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WO2001088174A1 - Nouvelles compositions et methodes permettant d'effectuer plusieurs reactions pcr sur un seul echantillon - Google Patents

Nouvelles compositions et methodes permettant d'effectuer plusieurs reactions pcr sur un seul echantillon Download PDF

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WO2001088174A1
WO2001088174A1 PCT/US2001/015981 US0115981W WO0188174A1 WO 2001088174 A1 WO2001088174 A1 WO 2001088174A1 US 0115981 W US0115981 W US 0115981W WO 0188174 A1 WO0188174 A1 WO 0188174A1
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pcr
dna
primers
target
primer
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Natalia Broude
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Boston University
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Boston University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors

Definitions

  • the present invention is directed to novel methods and compositions for use with multiplex polymerase chain reaction (PCR).
  • Multiplex PCR refers to the simultaneous amplification of different target nucleic acid sequences in a single PCR reaction. This method uses PCR suppression effect to obtain target-specific amplification with only a single target-specific primer for each target.
  • Polymerase chain reaction is a method whereby virtually any DNA or mRNA sequence can be selectively amplified.
  • the method involves using paired sets of oligonucleotides of predetermined sequence that hybridize to opposite strands of the template nucleic acid, e.g., DNA, and define the limits of the sequence to be amplified.
  • the template is duplicated.
  • PCR has gained widespread use for the diagnosis of inherited disorders and susceptibility to disease.
  • the genomic region of interest is amplified from either genomic DNA or from a source of specific cDNA encoding the desired gene product. Mutations or polymorphisms are then identified by subjecting the amplified DNA to analytical techniques such as DNA sequencing, hybridization with allele specific oligonucleotides, restriction endonuclease cleavage or single-strand conformational polymorphism (SSCP) analysis.
  • analytical techniques such as DNA sequencing, hybridization with allele specific oligonucleotides, restriction endonuclease cleavage or single-strand conformational polymorphism (SSCP) analysis.
  • SSCP single-strand conformational polymorphism
  • a significant limiting factor in being able to carry out multiplex PCR reactions is the complexity introduced by the use of multiple primers in the same reaction.
  • N targets typically 2N primers are required for a standard PCR reaction.
  • One complexity associated with the presence of increasing numbers of primers is that the primer sequences can cross anneal and are therefore not available for amplifying target DNA.
  • Five targets in a single reaction is the current maximum which can be amplified with routine ease; to amplify 20 targets requires considerable optimization of the reaction conditions.
  • a primer For use in multiplex PCR, a primer should be designed so that its predicted hybridization kinetics are similar to those of the other primers used in the same multiplex reaction. While the annealing temperatures and primer concentrations may be calculated to some degree, conditions generally have to be empirically determined for each multiplex reaction. Since the possibility of non-specific priming increases with each additional primer pair, conditions must be modified as necessary as individual primer sets are added. Moreover, artifacts that result from competition for resources (e.g., depletion of primers) are augmented in multiplex PCR, since differences in the yields of unequally amplified fragments are enhanced with each cycle. Given these limitations, the development of a new diagnostic test can be very labor- intensive and costly.
  • results obtained with multiplex PCR are frequently complicated by artifacts of the amplification procedure. These include “false- negative” results due to reaction failure and “false-positive” results such as the amplification of spurious products, which may be caused by annealing of the primers to sequences which are related to, but distinct from, the true recognition sequences.
  • Chenchik et al. (U.S. Patent Nos. 5,565,340 and 5,759,822) provides a method for decreasing artifacts generated during PCR by utilizing a PCR suppression effect (Lukyanov et al., Anal. Biochem. 229: 198-202 (1995); Siebert et al., Nucl. Acid. Res. 23: 1087-8 (1995)).
  • This method uses novel adapters that are ligated to the end of a DNA fragment prior to PCR amplification.
  • single- stranded DNA fragments having self-complementary adapters at the 5'- and 3'- ends of the strand can form suppressive "tennis racquet" shaped structures that suppress amplification of the fragments during PCR.
  • the present invention is directed to a method for multiplex polymerase chain reaction (mpxPCR), the simultaneous amplification of different target nucleic acid sequences in a single PCR reaction.
  • This method uses the PCR suppression effect to allow target-specific amplification with only a single target-specific primer for each target sequence.
  • This invention further provides primers that allow simultaneous amplification of multiple DNA target sequences present in a RNA or DNA sample.
  • the present invention provides increased multiplexing ability with a decreased number of sequence-specific primers required.
  • nucleic acid templates to be amplified in a multiplex PCR reaction are first prepared as tennis racquet structures to allow the PCR suppression effect.
  • the DNA sample in a single reaction mixture is contacted with a set of oligonucleotide primers, wherein the set of oligonucleotide primers is comprised of one universal adapter primer and one target-specific primer for each target sequence.
  • the total number of primers required to amplify a given number of targets in a single PCR reaction is N+l, not 2N, wherein N is the number of targets to be amplified.
  • the invention encompasses methods for high-throughput genetic screening.
  • the method allows the rapid and simultaneous detection of multiple defined target DNA sequences in DNA samples obtained from a multiplicity of individuals. It is carried out by simultaneously amplifying many different target sequences from a large number of desired samples, such as patient DNA samples, using oligonucleotide primers as above.
  • the present invention provides single-stranded oligonucleotide DNA primers for amplification of a target DNA sequence in a multiplex polymerase chain reaction.
