WO2017186117A1 - 扩增dna的方法 - Google Patents

扩增dna的方法 Download PDF

Info

Publication number
WO2017186117A1
WO2017186117A1 PCT/CN2017/081977 CN2017081977W WO2017186117A1 WO 2017186117 A1 WO2017186117 A1 WO 2017186117A1 CN 2017081977 W CN2017081977 W CN 2017081977W WO 2017186117 A1 WO2017186117 A1 WO 2017186117A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
primer
dna
temperature
universal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2017/081977
Other languages
English (en)
French (fr)
Inventor
高芳芳
陆思嘉
任军
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xukang (suzhou) Medical Science & Technology Co Ltd
Original Assignee
Xukang (suzhou) Medical Science & Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Xukang (suzhou) Medical Science & Technology Co Ltd filed Critical Xukang (suzhou) Medical Science & Technology Co Ltd
Priority to EP17788769.2A priority Critical patent/EP3450569B1/en
Priority to US16/093,975 priority patent/US20190106738A1/en
Publication of WO2017186117A1 publication Critical patent/WO2017186117A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • 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
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • 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
    • 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
    • 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
    • C12Q1/6869—Methods for sequencing
    • 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
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation

Definitions

  • the present invention relates to a method of amplifying DNA, and in particular to a method of amplifying and sequencing a single cell whole genomic DNA.
  • Single-cell whole-genome sequencing is a new technology for amplification and sequencing of whole genomes at the single-cell level.
  • the principle is to amplify a small amount of whole genome DNA of a single cell to obtain a high coverage complete genome and then perform high-throughput sequencing.
  • PEP-PCR Primer Extension Preamplification-Polymerase Chain Reaction
  • DOP-PCR Polymerase chain reaction
  • the first generation of DNA sequencing technologies include chemical degradation, dideoxy chain termination, and various sequencing technologies developed on them, the most representative of which was 1975 Chain termination method proposed by Sanger and Coulson.
  • the first generation technology has high accuracy and long read length. It is the only method that can be used for “snap-to-tail” sequencing. However, it has shortcomings such as high cost and slow speed, and it is not the most ideal sequencing method.
  • Subsequent second- and third-generation sequencing technologies share a common feature of high throughput, also known as "next generation sequencing technology (NGS)."
  • NGS next generation sequencing technology
  • the second-generation sequencing technology is represented by pyrosequencing technology, SBS technology, and ligation sequencing technology.
  • the second-generation sequencing technology is edge synthesis sequencing technology, semiconductor sequencing technology and CG sequencing technology.
  • Third generation sequencing The technology is roughly divided into two categories, one is single-molecule fluorescence sequencing, the representative technologies are TSMS technology and SMRT technology, and the other is nanopore single molecule technology. Compared with the previous two generations of technology, the biggest feature of the third generation sequencing technology is single molecule sequencing. Although the third-generation sequencing technology has made some progress, the mainstream sequencing technology at this stage is still the second-generation sequencing technology.
  • the whole genome sequence amplified by the current genome-wide amplification technology cannot be directly used in the second-generation sequencing technology. Therefore, whether the above-mentioned whole genome sequence is applied to the side synthesis sequencing technology, the semiconductor sequencing technology or the CG sequencing technology in the second generation sequencing technology, the library preparation process is required before the sequencing of the upper machine.
  • Each sequencing technology has its own corresponding library preparation method.
  • the library preparation of the side synthesis sequencing platform is mainly divided into two categories, one is the fragmented DNA after the end repair, the Y-joint technology or the neck ring joint technology is added, and the other One type is the transpson technology.
  • Library preparation for semiconductor sequencing platforms is also divided into two categories, one is the addition of linker technology after fragmentation of the fragmented DNA, and the other is transpson technology.
  • the preparation process of the CG platform library is complicated.
  • the fragmented DNA needs to be digested and twice cyclized after end-repairing, which is cumbersome and takes a long time.
  • the present invention provides a method of amplifying cellular genomic DNA and a kit for amplifying genomic DNA.
  • the first variable sequence and the third variable sequence further comprise a fixed sequence at their 3' end, the immobilized sequence being capable of increasing base combinations of genomic coverage.
