion PCR for double-ended sequencing

Example 11: template quality control

As shown in the past, the success of the emulsion PCR reaction was found to be related to the quality of the single-stranded template. Thus, two separate quality controls were used to assess the quality of the template material prior to starting the emulsion PCR method. First, an aliquot of single stranded template was run on a 2100BioAnalyzer (Agilient). The RNA Pico Chip was used to verify that the sample contained a heterogeneous population of fragments ranging in size from about 200 to 500 bases. Second, the library was quantified using a RiboGreen fluorescence assay on a Bio-Tek FL600 plate fluorometer. Samples determined to have DNA concentrations below 5 ng/. mu.l are too dilute to be used.

Example 12: DNA Capture bead Synthesis

Packed beads from a 1mL N-hydroxysuccinimide ester (NHS) activated agarose HP affinity column (Amersham Biosciences, Piscataway, NJ) were removed from the column. 30-25 μm size beads were selected by sequential passage through 30 and 25 μm pore filter mesh portions (Sefar America, Depew, NY, USA). The beads that passed through the first filter but were retained by the second were collected and activated as described in the product literature (Amersham Pharmacia Protocol # 71700600 AP). Two different amine-labeled HEG (hexaethyleneglycol) long capture primers were obtained corresponding to the 5 'ends of the sense and antisense strands of the template to be amplified, (5' -amine-3 HEG spacer gcttacctgaccgacctctgcctctgttgcgtc-3; SEQ ID NO: 12; and 5 '-amine-3 HEG spacer ccattccccagctcgtcttgccatctgttccctccctgtc-3'; SEQ ID NO: 13) (IDT Technologies, Coralville, IA, USA). The primers are designed to capture both strands of the amplification product to allow double-ended sequencing, i.e., sequencing of the first and second strands of the amplification product. The capture primer was dissolved in 20mM phosphate buffer, pH 8.0, to obtain a final concentration of 1 mM. 3 microliter of each primer was bound to the screened 30-25 μm beads. The beads were then stored in a bead storage buffer (50mM Tris, 0.02% Tween and 0.02% sodium azide, pH 8). The beads were quantified with a hemocytometer (hauser Scientific, Horsham, PA, USA) and stored at 4 ℃ until use.

Example 13: PCR reaction mixture preparation and formulation

For any single molecule amplification technique, residual amplicon reaction contamination from extraneous or other experiments can interfere with the sequencing run. To reduce the possibility of contamination, PCR reaction mixtures were prepared in a UV-treated laminar flow hood clean bench located in a PCR clean room. For every 600,000 bead emulsion PCR reactions, the following reagents were mixed in a 1.5ml tube: 225. mu.l of reaction mixture (1 XPlatin HiFi Buffer (Invitrogen)), 1mM dNTPs, 2.5mM MgSO₄(Invitrogen), 0.1% BSA, 0.01% Tween, 0.003U/. mu.l of thermostable pyrophosphatase (NEB), 0.125. mu.M forward primer (5'-gcttacctgaccgacctctg-3'; SEQ ID NO: 14) and 0.125. mu. lM reverse primer (5'-ccattccccagctcgtcttg-3'; SEQ ID NO: 15) (IDT Technologies, Coralville, IA, USA) and 0.2U/. mu.l of Platinum HiFi Taq polymerase (Invitrogen). Remove 25. mu.l of reaction mixture and store in a single 200. mu.l PCR tube for negative control. Both the reaction mixture and the negative control were stored on ice until use.

Example 14: binding template to DNA Capture beads

Successful amplification of cloned DNA for sequencing is associated with the delivery of a controlled number of templates onto each bead. For the experiments described herein below, a typical target template concentration of 0.5 template copies per capture bead was determined. At this concentration, the puavan distribution showed that 61% of the beads had no associated template, 30% had one species of template, and 9% had two or more templates. Delivery of excess template can result in binding and subsequent amplification of mixed populations (2 or more templates) on a single bead, preventing the generation of meaningful sequence data. However, delivering too little template will result in fewer wells containing template (one template/bead), reducing the extent of sequencing coverage. Therefore, the concentration of single-stranded library templates is considered important.

Template nucleic acid molecules are annealed to complementary primers on DNA capture beads by a method performed in a UV-treated laminar flow hood clean bench. 600,000 DNA capture beads suspended in bead storage buffer (see example 9 above) were transferred to a 200. mu.l PCR tube. The tube was centrifuged in a bench top mini centrifuge for 10 seconds, rotated 180 °, and centrifuged for an additional 10 seconds to ensure equal sedimentation. The supernatant was removed and the beads were washed with 200. mu.l of annealing buffer (20mM Tris, pH 7.5 and 5mM magnesium acetate). The tube was shaken for 5 seconds to resuspend the beads and the beads were pelleted as before. All but approximately 10. mu.l of the supernatant above the beads was removed and an additional 200. mu.l of annealing buffer was added. The beads were shaken for an additional 5 seconds, allowed to stand for 1 minute, and allowed to settle as before. All supernatants were discarded except 10. mu.l.

Next, 1.5. mu.l of 300,000 molecules/. mu.l template library was added to the beads. The tube was shaken for 5 seconds to mix the components and the template was annealed to the beads in a controlled denaturation/annealing program performed on an MJ thermocycler. The program allows for 80 degrees C temperature in 5 minutes, followed by 0.1 ℃/sec to reduce to 70 degrees C temperature in 1 minutes, at 70 degrees C temperature in 1/60 degrees C, 0.1 ℃/sec to reduce to 60 degrees C, at 60 degrees C for 1 minutes, 0.1 ℃/sec to reduce to 50 degrees C, at 50 degrees C for 1 minutes, reduced to 0.1 ℃/sec to 20 degrees C, maintained at 20 degrees C. After the annealing process was complete, the beads were removed from the thermal cycler, centrifuged as before, and the annealing buffer was carefully decanted. The capture beads comprise on average 0.5 copies of single stranded template DNA bound to each bead and are stored on ice until use.

Example 15: emulsification action

The emulsification process produced a heat stable water-in-oil emulsion containing 10,000 separate PCR microreactors per microliter. This acts as a matrix for single molecule clonal amplification of a single molecule of the target library. The reaction mixture and the DNA capture beads, which were subjected to a single reaction, were emulsified in the following manner. In a UV-treated laminar flow hood clean bench, 200. mu.l of PCR solution (from example 10) was added to a tube containing 600,000 DNA capture beads (from example 11). Resuspend the beads by repeated aspiration. Thereafter, the PCR-bead mixture was incubated at room temperature for at least 2 minutes and the beads were allowed to equilibrate with the PCR solution. At the same time, 450. mu.l of emulsion oil (4.5% (w: w) Span 80, 1% (w: w) Atlox 4912(Uniqema, Delaware) in light mineral oil (Sigma)) was aliquoted into a flat-topped 2ml centrifuge tube (Dot Scientific) containing a sterile 1/4 inch magnetic stir bar (Fischer). This tube was then placed in a custom made plastic tube holder and then placed in the middle of a FisherIsotemp digital stirring hot plate set at 450 RPM.

The PCR-bead solution was shaken for 15 seconds to resuspend the beads. The solution was then drawn into a 1ml disposable plastic syringe (Benton-Dickenson) with a plastic safety syringe needle (Henry Schein). The syringe was placed into a syringe pump modified with an aluminum base unit to position the pump vertically rather than horizontally (fig. 30). The tube with the emulsion oil was aligned on the stir plate so that it was centered under the plastic syringe needle and the magnetic stir bar rotated appropriately. The syringe pump was set to dispense 0.6ml at 5.5 ml/hr. The PCR-beads were added drop wise to the emulsion oil. Care was taken to ensure that the droplets did not contact the sides of the tube as they fell into the rotating oil.

Once the emulsion is formed, great care is taken to minimize agitation of the emulsion during the emulsification process and the post-emulsification sample separation step. It was found that shaking, rapid stirring or over-mixing can cause the emulsion to break, destroying the separated microreactors. During the formation of the emulsion, the two solutions turned into a homogeneous milky white mixture with the viscosity of mayonnaise. The contents of the syringe were emptied into the rotating oil. Subsequently, the emulsion tube was removed from the holder and flicked with the index finger until any residual oil layer on top of the emulsion disappeared. The tube was replaced in the holder and stirred with the magnetic stirring bar for another minute. The stir bar was removed from the emulsion by running a magnetic retrieval tool along the outside of the tube and discarded.

Using a P100 pipette, 20 microliters of emulsion was removed from the middle of the tube and placed on a microscope slide. Larger pipette tips are used to minimize shear forces. The emulsion was examined at 50 x magnification to ensure that it consisted primarily of single beads in microreactors 30-150 microns in diameter of the PCR solution in oil (fig. 33). Immediately after visual inspection, the emulsion was amplified.

Example 16: amplification of

The emulsion was aliquoted into 7-8 separate PCR tubes. Each tube contained approximately 75 μ l of emulsion. The tube was closed and placed in an MJ thermocycler along with the 25. mu.l negative control described above. The following cycle times were used: 1 cycle of temperature in 94 ℃ for 4 minutes (heat start), 30 cycles of temperature in 94 ℃ for 30 seconds, at 68 ℃ for 150 seconds (amplification), and 40 cycles of temperature in 94 ℃ for 30 seconds, and at 68 ℃ for 360 seconds (hybridization and extension). After the PCR procedure was completed, the tube was removed and the emulsion was broken immediately or the reaction was stored at 10 ℃ for 16 hours before initiating the breaking process.

Example 17: burst emulsion and bead recovery

After amplification, the degree of emulsion breaking (separation of oil and water phases) was checked. The uncracked emulsion was combined into a single 1.5ml microcentrifuge tube, while the sporadically broken emulsion was discarded. Since the emulsion samples were very viscous, a significant amount remained in each PCR tube. The remaining emulsion was recovered by adding 75. mu.l of mineral oil and aspirating the mixture to each PCR tube. The mixture was added to a 1.5ml tube containing a large block of emulsified material. The 1.5ml tube was shaken for 30 seconds. Thereafter, the tubes were centrifuged in a benchtop microcentrifuge for 20 minutes at 13.2K rpm (full speed).

After centrifugation, the emulsion separated into two phases with a large white interface. The clear oil phase above was discarded while leaving the cloudy interface material in the tube. In a chemical fume hood, 1ml of hexane was added to the lower phase and interface layer. The mixture was shaken for 1 minute and centrifuged at full speed for 1 minute in a benchtop microfuge. The top oil/hexane phase was removed and discarded. Thereafter, 1ml of 80% ethanol/1 × annealing buffer was added to the remaining aqueous phase, interface and beads. The mixture was shaken for 1 minute or until the white substance from the interface dissolved. The samples were then centrifuged at full speed for 1 minute in a benchtop microcentrifuge. The tube was rotated 180 ° and centrifuged for an additional 1 min. The supernatant was then carefully removed without disturbing the bead pellet.

The white bead pellet was washed twice with 1ml of annealing buffer containing 0.1% Tween 20. The wash solution was discarded after each wash as described above and the beads were precipitated. The precipitate was washed with 1ml of Picopure water. The beads were pelleted with the centrifugation-spin-centrifugation method used previously. The aqueous phase was carefully removed. The beads were then washed with 1ml of 1mM EDTA as before, except that the beads were briefly shaken on the middle setting for 2 seconds before pellet and supernatant were removed.