  • the primers to amplify each target are comprised of a universal adapter-primer and a target-specific primer.
  • the methods and compositions of the present invention can be applied to the diagnosis of genetic and infectious diseases, gender determination, genetic linkage analysis, and forensic studies.
  • Figure 1 provides a schematic outlining the PCR suppression effect which allows the selective amplification of targeted sequences from genomic DNA using one target-specific primer and one universal adapter-primer.
  • DNA is digested with a restriction enzyme, and the ends of the resultant DNA fragments are tagged by ligation with complementary adapter sequences. After filling in the ends of the adapter sequences all fragments are entirely double-stranded.
  • each single-stranded fragment has self-complementary ends which favors the formation of stem-loop structures due to intramolecular base pairing of the adapter sequences since the adapters contain long G/C rich oligonucleotides.
  • PCR amplification using a universal adapter-primer (which is complementary to the adapter sequence) is inhibited by the double-stranded nature of the stem portion of the template.
  • amplification with a target-specific primer is efficient if the target sequence is present within the single-stranded loop region.
  • the presence of the universal adapter-primer in the reaction subsequently allows the product of the first amplification reaction to be further amplified.
  • Figures 2A-C show the results of PS-based mpxPCR targeted at several anonymous sequences in chromosome 7 DNA. 5 ng of Rs ⁇ l-digested human genomic
  • DNA with ligated adapter was amplified in a 25 ⁇ l reaction containing lx PCR buffer
  • thermostable DNA polymerase 2.5 mM MgCl 2 , 250 ⁇ M dNTP, 2.5 U of thermostable DNA polymerase
  • FIG. 1 depicts Rs ⁇ l restriction maps of two fragments from human chromosome 7 with the PCR primers.
  • Figure 2B is a gel showing resolution of mpxPCR products using 2% agarose gel electrophoresis.
  • FIG. 1 PCR with the T-primers 1+2; 2: PCR with the T- primers 3+4; 3: PCR with the T-primers 1+2+3+4 (see Table 1), M: 100 bp size marker.
  • Figure 2C shows resolution of the 4-plex (top, primers 1-4, Table 1) and 5- plex (bottom, primers 1-5, Table 1) PCR products using 6% PAGE and an ALF sequencer. The numbers above the peaks show the amplicon lengths. The 1 kb-long PCR amplicon is beyond the displayed window.
  • Figures 3A-D show the results of 14-fold mpxPCR.
  • Figure 4 shows that different DNA polymerases display different specificity in mpxPCR.
  • 4-plex PCR used primers 1, 2, 3, and 4, Table 1.
  • 2.5 U of thermostable DNA polymerase or 2.5 U of a mixture of two DNA polymerases was used in PCR.
  • Other conditions were as in Fig. 2.
  • PD primer-dimers.
  • Figures 5A-C shows the results of allele-specific mpxPCR.
  • Figure 5 A shows the results of uniplex reactions with wild type (N) and mutated (M) primers targeting the interleukin-2 gene (IL2); neurofibromatosis (NFM) gene; and alpha-2- macroglobulin (MG) gene.
  • IL2 interleukin-2 gene
  • NFM neurofibromatosis
  • MG alpha-2- macroglobulin
  • 4-plex PCR was performed with an equimolar mixture of wild type ( ) or mutant (M) primers.
  • the fourth target in both reactions was a fragment of the integrin B 2 subunit gene (primer 8, Table 2).
  • the PCR products were resolved on a 2% agarose gel.
  • Figure 5C shows a window showing the absence of two targets (MG and IL2, marked by arrows) amplified with the mutant primers in a 14-plex PCR. PCR products were resolved using 6% PAGE in an ALF sequencing instrument.
  • Figures 6A-B show genotyping of cystic fibrosis (CF) human DNA samples.
  • Figure 6A shows uniplex reactions with primers targeting wild type (1), ⁇ F508
  • Figure 6B shows genotyping of the same DNA samples in a 4-plex PCR.
  • CFTR wild type (n) and CFTR mutant (m) primers primers 10 and 11, respectively, Table 2
  • primers 1, 2 and 4 Table 1
  • the present invention is directed to a method for multiplex polymerase chain reaction (PCR), the simultaneous amplification of different target nucleic acid sequences in a single PCR reaction.
  • This invention further provides primers that allow simultaneous amplification of multiple target sequences present in a nucleic acid sample.
  • the nucleic sample in a single reaction mixture is contacted with a set of oligonucleotide primers, wherein the set of oligonucleotide primers is comprised of one universal adapter-primer and one target-specific primer for each target sequence.
  • the total number of primers required to amplify a given number of targets in a single PCR reaction is N+l, wherein N is the number of targets to be amplified.
  • the method of the present invention is comprised of the following steps.
  • the nucleic acid of interest i.e. containing the putative target sequences
  • a restriction enzyme generating a population of double-stranded DNA fragments.
  • adapter sequences are ligated to both ends of each DNA fragment, such that following denaturation, the ends of the resultant single strands (or the ligated adapters) are complementary and anneal to form a double-stranded stem of the stem loop structure.
  • the region of the genomic DNA forms single-stranded loop.
  • a set of oligonucleotide primers is added, including one universal adapter- primer and one target-specific primer for each target sequence.
  • the PCR reaction is performed.
  • the universal adapter-primer alone cannot initiate DNA amplification, as its binding to its complementary sequence is highly inefficient due to the double-stranded nature of the stem region.
