HK1219117B - Methods for amplifying fragmented target nucleic acids utilizing an assembler sequence - Google Patents

Description

Method for amplifying fragmented target nucleic acid using assembler sequences

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional patent application of provisional patent application No. 61/789,984, filed on March 15, 2013.

Statement Regarding Federal Research or Development Funding

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Related reference materials submitted on CD-ROM

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Background of the Invention

(1) Field of the Invention

The present invention relates to a method for amplifying a fragmented target nucleic acid containing short nucleic acid fragments, specifically, using an assembly template to assemble and extend these short nucleic acids to produce larger amplicons for further manipulation.

(2) Description of Background Technology

There are many sample types where the target nucleic acid is severely degraded, making it difficult to detect using standard molecular diagnostic tests. More specifically, these target nucleic acids are shorter than the "footprint" of the test used to detect them. For example, to detect a target with very low copy number, the target is generally amplified. In addition, in many cases, it is desirable to detect the target with high specificity. This detection is often at the resolution of a single nucleotide change. When performing amplification, such as a PCR reaction, there are a forward primer, a reverse primer, and a detection probe. Typically, these primers and the detection probe are at least about 20 nucleotides in length. Assuming that there is no partial overlap between the primers and probe, the target is required to be at least 60 nucleotides in length to accommodate the combination of these three elements (i.e., the footprint of the test). Moreover, the desired single nucleotide change (SNV) to be detected can occur anywhere in the target nucleic acid sequence. For reliable detection, the SNV must be located within the amplified target nucleic acid (i.e., within a region of the amplicon within the primer binding site) so that the probe selectively hybridizes to this region. This effectively increases the potential footprint of the assay to at least about 100 nucleotides in length for such detection.

In many cases, detecting a known or unknown SNV in the target sequence requires fragments up to 160 nucleotides long for detection. This is largely due to the arrangement of probes, primers, and blocking probes on the amplicon.

The challenge of this demand is that many sample types contain fragments as short as 20-50 nucleotides in length, which are usually attributed to partial degradation of the target DNA or RNA. This is typical in RNA sequences because they are more easily degraded. This is also common in special sample types, such as urine, blood samples, and samples of formaldehyde-fixed paraffin-embedded (FFPE) and fine needle aspiration (FNA), where cross-linked nucleic acids become more degraded as they are stored. In the case of urine, nucleic acids naturally enter the urine in the form of small fragments due to the processing by the kidneys. Typically, the size of the nucleic acid fragments that pass through the kidneys is about 20-50 nucleotides or less.

Therefore, it is necessary to extend the length of short target fragments for more efficient and accurate detection. This requires that the enlarged target short fragments have the same sequence background as the sequence from which they were originally derived to enable appropriate processing. The present invention provides methods to meet this need.

Summary of the Invention

The present invention provides a method for amplifying a fragmented target nucleic acid using an assembling template that binds and extends short nucleic acid fragments of a target nucleic acid to produce larger amplicons for further manipulation. One aspect of the invention is a method for amplifying a fragmented target nucleic acid comprising short target nucleic acid fragments that are difficult to amplify using existing techniques due to their length. In one embodiment, the fragmented target nucleic acid is mixed with an assembling sequence comprising a sequence that is substantially complementary to the target nucleic acid but significantly different. A population of short target nucleic acid fragments is annealed to the assembling sequence, and the short target nucleic acid fragments are extended in the 3' direction by a polymerase to produce a population of first duplex nucleic acids comprising a population of first nucleic acids and the assembling sequence.

The group of first double nucleic acids dissociates from the assembly sequence, and the group of first nucleic acids anneals and binds to a first primer containing a sequence identical to a region of the assembly primer, and the first primer is located at the 5' end of the target region to be detected (the synonymous chain of the assembly sequence). In some cases, the primer site is located exactly at the 5' end of the assembly sequence. The primer is derived toward its 3' end to produce a group of second nucleic acids. The first and second nucleic acids dissociate and anneal again to bind to the assembly sequence and themselves, and are extended toward their 3' ends respectively by a polymerase. This step is repeated, thereby extending and linearly amplifying the short fragmented target nucleic acid within the boundary generated by the first primer at the 5' end of the assembly sequence and the target nucleic acid fragment that is complementary to most of the region closest to the 3' end of the assembly sequence.