The amplified DNA immobilized on the capture beads is treated to obtain single-stranded DNA. The second strand is removed by incubation in an alkaline melt solution. 1ml of a thawing solution (0.125M NaOH, 0.2M NaCl) was then added to the beads. Resuspend pellet by briefly shaking for 2 seconds on the middle setting and place tube in Thermolyne Lab shake tube roller for 3 minutes. The beads were then pelleted as above, and the supernatant carefully removed and discarded. The residual melting solution was neutralized by adding 1ml of annealing buffer. Thereafter, the beads were shaken at medium speed for 2 seconds. The beads were pelleted and the supernatant removed as before. The annealing buffer wash was repeated except that only 800. mu.l of annealing buffer was removed after centrifugation. The beads and remaining annealing buffer were transferred to a 0.2ml PCR tube. The beads were used immediately or stored at 4 ℃ for up to 48 hours before proceeding to the enrichment process.

Example 18: optional bead enrichment

The bead mass includes beads with amplified, immobilized DNA strands, and empty or null beads. As mentioned previously, it was calculated that 61% of the beads lacked template DNA during the amplification process. Enrichment is used to selectively isolate beads with template DNA, thereby maximizing sequencing efficiency. The enrichment process is described in detail below.

The single stranded beads from example 14 were pelleted by centrifugation-spin-centrifugation to remove as much supernatant as possible without disturbing the beads. To the beads were added 15. mu.l of annealing buffer followed by 2. mu.l of 100uM biotin-labeled 40 base enrichment primer (5 '-biotin-tetra-ethylene glycol spacer ccattccccagctcgtcttgccatctgttccctccctgtctcag-3'; SEQ ID NO: 16). The primers are complementary (20 bases each) to the combined amplification and sequencing sites at the 3' end of the bead immobilization template. The solution was mixed by briefly shaking for 2 seconds on the middle setting and the enrichment primers were annealed to the immobilized DNA strands in an MJ thermocycler using a controlled denaturation/annealing program. The program consisted of the following cycle times and temperatures: incubate at 65 ℃ for 30 seconds, decrease to 58 ℃ at 0.1 ℃/sec, incubate at 58 ℃ for 90 seconds, maintain at 10 ℃.

Resuspend DynalMyOne by gentle swirling when the primers anneal^TMStreptavidin beads. Next, 20. mu.l of DynalMyOne was added^TMThe beads were added to a 1.5ml microcentrifuge tube containing 1ml of enhancing fluid (2M NaCl, 10mM Tris-HCl, 1mM EDTA, pH 7.5). The MyOne bead mixture was shaken for 5 seconds and the tube placed in a Dynal MPC-S magnet. Paramagnetic beads were again precipitated onto the side of the microcentrifuge tube. Carefully remove and discard supernatant without disturbing MyOne ^TMBeads. The tube was removed from the magnet and 100. mu.l of enhancing fluid was added. Tubes were shaken for 3 seconds to resuspend beads and stored on ice until use.

After the annealing procedure was completed, 100. mu.l of annealing buffer was added to the PCR tube containing the DNA capture beads and the enriching primers. The tube was shaken for 5 seconds and the contents transferred to a new 1.5ml microcentrifuge tube. The PCR tube was washed once with 200. mu.l of annealing buffer and the wash solution was added to a 1.5ml tube in which the enriching primer was annealed to the capture beads. The beads were washed three times with 1ml of annealing buffer, shaken for 2 seconds, and precipitated as before. The supernatant was carefully removed. After the third wash, the beads were washed twice with 1ml of ice-cold enhancing fluid. The beads were shaken, pelleted and removed as beforeAnd (4) supernatant fluid. Resuspend beads in 150. mu.l ice-cold enhancing fluid and add the bead solution to washed MyOne^TMIn beads.

The bead mixture was shaken for 3 seconds and incubated on a LabQuake tube roller for 3 minutes at room temperature. Will coat the streptavidin MyOne^TMThe beads are bound to biotin-labeled enrichment primers that anneal to immobilized templates on the DNA capture beads. The beads were then centrifuged at 2,000RPM for 3 minutes, after which the beads were shaken with a 2 second pulse until resuspended. The resuspended beads were placed on ice for 5 minutes. After this, 500. mu.l of cold enhancing fluid was added to the beads and the tube was inserted into a Dynal MPC-S magnet. The beads were allowed to stand for 60 seconds to allow for settling against the magnet. After this time, carefully remove and discard the MyOne with excess ^TMAnd the supernatant of null DNA capture beads.

The tube was removed from the MPC-S magnet and 1ml of cold enhancing fluid was added to the beads. The beads were resuspended with a gentle flick of the finger. It is important not to shake the beads at this time, as intense mixing can disrupt MyOne^TMAnd a DNA capture bead. The beads were returned to the magnet and the supernatant removed. This wash was repeated three more times to ensure removal of all the ineffective capture beads. Enrichment primer and MyOne to remove annealing^TMThe beads, DNA capture beads heavy suspension in 400 u l melting solution, oscillation 5 seconds, and magnet precipitation. The supernatant with the enriched beads was transferred to a separate 1.5ml microcentrifuge tube. For maximum recovery of enriched beads, MyOne-containing cells were added^TMA second 400. mu.l aliquot of the thawing solution was added to the bead tube. The beads were shaken and pelleted as before. The supernatant from the second wash was removed and combined with the first pellet of enrichment beads. Discard the used MyOne^TMTest tubes for beads.

Place the enriched DNA capture bead microcentrifuge tube on a Dynal MPC-S magnet to precipitate any residual MyOne^TMBeads. The enriched beads in the supernatant were transferred to a second 1.5ml microcentrifuge tube and centrifuged. The supernatant was removed and the beads were washed 3 times with 1ml of annealing buffer to neutralize the residual molten solution . After the third wash, 800. mu.l of the supernatant was removed and the remaining beads and solution were transferred to a 0.2ml PCR tube. The enriched beads were centrifuged at 2,000RPM for 3 minutes and the supernatant discarded. Next, 20. mu.l of annealing buffer and 3. mu.l of two different 100. mu.M sequencing primers (5'-ccatctgttccctccctgtc-3'; SEQ ID NO: 17; and 5'-cctatcccctgttgcgtgtc-3' phosphate; SEQ ID NO: 18) were added. The tube was shaken for 5 seconds and placed in an MJ thermocycler for the following 4-stage annealing program: incubate at 65 ℃ for 5 minutes, then 0.1 ℃/sec to 50 ℃, 50 ℃ temperature in 1 minutes, 0.1 ℃/sec to 40 ℃, maintain at 40 ℃ for 1 minute, 0.1 ℃/sec to 15 ℃, and maintain at 15 ℃.

After the annealing process was complete, the beads were removed from the thermal cycler and pelleted by centrifugation for 10 seconds. The tube was rotated 180 ° and centrifuged for 10 seconds. The supernatant was decanted and discarded, and 200. mu.l of annealing buffer was added to the tube. The beads were resuspended with 5 seconds shaking and pelleted as before. The supernatant was removed and the beads were resuspended in 100. mu.l of annealing buffer. At this time, the beads were quantified using a Multisizer3Coulter counter (Beckman Coulter). The beads were stored at 4 ℃ and stabilized for at least 1 week.

Example 19: double-stranded sequencing

For double-stranded sequencing, two different sequencing primers were used: an unmodified primer MMP7A and a 3' phosphorylated primer MMP2 Bp. There are multiple steps in this process. This process is illustrated in fig. 28.

1. First strand sequencing. Sequencing of the first strand involves extension of the unmodified primer by a DNA polymerase by sequential addition of nucleotides for a predetermined number of cycles.

2. Capping: sequencing of the first strand was terminated by flowing a capping buffer containing 25mM Tricine, 5mM magnesium acetate, 1mM DTT, 0.4mg/ml PVP, 0.1mg/ml BSA, 0.01% Tween and 2pM of each dideoxynucleotide and 2. mu.M of each deoxynucleotide.

3. Clearing: residual dideoxynucleotides and deoxynucleotides were removed by flowing in apyrase buffer containing 25mM Tricine, 5mM magnesium acetate, 1mM DTT, 0.4mg/ml PVP, 0.1mg/ml BSA, 0.01% Tween and 8.5 units/L apyrase.

4. Shearing: the second blocked primer was deblocked by removing the phosphate group from the 3 'end of the modified 3' phosphorylated primer by running a cleavage buffer containing 5 units/ml of calf intestinal phosphatase.

5. Continuing: the second unblocked primer is activated by running 1000 units/ml of DNA polymerase to add polymerase to capture all existing primer sites.

6. Second strand sequencing: sequencing of the second strand is performed by the DNA polymerase by adding nucleotides in sequence for a predetermined number of cycles.

The genomic DNA of Staphylococcus aureus (Staphylococcus aureus) was sequenced by the method described above. The results are shown in FIG. 39. Based on 15770 reads of the first chain and 16015 reads of the second chain, a total of 31,785 reads were obtained. Of these, a total of 11,799 reads were paired and 8187 reads were unpaired, resulting in a total coverage of 38%.

Reads were 60-130 in length, averaging 95+/-9 genes (FIG. 40). The distribution of genome spans and the number of wells per genome span are shown in figure 41. Representative alignment from this genome sequencing is shown in figure 42.

Example 20: template PCR

30 μm NHS agarose beads were combined with 1mM of each of the following primers:

MMP1A：cgtttcccctgtgtgccttg(SEQ ID NO：19)

MMP1B：ccatctgttgcgtgcgtgtc(SEQ ID NO：20)

by mixing in a 1: 1 volume: volume ratio to PCR basic mix to add 50 u l washed combined with primer beads in the test tube to Drive beads (Drive-to-bead) PCR. The PCR master mix comprises:

1X PCR buffer;

1mM of each dNTP;

0.625 μ M primer MMP 1A;

0.625 μ M primer MMP 1B;

1 μ l of 1 unit/. mu.l Hi Fi Taq (Invitrogen, San Diego, Calif.); and 5-10ng template DNA (DNA to be sequenced).

The PCR reaction was performed by running the following program on an MJ thermocycler: incubation at 94 ℃ for 3 minutes, 39 cycles of 30 seconds at 94 ℃, 30 seconds at 58 ℃, 30 seconds at 68 ℃ and 30 seconds; followed by incubation at 94 ℃ for 30 seconds and 58 ℃ for 10 minutes; 10 cycles at 94 ℃ for 30 seconds, 58 ℃ for 30 seconds, and 68 ℃ for 30 seconds; and stored at 10 ℃.

Example 21: template DNA preparation and annealing sequencing primer

The beads from example 1 were washed twice with distilled water; washed once with 1mM EDTA and incubated for 5 minutes with 0.125M NaOH. This removed the DNA strand that was not attached to the bead. Subsequently, the beads were washed once with 50mM Tris acetate buffer and with annealing buffer: 200mM Tris-acetate, 50mM magnesium acetate, pH 7.5. Subsequently, 500pmol of sequencing primers MMP7A (ccatctgttccctccctgtc; SEQ ID NO: 21) and MMP2B-phos (cctatccctgttgcgtc; SEQ ID NO: 22) were added to the beads. Primers were annealed on an MJ thermocycler with the following program: incubation at 60 ℃ for 5 minutes; reduced to 50 ℃ at 0.1 ℃/sec; incubation at 50 ℃ for 5 minutes; reduced to 4 ℃ at 0.1 ℃/sec; holding at 40 deg.C for 5 min; decreasing to 10 c at 0.1 c/sec. The template was then sequenced using standard pyrosequencing methods.

Example 22: sequencing and stopping of first Strand

The beads were centrifuged at 3000rpm for 10 minutes into a 55 μm PicoTiter plate (PTP). The PTP was placed on a bench and run using a predetermined number of cycles for resequencing. Sequencing was stopped by capping the first strand. The first strand was capped by adding 100. mu.l of 1 × AB (50mM magnesium acetate, 250mM tricine), 1000 units/ml BST polymerase, 0.4mg/ml single stranded DNA binding protein, 1mM DTT, 0.4mg/ml PVP (polyvinylpyrrolidone), 10. mu.M of each ddNTP and 2.5. mu.M of each dNTP. Apyrase was then flowed over to remove excess nucleotides by adding 1 XaB, 0.4mg/ml PVP, 1mM DTT, 0.1mg/ml BSA, 0.125 units/ml apyrase and incubating for 20 minutes.