  • the fixed sequence is selected from the group consisting of CCC, AAA, TGGG, GTTT, GGG, TTT, TNTNG, or GTGG.
  • the universal sequence is selected such that it does not substantially bind to genomic DNA to produce amplification, the universal sequence being 6-60 bp in length. In some embodiments, the universal sequence is selected such that the amplification product is capable of direct sequencing. In some embodiments, the universal sequence is selected from the group consisting of SEQ ID NO: 1 [TTGGTAGTGAGTG], SEQ ID NO: 2 [GAGGTGTGATGGA], SEQ ID NO: 3 [GTGATGGTTGAGGTA], SEQ ID NO: 4 [AGATGTGTATAAGAGACAG], SEQ ID NO: 5 [GTGAGTGATGGTTGAGGTAGTGTGGAG] or SEQ ID NO: 6 [GCTCTTCCGATCT].
  • the m 1
  • the method further comprises the step of sequencing the amplification product obtained in step (d), wherein the second primer comprises a sequence that is complementary or identical to a portion or all of the primers for sequencing.
  • the universal sequence comprises a sequence that is complementary or identical to a portion or all of the primers for sequencing.
  • the specific sequence of the second primer comprises a sequence that is complementary or identical to a portion or all of the primers for sequencing.
  • the specific sequence of the second primer further comprises a sequence that is complementary or identical to a portion of the capture sequence of the sequencing platform.
  • sequence contained in a particular sequence of the second primer that is complementary or identical to a portion or all of the primers for sequencing comprises or consists of SEQ ID NO: 31 [ACACTCTTTCCCTACACGAC], or SEQ ID NO: 32 [GTGACTGGAGTTCAGACGTGT ]composition.
  • the sequence contained in a particular sequence of the second primer that is partially or identical to the capture sequence of the sequencing platform comprises or consists of SEQ ID NO: 33 [AATGATACGGCGACCACCGAGATCT], or SEQ ID NO: 34 [ CAAGCAGAAGACGGCATACGAGAT] composition.
  • the specific sequence of the second primer further comprises an identification sequence located in the sequence complementary or identical to the capture sequence of the sequencing platform and the portion of the primer for sequencing or All are complementary or identical between sequences.
  • the second primer comprises a primer mixture having the same universal sequence and a different specific sequence, respectively, complementary or identical to some or all of the different primers in the sequencing primer pair used in the same sequencing. .
  • the second primer comprises SEQ ID NO: 35 [AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT] and SEQ ID NO: 36 [CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGCTCT A mixture of the sequences shown in TCCGATCT].
  • the nucleic acid polymerase has thermostability and/or strand displacement activity.
  • the nucleic acid polymerase is selected from the group consisting of: Phi29 DNA polymerase, Bst DNA polymerase, Pyrophage 3137, Vent polymerase, TOPOTaq DNA polymerase, 9° Nm polymerase, Klenow Fragment DNA polymerase I, MMLV Transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, T7phase DNA polymerase variant, Ultra-fidelity DNA polymerase, Taq polymerase, Bst DNA polymerase, E.coli DNA polymerase, LongAmp Taq DNA polymerase, OneTaq DNA polymerase, Deep Vent DNA polymerase, Vent (exo-) DNA polymerase, Deep Vent (exo-) DNA polymerase, and any combination thereof.
  • step (b) enables the variable sequence of the first type of primer to pair with the genomic DNA and amplify the genomic DNA to obtain a genomic preamplification product, wherein the genomic preamplification
  • the 5' end of the product comprises the universal sequence and the 3' end comprises the complement of the universal sequence.
  • the first temperature cycling program comprises: (b1) a temperature program capable of opening the DNA duplex to obtain a DNA single-stranded template; (b2) enabling the first primer and an optional first a temperature program in which a triple primer binds to the DNA single-strand template; (b3) a first-type primer capable of binding to the DNA single-strand template by the action of the nucleic acid polymerase to extend a length to produce a pre-amplified product a temperature program; (b4) repeating steps (b1) through (b3) to a specified first number of cycles, wherein the specified first number of cycles is greater than one.
  • the DNA duplex in step (b1) is a genomic DNA duplex
  • the temperature program comprises a denaturation reaction between 90 and 95 ° C for 1-20 minutes.
  • the temperature program described in step (b1) comprises a melting reaction between 3 and 50 seconds between temperatures of 90-95 °C.