In a second aspect, once assembly is achieved, the method further comprises detecting genetic variants specific for the target sequence in the presence of the assembled sequence using an experimental method capable of detecting rare genetic events.

In one embodiment the assembler sequence is a wild-type assembler sequence.

In a second embodiment, the assembler sequence is a genomic DNA or a portion thereof that is specific to the target of interest. In some embodiments, the genomic DNA or a portion thereof is human genomic DNA. In other embodiments, the genomic DNA or a portion thereof is associated with a specific organism such as a virus, bacterium, parasite, or fungus.

In a third embodiment, the assembler sequence may be a fragment of the target nucleic acid sequence and/or other arbitrary but known sequences.

In a fourth embodiment, the assembled sequence contains genetic variations of mutant and wild-type sequences. In this application, once assembled, the mutant (genetic variation sequence) or wild-type sequence can be determined using downstream analytical methods when a wild-type sequence relative to the assembled sequence and a genetic variation relative to the assembled sequence are present. Similarly, the prevalence and/or copy number of wild-type and genetic variation copies can be determined in parallel reactions.

In all cases, the assembler sequence is designed to extend the short nucleic acid fragment of the target nucleic acid by 3' end extension to detect a region of a target nucleic acid.

In a sixth embodiment, the assembler sequence is double-stranded.

In a seventh embodiment, the assembly is performed under PCR conditions using a forward primer and a reverse primer simultaneously.

In an eighth embodiment, multiple linear amplification cycles are used to linearly assemble short nucleic acid fragments without amplifying the assembled sequences under exponential amplification conditions in the presence of both a forward primer and a reverse primer.

In a ninth embodiment, assembly is performed at a lower temperature to facilitate hybridization of short nucleic acids to the assembly sequence, and once assembly occurs, the amplification temperature can be increased and other reagents can be added to support exponential amplification.

In a tenth embodiment, two assembly reactions are performed, one reaction using primers at the 3' end of a detection region of interest and the other using primers at the 5' end of a detection region of interest. Once assembly occurs in the two separate reaction mixtures, they can be combined for exponential amplification using forward and reverse primers.

In an eleventh embodiment, when the concentration of the fragmented nucleic acid is higher, the assembly reaction can be performed simultaneously with the polymerase chain reaction (PCR).

In another embodiment, the assembler sequences are in excess of the expected concentration of nucleic acid fragments for assembly by 10, 100, 1000, 10,000, or 100,000-fold.

Other aspects of the invention are found throughout the specification.

Image Description

FIG1 is a schematic diagram of one aspect of the present invention.

Unless otherwise indicated, all terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. All patents, patent applications, and publications cited herein are incorporated by reference in their entirety. If a term is used in multiple definitions, the most commonly used definition in this section will prevail.

As used herein, the term "oligonucleotide" refers to a polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides, containing natural or non-natural nucleotides, and having a length of at least 2, typically between about 5 and about 200, or more typically about 100. Thus, the term encompasses double-stranded and single-stranded DNA and RNA. In addition, oligonucleotides may be resistant to nucleases and include, but are not limited to, 2'-O-methyl ribonucleotides, thionucleotides, dithionucleotides, phosphoramidite nucleotides, and methylphosphonate nucleotides.

As used herein, the terms "target," "target sequence," or "target nucleotide" refer to a nucleic acid comprising a polynucleotide of interest for which purification, isolation, capture, immobilization, amplification, identification, detection, quantification, mass determination, and/or sequencing, and the like, is desired. The target sequence may or may not be known in terms of its actual sequence and may be synthesized or obtained from a biological sample.

As used herein, the term "primer" or "primer sequence" refers to a nucleic acid containing a sequence selected to be substantially complementary to each specific sequence to be amplified. More specifically, primers are sufficiently complementary to their respective targets to hybridize. Thus, the primer sequence need not accurately reflect the precise sequence of the target. Non-complementary bases or longer sequences may be interspersed throughout the primer, as long as the primer sequence has sufficient complementarity to the sequence of the target nucleic acid to allow hybridization and extension.

In addition, primers can be resistant to nucleases and include primers that have been modified to prevent degradation by exonucleases. In some embodiments, the primers have been modified to protect against degradation by the activity of 3' or 5' end exonucleases. These modifications include, but are not limited to, 2'-O-methyl ribonucleotide modifications, thio backbone modifications, disulfide backbone modifications, phosphoramidite backbone modifications, methylphosphonate backbone modifications, 3' end phosphate modifications, and 3' alkyl substitutions. In some embodiments, the primers and/or probes used in an amplification reaction are protected from degradation by the activity of 3' or 5' end exonucleases by one or more modifications.