Example 23: preparation of the second Strand for sequencing

The second strand was deblocked by adding 100. mu.l of 1 × AB, 0.1 unit/ml polynucleotide kinase, 5mM DTT. The resulting templates are sequenced using standard pyrosequencing methods (described, for example, in U.S. Pat. Nos. 6,274,320 and 6,258,568, 6,210,891, which are incorporated herein by reference). The results of the sequencing method can be seen in FIG. 10F, where a 174bp fragment was sequenced at both ends using pyrosequencing and the methods described in these examples.

Example 24: nucleic acid sequence analysis on Picotiter plates

The picotiter plate containing the amplified nucleic acid as described in example 2 was placed in a perfusion chamber. The sulfurylase, apyrase and luciferase were then delivered to picotiter plates. As shown in FIGS. 11A-11D, the sequencing primer directs DNA synthesis extension into an insert suspected of having a polymorphism. The sequencing primer is first extended by delivering the sequencing primer into a perfusion chamber followed by a wash solution, DNA polymerase and one of dTTP, dGTP, dCTP, or thio-dATP (a dATP analog). Sulfurylase, luciferase and apyrase attached to the ends convert any PPi released as part of the sequencing reaction to detectable light. The apyrase present degrades any unreacted dNTPs. Light is typically allowed to collect for 3 seconds (although 1-100, e.g., 2-10 seconds is also suitable) by a CCD camera attached to the fiber imaging bundle, after which the wash solution is again added to the perfusion chamber to remove excess nucleotides and byproducts. The next nucleotide is then added along with the polymerase, thereby repeating the cycle.

The collected light image is transferred from the CCD camera to a computer during the washing process. The light emission is analyzed by computer and used to determine whether the corresponding dNTP has been incorporated into the extended sequence primer. The addition of dNTPs and pyrophosphate sequencing reagents is repeated until a sequence is obtained that contains the region of the insertion suspected of being polymorphic.

Example 25: PCR amplification on Picotiter plates

Preparation of Picotiter plates: in another embodiment, the single stranded library attached to the beads is dispensed directly onto a picotiter plate and the nucleic acid template on each bead is subsequently amplified (using PCR or other known amplification techniques) to generate a sufficient copy number of template that is capable of generating a detectable signal in the pyrophosphate-based sequencing methods disclosed herein.

Example 26: sequence analysis of nucleic acids on PTP

The reagents used for sequence analysis and as a control were four nucleotides and 0.1. mu.M pyrophosphate (PPi) was made in the substrate solution. The substrate solution refers to a mixture of 300uM luciferin and 4. mu.M adenosine 5' -phosphothioester APS, which are substrates for the reaction cascade involving PPi, luciferase and sulfurylase. The substrate is made in assay buffer. The background level of PPi used to detect the enzyme and determine the reagent passing through the chamber was 0.1. mu.M. The concentration of the nucleotides dTTP, dGTP, dCTP was 6.5. mu.M and the concentration of alpha dATP was 50. mu.M. Each nucleotide was mixed with 100U/mL of DNA polymerase, Klenow.

The PTP was placed in the flow cell of the embodied instrument and the flow cell was attached to the panel of the CCD camera. The PTP was washed by flowing the substrate (3ml/min, 2min) through the chamber. Thereafter, a series of reagents are flowed through the chamber by a pump connected to an actuator that has been programmed to shift positions with conduits inserted into different reagents. The order, flow rate and flow time of the reagents are determined. The camera is set to the fast acquisition mode and the exposure time is 2.5 s.

The signal output from the pad (pad) is measured as the average count of all pixels within the pad. The number of frames is equal to the time elapsed during the experiment. Images were used to represent the flow of different reagents.

Example 27: plate-based platform for Picoliter-grade PCR reactions

Materials and methods

Unless otherwise specified, all common laboratory chemicals were purchased from Sigma (Sigma-Aldrich Corporation, St. Louis, MI) or Fisher (Fisher Scientific, Pittsburgh, Pa.).

Production of PicoTiter boards by anisotropic etching of fiber optic faceplates^TM(25X 75X 2mm) in a manner similar to that previously described (Pantano, P. and Walt, D.R., chemistry of materials 1996, 8, 2832-2835). The plate was etched at three different hole depths, 26, 50 and 76 μm. The center-to-center spacing of the micropores was 50 μm, the pore diameter was between 39 and 44 μm (see FIG. 14), and the calculated pore density was 480 pores/mm²。

Solid phase immobilization of oligonucleotide primers: coated beads from 1ml NHS-activated Sepharose HP affinity column (Amersham Biosciences, Piscataway, N.J.) were removed from the column and activated according to the manufacturer's instructions (Amersham Pharmacia Protocol # 71700600 AP). 25 microliters of 1mM amine-labeled HEG capture primer (5 '-amine-3 hexaethylene glycol spacer ccatcctgttgcgtgcgtgtcgtc-3'; SEQ ID NO: 23) (IDT Technologies, Coralville, IA) in 20mM phosphate buffer pH 8.0 were bound to the beads. Thereafter, 36-25 μm sized beads were selected by serial passage through 36 and 25 μm pore filter mesh fractions (Sefar America, Depew, NY). The DNA capture beads that passed through the first filter but were retained by the second were collected in bead preservation buffer (50mM Tris, 0.02% Tween and 0.02% sodium azide, pH8), quantified with a hemocytometer (Hausser Scientific, Horsham, Pa., USA) and stored at 4 ℃ until use.

Generation of test DNA fragments: the amplified test fragment was derived from a commercially available adenovirus serotype 5 vector, pAdEasy (Stratagene, La Jolla, Calif.). Fragments were amplified using a two-part PCR primer, the 5 'end of which contained a 20 base amplification region, and a 20 base 3' portion, complementary to a specific region of the adenovirus genome. Using these primers, two fragments were amplified from 12933-13070 and 5659-5767 positions of the adenovirus genome and assigned marker fragment A and fragment B, respectively.

The sequences of the forward and reverse primers for fragment A are as follows. Slash (/) indicates the separation between the two regions of the primer: forward (5 '-cgtttcccctgtgtgccttg/catcttgtccactaggctct-3'; SEQ ID NO: 24-SEQ ID NO: 25) and reverse (5 '-ccatctgttgcgtgtc/accagcactcgcacccc-3'; SEQ ID NO: 26-SEQ ID NO: 27). The primers for fragment B include: forward (5 '-cgtttcccctgtgtgccttg/tacctctccgcgtaggcg-3'; SEQ ID NO: 28-SEQ ID NO: 29), and reverse (5 '-ccatctgttgcgtgtgtc/ccccccggacgagacgcag-3'; SEQ ID NO: 30-SEQ ID NO: 31).

The reaction conditions included 50mM KCl, 10mM Tris-HCl (pH9.0), 0.1% TritonX-100, 2.5mM MgCl ₂0.2mM dNTP, 1. mu.M of each of the forward and reverse primers, 0.1U/. mu.l Taq (Promega, Madison, Wis.) and 50nmol template DNA. Both templates were amplified using a PCR program comprising 35 cycles of incubation at 94 ℃ for 30 seconds, 56 ℃ for 30 seconds, and 72 ℃ for 90 seconds. Using PCR primers, the total length of the amplified fragment of fragment A was 178bp and fragment B was 148 bp.

To produce fluorescent probes, biotin-labeled double-stranded fluorescent probes were prepared from the pAdEasy vector by PCR amplification as described above. However, the primer sequence is altered to avoid hybridization between the test fragment and the probe primer region. In addition, the reverse primers for both fragments utilized 5' biotin followed by a 3 × hexaethylene glycol spacer to allow the product to be immobilized to the beads before the single stranded probe is eluted.

The forward primer sequence of the fluorescent fragment A probe is as follows. The slash (/) indicates the separation between the two regions of the primer (5 '-atctctgcctactaaccatgaag/catcttgtccactaggctct-3'; SEQ ID NO: 32-SEQ ID NO: 33). The sequence of the reverse primer is 5 '-biotin-3 Xhexaethylene glycol spacer-gtttctctctccagcctctcaccga/accagcactcgcacccc-3'; SEQ ID NO: 34-SEQ ID NO: 35. primers for fragment B were as follows: forward (5 '-atctctgcctactaaccatgaag/tacctctccgcgtaggcg-3'; SEQ ID NO: 36-SEQ ID NO: 37), and reverse (5 '-biotin-3 Xhexaethylene glycol spacer-gtttctctctcacga/cccggacgacgcag-3'; SEQ ID NO: 38-SEQ ID NO: 39).

The fluorescent moiety is incorporated by the nucleotide mixture. This included 0.2mM dATP/dGTP/dCTP, 0.15mM TTP and 0.05mM Alexa Fluor488-dUTP for fragment A (Molecular Probes, Eugene, OR). Alternatively, 0.2mM dATP/dGTP/TTP, 0.15mM dCTP and 0.05mM Alexa Fluor 647-dCTP (Molecular Probes, Eugene, OR) were used for amplified fragment B. The fluorescent product was purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). The biotin-labeled DNA was then bound to 100. mu.l (approximately 8.1 million) of streptavidin agarose high-performance beads (Amersham Biosciences) in 1 Xbinding wash (5mM Tris HClpH7.5, 1M NaCl, 0.5mM EDTA, 0.05% Tween-20) for 2 hours at room temperature. After incubation, the beads were washed three times in TE buffer (10mM Tris, 1mM EDTA, pH8.0) and incubated with 250. mu.l of a thawing solution (0.125N NaOH/0.1M NaCI) for 2 minutes, releasing the single-stranded probe from the beads.

The beads were pelleted with brief centrifugation on a bench top centrifuge and the supernatant neutralized in 1.25ml buffer PB (Qiagen) with 1.9. mu.l glacial acetic acid. This mixture was purified again on a QiaQuick column (Qiagen) and the concentration of purified probe was determined by TaqMan quantification using a BioRadiCycler (BioRad, Hercules, Calif.).

Solution phase PTPCR was performed as follows. The PCR reaction mixture was loaded onto a single 14mm by 43mm PicoTiter plate^TMIn each well. For this purpose, 500 μl PCR reaction mixture (1 XPlatinum HiFi Buffer (Invitrogen, Carlsbad, Calif.), 2.5mM MgSO₄0.5% BSA, 1mM dNTPs (MBI Fermentas, Hanover, Md.), 1. mu.M forward (5'-cgtttcccctgtgtgccttg-3'; SEQ ID NO: 40) and reverse (5'-ccatctgttgcgtgcgtgtc-3'; SEQ ID NO: 41) primers, 0.05% Tween-80, 1U/. mu.l platinum High Fidelity DNA polymerase (Invitrogen), 0.003U/. mu.l thermostable pyrophosphatase (USB, Cleveland, OH) and a calculated 5-copy fragment B template/well) were combined in a 1.5ml microcentrifuge tube. Tubes were fully shaken and stored on ice until the PicoTiter plate was assembled^TMAnd (4) adding a sample box.

With two plastic clips holding the inner PicoTiter plate^TMThe sample application cassette is attached to the PicoTiter plate^TMIn the above, the silicon box pad is firmly seated on the PicoTiter plate^TMOn the surface (see fig. 20). The PCR mixture was aspirated into a 1ml disposable syringe, and the mouth of the syringe was inserted into the input tube of the loading cassette. The sample application cartridge was placed at the end such that the input port was positioned at the bottom of the cartridge, and the PCR mixture was slowly applied to the chamber. While loading, pass through PicoTiter plate ^TMThe transparent back side was inspected to ensure uniform, foam-free delivery.