  • the preamplification product upon proceeding to the second cycle, comprises a genomic preamplification product comprising the universal sequence at the 5' end and a complementary sequence of the universal sequence at the 3' end.
  • step (b2) comprises placing the reaction mixture in more than one temperature program to facilitate efficient binding of the first type of primer to the DNA template.
  • the more than one temperature program comprises: a first temperature between 10-20 °C, a second temperature between 20-30 °C, and between 30-50 °C The third temperature between.
  • the step package in the step (b2) The annealing reaction is performed at a first temperature for 3 to 60 seconds, at the second temperature for 3 to 50 seconds, and at the third temperature for 3 to 50 seconds.
  • the temperature program described in step (b3) comprises extending the reaction between temperatures of 60-80 ° C for 10 seconds to 15 minutes.
  • the first number of cycles of step (b4) is 2-40.
  • the step (d) enables the universal sequence of the second primer to pair with the 3' end of the genomic preamplification product and amplify the genomic preamplification product for amplification Genomic amplification products.
  • the step (d) comprises: (d1) a temperature program capable of opening a DNA duplex; (d2) a temperature program further capable of opening the DNA duplex; (d3) enabling the second primer to a temperature program for single-strand binding of the genomic pre-amplification product obtained in step (b); (d4) a second primer capable of binding a single strand to the genomic pre-amplification product at the action of the nucleic acid polymerase a temperature program of the lower extension length; (d5) repeating steps (d2) through (d4) to a specified second number of cycles, wherein the specified second number of cycles is greater than one.
  • the DNA duplex in step (d1) is the genomic preamplification product, and the DNA duplex comprises a double strand contained in a DNA hairpin structure, the temperature program comprising 90-95 ° C Denaturation reaction between the temperatures of 5 seconds - 20 minutes.
  • the temperature program described in step (d2) comprises a melting reaction between 3 and 50 seconds between temperatures of 90-95 °C. In some embodiments, the temperature program described in step (d3) comprises annealing the reaction between temperatures of 45-65 ° C for 3-50 seconds. In some embodiments, the temperature program described in step (d4) comprises extending the reaction between temperatures of 60-80 ° C for 10 seconds to 15 minutes.
  • the method further comprises analyzing the amplification product to identify sequence features associated with a disease or phenotype.
  • the sequence features associated with a disease or phenotype include chromosomal abnormalities, ectopic chromosomes, aneuploidy, deletions or duplications of some or all of the chromosomes, fetal HLA haplotypes, and paternal mutations
  • the disease or phenotype is selected from the group consisting of beta-thalassemia, Down's syndrome, cystic fibrosis, sickle cell disease, Thai-sax disease, fragile X syndrome, spinal muscular atrophy, hemoglobin Disease, alpha-thalassemia, X-linked disease (a disease dominated by genes on the X chromosome), spina bifida, no brain malformation, congenital heart disease, obesity, diabetes, cancer, fetal sex, fetal RHD.
  • the genomic DNA is derived from a blastomere
  • An aspect of the present application provides a method of amplifying genomic DNA, the method comprising: (a) providing a first reaction mixture, wherein the first reaction mixture comprises a sample comprising the genomic DNA, a first primer, a mixture of nucleotide monomers, and a nucleic acid polymerase, wherein the first primer comprises a universal sequence and a variable sequence from the 5' end to the 3' end, wherein the first primer comprises a universal from the 5' end to the 3' end a sequence and a first variable sequence, the first variable sequence comprising a first random sequence, wherein the first random sequence is X a1 X a2 ...
  • the variable sequence and optionally the third variable sequence of the third primer are capable of pairing with the genomic DNA and amplifying the genomic DNA to obtain a genomic preamplification product, wherein the 5' end of the genomic preamplification product Including the universal sequence, the 3' end comprises a complementary sequence of the universal sequence;
  • the first temperature cycling procedure comprises: (b1) the first cycle is the first between temperatures between 90-95 ° C Denaturation temperature reaction for 1-20 minutes, after the first cycle is a reaction at a second melting temperature between 90-95 ° C for 3-50 seconds; (b2) between 10-20 ° C The first annealing temperature is reacted for 3 to
  • the amplification product obtained in step (d) has completed library construction.