A skilled artisan is capable of designing and preparing primers suitable for extending a target sequence. The length of primers used in the methods and compositions provided herein depends on a variety of factors, including the identity of the nucleotide sequence and the temperature at which the nucleic acids are hybridized or subjected to in vitro nucleic acid extension. Those skilled in the art will appreciate the considerations for determining the preferred length of primers for a particular sequence identity.

As used herein, the term "blocker oligonucleotide" or "blocker" refers to a modified oligonucleotide or agent that binds to a nucleic acid, or an agent that binds to a modified nucleic acid, that is capable of preventing or inhibiting replication and that is fused to a primer and/or probe in an amplification reaction. Blocker nucleotides may include 2'-fluoro (2'-deoxy-2'-fluoro-nucleosides) modifications, nuclease-resistant nucleotides, or nucleotides with a modification at the 3' end that inhibits or prevents replication.

As used herein, the term "sample" refers to essentially any sample containing the desired target nucleic acid, including but not limited to tissue or body fluid isolated from a human or animal, including but not limited to, for example, blood, plasma, serum, spinal fluid, lymph, tears or saliva, urine, sperm, feces, sputum, vomitus, gastric aspirate, bronchial aspirate, swabs (nasopharyngeal, rectal, ocular, genitourinary, etc.), organs, muscle, bone marrow, paraffin-embedded tissue, skin, tumors and/or cells obtained from any part of the organism; plant material, cells, fluids, etc.; an individual bacterium, bacterial population and culture thereof, food, cosmetics, pharmaceuticals/drugs, materials prepared by bioprocessing (final products and intermediate materials), water, environmental samples, including but not limited to, for example, soil, water and air; semi-purified or purified nucleic acids obtained from the sources listed above, such as nucleic acids obtained as a result of a process, such as template formation for sequencing, including next-generation sequencing, sample processing, nuclease digestion, restriction enzyme digestion, replication or the like.

As used herein, the term "amplification" refers to the process of generating a nucleic acid chain that is identical or complementary to a complete target nucleic acid sequence or a portion thereof or a universal sequence that is a surrogate of the target nucleic acid sequence.

As used herein, the term "nucleic acid" refers to a polynucleotide compound, including oligonucleotides, comprising nucleosides or nucleoside analogs containing nitrogenous heterocyclic bases or base analogs thereof, covalently linked by standard phosphodiester bonds or other linkages. Nucleic acids include RNA, DNA, chimeric DNA-RNA polymers, and analogs thereof. In a nucleic acid, the backbone is composed of a variety of linkages, including one or more sugar-phosphodiester bonds, peptide nucleic acid bonds (PCT No. WO 95/32305), phosphorothioate bonds, methylphosphonate bonds, or combinations thereof. In a nucleic acid, the sugar moiety can be ribose, deoxyribose, or similar compounds with substituents such as 2'-methoxy and 2'-halogen (e.g., 2'-F).

The nitrogenous bases may be conventional bases (A, G, C, T, U), non-natural nucleotides such as isocytosine and isoguanine and their analogs (e.g., inosine, The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992), derivatives of purine and pyrimidine bases, such as N4-methyldeoxyguanosine, aza or azapurine, aza or azapyrimidine, purines or pyrimidines having substituents that are altered or substituted at any of a range of chemical positions, such as 2-amino-6-methylaminopurine, ^O6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine and ^O4 -alkyl-pyrimidine or pyrazolone compounds, such as unsubstituted or 3-substituted pyrazolone [3,4-d] pyrimidine (eg, US Pat. Nos. 5,378,825, 6,949,367 and PCT No. WO 93/13121).

As used herein, the terms "hybridization" and "annealing" refer to the ability to bind to nucleic acids of substantially complementary sequences by Watson & Crick base pairing under appropriate conditions. Nucleic acid annealing or hybridization techniques are known in the art. See, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y. (1989); Ausubel, F.M., et al., Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus, N.J. (1994). As used herein, the term "substantially complementary" refers to complete complementarity with the nucleic acid to be bound, in some cases an identical sequence, and sufficient complementarity to achieve binding to the desired nucleic acid. Accordingly, the term "complementary hybridization" includes substantially complementary hybridization.