After loading, the PCR mixture was incubated for 5 minutes, at which time the reaction mixture was removed from the PicoTiter plate^TMSucking out the sample adding box. The PicoTiter plate^TMRemoved from the loading cassette and immediately placed in the amplification chamber (see FIG. 21). The PicoTiter plate was covered with a 0.25mm thick Silpad A-2000 silicon wafer (The Bergquist company, Chanhassen, MN)^TMA surface. On top of this, a 25mm by 75mm standard glass microscope slide (Fisher) was placed. A closed cell foam spacer pad (Wicks air Supply, Highland, IL) was placed on top of the microscope slide. An aluminum lid was attached to the base of the chamber by six 25mm bolts, sealing the amplification chamber.

Once sealed, the amplification chamber was placed on a thermal cycler MJ PTC 225 Tetrad (MJ Research, Waltham, Mass.), equipped with Flat Block Alpha Units. The amplification procedure included incubation at 94 ℃ for 3 minutes(hot start), followed by 40 cycles of incubation at 94 ℃ for 12 seconds, at 58 ℃ for 12 seconds, at 68 ℃ for 12 seconds, and finally at 10 ℃. After the PCR procedure was completed, the PicoTiter plate was plated^TMRemoved from the amplification chamber and reattached to the loading cassette. The cassette compartment was filled with 1ml of H using a disposable syringe ₂O, and allowed to incubate at 10 ℃ for 20 minutes at room temperature.

After incubation was complete, the recovery solution was aspirated from the sample addition cassette and transferred to a 1.5ml microcentrifuge tube. The PCR products were quantified using an iCycler RealTime PCR apparatus (BioRad) and a FAM-labeled reporter probe (Epoch Biosciences, Bothell, WA). TaqMan Universal PCR MasterMix (Applied Biosystems, Foster City, Calif.) was mixed with 0.3. mu.M of forward and reverse primers, and 0.15. mu.M of FAM-labeled probe, and 27. mu.l of reaction mixture were added to a 96-well PCR plate.

Using the purified fragments, a standard curve (from 1X 10) was generated⁹To 1X 10⁴Six standards of molecules/well) which were run in triplicate. PCR amplification was run with the following parameters: incubate at 94 ℃ for 5 minutes (hot start), 60 cycles of 15 seconds at 94 ℃, 45 seconds at 68 ℃, and finally 4 ℃. Data were analyzed using iCycler Optical Systems Software version2.3(BioRad) and PCR yields were quantified using iCycler data and Microsoft Excel (Microsoft, Redmond, WA).

Solid phase PTPCR was performed similarly to solution phase PTPCR, except that the DNA capture beads were loaded onto the PicoTiter plate by centrifugation as described below prior to amplification ^TMIn the hole. In addition, the PCR mixture is loaded into the microwells after the bead deposition is complete. To facilitate retention of the capture beads during the washing step, the solid phase experiment utilized a 50 μm deep PicoTiter plate^TM. The PicoTiter plate^TMIs placed in an internally built plexiglass bead loading clamp. This is in contrast to the PicoTiter plate depicted in FIG. 20^TMThe application clamp is similar except that the PicoTiter plate^TMSandwiched between a bottom plexiglas plate and a top clamping plate, containing the inlet and outlet ports, and sealed off by a silicone gasket with plastic screws.

Template DNA was preannealed to DNA capture beads at 5 templates/bead by incubation at 80 ℃ for 3 minutes, after which the beads were allowed to cool to room temperature for 15 minutes. The beads were then spun into a PicoTiter plate before loading the PCR reaction mixture^TMIn the hole. Through one of the inlets by a pipette containing 100,000 agarose DNA capture beads (approximately 1 bead/3 PicoTiter plate)^TMWells) of bead loading buffer (450 μ l; 1 XPlatin HiFi PCR buffer (Invitrogen), 0.02% Tween-80) was injected into the jig. Each inlet hole was then sealed with a circular adhesive gasket (3M VHS, st. paul, MN). The fixture holds the PicoTiter plate^TMThe wells were faced up and covered with the bead suspension. This was centrifuged in an Allegra 6 centrifuge (Beckman Coulter, Fullerton, Calif.) using a Microtiter rotor at 2000rpm for 5 minutes at room temperature.

After centrifugation, the PicoTiter plate was removed^TMAnd removing the clamp from the clamp. The PCR mix was loaded onto the PicoTiter plate as described for solution phase PCR^TMThe above. However, the solid phase PCR mixture contains no template because the template is pre-annealed to the DNA capture beads. The solid phase PCR amplification procedure includes additional hybridization/extension cycles to compensate for the slower kinetics of the immobilized primers. The procedure included a hot start at 94 ℃ for 3 minutes, 40 cycles of 94 ℃ for 12 seconds, 58 ℃ for 12 seconds, 68 ℃ for 12 seconds, followed by 10 cycles of 94 ℃ for 12 seconds, 68 ℃ for 10 minutes for hybridization and extension, and finally a hold at 10 ℃.

After the PCR procedure was completed, the PicoTiter plate was plated^TMRemoved from the amplification chamber and used 1ml of H as described for solution phase PCR₂And O, washing. Then prepare the PicoTiter plate^TMAnd carrying out hybridization detection of the fixed PCR product.

Hybridization was performed with the fluorescently labeled probe as follows. After the PTPCR is finished, the strand complementary to the fixed strand is removed. To do this, the entire PicoTiter plate is put in^TMIncubate for 8 minutes at room temperature in 0.125M NaOH. This solution was neutralized by two 5 minute washes in 50ml of 20mM Tris-acetate pH 7.5. Followed by adding PicoTiter board ^TMPlace in a custom made 800. mu.l hybridization chamber and block with hybridization buffer (3.5 XSSC, 3.0% SDS, 20 XSSC 3M NaCl; 0.3M sodium citrate) for 30 min at 65 ℃. Using a probe-containing probe: 20nM of fluorescent fragment A (Alexa-488) and fragment B (Alexa-647) in fresh hybridization buffer was substituted for the chamber contents. The probes are allowed to hybridize to their targets. Incubation was carried out at 65 ℃ for 4 hours while shaking on a orbital shaker (Barnstead International, Dubuque, IA) at 200 RPM.

After hybridization, the PicoTiter plate was washed with 2 XSSC, 0.1% SDS at 37 ℃^TM15 minutes, followed by a 15 minute wash in 1 XSSC at 37 ℃ and finally a 15 minute wash in 0.2 XSSC at 37 ℃ twice. After post-hybridization washes, PicoTiter plates^TMAir dried and placed in a FLA-8000 fluorescence image analyzer (Fujifilm Medical Systems USA, Stamford, CT) and scanned at 635 and 473nm wavelengths. The resulting 16-bit tiff image was imported into Genepix 4.0(Axon Instruments, Union City, Calif.). A set of 100 analytical features is taken at the target area and the fluorescence intensity is recorded 635 and 473 for each feature. The data was then exported into Microsoft Excel for further analysis.

Control beads were prepared as follows. Biotin-labeled test templates A and B were prepared from the pAdEasy vector by PCR amplification, purified, immobilized on streptavidin Sepharose high performance beads and strands isolated as described in "preparation of fluorescent probes". However, no fluorescently labeled dNTPs were included in the PCR reaction. The precipitated beads were washed 3 times with TE buffer and stored in TE at 4 ℃ until being deposited on a PicoTiter plate^TMThe above.

Results

Solution phase amplification was demonstrated by loading the PicoTiter plate with a PCR master mix containing calculated 5 template copies/PicoTiter plate^TMAnd (4) a hole. The reactions were run in duplicate on PicoTiter plates with wells 26, 50 and 76 μm deep. PTPCR amplification was performed for 40 cycles as described in materials and methods. Incorporation of additives to avoidDeleterious surface effects conventionally reported for silicon reaction vessels (Kalinna, O. et al, Nucleic Acids Res.1997, 25, 1999-2004; Witter, C.T. and Garling, D.J., Biotechniques1991, 10, 76-83; Taylor, T.B. et al, Nucleic Acids Res.1997, 25, 3164-3168).

The inclusion of 0.5% BSA and 0.05% Tween-80 in the reaction mixture was not only effective in reducing surface effects, it also facilitated amplification. Reducing the relative concentration of either reagent has a negative effect on amplification. In addition, increased Taq concentrations have proven beneficial due to the polymerase inactivating properties of silicon surfaces (Taylor, T.B. et al, Nucleic Acids Res.1997, 25, 3164-. Concentrations above 1U/. mu.l are most suitable for increasing amplicon production.

After PTPCR, recovery from each PicoTiter plate^TMAnd triplicate samples of each solution were quantified by TaqMan assay. One standard curve (from 1X 10) using diluted template⁹To 10⁴Linearity of the molecule, r²0.995) the concentration of the amplification product was determined. By dividing the amount of amplification product by the PicoTiter plate^TMThe total number of wells (372,380) gives the number of amplified molecules per well. The amount of amplification per well was calculated by dividing this number by the starting template concentration per well. In all PicoTiter boards^TMThe PTPCR amplification was successful, with yields of 2.36X 10 in 39.5pl wells⁶1.28X 10 times to 50pl well⁹Fold (see table below).

PicoTiter plate depth [ mu.m ]]	Pore volume [ pl]	Mean fold amplification of N6	Fold amplification of SD	Final product concentration [ M]
					26	39.5	2.36E+06	1.02E+06	4.96E-07
50	76.0	1.28E+09	1.03E+09	1.40E-04
					76	115.6	9.10E+08	4.95E+08	6.54E-05

The table shows the PicoTiter plate as determined by Taq assay^TMAnd (4) PCR amplification. Values reflect triplicate measurements made from duplicate PicoTiter plates. (N ═ 6); SD-standard deviation.

Yield is affected by pore volume. Concentration of the final product obtained for 50 μm deep wells (1.4X 10)^-4M) than obtained in wells 76 μ M deep (6.54X 10)^-5M) significantly large (p of ANOVA)Value 0.023), both of which gave a yield (4.96 × 10) greater than that obtained in the 26 μm deep wells ^-7M) is two orders of magnitude larger. The yield of 50 μm deep microwells represents the best balance of cost and benefit associated with low volume PCR. In this case, the maximum increase in effective concentration and low heat of the reagents is obtained, but the surface-to-volume ratio is still low enough to avoid detrimental surface effects significantly reducing amplification efficiency.

Final concentration of PTPCR obtained at each different well depth (4.96X 10)^-7To 1.4X 10^-4M) are all above 10 of the maximum values that are generally reported to be obtainable before the PCR plateau effect occurs^-8Concentration of M (Sardelli, A., amplifications 1993, 9, 1-5). Higher effective concentrations of primer and template molecules due to low microwell volumes increase overall reaction efficiency and delay the onset of plateau phase until higher molar yields are obtained. Alternatively, this effect is caused by the high concentration of Taq used in the PTPCR reaction, since elevated polymerase concentrations have been shown to be effective in delaying the plateau effect (Kainz, P., Biochim. Biophys. acta 2000, 1494, 23-27; Collins, F.S. et al, Science 2003, 300, 286-. The amplification efficiency in 40 cycles was 44.3, 68.9 and 67.5% for 26, 50 and 76 μm deep wells, respectively, providing a high final concentration of amplicons. The maximum yield was observed in wells 50 μm deep. However, it should be appreciated that no optimization of the number of cycles is performed; it is possible to achieve similar amplification yields with much fewer cycles, thereby increasing the efficiency of PTPCR amplification.