  • a kit for amplifying genomic DNA comprising a first primer, wherein the first primer comprises a universal sequence from the 5' end to the 3' end a first variable sequence, the first variable sequence comprising a first random sequence, wherein the first random sequence is X a1 X a2 ...
  • said first reaction mixture further comprises a third a primer, wherein the third primer comprises the universal sequence and a third variable sequence from a 5' end to a 3' end, the third variable sequence comprising a third random sequence, wherein the third random sequence is from 5 'End to 3' end is X b1 X b2 ;
  • the kit is used to construct a whole genome DNA library.
  • the kit further comprises a nucleic acid polymerase, wherein the nucleic acid polymerase is selected from the group consisting of: Phi29 DNA polymerase, Bst DNA polymerase, Pyrophage 3137, Vent polymerase, TOPOTaq DNA polymerase, 9° Nm polymerization Enzyme, Klenow Fragment DNA Polymerase I, MMLV Reverse Transcriptase, AMV Reverse Transcriptase, HIV Reverse Transcriptase, T7phase DNA Polymerase Variant, Ultra-fidelity DNA polymerase, Taq polymerase, Bst DNA polymerase, E.coli DNA polymerase, LongAmp Taq DNA polymerase, OneTaq DNA polymerase, Deep Vent DNA polymerase, Vent (exo-) DNA polymerase, Deep Vent (exo-) DNA polymerase, and any combination thereof.
  • the nucleic acid polymerase is selected from the group consisting of: Phi29 DNA polymerase, Bst DNA polymerase, Pyroph
  • the kit further comprises comprises one or more components selected from the group: a mixture of nucleotide monomers, Mg 2+, dTT, bovine serum albumin, pH adjusting agents, DNase inhibitors One or more reagents of RNase, SO 4 2- , Cl - , K + , Ca 2+ , Na + , (NH 4 ) + .
  • the mixture further comprises a cell lysing agent selected from the group consisting of proteinase K, pepsin, papain, NP-40, Tween, SDS, Triton X-100, EDTA, and isothiocyanate.
  • a cell lysing agent selected from the group consisting of proteinase K, pepsin, papain, NP-40, Tween, SDS, Triton X-100, EDTA, and isothiocyanate.
  • a cell lysing agent selected from the group consisting of proteinase K, pepsin, papain, NP-40, Tween, SDS, Triton X-100, EDTA, and isothiocyanate.
  • acid bismuth selected from the group consisting of proteinase K, pepsin, papain, NP-40, Tween, SDS, Triton X-100, EDTA, and isothiocyanate.
  • Figure 1 shows the basic principle of the amplification method of the present application.
  • Figure 2 is a schematic view showing the structure of a first type of primer (linear amplification primer) used in the amplification method of the present application.
  • Figure 3 shows the results of gel electrophoresis of 50 pg of human genomic DNA using different first-class primer mixtures, and the obtained amplification products were respectively subjected to gel electrophoresis, wherein the first lane from left to right is a molecular weight marker (M).
  • Lanes 2-13 are amplified samples obtained by amplification of gDNA using primer mixtures of experimental groups 1-12 (see Table 1 for details), and lane 14 is a molecular weight marker.
  • Figure 4 shows the distribution of A, T, C, G for each reading position of the amplified products obtained in experimental groups 1-12 in SBS sequencing.
  • Figure 5 shows the amplification results of amplification of normal human epidermal fibroblasts (AFP cells) using primer mixtures of 1-12 experimental groups shown in Table 1, starting from left to right. Lanes are molecular weight markers, lanes 2-11 are single cell amplification samples, and lane 12 is molecular weight markers.
  • Figure 6 shows that the primer mixture of the experimental group 9/10 and the experimental group 11/12 shown in Table 1 was used to amplify normal human epidermal fibroblasts (AFP cells) as a starting sample, and the obtained amplification was obtained. The results of the gel electrophoresis were respectively performed on the product.
  • the first lane from left to right is the molecular weight marker
  • the lanes 2-11 are the amplified samples obtained by amplifying single cells using the primer mixture of the experimental group 11/12
  • the 12th lane is the molecular weight marker, lanes 13-22.
  • An amplified sample obtained by amplifying a single cell using a primer mixture of the experimental group of 9/10, the 23rd lane is a molecular weight marker.