Conventional methods for amplifying nucleic acid sequences are well described and well known in the art. Any such method can be used in conjunction with the methods described herein. In some embodiments, the amplification is performed using digital PCR methods, as described in the literature, for example, by Vogelstein and Kinzler ("Digital PCR," PNAS, 96:9236-9241 (1999), which is incorporated herein by reference in its entirety). These methods involve diluting the sample containing the target region prior to amplifying the target region. Dilution can include dilution into conventional flat plates, multiwell plates, and nanowells, as well as dilution into microplates and microdroplets. (See, e.g., Beer NR, et al "On-chip, real time, single copy polymerase chain reaction in picoliter droplets," Anal. Chem. 79(22):8471-8475 (2007); Vogelstein and Kinzler, "Digital PCR," PNAS, 96:9236-9241 (1999); and Pohl and Shih, "Principle and applications of digital PCR," Expert Review of Molecular Diagnostics, 4(1):41-47 (2004), which are incorporated herein by reference in their entireties.) In some embodiments, the amplification is performed by digital PCR.

In some embodiments, the enzymes used to amplify the target region using the methods of the present invention include, but are not limited to, high-fidelity DNA polymerases, such as DNA polymerases with 3'-5' proofreading capabilities. Enzymes that can be used in the methods include, but are not limited to, AmpliTaq, Phusion HS II, Deep Vent, and Kapa HiFi DNA polymerases.

High-fidelity enzymes are capable of amplifying a target sequence with high fidelity (high accuracy). In some embodiments, the enzymes used include high-fidelity DNA polymerases, such as DNA polymerases with 3'-5' end exonuclease proofreading capabilities. Enzymes that can be used in the methods include, but are not limited to, AmpliTaq, Phusion HS II, Deep Vent, and Kapa HiFi DNA polymerases.

The amplified product can be detected or analyzed using methods known to those skilled in the art, including but not limited to fluorescence, electrochemical detection, gel analysis, and sequencing. Further, the product can be quantified using methods known to those skilled in the art, such as real-time amplification. Quantification can be normalized by comparison with so-called housekeeping genes, such as actin, GAPDH, or with an internal reference of known amount added to the reaction. These methods are known and described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Ed.) (2001).

Instruments for performing the methods described herein can be readily available. These instruments may include those for performing real-time and endpoint PCR assays, emulsion PCR, solid-phase PCR, melting curve analysis, and sequencing analysis. These instruments include the 7500Fast Dx real-time instrument (which also performs high-resolution melting curve analysis) (Life Technologies, Carlsbad, USA) and the 3500xl capillary gel analyzer. Other instruments known in the art that would be beneficial to the methods of the present invention should also be considered for use by those skilled in the art when performing the methods of the present invention.

The present invention provides a method for amplifying a fragmented target nucleic acid using an assembling template that binds and extends short nucleic acid fragments of a target nucleic acid to produce larger amplicons for further manipulation and analysis. The target nucleic acid fragments can be DNA, RNA, or a combination of both.

In one aspect of the invention, an assembling primer or assembling sequence is used to convert short nucleic acid fragments into longer sequences to identify and detect them. This is particularly important when attempting to identify small genetic variations such as SNVs in highly fragmented nucleic acid samples. This is achieved by hybridizing the short target nucleic acid fragment sequence of interest with a longer assembling sequence, where the short sequence serves as a primer for extension. Because the fragmented target nucleic acid containing SNVs is used as a primer on the assembling sequence, the SNVs are protected and can be detected.

For example, the methods of the present invention provide for amplification of a fragmented target nucleic acid comprising short nucleic acid fragments that are difficult to amplify using existing techniques due to their length. The fragmented target nucleic acid is mixed with a single-stranded assembler sequence comprising a sequence that is substantially complementary to the target nucleic acid but significantly different from the target nucleic acid. A population of short target nucleic acid fragments is annealed and hybridized to the assembler sequence, and the short target nucleic acid fragments are extended in the 3' direction by a polymerase to produce a population of first duplex nucleic acids comprising a population of first nucleic acids and the assembler sequence.