The experimental strategy of clonal solid-phase PTPCR, starting with a single valid copy of a single-stranded DNA fragment and ending with a specific bead-immobilized DNA amplicon detected by fluorescent probe hybridization, is illustrated in fig. 22 and described in detail below:

stage 1: each PicoTiter plate^TMThe wells all contain a PCR reaction mixture consisting of single stranded template molecules (single stranded or annealed to DNA capture beads, or free in solution, as shown here), forward "F" (red) and reverse in solution "R "(blue) primer, and an R primer attached to the DNA capture bead. Solution phase primers were run at 8: 1, F primer is present in excess. Arrows indicate DNA orientation of 5 '→ 3'.

And (2) stage: the initial thermal cycle denatures the DNA template, allowing the R primer in solution to bind to a complementary region on the template molecule. A thermostable polymerase initiates elongation at the primer site (dashed line) and in subsequent cycles, solution phase exponential amplification continues. At this stage the bead immobilized primers did not become a major contributor to amplification.

And (3) stage: early PCR. Both F and R primers amplified the template equally in early exponential amplification (cycles 1-10), although the F primer was in excess in solution.

And (4) stage: and (5) middle-term PCR. Between cycles 10 and 30, the R primer was depleted, stopping exponential amplification. The reaction then enters an asymmetric pay-off period, with the amplicon population gradually dominated by the F strand.

And (5) stage: and (4) performing late PCR. After 30-40 cycles, asymmetric amplification continues to increase the concentration of F strands in solution. Excess F strands without the R strand complement begin to anneal to the bead immobilized R primer. Thermostable polymerases utilize the F strand as a template for the synthesis of the immobilized R strand of the amplicon.

And 6: and (4) final-stage PCR. Continued thermal cycling forces further annealing to the bead-bound primers. Solution phase amplification may be minimal at this stage, but the concentration of immobilized R strands continues to increase.

And (7) stage: the non-immobilized F strand, complementary to the immobilized R strand, is removed by alkaline denaturation. The DNA capture beads are now clustered by the single stranded R strands of the amplicon.

And (8): a fluorescently labeled probe (green) complementary to the R strand was annealed to the immobilized strand. Probes specific for a particular strand sequence are labeled with a unique fluorophore, according to the protocol in a given PicoTiter plate^TMThe number of amplified separate templates within a well results in a range of homogeneous and heterogeneous fluorescent signals.

Initially, the DNA fragments were tested by binding biotin-labeled fragment A or fragment B to streptavidin agarose beads, which were loaded by centrifugation to a 50 μm deep PicoTiter plate ^TMAnd hybridizing a mixed population of fluorescently labeled probes to fragment a or fragment B to confirm the specificity of the fluorescently labeled probes. No mixed signal or non-specific hybridization was observed; the beads with fragment A product exhibited a 488nm signal, while the fragment B beads exhibited a 635nm signal (see FIGS. 23A and 23B). Close examination of FIGS. 23A and 23B revealed several fragment A beads in the fragment B pad and vice versa. Given the purity of the signal presented by these free beads, it is possible that they are some cross-contaminating product during loading, or are washed from one pad to another in a subsequent washing step.

As shown in fig. 23C, the fluorescent probe detects successful solid-phase PTPCR amplification of both fragment a and fragment B templates. The signal generated by the hybridized probe depends on the relative efficiency of dye incorporation into the probe, the sensitivity of the reaction to unequal amounts of template DNA, and the total and relative amounts of amplification product present on each bead. Furthermore, it is possible that the amount of template generated and retained on the DNA capture beads varies from well to well, and the number of capture primers bound to each bead may also vary due to the distribution of bead sizes. As a result, non-normalized ratios resulting from probe hybridization should be considered semi-qualitative rather than quantitative data. However, the fluorescent signal generated by the hybridized probe varied from a homogeneous fragment B signal (red) to the same homogeneous fragment a signal (green), and a heterogeneous mixture of the two signal events (degree of yellow).

Probe specificity due to control, and PicoTiter plate^TMWith the relatively large number of homogeneous red and green beads, non-specific probe hybridization is unlikely to trigger heterogeneous signals. The approximation of homogeneous beads for both templates suggests that heterogeneous beads are unlikely to result from inter-pore amplicon leakage during amplification; if communication is responsible within the well, it is expected that heterogeneous beads will be seen between homogeneous beads of two templates, and oneThe homogeneous signal is generally distributed in a point shape. More precisely, it is possible that the template molecules dissociate from their original beads and re-anneal to the PicoTiter plate before being centrifuged into the microwells^TMAdding new beads to the mixture, or when applying PCR mixtures to PicoTiter plates^TMWhen washed from one bead to another. Regardless of the reason for mixing the template beads, the hybridization results are shown on the PicoTiter plate^TMPCR amplification in microwells can drive enough product onto DNA capture beads to enable fluorescent probe hybridization and detection.

Discussion of the related Art

The results display in this example is based on the PicoTiter board^TMThe PCR of (a) reduces many factors associated with the DNA amplification process, such as high cost of reagents, large number of reactions and long reaction times, leading to another "evolutionary jump" in PCR technology. On a single PicoTiter board ^TMThe above microwells can function as a vessel for up to 370,000 separation reactions, and high yields (2.3X 10) can be obtained even at reaction volumes as low as 39.5 picoliters⁶To 1.2X 10⁹Double) amplification. As a result, throughput is improved and the total reagent cost of PTPCR is reduced; PicoTiter plate contained in the entire 26 or 76 μm depth^TMThe reaction volumes in (1) were 15.3 and 43. mu.l, respectively. PicoTiter board^TMThe increase in size can further increase the maximum flux. For example, the PicoTiter plate^TMThe increase of the dimension of (A) to 40mm by 75mm provides about 1.4 by 10⁶A separate reaction vessel and a PicoTiter plate having the same perimeter (85.47 mm. times. 127.81mm) as a commercially available 96-well PCR plate^TMCan contain up to 5.24 x 10⁶And (4) holes.

Despite the number and volume in which they are performed, solution phase PCR amplification has limited usefulness unless the product can be easily and efficiently recovered. Previous attempts in parallel PCR (Nagai, H. et al, anal. chem.2001, 73, 1043-Do this. The methods disclosed herein avoid the problem of product recovery by immobilizing the PCR products on DNA capture beads, including solid phase amplification. Thus, the PicoTiter plate ^TMThe product of the microwell reaction is not 370,000 wells containing solution phase PCR products, but up to 370,000 beads to which immobilized PCR products are bound. These PCR products are suitable for a variety of solid phase methods of nucleic acid research, including the potential to support massively parallel methods of sequencing a whole genome containing up to hundreds of millions of bases. The simplicity of the disclosed method will significantly reduce the cost of sequencing and other applications that now require robotics to maintain large-scale cloning and PCR.

The disclosure of one or more embodiments of the invention is set forth in the accompanying description. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and the appended claims, a singular form includes plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless specifically stated otherwise, the techniques employed or claimed herein are standard methodologies well known to those of ordinary skill in the art. The examples of embodiments are for illustrative purposes only. All patents and publications cited in this specification are incorporated herein by reference.

Example 28: complete set of sequencing method

Step 1: pAdEasy PCR DNA beads

This procedure was used for 384-well plate PCR of adenovirus clones. Streptavidin-agarose beads (12mls) were prepared for binding the PCR fragments by washing once with 2M NaCl solution and resuspending in 288ml of 2M NaCl. The washed beads were transferred to 15 96-well plates at 200. mu.l bead suspension/well. PCR products (25. mu.l) were transferred to 384-deep well plates using a Tecan TeMo robot. To bind the DNA to the solid support, 25 μ Ι of bead suspension (15,000 beads) was added to each well of the 384-deep well plate using a TecanTeMo robot and mixed. The final concentration of NaCl in the binding reaction was 1M. The binding reactions were incubated for 3 hours at room temperature on a shaker with shaking. The contents of the microtiter plates were pooled by inverting the 384-well plate onto the reservoir and centrifuging at 1000g in a Beckman Allegra bench centrifuge. The pooled beads were transferred to a 50ml Falcon tube, centrifuged at 1000g, and the supernatant removed.

Approximately one million beads (moving solid support) were washed once with 100. mu.l of 2M NaCl, followed by two washes with distilled water (100. mu.l each). The washed beads were incubated in 300. mu.l of thawing reagent (0.1M NaCl and 0.125M NaOH) for 10 minutes in a rotator to remove DNA strands that were not biotin-labeled. The tube was centrifuged at maximum speed to pellet the beads and the molten solution was removed and discarded. The beads were washed with 100. mu.l of the melting solution and then three more washes with 1 × annealing buffer. After washing, the beads were resuspended in 25. mu.l of 1 × annealing buffer.

Primer P2(500pmol) was added to the bead mixture and mixed. The bead mixture in the tube is placed into an automated incubator (in this case a PCR thermal cycler) with the following temperature profile: incubate at 60 ℃ for 5 minutes, decrease to 50 ℃ at 0.1 ℃/sec, incubate at 50 ℃ for 5 minutes, decrease to 40 ℃ at 0.1 ℃/sec, incubate at 40 ℃ for 5 minutes, decrease to 4 ℃ at 0.1 ℃/sec, and incubate at 4 ℃.

After annealing, the beads were carefully washed and resuspended in 200. mu.l of Bst DNA polymerase binding solution. Subsequently, 10 μ l aliquots (50,000 beads) of the bead suspension were processed for sequencing on the instrument described below.

Step 2: preparation of control DNA beads

Six control DNA sequences TF 2, 7, 9, 10, 12 and 15 were cloned into pBluescriptIIKS + vector and plasmid DNA was used as template for PCR with a biotin-labeled primer for solid phase immobilization of the amplicon.

The following reagents were added to a 1.7ml tube to generate a PCR mixture.

10 XHIFI buffer	100μl
		10mM dNTP mix	100μl
50mM MgSO4	60μl
		5′-Bio-3HEG-MMP1B	10μl
MMP1A	10μl
		HIFI Taq polymerase	10μl
Molecular biological grade water	690μl

Add 20. mu.l of plasmid template DNA and dispense the mixture in 50. mu.l aliquots into 0.2ml PCR tubes. Thermal cycling was performed using the following procedure: incubation at 94 ℃ for 4 minutes; 39 cycles of incubation at 94 ℃ for 15 seconds, 58 ℃ for 30 seconds, 68 ℃ for 90 seconds, and 68 ℃ for 120 seconds; the temperature was maintained at 10 ℃.

The amplified DNA of each test fragment was purified using the Qiagen MinElute PCR Clean-Up kit according to the manufacturer's instructions. The purity and yield of DNA for each test fragment was evaluated using the Agilent 2100 Bioanalyzer and DNA 500 kit and chip. The biotin-labeled PCR product was immobilized on agarose streptavidin beads at 10,000,000DNA copies per bead.

The beads were washed 1 time with 2M NaCl solution. This was done by adding 100 μ l, briefly shaking to resuspend the beads, centrifuging at maximum speed for 1 minute to pellet the beads, and then removing the supernatant. Thereafter a second wash with 2M NaCl was performed. The beads were resuspended in 30. mu.l of 2M NaCl. The PCR product was added to the beads. The mixture was shaken to resuspend the beads in solution and then placed on a rack of a microtiter plate shaker at speed 7 for 1 hour at room temperature.

The second strand, which was not biotin-labeled, was removed by incubation with an alkaline melting solution (0.1M NaOH/0.15M NaCl) on an overhead rotator for 10 minutes at room temperature. After which the beads were washed once with 100. mu.l of the melting solution and 100. mu.l of 1 × annealing buffer (50mM Tris-acetate, pH 7.5; 5mM MgCl)₂) Washed three times. The sequencing primer was annealed to the immobilized single-stranded DNA by centrifugation at maximum speed for one minute. The supernatant was removed and the beads were resuspended in 25. mu.l of 1 × annealing buffer. Next, 5. mu.l of sequencing primer MMP7A (100 pmol/. mu.l) was added to the bead suspension and the sequencing primers hybridized using the following temperature profile:

Incubation at 60 ℃ for 5 minutes;

reducing the temperature to 50 ℃ at a speed of 0.1 ℃/second;

incubation at 50 ℃ for 5 minutes;

reducing the temperature to 40 ℃ at the speed of 0.1 ℃/second;

incubation at 40 ℃ for 5 minutes;

reduced to 4 ℃ at 0.1 ℃/sec;

the temperature was maintained at 4 ℃.