  • the target-specific primer however is able to bind its complement if the target sequence is present within the loop region, leading to amplification of the target which continues into the stem region (albeit with reduced efficiency).
  • the universal adapter- primer (which recognizes the stem region) provides the second primer for a traditional PCR reaction, and the segment is amplified exponentially.
  • Amplification of DNA denotes the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixture of DNA sequences.
  • Multiplex PCR refers to the simultaneous amplification of multiple nucleic acid targets in a single polymerase chain reaction (PCR) mixture.
  • High-throughput denotes the ability to simultaneously process and screen a large number of nucleic acid samples (e.g. in excess of 100 genomic DNAs) in a rapid and economical manner, as well as to simultaneously screen large numbers of different genetic loci within a single nucleic acid sample.
  • the DNA sample to be analyzed is first cleaved with a restriction enzyme. Any restriction enzyme which allows ligation to the adapter sequences may be used. Adapter sequences are ligated to both ends of each DNA fragment. Any overhanging single-stranded ends will be filled in during the first seconds of the first PCR cycle.
  • the ends of the resultant single strands are complementary and anneal to form a stem-loop structure.
  • the adapter sequences anneal to form the double-stranded loop, and the region of the genomic DNA fragment forms the single-stranded loop.
  • This stem-loop structure is the template used for the multiplex PCR reaction.
  • the adapters of the present invention are oligonucleotides which may be partially double-stranded.
  • the adapters are at least partially double- stranded to aid in ligation of the adapter to the nucleic acid fragment.
  • the adapters can be attached to the ends of DNA fragments using a variety of techniques that are well known in the art, including DNA ligase-mediated ligation of the adapters to sticky- or blunt-ended DNA and T4 RNA ligase-mediated ligation of a single- stranded adapter to single-stranded DNA.
  • the term "attach,” when used in the context of attaching the adapter to a nucleic acid fragment refers to bringing the adapter into covalent association with the nucleic acid fragment regardless of the manner or method by which the association is achieved.
  • DNA fragments can be cloned into plasmid vectors that have the adapters of the present invention inserted in the appropriate orientation upstream and downstream of a cloning site in the vector.
  • the adapters per se consist of a single vector sequence in the plasmid that duplicates the adapter sequence upstream and downstream from a cloning site.
  • the vector can include any plasmid sequence that is necessary for maintenance of the recombinant DNA in a host cell.
  • the adapters of the present invention are comprised of nucleic acid sequences typically not found in the population of nucleic acid templates.
  • sequence of the nucleic acid templates e.g. genomic DNA of certain organisms
  • the lack of homology between the adapter sequence and the nucleic acid template(s) may ⁇ be determined using sequence comparison analysis programs well known in the art (e.g. BLAST).
  • sequence comparison analysis programs well known in the art
  • such lack of homology can be determined empirically, for example by nucleic acid hybridization techniques such as Southern blotting.
  • there is less than 35% identity (homology) between the adapter sequence and the template more preferably less than 30% identity, still more preferably less than 25% identity.
  • the sequence analysis programs used to determine homology are run at the default setting.
  • the adapter sequence of the present invention includes a specific recognition sequence for a restriction enzyme.
  • the sequence is for a type IIs restriction enzyme near its 3' end. For example, a Hgal site.
  • adapter structures are contemplated for use with the present invention. Two types of adapters are referred to herein as “Type 1" and “Type 2" adapter structures. Using the teachings contained herein, the skilled artisan could readily construct other adapters that have different sequences from those adapters exemplified herein, including variants of the subject adapters, that would be operable with the present invention. Any polynucleotide sequence that comprises a primer binding portion and an effective suppressor sequence portion and which when associated with a DNA or RNA fragment can form a suppressive stem-loop structure during PCR as described herein is contemplated by the subject invention. Such adapters are within the scope of the present invention.
  • the Type 1 adapter structure typically has a length of about 40-50, more preferably 42-50 nucleotides. However, the adapter length can vary from as few as about 25 nucleotides, up to 80 or more nucleotides. Generally, the Type 1 adapter does not contain any homopolymer sequence.
  • the Type I adapter typically has a high GC content. A high GC content means that at least 40% of the base pairs are G or C, more preferably at least 45% of the base pairs, still more preferably at least 50%).
  • the Type 1 adapter is typically at least partially double-stranded and generally comprises one long oligomer and one short oligomer, resulting in a 5' overhang at one end of the adapter. The length of the shorter oligomer is not critical to the function of the PCR suppression method of the present invention, and can be shorter or equal to the length of the longer oligomer.
  • Type I adapter sequences include the following:
  • Type 1 adapter allows it to be ligated to any blunt-ended DNA fragment using T4 DNA ligase.
  • Adapters having "sticky ends" that are compatible with certain restriction sites can also be used to attach the adapter to DNA that has been digested with appropriate restriction endonucleases.
  • the upper (and typically longer) oligomer of the adapter can be ligated to DNA.
  • the lower (and typically shorter) oligomer usually is not ligated because it lacks the requisite 5'-phosphate group.
  • the lower oligomer portion of the adapter does increase the efficiency of ligation of the adapter to double-stranded DNA (dsDNA).
  • dsDNA double-stranded DNA
  • tliese modifications include adding a 5'-phosphate for more efficient ligation.