The first duplex nucleic acid population dissociates from the assembler sequence. The first nucleic acid population anneals to a first primer containing a sequence identical to a region of the assembler primer, with the first primer located at the 5' end of the target region to be detected (the synonymous strand of the assembler sequence). For example, if the region of interest is multiple medically relevant mutations in the EGFR gene, the first primer should be selected to be located at the 5' end of the EGFR gene region (the synonymous strand of the assembler sequence) so that sequences located at the 3' end relative to the first primer can be detected. In some examples, the first primer site is located exactly at the 5' end of the assembler sequence. The first and second nucleic acids dissociate, anneal, and bind to each other, where they are then extended by a polymerase from their 3' ends. Similarly, the first nucleic acid can also anneal to the assembler sequence, in which case the first nucleic acid is extended by a polymerase from its 3" end. Similarly, the first primer can also anneal to the first nucleic acid, in which case the first primer is extended by a polymerase from its 3' end.

Repeat this step, thereby extend and linearly amplify the target nucleic acid of described short fragmentation in the boundary produced by described first primer at the 5 ' end of described assembling sequence and described target nucleic acid fragment that is complementary to most part of region closest to 3 ' end with described assembling sequence.Several rounds of linear amplification (for example 10-15 rounds or more) can be implemented as needed.Correspondingly, the sequence that extends and extends again also can rebind by oneself, also can be combined with described assembling sequence to extend their length and produce larger target nucleic acid sequence.If described primer is not always positioned at the 3 ' end of described assembling sequence, the extension product of some described target nucleic acid fragments will be longer than above-mentioned boundary.

If it is desired to stably bind the short target nucleic acid fragment to the assembler sequence, the initial annealing step of the target nucleic acid fragment to the assembler sequence (or sequences, if both strands are present) can be performed at a lower temperature. After the initial round or rounds of extension are completed, the temperature can be raised to complete the process.

Another aspect of the present invention also provides the second primer of the negative sense with described first primer, and its direction is the 3 ' end (synonymous strand of assembling sequence) of described target nucleic acid region to be detected.However, described second primer can not be closer to 3 ' end than the fragment closest to 3 ' end in described mixture.Described method is carried out as above-mentioned, but now described second primer will be combined with the second nucleic acid of length enough to cross described second primer binding site.In addition, length becomes enough to cross the assembling fragment of two primer sites and will be amplified exponentially.

In another aspect of the method, the assembler sequence is double-stranded. At this point, the assembler sequence is denatured, and the target nucleic acid fragments anneal to the complementary strand of the assembler sequence (i.e., if the fragments are negative sense, they will anneal to the positive strand of the assembler sequence, and vice versa). The method proceeds as described above to produce a first and a second nucleic acid (if both positive and negative sense fragments are present, both positive and negative sense strands of the first nucleic acid are produced; however, if the first primer is positive sense, only a positive sense second nucleic acid is produced). At this point, the first and second nucleic acids dissociate and anneal to each other (guided by the complementarity of positive and negative sense), and/or the first nucleic acid anneals to the positive strand of the assembler sequence, and/or the second nucleic acid binds to the negative strand of the assembler sequence, and/or the first primer binds to the first nucleic acid (the opposite sense strand), and the first and second nucleic acids and the first primer are extended toward their respective 3' ends by a polymerase. This step is repeated, thereby extending and linearly amplifying the short fragmented target nucleic acid.

In another aspect of the method, the assembly sequence is double-stranded and a second primer is also used in the process. The second primer is similar in design and use to the first primer, but is designed relative to the other chain of the assembly sequence, and the other chain of the assembly sequence is now present in the reaction mixture. The process is carried out as described above, with the difference that a positive and negative second nucleic acid will be produced, and the two second nucleic acids will bind to the chains opposite to the sense of the chain of the assembly sequence, while the second primer will bind to the second nucleic acid with the appropriate sense of the chain. Repeating the steps of dissociation, annealing and extension will extend and linearly amplify the fragment. In addition, when the first and second primers are present at the same time, the fragment that is assembled and becomes long enough to span two primer sites will be exponentially amplified.

In another aspect of the method, the positive and negative sense strands of the assembler sequence are used, but the method of extending and linearly amplifying the target nucleic acid fragment is performed in two different tubes, one containing the positive sense assembler sequence and a positive first primer, and one containing the negative sense assembler sequence and a negative second primer. After the reaction is completed, the contents of the tubes can be manipulated separately (e.g., for exponential amplification) or combined (e.g., for exponential amplification).

In another aspect of the method, according to any of the above-mentioned sequence assembly methods, one or more PCR reactions or one or more additional PCR reactions containing primer pairs (if a first and a second primer are already present) can be spontaneously performed within the boundaries of the target nucleic acid to be detected.