The beads were washed twice with 100. mu.l of 1 × annealing buffer and then resuspended with 1 × annealing buffer to a final volume of 200. mu.l and stored at 4 ℃ in 10. mu.l aliquots of labeled tube strips.

And step 3: sequencing chemistry

Agarose beads with immobilized single-stranded DNA template and annealed sequencing primer were incubated with E.coli (E.coli) single-stranded binding protein (Amersham Biosciences) (5. mu.l of 2.5. mu.g/. mu.lsb stock solution per 50,000 beads) and 500U (10. mu.l of 50U/. mu.l) of Bst DNA polymerase (NEB) in 200. mu.l of Bst polymerase binding solution (25mM Tricine pH 7.8; 5mM magnesium acetate; 1mM DTT; 0.4mg/ml PVP molecular weight 360,000) at room temperature for 30 minutes on a rotator. After this, the DNA beads were mixed with SL beads and deposited in the wells of a PicoTiter plate as follows. Reagents required to run sequencing on the 454 instrument included 1) substrate wash solution; 2) apyrase comprising a wash solution; 3)100nM inorganic pyrophosphate calibration standard; 4) single nucleotide triphosphate solutions.

All solutions were prepared in sulfurylase-luciferase assay buffer with enzyme substrate (25mM tricinepH 7.8; 5mM magnesium acetate; 0.4mg/ml PVP molecular weight 360,000; 0.01% Tween 20; 300. mu. M D-luciferin; 4. mu.M APS). The substrate wash solution was the same as the luciferase assay buffer. Apyrase containing wash solution was based on luciferase assay buffer except that no enzyme substrate (APS and D-luciferin) was added and this wash contained apyrase (Sigma St. Lous, MO; Pyrosequencing AB, Pyrosequencing, Inc. Westborough, MA) at a final concentration of 8.5U/1.

Sodium pyrophosphate (PPi) standards were prepared by adding sodium pyrophosphate tetrahydrate (Sigma, st. louis, MO) to luciferase assay buffer to a final concentration of 100 nM. Nucleotide triphosphates (dCTP, dGTP, TTP; lowest bisphosphate grade) (Amersham Biosciences AB, Uppsala, Sweden) were diluted in luciferase assay buffer to a final concentration of 6.5. mu.M. The dideoxynucleotide triphosphate analogue, 2 '-deoxyadenosine-5' -O- (l-thiotriphosphate), Sp-isomer (Sp-dATP-. alpha. -S, Biolog Life Science Institute, Bremen, Germany) was diluted to a final concentration of 50. mu.M in luciferase assay buffer.

And 4, step 4: clones His 6-BCCP-sulfurylase and His 6-BCCP-sulfurylase

Bacillus stearothermophilus (Bst) ATP sulfurylase (E.C.2.7.7.4) and firefly (Photinus pyralis) luciferase (E.C.1.13.12.7) were cloned into the Nhe I-BamHI digested pRSET-A vector (Invitrogen). PCR primers were designed to amplify a fragment corresponding to amino acids 87-165 of the BCCP protein using the coding sequence of the BCCP (Biotin carboxyl Carrier protein) gene (Alix, J.H., DNA 8(10), 779-789 (1989); Muramatsu, S. and Mizuno, T., nucleic acids Res.17(10), 3982 (1989); Jackowski, S. and Alix, J.H., J.Bacteriol.172(7), 3842-3848 (1990); Li, S.J. and Cronan, J.E.Jr., J.biol.chem.267(2), 855-863(1992), Genbank accession number M80458). The forward primer is 5'-ctagctagcatggaagcgccagcagca-3'; SEQ ID NO: 42 and the reverse primer 5'-ccgggatccctcgatgacgaccagcggc-3'; SEQ ID NO: 43. the PCR Mix was prepared as Mix1 and Mix2, 25. mu.l each. Mix1 included 75pmol of primer, 100ng of E.coli genomic DNA and 5. mu. mol of dNTPs.Mix2 included 1 unit of Fidelity extended DNA polymerase (Boehringer Mannheim/Roche diagnostics corporation, Indianapolis, IN, Cat. No.1732641) and 5. mu.l of 10 XFidelity extended buffer (Boehringer Mannheim/Roche diagnostics corporation, Indianapolis, IN). For PCR hot start, Mix1 and Mix 220 seconds were heated separately at 96 ℃ before combining them. The combined reactants were recycled as follows: incubation at 96 ℃ for 3 minutes, 10 cycles of 30 seconds at 96 ℃, 1 minute at 60 ℃ and 2 minutes at 68 ℃ followed by a fill-in step of 7 minutes at 72 ℃. After PCR, a single 250bp fragment was obtained. The BCCP fragment was digested with NheI-BamHI and subcloned into NheI-BamHI digested pRSET-A.

And 5: expression of sulfurylase and luciferase

PCR was performed with primers containing PstI/Hind IH and BamHI/XhoI sites (first enzyme at the 5 'end and second enzyme at the 3' end) to amplify the open reading frames of Bst ATP sulfurylase and firefly luciferase, respectively. This resulted in a 6 × His and BCCP domain fused to the N-terminus of ATP sulfurylase and luciferase. The enzyme is expressed in E.coli using growth medium supplemented with biotin to allow in vivo biotin labeling by the BCCP domain. The enzyme was purified to near homogeneity using a combination of IMAC and size exclusion column chromatography. Purification was assessed by electrophoresis on a Protein 200Plus chip using an Agilent 2100 Bioanalyzer.

Step 6: solid phase immobilization of luciferase and sulfurylase

The enzymes were immobilized onto Dynal M-280 streptavidin coated magnetic microparticles (Dynal, Oslo, Norway) and Bangs microspheres (300nm) by incubation with a 1: 3 mixture of ATP sulfurylase and luciferase, respectively. Binding was performed by mixing 50. mu.g of ATP sulfurylase and 150. mu.g of luciferase with 1mg of Dynal M-280 beads or 0.6mg of Bangs microspheres in TAGE buffer (25mM Tris-Acetate pH 7.8, 200mM ammonium sulfate, 15% v/v glycerol and 30% v/v ethylene glycol). The mixture was incubated at 4 ℃ for 1 hour on a rotator. After binding, the beads can be stored in enzyme solution for 3 months at-20 ℃. Prior to use, beads were washed extensively in luciferase assay buffer containing 0.1mg/ml bovine serum albumin (Sigma, St Louis, Mo.). The activity of the immobilized enzyme was tested using a luminometer (Turner, Sunnyvale, California). The washed beads were stored on ice until deposited on a PTP slide.

And 7: PicoTiter board^TM(PTPs)

Production of PicoTiter boards by anisotropic etching of fiber optic faceplates^TM(25X 75X 2mm) by etching in a similar manner to that described in the literature. The plate was etched at three different depths, 26, 50 and 76 μm. The center-to-center distance of the micropores was 50 μm, and the diameter of the pores was mediumBetween 39 and 44 μm, the calculated pore density is 480 pores/mm²。

And 8: PTP loading

Agarose beads carrying DNA template and DynalM-280/Bangs 0.3 μm bead mixture with immobilized sulfurylase and luciferase were deposited in individual wells of a PicoTiter plate using a centrifugation-based method. The method uses an internal polycarbonate clamp comprising a bottom plate (with slide placement pegs), an elastomeric sealing gasket, and a top plate with two sample ports. The PTP slide was placed on the bottom plate with the etched side up and the top plate with the sealing gasket clamped on top of the PTP slide. The entire device was tightened with four plastic screws to provide a water-tight closure. The occlusive gasket was designed to form a cap of bead deposition resulting in a hexagonal area (14 x 43mm) covering approximately 270,000 PTP holes.

The beads are deposited in a custom layer. The PTP was removed from incubation in bead wash buffer. A mixture of layer 1, DNA and enzyme beads was deposited. After centrifugation, layer 1 supernatant was aspirated off the PTP and layer 2, Dynal enzyme beads, were deposited.

A bead suspension was prepared by mixing 150,000 DNA-carrying agarose beads in 120. mu.l of the ssb/Bst pol binding mixture (see above) with 270. mu.l Dynal-SL and Bangs-SL beads (both 10mg/ml) in a total volume of 500. mu.l of luciferase assay buffer containing 0.1mg/ml bovine serum albumin. The bead slurry was oscillated and flowed into the bead deposition nip through the suction port. Care was taken to avoid introducing air bubbles. The clamp/PTP assembly was centrifuged at 2000rpm for 8 minutes in a Beckman Allegra 6 centrifuge equipped with a 4-position plate oscillating rotor. After centrifugation, the supernatant was carefully removed from the clamp chamber using a pipette. A second layer of Dynal-SL beads only was deposited. This layer included 125. mu.l of Dynal-SL (at 10mg/ml) and 375. mu.l of bead wash buffer (2.5mg/ml Dynal beads) in a 1.5ml tube. The Dynal bead mixture was aspirated into the PTP major active region and centrifuged at 2000rpm for 8 minutes. The layer 2 mixture was aspirated and the PTP was placed back in bead wash buffer (luciferase assay buffer with 0.1mg/ml bovine serum albumin and 8.5U/1 apyrase) until ready to be loaded onto a sequencer.

And step 9: sequencing instrument

The internal sequencing instrument includes three main devices: a fluidics subsystem, a PTP cartridge/flow cell, and an imaging subsystem. The fluidics subsystem includes a reagent reservoir, a reagent input line, a multi-valve manifold, and a peristaltic pump. It allows reagents to be delivered to the flow chamber one at a time, at a pre-programmed flow rate and duration. The PTP cassette/flow cell was designed in such a way that after attaching the PTP, there would be a 300 μm gap between the PTP top (etched side) and the chamber top plate. It includes the temperature control means of the reagents and PTP, and a light-tight housing. The polished side of the PTP was exposed to the backside of the PTP cassette and placed in direct connection with the imaging system. The imaging system includes a CCD camera with 1-1 imaging fiber bundle, as well as a camera cool down system and camera control electronics. The camera used was a Spectral Instruments (Tucson, AZ) series 600 camera with a Fairchild Imaging LM485CCD (16,000,000 pixels, 15 μm pixel size). This was directly connected to the imaging fiber bundle with a fiber pitch of 6 μm. The camera was cooled to-70 ℃ and operated in frame transition mode. In this way, imaging is performed using the central portion of the CCD, while image saving and reading are performed using the outer portion of the CCD. The reading is done through 4 ports at each corner of the CCD. The data acquisition speed was set to 1 frame every 30 seconds. The frame transfer transition time is about 0.25 seconds. All camera images were saved on a computer hard drive in UTIFF 16 format (IBM serverxseries 335, IBM, White Plains, NY).

Step 10: sequencing run conditions

Cycling the delivery of sequencing reagents to the PTP wells and washing sequencing reaction byproducts from the wells is accomplished by operation of a fluidics system preset program. The program was written in the form of a Microsoft Excel script, specifying the reagent name (wash, dATP α S,dCTP，dGTP，dTTP，PP_istandard), flow rate and duration of each script (script) step. The flow rate was set at 3ml/min for all reagents and the linear rate in the flow cell was approximate. One initial washing step (5 min) followed by PP_iStandard flow (2 min), followed by 21 or 42 cycles (Wash-C-Wash-A-Wash-G-Wash-T), where each nucleotide flow was 0.5 min and the wash step was 2 min. After all cycles of nucleotide addition and washing, a second PP was delivered_iStandard flow (2 min), followed by a final 5 min wash step. The total run time was 4 hours. The reagent volumes needed to complete this run script are as follows: 300ml of each wash solution, 50ml of each nucleotide solution, 20ml of PPi standard solution. During the run, all reagents were kept at room temperature. Since the flow cell and flow cell input tube were maintained at 3 ℃, all reagents entering the flow cell were at 30 ℃.