  • Figure 7 shows the amount of data (sequencing in equal volumes of amplification products) of each sample 1_1, 1_2...1_10 and 2_1, 2_2...2_10 in Figure 6 in SBS sequencing.
  • Figure 8 shows the copy number variation coefficients of each of samples 1_1, 1_2...1_10 and 2_1, 2_2...2_10 in Figure 6 in SBS sequencing.
  • Figure 9 shows the copy number of each chromosome in SBS sequencing for each of samples 1_1, 1_2...1_10 and 2_1, 2_2...2_10 in Figure 6.
  • FIG. 10 shows that the amplified samples 1_1, 1_2, and 2_1, 2_2 in FIG. 6 are further subjected to PCR amplification for the 35 disease-causing loci genes listed in Table 8, respectively, and the amplified products are subjected to gel electrophoresis. result.
  • Each lane from left to right sequentially represents a molecular weight marker, an amplification result for the disease-causing sites 1-23 shown in Table 8, a molecular weight marker, and a disease-causing site 24-35 shown in Table 8.
  • Figure 11 shows that primer mixtures of the experimental group 9/10 shown in Table 1 were used to amplify normal human epidermal fibroblasts (AFP cells), and the obtained amplification products were separately gelled. The result of electrophoresis.
  • Each lane from left to right sequentially represents a molecular weight marker, an amplified sample (four parallel experimental wells) obtained by amplifying a single cell using a primer mixture of an experimental group of 9/10, and a molecular weight marker.
  • Fig. 12 shows the results of PCR amplification of the amplification products of the amplified samples 1 and 2 in Fig. 11 further against the 35 disease-causing site genes listed in Table 8, and the amplification products were subjected to gel electrophoresis.
  • Each lane from left to right sequentially represents a molecular weight marker, an amplification result for the disease-causing sites 1-23 shown in Table 8, a molecular weight marker, and a disease-causing site 24-35 shown in Table 6.
  • Figure 13 shows the number of copies of each chromosome in the semiconductor sequencing of the amplified sample of Figure 11.
  • Figure 14 shows the chromosome copy number obtained by amplifying the DNA in the blastocyst culture solution using the primer mixture of the experimental group 9/10 shown in Table 1, and performing SBS sequencing on the amplified sample.
  • the invention provides methods for amplifying genomic DNA, particularly methods for amplifying single cell whole genomic DNA.
  • the database was usually built after the completion of the gene amplification, and then sequenced after the completion of the database construction. This method is complicated and takes a long time.
  • the inventors of the present application by designing primers of a specific structure and optimizing the process of amplification, enable direct library formation after single cell amplification to substantially reduce the time required for single cell whole genome DNA library construction. Although some designs for primers have been reported in some literature, these designs have one or the other drawbacks.
  • the random sequence of the primer is selected from four bases (ie, A, T, C, and G), but using this method for direct amplification It is inevitable to automate or form a dimer or form a dimer with each other, thereby significantly reducing the efficiency of amplification. Further, for example, it is reported in US Pat. No.
  • the random sequence in the primer is selected from two bases (ie, G and T, G and A, A and C, C and T) to avoid auto- or inter-ring formation, but Since the bases before the target sequence in the sequence amplified by using such primers are very random, it is necessary to add a positive control to correct the base randomness when performing SBS sequencing on the whole board, otherwise the detection cannot be performed, so This method is bound to waste a certain amount of data.
  • the primers involved in the present invention contain high base randomness, the primers themselves or primers are substantially not formed or form very few loops or compared with the four base random primers.
  • Dimer, and the library constructed by the present invention has high base randomness before the target sequence, and thus the amplification product obtained by the method of the present invention has few dimers of amplification products, can be directly formed into a library, and can be used for The whole version was on the machine and the sequencing results were good.
  • the first reaction mixture further comprises a third primer, wherein The third primer comprises the universal sequence and a third variable sequence from the 5' end to the 3' end, the third variable sequence comprising a third random sequence, wherein the third random sequence is from the 5' end to the 3' end End
  • the second is X b1 X b2 ...
  • the methods of the present application are broadly applicable to the amplification of genomic DNA, particularly the amplification of trace amounts of genomic DNA.
  • the methods of the present application are preferably applicable to genomic DNA.