  • the efficiency of suppression can be regulated through varying the length and GC content (which in turn determines the melting temperature of the dsDNA) of the suppressor portion of the adapter.
  • the Type 2 adapter structure is similar to the Type 1 structure but contains a homopolymer sequence in the suppressor portion of the adapter.
  • the Type 2 adapter is incorporated into DNA fragments that have been tailed with oligo (dA) using terminal deoxynucleotidyl transferase, followed by PCR using an appropriate primer.
  • the primer becomes incorporated into the DNA as an adapter.
  • the PCR product can be subsequently treated with exonuclease III to remove the lower strand of the adapter.
  • the skilled artisan will readily recognize that certain of the primers of the subject invention can also be used as adapters. The use of primers as adapters in the present method, and vice versa, is contemplated by the present invention.
  • the adapter should not contain any sequences that can result in the formation of "hairpins" or other secondary structures which can prevent adapter ligation and intramolecular annealing.
  • the universal adapter primer binding portion of the adapter can be complementary with a PCR primer capable of priming for PCR amplification of a target DNA.
  • the universal adapter primers of the present invention comprise a polynucleotide sequence that is complementary to a portion of the polynucleotide sequence of a suppression adapter of the invention.
  • adapters and primers used in the subject invention can be readily prepared by the skilled artisan using a variety of techniques and procedures.
  • adapters and primers can be synthesized using a DNA or RNA synthesizer.
  • adapters and primers may be obtained from a biological source, such as through a restriction enzyme digestion of isolated DNA.
  • the primers are single-stranded.
  • the present invention has an increased multiplexing ability due to the decreased number of sequence-specific primers required.
  • the term "primer” has the conventional meaning associated with it in standard PCR procedures, i.e., an oligonucleotide that can hybridize to a polynucleotide template and act as a point of initiation for the synthesis of a primer extension product that is complementary, to the template strand.
  • the universal adapter-primers of the present invention comprise a polynucleotide sequence that is complementary to a portion of the polynucleotide sequence of a suppression adapter of the invention.
  • the universal adapter primer of the present invention has exact complementarity with a portion of the adapter sequence.
  • primers used in the present invention can have less than exact complementarity with the primer binding sequence of the adapter as long as the primer can hybridize sufficiently with the adapter sequence so as to be extendible by a DNA polymerase.
  • the target-specific primers of the present invention comprise a polynucleotide sequence that is complementary to a portion of the polynucleotide sequence of the target sequence found within the loop region of the stem-loop structure.
  • the target-specific primer of the present invention is completely complimentary to the target sequence.
  • primers used in the present invention can have less than exact complementarity with the primer binding sequence of the target sequence as long as the primer can hybridize sufficiently with the target sequence so as to be extendible by a DNA polymerase.
  • the universal adapter and the target-specific primer sequences are typically analyzed as a group to evaluate the potential for fortuitous dimer formation between different primers. This evaluation may be achieved using commercially available computer programs for sequence analysis, such as Gene Runner, Hastings Software Inc. Other variables, such as the preferred concentrations of Mg +2 , dNTPs, polymerase, and primers, are optimized using methods well-known in the art (Edwards et al., PCR Methods and Applications 3:565 (1994)).
  • the target nucleic acid represents a sample of genomic DNA isolated from a patient.
  • This DNA may be obtained from any cell source or body fluid.
  • Non-limiting examples of cell sources available in clinical practice include blood cells, buccal cells, cervicovaginal cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy.
  • Body fluids include blood, urine, cerebrospinal fluid, semen and tissue exudates at the site of infection or inflammation.
  • DNA is extracted from the cell source or body fluid using any of the numerous methods that are standard in the art. It will be understood that the particular method used to extract DNA will depend on the nature of the source.
  • the preferred amount of DNA to be extracted for use in the present invention is at least 5 pg (corresponding to about 1 cell equivalent of a genome size of 4x10 9 base pairs).
  • the present method can be used with polynucleotides comprising either full- length RNA or DNA, or their fragments.
  • the RNA or DNA can be either double- stranded or single-stranded, and can be in a purified or unpurified form.
  • the polynucleotides are comprised of DNA.
  • the DNA fragments used in the present invention can be obtained from DNA by random shearing of the DNA, by digestion of DNA or cDNA with restriction endonucleases, or by amplification of DNA fractions from DNA using arbitrary or sequence-specific PCR primers.
  • the present invention can also be used with full-size cDNA polynucleotide sequences, such as can be obtained by reverse transcription of RNA.
  • the DNA can be obtained from a variety of sources, including both natural and synthetic sources.
  • the DNA can be from any natural source including viruses, bacteria, yeast, plants, insects and animals.
  • the DNA can also be prepared from any RNA source.
  • a nucleic acid sample is contacted with pairs of oligonucleotide primers under conditions suitable for polymerase chain reaction.
  • Standard PCR reaction conditions may be used, e.g., 1.5 mM MgCl.sub.2, 50 mM KC1, 10 mM Tris-HCl, pH 8.3, 200 ⁇ M deoxynucleotide triphosphates (dNTPs), and 25-100 U/ml Taq polymerase (Perkin-Elmer, Norwalk, Conn.).
  • each primer in the reaction mixture can range from about 0.05 to about 4 ⁇ M.