Reference to the literature

Hamilton，S.C.，J.W.Farchaus and M.C.Davis.2001.DNA polymerases as enginesfor biotechnology.BioTechniques 31：370.

QiaQuick Spin Handbook(QIAGEN，2001)：hypertext transfer protocol://world wideweb.qiagen.com/literature/handbooks/qqspin/1016893HBQQSpin_PCR_mc_prot.pdf.

Quick Ligation Kit(NEB)：hypertext transfer protocol://world wideweb.neb.com/neb/products/mod_enzymes/M2200.html.

MinElute kit(QIAGEN)：hypertext transfer protocol://world wideweb.qiagen.com/literature/handbooks/minelute/1016839_HBMiuElute_Prot_Gel.pdf.

Biomagnetic Techniques in Molecular Biology，Technical Handbook，3rd edition(Dynal，1998)：hypertext transfer protocol://world wideweb.dynal.no/kunder/dynal/DynalPub36.nsf/cb927fbab127a0ad4125683b004b011c/4908f5b1a665858a41256adf005779f2/$FILE/Dynabeads M-280 Streptavidin.pdf.

Bio Analyzer User Manual(Agilent)：hypertext transfer protocol://world wideweb.chem.agilent.com/temp/rad31B29/00033620.pdf

BioAnalyzer DNA and RNA LabChip Usage(Agilent)：hypertext transferprotocol://world wide web.agilent.com/chem/labonachip

BioAnalyzer RNA 6000 Ladder(Ambion)：hypertext transfer protocol://world wideweb.ambion.com/techlib/spec/sp_7152.pdf

Claims (84)

1. A method of sequencing a nucleic acid, comprising:

(a) fragmenting a large template nucleic acid molecule to produce a plurality of fragmented nucleic acids;

(b) delivering the fragmented nucleic acids into aqueous microreactors in a water-in-oil emulsion such that a plurality of aqueous microreactors comprise a single copy of fragmented nucleic acids, a single bead capable of binding to the fragmented nucleic acids, and an amplification reaction solution containing reagents necessary to perform nucleic acid amplification;

(c) amplifying the fragmented nucleic acids in microreactors to form amplified copies of the nucleic acids and binding the amplified copies to beads in microreactors;

(d) delivering the beads to an array of at least 10,000 reaction chambers on a planar surface, wherein a plurality of the reaction chambers comprises no more than a single bead; and

(e) sequencing reactions are performed simultaneously in multiple reaction chambers.

2. The method of claim 1, wherein the reaction chambers have a center-to-center spacing of 20-100 μm.

3. The method of claim 1, wherein the fragmented nucleic acids are 30-500 bases.

4. The method of claim 1, wherein the plurality of beads bind at least 10,000 amplified copies.

5. The method of claim 1, wherein step (c) is accomplished using polymerase chain reaction.

6. The method of claim 1, wherein the sequencing reaction is a pyrophosphate-based sequencing reaction.

7. The method of claim 1, wherein the sequencing reaction comprises the steps of:

(a) annealing an effective amount of a sequencing primer to the amplified copy of the nucleic acid and extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to produce a sequencing product and a sequencing reaction byproduct if the predetermined nucleotide triphosphate is incorporated onto the 3' end of the sequencing primer; and

(b) identifying the sequencing reaction by-product, thereby determining the nucleic acid sequence in the plurality of reaction chambers.

8. The method of claim 1, wherein the sequencing reaction comprises the steps of:

(a) hybridizing two or more sequencing primers to a single strand of one or more nucleic acid molecules, wherein all but one of the primers are reversibly blocked primers;

(b) incorporating at least one base into a nucleic acid molecule by polymerase extension from an unblocked primer;

(c) preventing further elongation of the unblocked primer;

(d) deblocking one of said reversibly blocked primers into an unblocked primer; and

(e) repeating steps (b) to (d) until at least one of said reversibly blocked primers is deblocked and used for sequencing.

9. The method of claim 1, wherein the reaction chamber is a cavity formed by etching an end of a fiber optic bundle.

10. An array comprising a planar surface having a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center-to-center spacing of between 20-100 μm and each cavity has a width in at least one dimension of between 20 μm and 70 μm, and wherein there are at least 10,000 reaction chambers.

11. The array of claim 10, wherein the plurality of reaction chambers comprise at least 100,000 copies of a single species of single-stranded nucleic acid template.

12. The array of claim 11, wherein the single stranded nucleic acid templates are immobilized on a mobile solid support disposed in the reaction chamber.

13. The array of claim 10, wherein the center-to-center spacing is between 40 μ ι η and 60 μ ι η.

14. The array of claim 10, wherein each cavity has a depth of between 20 μm and 60 μm.

15. An array comprising a flat top surface and a flat bottom surface, wherein the flat top surface has at least 10,000 cavities thereon, each cavity forming an analyte reaction chamber, the flat bottom surface optionally being conductive such that optical signals from the reaction chambers can be detected through the flat bottom surface, wherein the distance between the flat top surface and the flat bottom surface is no more than 5mm, wherein the reaction chambers have a center-to-center spacing of between 20-100 μm and each chamber has a width in at least one dimension of between 20 μm and 70 μm.

16. The array of claim 15, wherein the distance between the top surface and the bottom surface is no more than 2 mm.

17. The array of claim 10 or 15, wherein the number of cavities is greater than 50,000.

18. The array of claim 10 or 15, wherein the number of cavities is greater than 100,000.

19. The array of claim 10 or 15, wherein each reaction chamber is substantially hexagonal in shape.

20. The array of claim 10 or 15, wherein each cavity has at least one irregular wall surface.

21. The array of claim 10 or 15, wherein the array is formed in a fused fiber bundle.

22. The array of claim 10 or 15, wherein each cavity has a smooth wall surface.

23. The array of claim 10 or 15, wherein the cavities are formed by etching one end of a fiber bundle.

24. The array of claim 10 or 15, wherein each chamber contains reagents for analyzing nucleic acids or proteins.

25. The array of claim 10 or 15, further comprising a second surface, separate from and in opposing contact with said planar array, thereby forming a flow chamber on the array.

26. An array tool for performing separate parallel co-reactions in an aqueous environment, wherein the array tool comprises a substrate comprising at least 10,000 separate reaction chambers containing a starting material capable of reacting with a reagent, each reaction chamber being measured such that when one or more fluids containing at least one reagent are delivered into each reaction chamber, the diffusion time for the reagent to diffuse out of the well exceeds the time required for the starting material to react with the reagent to form a product.

27. The array of claim 26, wherein each chamber contains reagents for analyzing nucleic acids or proteins.

28. The array of claim 26, further comprising a population of mobile solid supports disposed in the reaction chamber, each mobile solid support having one or more bioactive agents attached thereto.

29. The array of claim 26, wherein the cavities are formed in the matrix by etching, molding, or micromolding.

30. The array of claim 17, wherein the substrate is a fiber optic bundle.

31. The array of claim 10, 15 or 26, wherein at least 5% to 20% of the reaction chambers contain at least one mobile solid support having at least one reagent immobilized thereon.

32. The array of claim 10, 15 or 26, wherein at least 20% to 60% of the reaction chambers have at least one mobile solid support having at least one reagent immobilized thereon.

33. The array of claim 10, 15 or 26, wherein at least 50% to 100% of the reaction chambers have at least one mobile solid support having at least one reagent immobilized thereon.

34. The array of claim 31, wherein the reagent immobilized on the mobile solid support is a polypeptide having sulfatase activity.

35. The array of claim 31, wherein the reagent immobilized on the mobile solid support is a polypeptide having luciferase activity.

36. The array of claim 31, wherein the mobile solid support has immobilized sulfurylase and luciferase.

37. The array of claim 31, wherein a plurality of the reaction chambers each comprise at least 100,000 copies of a single species of single-stranded nucleic acid template.

38. The array of claim 31, wherein the single stranded nucleic acid templates are immobilized on a mobile solid support disposed in the reaction chamber.

39. The array of claim 10, 15 or 26, wherein the nucleic acids are suitable for use in a pyrosequencing reaction.

40. A method of delivering a biologically active agent to an array comprising dispersing a plurality of moving solid supports on the array, each moving solid support having at least one reagent immobilized thereon, wherein the reagents are suitable for use in a nucleic acid sequencing reaction, wherein the array comprises a planar surface on which a plurality of reaction chambers are arranged, wherein the reaction chambers have a center-to-center spacing of between 20-100 μ ι η and each reaction chamber has a width in at least one dimension of between 20 μ ι η and 70 μ ι η.

41. An apparatus for simultaneously monitoring an array of reaction chambers for light indicative of a reaction occurring at a particular site, the apparatus comprising:

(a) An array of reaction chambers formed from a planar substrate, the planar substrate comprising a plurality of cavitated surfaces, each cavitated surface forming a reaction chamber for containing an analyte and wherein the reaction chambers have a center-to-center spacing of between 20-100 μm, each reaction chamber having a volume of between 10-150pL, the array comprising more than 10,000 separate reaction chambers;

(b) an optically sensitive device arranged such that light from a particular reaction chamber is directed to a particular predetermined area of the optically sensitive device;

(c) means for determining the level of light striking each of said predetermined areas; and

(d) means for recording said light level changes of each of said reaction chambers over time.

42. An analytical sensor, comprising:

(a) an array formed from a first bundle of optical fibers having a plurality of cavitated surfaces at one end thereof, each cavitated surface forming a reaction chamber for containing an analyte and wherein the reaction chambers have a center-to-center spacing of between 20-100 μm, a width of between 20-70 μm, the array comprising more than 10,000 separate reaction chambers;

(b) an enzymatic or fluorescent means for generating light in the reaction chamber; and

(c) light detection means comprising a light capture means and a second fiber bundle for delivering light to the light detection means, said second fiber bundle being in optical contact with said array such that light generated in a single reaction chamber is captured by a single fiber or individual fiber cluster of said second fiber bundle for delivery to the light capture means.

43. The sensor of claim 42, wherein the sensor is adapted for use in a biochemical test.

44. The sensor of claim 42, wherein the sensor is adapted for use in a cell-based assay.

45. The sensor of claim 42, wherein the light capture means is a CCD camera.

46. The sensor of claim 42, wherein the reaction chamber comprises one or more mobile solid supports having a bioactive agent immobilized thereon.

47. A method of performing separate parallel co-reactions in an aqueous environment, comprising:

(a) delivering a fluid containing at least one reagent to an array, wherein the array comprises a matrix comprising at least 10,000 separate reaction chambers, each reaction chamber being adapted to contain an analyte, wherein the reaction chambers have a volume between 10-150pL and comprise a starting material capable of reacting with a reagent, each reaction chamber being dimensioned such that when the fluid is delivered to each reaction chamber, the diffusion time of the reagent out of the well exceeds the time required for the starting material to react with the reagent to form a product; and

(b) after (i) the starting material reacts with the reagents to form a product in each reaction chamber, but before (ii) the reagents delivered to any one reaction chamber diffuse out of that reaction chamber into the other reaction chambers, the fluid is washed from the array.

48. The method of claim 47, wherein the product formed in any one reaction chamber is independent of the product formed in any other reaction chamber but generated using one or more co-reagents.

49. The method of claim 47, wherein the starting material is a nucleic acid sequence and at least one reagent in the fluid is a nucleotide or nucleotide analog.