  • the initial amount of genomic DNA contained in the reaction mixture is no more than 10 ng, no more than 5 ng, no more than 1 ng, no more than 500 pg, no more than 200 pg, no more than 100 pg, no more than 50 pg, no more than 20 pg, Or no more than 10pg.
  • Genomic DNA can be derived from biological samples, such as biological tissues or body fluids containing cells or free DNA. Samples containing genomic DNA can be obtained by known methods, for example, by oral mucosa samples, nasal samples, hair, mouthwash, cord blood, plasma, amniotic fluid, embryonic tissue, endothelial cells, nail samples, hoof samples, and the like.
  • the biological sample can be provided in any suitable form, such as in a paraffin embedded form, in a freshly isolated form, and the like.
  • Genomic DNA can be from any species or species of organism such as, but not limited to, humans, mammals, cows, pigs, sheep, horses, rodents, birds, fish, zebrafish, shrimp, plants, yeast, viruses or bacteria.
  • the genomic DNA is genomic DNA from a single cell, or genomic DNA from two or more cells of the same type.
  • a single cell or similar cell can be derived, for example, from an embryo prior to implantation, an embryonic cell in a peripheral blood of a pregnant woman, a single sperm, an egg cell, a fertilized egg, a cancer cell, a bacterial cell, a tumor circulating cell, a tumor tissue cell, or from any tissue.
  • Single or multiple cells of the same type obtained.
  • the method of the present application can be used to amplify some valuable samples or DNA in a low amount of samples, such as human egg cells, germ cells, tumor circulating cells, tumor tissue cells, and the like.
  • the genomic DNA is derived from a blastomere, a blastocyst trophoblast, a cultured cell, an extracted gDNA, or a blastocyst culture.
  • Methods for obtaining single cells are also well known in the art, for example, by flow cytometric sorting (Herzenberg et al. Proc Natl Acad Sci USA 76: 1453-55, 1979; lverson et al. Prenatal Diagnosis 1: 61-73). , 1981; Bianchi et al. Prenatal Diagnosis 11: 523-28, 1991), fluorescence activated cell sorting, separation by magnetic beads (MACS, Ganshirt-Ahlert et al. Am J Obstet Gynecol 166: 1350, 1992), use semiautomatic cell picking device (e.g. Stoelting produced cell transfer system Quixell TM) or a combination of methods described above.
  • flow cytometric sorting Herzenberg et al. Proc Natl Acad Sci USA 76: 1453-55, 1979; lverson et al. Prenatal Diagnosis 1: 61-73). , 1981; Bianchi et al. Prenatal Diagnosis
  • gradient centrifugation and flow cytometry techniques can be used to increase the efficiency of separation and sorting.
  • a particular type of cell can be selected based on the different properties of a single cell, such as a cell that expresses a particular biomarker.
  • the cells can be lysed from a biological sample or in a single cell and released to obtain genomic DNA.
  • Cleavage can be carried out using any suitable method known in the art, for example by thermal cleavage, alkaline lysis, enzymatic cleavage, mechanical lysis, or any combination thereof (see, for example, US 7,521,246, Thermo Scientific Pierce Cell). Lysis Technical Handbook v2 and Current Protocols in Molecular Biology (1995). John Wiley and Sons, Inc. (supplement 29) pp. 9.7.1-9.7.2.).
  • Mechanical cracking involves the use of mechanical force to destroy cells using ultrasound, high speed agitation, homogenization, pressurization (eg, French filter press), decompression, and grinding.
  • the most common mechanical lysis method is the liquid homogenization method, which forces the cell suspension through a very narrow space to apply shear to the cell membrane (e.g., as described in WO2013153176A1).
  • a mild lysis method can be used.
  • cells can be heated in a solution containing Tween-20 at 72 ° C for 2 minutes and in water at 65 ° C for 10 minutes (Esumi et al, Neurosci Res 60 (4): 439-51 (2008), containing 0.5% NP -40 of PCR Buffer II (Applied Biosystems) heated at 70 ° C for 90 seconds (Kurimoto et al, Nucleic Acids Res 34 (5): e42 (2006), or use protease (such as proteinase K) or salt solution (such as different The guanidinium thiocyanate is subjected to cleavage (for example, as described in U.S. Patent Application No. US 20070281313).