In each of the above aspects, the one or more assembler strands preferably outnumber the target nucleic acid fragments by 10, 100, 1000, 10,000, 100,000, or more. In some cases, no more than 10 times. In some cases, all or part of the fragments exceed the assembler sequence.

The assembling sequence and the target nucleic acid of the fragmentation are derived from the same genetic species and are highly correlated. For example, if the target nucleic acid of the fragmentation being assessed is human, the assembling sequence will contain a closely related genomic target sequence in the region of interest. In some cases, the assembling sequence is human genomic DNA or a portion thereof. In other cases, the assembling sequence and the genomic DNA or a portion thereof associated with a specific organism, such as a virus, bacterium, parasite and fungus. In some cases, the assembling sequence is an arbitrary but known sequence.

The assembling sequence can be prepared from a natural or synthetic sequence. Natural sequences include, for example, genomic DNA, mRNA, plasmid DNA, and DNA or its analogs obtained using DNA synthesis. Synthetic sequences can be prepared DNA, RNA or its analogs that can meet the requirements of the template for the DNA or RNA polymerase extension reaction. In one embodiment, human genomic DNA is used as the assembling sequence. In other embodiments, a human sequence equivalent is obtained using DNA synthesis or cloning methods. Typically, the assembling sequence may contain slight genetic variations of the wild type and mutant sequences, but retains sufficiently high sequence complementarity to hybridize with the fragmented target nucleic acid, thereby maintaining the specificity of the gene sequence region of the fragmented target nucleic acid.

In another embodiment, the assembler sequence is a wild-type sequence that does not contain rare mutations such as SNVs. These mutations can be reliably detected using a wild-type assembler sequence. In each case, the fragmented target nucleic acid sequence has appropriate complementarity so that it can hybridize with the assembler sequence with high specificity.

Once assembled according to the methods of the present invention, analysis of the now longer target nucleic acids can be performed using a variety of methods known in the art, both non-amplification and amplification based methods, and including methods designed to detect rare genetic events and rare variants.

If the method for subsequent analysis of the assembled target nucleic acid is based on amplification, such as PCR, the amplification of the assembled sequence can be inhibited by a Selector test described in U.S.Pat.APP.NO.WO/2012/151560. The Selector test uses a blocker to inhibit the wild-type sequence while minimizing the effect on the amplification of the mutant sequence. Implementation of this test can detect rare mutations in a "wild-type" background, which account for 0.1-0.01% or even less of the target species present. Therefore, using the Selector test, mutation sites with a prevalence ranging from 1:10,000 to 1 to 100,000 can be detected. The Selector test can be used to inhibit the amplification of the assembled sequence, whether it is a wild-type sequence or a variant of a wild-type sequence, as long as the assembled sequence is known or can be determined.

In some cases, the assembled sequence contains one or more SNVs compared to the target nucleic acid sequence. In another post-assembly analysis method, a Selector-like test can be used to inhibit the amplification of the assembled sequence, while allowing the amplification of SNVs associated with the short target sequence, and a separate test can be used to detect the wild-type sequence, and the above tests all need to be performed when the assembled sequence is inhibited. If you want to know how many wild types and mutants there are, you can prepare an assembled sequence that has mismatches with both mutants and wild types. You can assemble possible mutant sequences and possible wild-type sequences. The assembled sequence assembles both. A Selector test (or equivalent) is applied to inhibit the assembled sequence, but not the mutant or the wild type. Then an allele-specific Selector test is applied to determine the number of mutants and the number of wild types at each SNV site. Inhibition is achieved using a blocking oligomer that is different from the assembled sequence and specific to the assembled sequence region (i.e., the region where one or more nucleotide gene variations are found).