50. The method of claim 47, wherein the fluid further comprises a polymerase capable of reacting the nucleic acid sequence with the nucleotide or nucleotide analog.

51. The method of claim 47, further comprising sequentially repeating steps (a) and (b).

52. A method of delivering a nucleic acid sequencing enzyme to an array, said array having a planar surface with a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein said reaction chambers have a center-to-center spacing of between 20-100 μm; the method comprises dispersing a plurality of moving solid supports having one or more nucleic acid sequencing enzymes immobilized thereon over the array, such that a plurality of reaction chambers comprise at least one moving solid support.

53. The method of claim 52, wherein one nucleic acid sequencing enzyme is a polypeptide having sulfurylase activity, luciferase activity, or both.

54. A method of delivering a plurality of nucleic acid templates to an array, said array having a planar surface with a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein said reaction chambers have a center-to-center spacing of between 20-100 μm and said array has at least 10,000 reaction chambers; the method comprises dispersing a plurality of mobile solid supports on an array, each mobile solid support having no more than one species of nucleic acid template immobilized thereon, said dispersing resulting in the loading of no more than one mobile solid support in any one reaction chamber.

55. The method of claim 54, wherein the nucleic acid sequence is a single-stranded nucleic acid.

56. The method of claim 54, wherein at least 100,000 copies of a single species of nucleic acid template are immobilized on a plurality of mobile solid supports.

57. The method of claim 54, wherein each single species of nucleic acid template is amplified on a picotiter plate to produce at least 2,000,000 copies/well of the nucleic acid template after being dispensed into a reaction chamber.

58. The method of claim 57, wherein the nucleic acid sequence is amplified using an amplification technique selected from the group consisting of polymerase chain reaction, ligase chain reaction, and isothermal DNA amplification.

59. A method of sequencing a nucleic acid, the method comprising:

(a) providing a plurality of single-stranded nucleic acid templates disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center-to-center spacing of between 20-100 μm and the planar surface has at least 10,000 reaction chambers;

(b) performing a pyrophosphate-based sequencing reaction simultaneously in all reaction chambers by annealing an effective amount of a sequencing primer to a nucleic acid template and extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to produce a sequencing product, a sequencing reaction byproduct being produced if the predetermined nucleotide triphosphate is incorporated onto the 3' end of the sequencing primer;

(c) identifying the sequencing reaction by-products, thereby determining the nucleic acid sequence in each reaction chamber.

60. The method of claim 59, wherein the sequencing reaction byproduct is PPi and detection is performed using a coupled sulfurylase/luciferase reaction to generate light.

61. The method of claim 60 wherein either or both of the sulfurylase and luciferase are immobilized on one or more mobile solid supports disposed at each reaction site.

62. A method of determining the base sequence of a plurality of nucleotides on an array, the method comprising

(a) Providing at least 10,000 DNA templates, each individually disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center-to-center spacing of between 20-100 μm and a volume of between 10-150 pL; wherein

(b) Adding an activated nucleoside 5 ' triphosphate precursor of a known nitrogenous base to the reaction mixture in each reaction chamber, each reaction mixture comprising a template-directed nucleotide polymerase and a single-stranded polynucleotide template hybridized to a complementary oligonucleotide primer strand at least one nucleotide residue shorter than the template to form at least one unpaired nucleotide residue on each template at the 3 ' end of the primer strand, under reaction conditions permitting incorporation of the activated nucleoside 5 ' triphosphate precursor into the 3 ' end of the primer strand, provided that the nitrogenous base of the activated nucleoside 5 ' triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the template;

(c) detecting whether the nucleoside 5 ' triphosphate precursor is incorporated into the primer strand, wherein incorporation of the nucleoside 5 ' triphosphate precursor indicates that the unpaired nucleotide residue of the template has a nitrogenous base composition that is complementary to the nitrogenous base of the incorporated nucleoside 5 ' triphosphate precursor;

(d) Sequentially repeating steps (b) and (c), wherein each sequential repetition adds and detects incorporation of one type of activated nucleoside 5' triphosphate precursor of a known nitrogenous base composition; and

(e) the base sequence of the unpaired nucleotide residues of the template in each reaction chamber is determined from the incorporation sequence of the nucleoside precursor.

63. A method of identifying a base at a target position in a DNA sequence of a template DNA, wherein:

(a) disposing at least 10,000 individual DNA templates in a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center-to-center spacing of between 20-100 μm, the DNA being made single-stranded before or after dispensing into the reaction chambers;

(b) providing an extension primer that hybridizes to the immobilized single-stranded DNA at a location immediately adjacent to the target location;

(c) subjecting the immobilized single-stranded DNA to a polymerase reaction in the presence of a predetermined deoxynucleotide or dideoxynucleotide, wherein a sequencing reaction byproduct is formed if the predetermined deoxynucleotide or dideoxynucleotide is incorporated at the 3' end of the sequencing primer; and

(d) identifying the sequencing reaction by-product, thereby determining the nucleotides complementary to the bases at the target position of each of the 10,000 DNA templates.

64. The method of claim 63, wherein a dATP or ddATP analogue that is capable of acting as a substrate for a polymerase but not said PPi detection enzyme is used in place of deoxy or dideoxy Adenosine Triphosphate (ATP).

65. An apparatus for analyzing nucleic acid sequences. The device includes:

(a) a reagent delivery cuvette, wherein the cuvette comprises an array comprising a planar surface having a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center-to-center spacing of between 20-100 μm and there are more than 10,000 reaction chambers, and wherein the reagent delivery cuvette contains reagents for a sequencing reaction;

(b) an agent delivery means associated with said agent delivery capsule;

(c) an imaging system in communication with the reagent delivery chamber; and

(d) a data collection system is associated with the imaging system.

66. An apparatus for determining a base sequence of a plurality of nucleotides on an array, the apparatus comprising:

(a) a reagent cuvette comprising a plurality of cavities on a flat surface, each cavity forming an analyte reaction chamber, wherein there are more than 10,000 reaction chambers, each reaction chamber having a center-to-center spacing of between 20-100 μm and a volume of between 10-150 pL;

(b) Simultaneously adding to the reaction mixture in each reaction chamber a reagent delivery means for an activated nucleoside 5 ' triphosphate precursor of a known nitrogenous base, each reaction mixture comprising a template-directed nucleotide polymerase and a single-stranded polynucleotide template hybridized to a complementary oligonucleotide primer strand at least one nucleotide residue shorter than the template to form at least one unpaired nucleotide residue on each template at the 3 ' end of the primer strand, under reaction conditions that allow incorporation of the activated nucleoside 5 ' triphosphate precursor into the 3 ' end of the primer strand, provided that the nitrogenous base of the activated nucleoside 5 ' triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the template;

(c) a detection means for detecting in each reaction chamber whether said nucleoside 5 ' triphosphate precursor is incorporated into the primer strand, wherein incorporation of said nucleoside 5 ' triphosphate precursor indicates that the unpaired nucleotide residue of the template has a nitrogenous base composition that is complementary to the nitrogenous base of said incorporated nucleoside 5 ' triphosphate precursor; and

(d) means for sequentially repeating steps (b) and (c), wherein each sequential repetition adds and detects incorporation of an activated nucleoside 5' triphosphate precursor of one type of known nitrogenous base composition; and

(e) Data processing means for simultaneously determining the base sequence of unpaired nucleotide residues of the template in each reaction chamber from the incorporation sequence of the nucleoside precursors.

67. A device for processing a plurality of analytes, the device comprising:

(a) a flow cell configured with a matrix comprising at least 50,000 cavitated surfaces on a fiber optic bundle, each cavitated surface forming a reaction chamber for containing an analyte, wherein the reaction chamber has a center-to-center spacing of between 20-100 μm and a diameter of between 20-70 μm;

(b) a fluidic means for delivering a processing reagent from one or more reservoirs to the flow chamber, whereby the analyte dispensed in the reaction chamber is contacted with the reagent; and

(c) and a detection means for simultaneously detecting a sequence of optical signals from each reaction chamber, each optical signal of the sequence being indicative of an interaction between a processing reagent and an analyte dispensed in the reaction chamber, wherein the detection means is associated with the cavitated surface.

68. The apparatus of claim 67, wherein the detection means is a CCD camera.

69. The device of claim 67, wherein the analyte is a nucleic acid.

70. The device of claim 67, wherein the analytes are immobilized on one or more mobile solid supports disposed in the reaction chamber.

71. The device of claim 67, wherein the treatment reagents are immobilized on one or more mobile solid supports.

72. A method of sequencing a nucleic acid, the method comprising:

(a) providing a plurality of single-stranded nucleic acid templates in an array, the array having at least 50,000 isolated reaction sites;

(b) contacting the nucleic acid template with reagents necessary to perform a pyrophosphate-based sequencing reaction coupled with light emission;

(c) detecting light emitted by the plurality of reaction sites on portions of the optically sensitive device;

(d) converting light striking each portion of the optically sensitive device into an electrical signal that is distinguishable from signals from all other reaction sites;

(e) determining the sequence of the nucleic acid template for each of the isolated reaction sites from the corresponding electrical signal based on the light emission.

73. The method of claim 1, further comprising the step of:

(a) uniquely tagging fragmented nucleic acids from different biological source libraries to generate libraries of fragmented nucleic acids having different detectable sequence tags;

(b) sequencing the fragmented nucleic acids and detecting the detectable sequence tags from each of the labeled nucleic acid fragments.

74. The method of claim 1, wherein the libraries are delivered individually or wherein the libraries are mixed and delivered simultaneously.

75. The method of claim 1, wherein the detectable sequence tag comprises an oligonucleotide between 2 and 50 bases.

76. A method of sequencing a nucleic acid, comprising:

(a) fragmenting a large template nucleic acid molecule to produce a plurality of fragmented nucleic acids;

(b) attaching one strand of the plurality of fragmented nucleic acids to beads, respectively, to generate single-stranded nucleic acids attached to the beads, respectively;

(c) delivering a population of single-stranded fragmented nucleic acids individually attached to beads to an array of at least 10,000 reaction chambers on a planar surface, respectively, wherein a plurality of wells comprises no more than one bead having single-stranded fragmented nucleic acids;

(d) sequencing reactions are performed simultaneously on multiple reaction chambers.

77. The method of claim 76, wherein the reaction chambers have a center-to-center spacing between 20-100 μm.

78. The method of claim 76, wherein said fragmented nucleic acids are between 30-500 bases.

79. The method of claim 76, wherein the fragmented nucleic acids are amplified in a reaction chamber prior to step (d).

80. The method of claim 76, wherein the step of amplifying is accomplished using polymerase chain reaction.

81. The method of claim 76, wherein the sequencing reaction is a pyrophosphate-based sequencing reaction.

82. The method of claim 76, wherein the sequencing reaction comprises the steps of:

(e) annealing an effective amount of a sequencing primer to a single-stranded fragmented nucleic acid template and extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to produce a sequencing product and, if the predetermined nucleotide triphosphate is incorporated onto the 3' end of the sequencing primer, a sequencing reaction byproduct; and

(f) identifying the sequencing reaction by-product, thereby determining the nucleic acid sequence in the plurality of reaction chambers.

83. The method of claim 76, wherein the sequencing reaction comprises the steps of:

(a) hybridizing two or more sequencing primers to one or more single strands of a nucleic acid molecule, wherein all but one of the primers are reversibly blocked primers;

(b) incorporating at least one base into a nucleic acid molecule by polymerase extension from an unblocked primer;

(c) preventing further elongation of the unblocked primer;

(d) deblocking one of said reversibly blocked primers into an unblocked primer; and

(e) repeating steps (b) to (d) until at least one of said reversibly blocked primers is deblocked and used for sequencing.

84. The method of claim 76, wherein the reaction chamber is a cavity formed by etching at an end of a fiber optic bundle.