  • Each target primer is evaluated by performing single PCR reactions using each primer pair (a universal adapter-primer and a target-specific primer) individually. Similarly, each primer pair is evaluated independently to confirm that all primer pairs to be included in a single multiplex PCR reaction generate a product of the expected size. As the number of targets in a single reaction increases, certain targets may not be amplified as efficiently as other targets. The concentration of the primers for such underrepresented targets may be increased to increase their yield. For example, when multiplying 15 or more targets; more preferably, when multiplying 30 or more targets.
  • Multiplex PCR reactions are carried out using manual or automatic thermal cycling. Any commercially available thermal cycler may be used, such as, e.g., Perkin-Elmer 9600 cycler.
  • the present invention is preferably used with at least 5 targets, more preferably at least 10 targets, still more preferably with at least 14 targets, even more preferably with at least 20 targets, yet more preferably with at least 30 targets, still more preferably with at least 50 targets, and even more preferably with at least 100 targets.
  • the polymerase is a thermostable DNA polymerase such as may be obtained from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermus flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu). Many of these polymerases may be isolated from the bacterium itself or obtained commercially. Polymerases to be used with the present invention can also be obtained from cells which express high levels of the cloned genes encoding the polymerase. Preferably, a combination of several thermostable polymerases can be used.
  • the PCR conditions used to amplify the targets are standard PCR conditions, as described below in Protocol B. Typical conditions use 35-40 cycles, with each cycle comprising a denaturing step (e.g. 10 seconds at 94°C), an annealing step (e.g. 15 sec at 68°C), and an extension step (e.g. 1 minute at 72°C). As the number of targets in a single reaction increases, the length of the extension time may be increased. For example, when amplifying 30 or more targets, the extension time may be three times as longer than when amplifying 10-15 targets (e.g. 3 minutes instead of 1 minute).
  • reaction products are analyzed using any of several methods that are well-known in the art.
  • agarose gel electrophoresis is used to rapidly resolve and identify each of the amplified sequences.
  • different amplified sequences are preferably of distinct sizes and thus can be resolved in a single gel.
  • the reaction mixture is treated with one or more restriction endonucleases prior to electrophoresis.
  • Alternative methods of product analysis include without limitation dot-blot hybridization with allele-specific oligonucleotides and SSCP.
  • kits which contain, in separate packaging or compartments, the reagents such as adapters and primers required for practicing the multiplex PCR method of the subject invention.
  • Such kits may optionally include the reagents required for performing PCR reactions, such as DNA polymerase, DNA polymerase cofactors, and deoxyribonucleotide-5'-triphosphates.
  • the kit may also include various polynucleotide molecules, DNA or RNA ligases, restriction endonucleases, reverse transcriptases, terminal transferases, various buffers and reagents, and antibodies that inhibit DNA polymerase activity.
  • the kits may also include reagents necessary for performing positive and negative control reactions. Optimal amounts of reagents to be used in a given reaction can be readily determined by the skilled artisan having the benefit of the current disclosure.
  • the multiplex PCR methods of the subject invention can be used in a wide variety of procedures. Several of these procedures are discussed herein. Other procedures would become apparent to one skilled in the art having the benefit of this disclosure.
  • the multiplex PCR method of the present invention can be used to simultaneously amplify difference disease-related sequences under identical conditions.
  • the subject invention can also be used with long distance (LD) PCR technology (Barnes, 1994; Cheng et al., 1994).
  • LD PCR which uses a combination of thermostable DNA polymerases, produces much longer PCR products with increased fidelity to the original template as compared to conventional PCR performed using Taq DNA polymerase alone.
  • the method of the present invention can also be used in conjunction with antibodies that bind to DNA polymerase and thereby inhibit polymerase function (Kellogg et al., 1994). These antibodies reversibly bind to DNA polymerase in a temperature-specific manner, and thereby increase the specificity of a PCR reaction by inhibiting the formation of non-specific amplification products prior to initiation of PCR amplification.
  • the ability to simultaneously amplify multiple DNA targets using PCR suppression was tested for several unique targets by using a mixture of gene-specific primers.
  • genomic DNA should be digested with an appropriate restriction enzyme and ligated with PS-adapters. That means that the design of the gene-specific primers will depend not only on the location of the tentative target, but also on the availability of the convenient restriction site/s in proximity to the target.
  • One problem, which is inherent to all PCR-based methods with multiple targeting, is biased amplification of certain templates, while others are lost during multiple PCR cycles. These examples look at such issues.
  • Non-phosphorylated oligonucleotides unlabeled or fluorescently labeled, were purchased as custom synthesis products from Integrated DNA Technologies, Inc. (Coralville, IA). DNA from peripheral blood lymphocytes from anonymous donors was isolated using a Qiagen Blood kit according to the manufacturer's protocol (Qiagen, Chatsworth, CA). DNA samples from cystic fibrosis affected individuals were kindly provided by Drs. R. Nelson and B. Allitto (Genzyme Genetics, Framingham, MA). AmpliTaq and AmpliTaq Gold DNA polymerases were from Applied Biosystems (Foster City, CA), KlenTaq DNA polymerase from Ab peptides (St.
  • Adapter-ligated DNA (2-5 ng) was amplified by PCR in a 25 ⁇ l reaction volume containing lx PCR buffer (depending on the DNA polymerase), 2.5 mM
  • DNA polymerase DNA polymerase.
  • the PCR mixtures containing all components but primers were denatured at 95°C for 3-10 min; the primer mixture (5-10 pmol of each) was added at 95°C; and 38 cycles of PCR (94°C, 10 sec, 68°C, 15 sec and 72°C, 1 min) were performed.