Post-assembly analysis methods are performed with or without the binding sequence or sequences, or when the binding sequence or sequences are selectively removed from the mixture, with or without the reduced level of the binding sequence or sequences. This provides a further benefit to the analysis of the target nucleic acid by providing more and more differentiation between the target nucleic acid and the binding sequence or sequences. Methods for separating the assembler sequence or sequences from the target nucleic acid sequence include, but are not limited to, for example, separation by size using methods known in the art (e.g., gel electrophoresis, spin columns, magnetic bead purification techniques, filtration, and precipitation), when there is a distinguishable size difference between the target nucleic acid and the assembler sequence, or specific separation using, for example, biotin and streptavidin (e.g., the assembler sequence or sequences can be pre-labeled with biotin, and after assembly, specifically captured and removed from the mixture using streptavidin-coated microparticles), digoxigenin and anti-digoxigenin antibodies (e.g., the assembler sequence or sequences can be pre-labeled with digoxigenin, and after assembly, specifically captured and removed from the mixture using anti-digoxigenin antibody-coated microparticles), or specific nucleic acid sequence capture (e.g., one or more sequences in the assembler sequence that are not related to the target nucleic acid sequence can bind to the complementary sequence or sequences on the microparticles and be removed from the reaction mixture). Optionally, the assembled target nucleic acid sequence can be labeled during the assembly process, for example, by labeled nucleotide triphosphates or a labeled primer.

The above information provides a complete disclosure for those skilled in the art and describes embodiments of how to make and use the apparatus and methods, and is not intended to limit the scope of the invention disclosed by the inventors. Modifications to the above modes that would be obvious to those skilled in the art in practicing the invention are intended to be within the scope of the following claims. All publications, patents, and patent applications cited in this specification are incorporated herein by reference. For example, many of the wash steps cited in the various methods described are optional, as are steps that remove and/or separate the two nucleic acid strands from each other. Omitting at least some of the wash and/or separation steps can make the workflow faster, simpler, and more economical, while still achieving the desired results. In another example, the stepwise addition/incorporation of certain oligonucleotides and/or target nucleic acids into the exemplified methods can also be incorporated. Furthermore, a variety of polymerases, extension conditions, and other amplification methods known to those skilled in the art can be used in various steps or combinations of steps in the methods described above. Other modifications to the disclosed methods that would be obvious to those skilled in the art are also encompassed by the present invention.

Claims (3)

1. A method for amplifying a fragmented target nucleic acid containing a short target nucleic acid fragment, the method comprising the steps of: A. Mix the fragmented target nucleic acid with an assembled sequence, wherein the assembled sequence is complementary to the fragmented target nucleic acid, and the assembled sequence and the fragmented target nucleic acid originate from the same genetic species and are highly related; B. The short target nucleic acid fragment is annealed and combined with the assembled sequence, and the short target nucleic acid fragment is extended in the 3' direction by polymerase to generate a group of first dual nucleic acids containing a group of first nucleic acids and the assembled sequence; C. Dissociate the assembled sequence from the first nucleic acid group; D. A primer is annealed to bind to the first group of nucleic acids, wherein the primer is identical to the assembled sequence and is located in the detection region of interest in the direction of the 3' end relative to the bound nucleic acid fragment, and the primer is extended in the direction of the 3' end with a polymerase to produce a second dual nucleic acid containing a group of second nucleic acids and the first group of nucleic acids; E. Separate the first and second nucleic acid groups; F. Anneal the first and second groups of nucleic acids to allow them to bind to each other and to the assembled sequence, extending the first and second groups of nucleic acids in the 3' direction; and G. Repeat steps E and F to amplify the fragmented target nucleic acid. 2. The method of claim 1, wherein the assembly sequence is a wild-type assembly sequence. 3. A method for amplifying and detecting a target nucleic acid containing a fragmentation of a short target nucleic acid fragment, the method comprising the steps of: A. In a mixture containing a short target nucleic acid fragment and an assembled sequence, the short target nucleic acid fragment is annealed to bind to the assembled sequence, wherein the assembled sequence is complementary to the fragmented target nucleic acid, and the short target nucleic acid fragment is extended at the 3' end by a polymerase to produce a first dual nucleic acid containing a first nucleic acid and the assembled sequence, optionally dissociating the first nucleic acid from the assembled sequence; B. A primer is annealed to bind to the first group of nucleic acids, wherein the primer is identical to the assembled sequence and is located in the detection region of interest in the direction of the 3' end relative to the bound nucleic acid fragment, and the primer is extended in the direction of the 3' end with a polymerase to produce a second dual nucleic acid containing a group of second nucleic acids and the first group of nucleic acids; C. Anneal the first and second groups of nucleic acids to allow them to bind to each other and to the assembled sequence, extending the first and second groups of nucleic acids in the 3' direction; and D. Repeat steps B and C to amplify the fragmented target nucleic acid; and E. Detect the amplified target nucleic acid using an assay capable of detecting genetic variations down to the single nucleotide level.