  • Thermal cracking includes heating and repeated freeze-thaw methods.
  • the thermal cracking comprises a temperature between 20-100 ° C and cleavage for 10-100 minutes.
  • the temperature of the thermal cracking can be between 20-90, 30-90, 40-90, 50-90, 60-90, 70-90, 80-90, 30-80, 40-80. Any temperature between 50-80, 60-80 or 70-80 °C.
  • the temperature of the thermal cracking is not less than 20, 30, 40 or 50 °C. In some embodiments, the temperature of the thermal cracking is no higher than 100, 90 or 80 °C.
  • the thermal cracking time can be between 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-100, 30. -90, 30-80, Any time between 30-70, 30-60, 30-50 or 30-40 minutes.
  • the thermal cracking time is no less than 20, 30, 40, 50, 60, 70, 80 or 90 minutes.
  • the thermal cracking time is no more than 90, 80, 70, 60, 50, 40, 30 or 20 minutes.
  • the thermal cracking temperature is a function of time. In some embodiments, the thermal cracking is maintained at a temperature of 30-60 ° C for 10-30 minutes, followed by a temperature of 70-90 ° C for 5-20 minutes.
  • the thermal cleavage is carried out in the presence of a lysis reagent.
  • a lysis reagent When the lysis reagent is present, the time required for cleavage can be reduced or the temperature required for cleavage can be reduced.
  • the cleavage reagent can disrupt protein-protein, lipid-lipid and/or protein-lipid interactions, thereby promoting the release of genomic DNA by the cells.
  • the lysis reagent comprises a surfactant and/or a lytic enzyme.
  • Surfactants can be classified into ionic, amphoteric and nonionic surfactants. In general, the cleavage potency of amphoteric and nonionic surfactants is weaker than ionic surfactants.
  • Exemplary surfactants include, but are not limited to, one of NP-40, Tween, SDS, GHAPS, Triton X-100, Triton X-114, EDTA, sodium deoxycholate, sodium cholate, and guanidinium isothiocyanate. kind or more.
  • One skilled in the art can select the type and concentration of the surfactant according to actual needs. In some embodiments, the working concentration of the surfactant is from 0.01% to 5%, from 0.1% to 3%, from 0.3% to 2%, or from 0.5% to 1%.
  • Exemplary lytic enzymes can be proteinase K, pepsin, papain, and the like, or any combination thereof.
  • the working concentration of the lytic enzyme is from 0.01% to 1%, from 0.02% to 0.5%, from 0.03% to 0.2%, or from 0.4 to 0.1%.
  • a cleavage product containing genomic DNA can be directly used in the first reaction mixture, for example, the biological sample can be subjected to a cleavage treatment in advance to obtain a cleavage product, and then the cleavage product and the first reaction mixture are further Ingredients are mixed. If necessary, the cleavage product may be subjected to further treatment to isolate the genomic DNA therein, and the isolated genomic DNA is mixed with other components of the first reaction mixture to obtain a first reaction mixture.
  • the lysed nucleic acid sample can be amplified without purification. In some embodiments, the lysed nucleic acid sample is amplified after purification. In some embodiments, the DNA has undergone varying degrees of fragmentation during lysis without the need for a special disruption step for amplification. In some embodiments, the lysed nucleic acid sample is amplified after disruption.
  • the present application also provides a more convenient method of directly mixing cells comprising genomic DNA with other components required for amplification to obtain a first reaction mixture, that is, genomic DNA in the first reaction mixture.
  • a first reaction mixture that is, genomic DNA in the first reaction mixture.
  • the first reaction mixture may further contain a fine particle capable of cracking Cellular surfactants (such as, but not limited to, NP-40, Tween, SDS, Triton X-100, EDTA, one or more of guanidinium isothiocyanate) and/or lytic enzymes (eg, proteinase K, stomach) One or more of protease, papain).
  • Cellular surfactants such as, but not limited to, NP-40, Tween, SDS, Triton X-100, EDTA, one or more of guanidinium isothiocyanate
  • lytic enzymes eg, proteinase K, stomach
  • protease, papain One or more of protease, papain
  • the method provided herein may further comprise, after completion of step (a) and prior to performing step (b), placing the reaction mixture in a lysis temperature cycle program such that the cells lyse and release The genomic DNA is derived.