  • the A-primer common for all targets was fluorescein-labeled, and the PCR products were analyzed by electrophoresis on a 2% agarose gel and by 6% denaturing PAGE.
  • CFTR cystic fibrosis transmembrane regulator
  • PCR products (2-3 ⁇ l) were denatured for 3 min at 90°C in a stop solution (Amersham Pharmacia Biotech), and analyzed using a 6% denaturing PAGE and an ALF sequencing instrument (Amersham Pharmacia Biotech) as described (17, 18).
  • CFTR DNA fragments were amplified from several DNAs using direct fluorescein-labeled GGGAGAACTGGAGCCTTCAGAG (SEQ ID NO:29) and reverse GGGTAGTGTGAAGGGTTCATATGC (SEQ ID NO:30) primers. 5-10 finol of the PCR product was sequenced using afitol DNA sequencing kit (Promega, Madison, WI) according to the manufacturer's protocol and the products were resolved by 6% PAGE in an ALF sequencing instrument (Amersham Pharmacia Biotech).
  • PS PCR relies on hairpin structures formed by all genomic fragments after DNA is digested with a restriction enzyme, ligated with long GC-rich PS adapters, denatured and re-annealed (Fig. 1).
  • PS PCR usually uses primers that tolerate high primer-annealing temperatures (13) As a result, the specificity of PS PCR is extremely high (13, 17, 18).
  • PS PCR is well suited for multiplex PCR.
  • genomic DNA is digested with an appropriate restriction enzyme and ligated with PS-adapters.
  • PS-adapters the design of the gene-specific primers depends on the location of the target and also on the availability of a proximal restriction site.
  • Protocol A Preparation of genomic DNA with ligated ada p ters.
  • Protocol B multiplex PCR with multiple sequence-specific primers
  • dNTPs 2.5 mM each dATP, dCTP, dGTP, dCTP 2.5 ⁇ l
  • 2C presents 4-plex and 5-plex PCR products after resolution on polyacrylamide gels. All targeted amplicons were detected as PCR products, and the amounts of amplicons in the range 100-400 base pairs were similar. However, a -500 bp amplicon was synthesized in smaller amounts (see also Fig. 4 and below). Table 1. PCR target specific primers targeted at several anonymous sequences in chromosome 7 used in the multiplex PCR.
  • the primer for the aldolase B gene (primer 4, Table 2) generated a PCR product, which appeared as a double peak on a polyacrylamide gel.
  • Fig. 3a, b and c show the patterns obtained in 5- plex PCR (primers 1-5, Table 1), 4-plex PCR (primers 2, 3, 5 and 8, Table 2) and 5- plex PCR (primers 1, 4, 6, 7, and 9, Table 2).
  • we performed a 14-plex PCR in which we combined the primers for the 5 fragments from chromosome 7 (Table 1) and 9 gene-specific primers from Table 2 (primers 1-9).
  • PCR primers targeted at bialleleic loci and the CFTR locus used in PS mpxPCR. Primers targeted at the interleuldn 1 alpha gene, neurofibromatosis 1 locus and alpha 2 -macroglobulin were used as a normal (n) and a double mismatch (m) variant. ND-not determined.
  • Fig. 4 presents the results obtained with two different DNA polymerases, AmpliTaq and Taq DNA polymerase, in an experiment designed to amplify four amplicons (Table 1, primers 1-4). The data show that Taq DNA polymerase amplifies 100-400 bp amplicons more uniformly than AmpliTaq; however, this is accompanied by a greater amount of primer-dimers (Fig. 4).
  • the 510 bp fragment is amplified by Taq DNA polymerase much less efficiently than by AmpliTaq.
  • AmpliTaq DNA polymerase generates much higher non-specific background and amplifies 100-400 bp amplicons less uniformly that Taq DNA polymerase.
  • Fig. A we also tested KlenTaq and AmpliTaq Gold DNA polymerases and found that the combination of these DNA polymerases with AmpliTaq or Taq DNA polymerase also generates a good amplification pattern with low background (data not shown).
  • a mixture (1:1 :1) of three different DNA polymerases, AmpliTaq, AmpliTaq Gold and Taq DNA polymerases are examples of three different DNA polymerases.
  • PCR should discriminate between alleles, and single-base discrimination should be routinely attainable.
  • a penultimate base was mismatched to increase the impact of the 3 '-mismatch (20-22).
  • Fig. 5a shows that uniplex PS PCR with such mismatched primers targeting interleukin, neurofibromatosis and alpha-2-macroglobulin genes perfectly discriminated against a 3 '-end mismatch nucleotide.
  • Fig. 5b shows an agarose gel where three bands, corresponding to interleukin, neurofibromatosis and alpha-2-macroglobulin gene fragments are essentially absent, when the corresponding mismatched primers were used for amplification.
  • Fig. 5c shows the absence of the interleukin and macroglobulin gene fragments amplified in a 14-plex PCR with the corresponding mutated primers.
  • mpx PS PCR in disease diagnostics was shown by genotyping of DNA samples from CF-affected individuals with the ⁇ F508 mutation consisting in a deletion of three nucleotides in exon 10 of the CFTR gene (23).
  • the region around the ⁇ F508 mutation is extremely AT-rich; therefore 39-40 base-long reverse CFTR-primers were designed to survive the 68°C annealing temperature (Table 2).
  • the choice of the reverse primer was determined by the location of an Rsal site about 300 bp upstream of the ⁇ F508 mutation point.
  • the normal and mutated primers were designed to differ by one nucleotide in length and also by the 3'- terminal nucleotide (Table 2). However, preliminary experiments showed that neither primer provided adequate specificity. Therefore, in this particular case we performed nested PCR and used in the first step a 29-mer nested CFTR primer (primer 12, Table
  • Fig. 6A shows that only the normal CFTR-primer amplifies an expected 350-bp fragment from unaffected DNA; both primers generate products from CFTR- heterozygous DNA; and only a mutant primer amplifies a product from ⁇ F508 CFTR- homozygous DNA.
  • the lower efficiency of PCR with the CFTR mutant primer Fig.
  • mpx PCR was used to amplify 30 target sequences, 14 of the target sequences and the target primers were those used for the 14-plex PCR described above (Example 2); 16 additional target sequences and target primers are shown in Table 3.
  • the extension time of the PCR reaction was tripled, to 3 minutes.
  • the concentration of primers was increased, particularly for those targets underrepresented in the final reaction product.
  • WIAF-2012 cactgtagag aaaagtgaag tataaaatgg ggtc SEQ ID NO: :37 34 60.7 530
  • This data shows the value of the present invention comprising mpxPCR utilizing the PS-effect (13, 24).
  • This method requires about half the number of primers as compared to conventional mpxPCR. This substantially simplifies primer design and brings down primer costs. Primer cost savings are typically at least 1.5 fold.
  • the average length of the sum of the two gene-specific primers was 45 (38-62) nt
  • the same targets were amplified with an average primer that was 31 (30- 33) nt long (see Table 2, primers 1-9), and only a single labeled primer was required.
  • the preparation of DNA samples for PS PCR includes digestion of genomic DNA with an appropriate restriction enzyme and ligation with the PS-driving adapters. These extra-steps, however, are not burdensome since the DNA samples prepared can be used in multiple experiments. It is preferably to choose one restriction enzyme for as many of the targets as possible. Preferably, all targets. In most of these examples, targets were not dropped because of the lack of a Rsal site in the range accessible to amplification. Therefore, we believe that the choice of a single restriction enzyme for most of the targets should be not a problem, and a suitable site will be available in proximity to practically every target.
  • PCR conditions which we used for mpxPCR, did not require special optimization and should be considered as default conditions applicable for amplification of any target, provided the primer's Tm tolerates 65-68°C.
  • the hairpin-loop structures acquired by all genomic fragments we believe present a positive factor for efficient target amplification. Indeed, all the targets are located within the ss regions of the hairpin-loop structures and are, therefore, available to corresponding primers for binding. This may explain the fact that the relative amplification efficiency of similar sized targets in mpxPCR correlates with the Tm of the primers (Table 4).
  • the size of the ss loop seems to be another factor affecting the efficiency of PS PCR.
  • a target residing in a larger ss loop (-600 nt) is amplified less efficiently than a target located in a smaller ss loop (227 nt) (see Table 4). This may occur because larger ss loops form stable secondary structures more easily than shorter ones; this can inhibit annealing of the corresponding T- primer and decrease the yield of the amplicon.
  • the size of the ss loop is determined by the distance between the two restriction sites and can not be regulated by experimentator. However, the decreased efficiency of amplification of targets with large ss loops can be partly compensated by increasing the length of the primer and consequently its Tm.
  • the Tm's of the primers were calculated using a nearest-neighbor thermodynamic parameter set (19, see also http://www.idtdna.com/technotes_facs/Calculating_Tm). Peak areas were determined by analyzing a 14-plex PCR pattern using Fragment manager software provided with the ALF sequencing instrument.
  • thermostable DNA polymerases displayed different specificity in PS mpxPCR. Mixtures of DNA polymerases generated more uniform amplification patterns than individual DNA polymerases. (Fig. 4). We speculate that minor differences in strand displacement activity and processivity of different DNA polymerases may cause differences in specificity. Mixes of DNA polymerases have been already used in PCR for other purposes. For example, a combination of two thermostable DNA polymerases, one of which had 3 '-5' proofreading activity, has been shown to increase the length of PCR amplification (27).
  • the criteria for primer selection are, in general, the same as in conventional mpxPCR: the primers should not be self- or mutually complementary and should not form homo- and hetero-dimers (28). Because PS PCR requires only one specific primer, its uniqueness is crucial. Therefore, the primers are usually relatively long oligonucleotides with high GC contents. This allows high annealing temperatures and thereby increases the specificity (13). In order to increase the specificity further, one can perform nested PCR (13, 18, 24). For the purposes of multiplexing, however, nested PCR may not be required.
  • Another advantage of this technique consists in its amenability to automation and development of high-throughput genetic diagnostics. For example, by performing 14- plex PCR in 96-wells microtitre plate, one will be able to analyze 192 chromosomes at 14 loci simultaneously.
  • This method can be easily combined with various advanced techniques, e. g., two-color detection and microarray-based analysis of the PCR products, which will increase further the throughput and information content of every experiment.

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Abstract

L'invention concerne une méthode multiplex de réaction en chaîne de la polymérase (mpxPCR) permettant de réaliser l'amplification simultanée de différentes séquences d'acide nucléique cible en une seule réaction PCR. Cette méthode fait intervenir l'effet de suppression PCR afin de réaliser une amplification spécifique de cible avec une seule amorce spécifique de cible pour chaque séquence cible. L'invention concerne également des amorces qui permettent de réaliser une amplification simultanée de plusieurs séquences cibles d'ADN présentes dans un échantillon d'ADN ou d'ARN.
PCT/US2001/015981 2000-05-17 2001-05-17 Nouvelles compositions et methodes permettant d'effectuer plusieurs reactions pcr sur un seul echantillon Ceased WO2001088174A1 (fr)

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EP1310569A3 (fr) * 2001-11-09 2004-01-02 President of Gifu University Méthode et kit d'essai pour la détection des gènes
US7108976B2 (en) 2002-06-17 2006-09-19 Affymetrix, Inc. Complexity management of genomic DNA by locus specific amplification
JP2006320217A (ja) * 2005-05-17 2006-11-30 Olympus Corp マルチプレックスｐｃｒ法
US7452671B2 (en) 2005-04-29 2008-11-18 Affymetrix, Inc. Methods for genotyping with selective adaptor ligation
US7459273B2 (en) * 2002-10-04 2008-12-02 Affymetrix, Inc. Methods for genotyping selected polymorphism
WO2009021518A1 (fr) * 2007-08-10 2009-02-19 Tina Holding Aps Procédé d'évaluation de la longueur de télomères
US8114978B2 (en) 2003-08-05 2012-02-14 Affymetrix, Inc. Methods for genotyping selected polymorphism
US9074244B2 (en) 2008-03-11 2015-07-07 Affymetrix, Inc. Array-based translocation and rearrangement assays
CN105219766A (zh) * 2015-11-10 2016-01-06 东华大学 一种三轮扩增的多重pcr方法
US9388459B2 (en) 2002-06-17 2016-07-12 Affymetrix, Inc. Methods for genotyping
US9388457B2 (en) 2007-09-14 2016-07-12 Affymetrix, Inc. Locus specific amplification using array probes
US11306351B2 (en) 2005-12-21 2022-04-19 Affymetrix, Inc. Methods for genotyping

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US5565340A (en) * 1995-01-27 1996-10-15 Clontech Laboratories, Inc. Method for suppressing DNA fragment amplification during PCR
US5582989A (en) * 1988-10-12 1996-12-10 Baylor College Of Medicine Multiplex genomic DNA amplification for deletion detection

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US5582989A (en) * 1988-10-12 1996-12-10 Baylor College Of Medicine Multiplex genomic DNA amplification for deletion detection
US5565340A (en) * 1995-01-27 1996-10-15 Clontech Laboratories, Inc. Method for suppressing DNA fragment amplification during PCR
US5759822A (en) * 1995-01-27 1998-06-02 Clontech Laboratories, Inc. Method for suppressing DNA fragment amplification during PCR

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1310569A3 (fr) * 2001-11-09 2004-01-02 President of Gifu University Méthode et kit d'essai pour la détection des gènes
US9388459B2 (en) 2002-06-17 2016-07-12 Affymetrix, Inc. Methods for genotyping
US7108976B2 (en) 2002-06-17 2006-09-19 Affymetrix, Inc. Complexity management of genomic DNA by locus specific amplification
US7459273B2 (en) * 2002-10-04 2008-12-02 Affymetrix, Inc. Methods for genotyping selected polymorphism
US8114978B2 (en) 2003-08-05 2012-02-14 Affymetrix, Inc. Methods for genotyping selected polymorphism
US8808995B2 (en) 2003-08-05 2014-08-19 Affymetrix, Inc. Methods for genotyping selected polymorphism
US10155976B2 (en) 2003-08-05 2018-12-18 Affymetrix, Inc. Methods for genotyping selected polymorphism
US9388461B2 (en) 2003-08-05 2016-07-12 Affymetrix, Inc. Methods for genotyping selected polymorphism
US7452671B2 (en) 2005-04-29 2008-11-18 Affymetrix, Inc. Methods for genotyping with selective adaptor ligation
US8133667B2 (en) 2005-04-29 2012-03-13 Affymetrix, Inc. Methods for genotyping with selective adaptor ligation
JP2006320217A (ja) * 2005-05-17 2006-11-30 Olympus Corp マルチプレックスｐｃｒ法
US11306351B2 (en) 2005-12-21 2022-04-19 Affymetrix, Inc. Methods for genotyping
WO2009021518A1 (fr) * 2007-08-10 2009-02-19 Tina Holding Aps Procédé d'évaluation de la longueur de télomères
US9388457B2 (en) 2007-09-14 2016-07-12 Affymetrix, Inc. Locus specific amplification using array probes
US10329600B2 (en) 2007-09-14 2019-06-25 Affymetrix, Inc. Locus specific amplification using array probes
US11408094B2 (en) 2007-09-14 2022-08-09 Affymetrix, Inc. Locus specific amplification using array probes
US12139817B2 (en) 2007-09-14 2024-11-12 Affymetrix, Inc. Locus specific amplification using array probes
US9932636B2 (en) 2008-03-11 2018-04-03 Affymetrix, Inc. Array-based translocation and rearrangement assays
US9074244B2 (en) 2008-03-11 2015-07-07 Affymetrix, Inc. Array-based translocation and rearrangement assays
CN105219766A (zh) * 2015-11-10 2016-01-06 东华大学 一种三轮扩增的多重pcr方法

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