  • a person skilled in the art can select an appropriate cleavage temperature cycle program depending on the lysing component contained in the reaction mixture, the type of the cell, and the like.
  • An exemplary cleavage temperature cycling procedure includes placing the reaction mixture at 50 ° C for 3 minutes to 8 hours (eg, at 3 minutes to 7 hours, 3 minutes to 6 hours, 3 minutes to 5 hours, 3 minutes to 4 hours, 3 minutes) Up to 3 hours, 3 minutes to 2 hours, 3 minutes to 1 hour, 3 minutes to 40 minutes, any time between 3 minutes and 20 minutes, such as 10 minutes, 20 minutes, 30 minutes, etc.), then placed at 80 ° C 2 minutes to 8 hours (for example, 2 minutes to 7 hours, 2 minutes to 6 hours, 2 minutes to 5 hours, 2 minutes to 4 hours, 2 minutes to 3 hours, 2 minutes to 2 hours, 2 minutes to 1 hour) , any time between 2 minutes and 40 minutes, between 2 minutes and 20 minutes, such as 10 minutes, 20 minutes, 30 minutes, etc.).
  • the cleavage temperature program can be run in one cycle and, if desired, two or more cycles, depending on the particular lysis conditions.
  • the methods described herein involve two broad classes of different primers, wherein the first type of primer comprises a universal sequence and a variable sequence from the 5' end to the 3' end, and the second type of primer comprises a specific sequence and a universal sequence, but Does not contain any variable sequences.
  • the "first primer” and the “third primer” described herein belong to the first type of primer described above.
  • the first primer included in the first reaction mixture comprises a universal sequence and a first variable sequence from the 5' end to the 3' end; and the third primer optionally included in the first reaction mixture is from the 5' end to the 3'
  • the 'end includes a universal sequence and a third variable sequence.
  • the first type of primer consists of a universal sequence and a variable sequence.
  • the first class of primers consists of a universal sequence, a variable sequence, and a spacer sequence.
  • a universal sequence in this application refers to a nucleotide sequence possessed by a first type of primer and a second type of primer at its 5' end.
  • the length of the universal sequence can be, for example, 6-60, 8-50, 9-40, 10-30, 10-15 or 25-30 bases.
  • an appropriate universal sequence is selected such that amplification is substantially not effected by binding to genomic DNA, and polymerization between the first type of primer and the first type of primer is avoided (eg, first primer and first primer) Between, between the third primer and the third primer or between the first primer and the third primer) and the loop of the first primer itself (for example, the portion of the first primer at the 5' end).
  • the sequence is complementary to a partial sequence at the 3' end and the first primer itself forms a hairpin structure, or the partial sequence at the 5' end of the third primer is complementary to the partial sequence at the 3' end and the third primer forms a hairpin structure itself, and the first primer and the first primer The case of polymerization or ringing between two types of primers.
  • the universal sequence comprises only three or two bases with weaker self-complementary pairing ability, and no other or two bases.
  • the universal sequence consists of three bases, G, A, and T, ie, the universal sequence does not contain a C base.
  • the universal sequence consists of three bases, C, A, and T, ie, the universal sequence does not contain a G base.
  • the universal sequence consists of two bases, A and T, A and C, A and G, T and C, or T and G, ie, the G and C bases are not simultaneously included in the universal sequence.
  • the inclusion of a C or G base in a universal sequence may result in mutual polymerization between the primer and the primer, resulting in a multimer, thereby impairing the ability to amplify the genomic DNA.
  • the universal sequence does not have a sequence capable of self-pairing, a sequence that results in pairing between the primer and the primer, or a plurality of consecutive bases of the same species.
  • the base sequence of the appropriate universal sequence and the ratio of bases therein can be selected to ensure that the universal sequence itself does not base pair or generate amplification with the genomic DNA template sequence.
  • the universal sequence can be selected such that the amplification product can be directly sequenced.
  • the universal sequence may be designed to include sequences that are complementary or identical to portions or all of the primers for sequencing (eg, sequences identical, all identical, partially complementary, or fully complementary to portions of the primers used for sequencing) .
  • the universal sequence is selected in a targeted manner according to different sequencing platforms.
  • the universal sequence is selected in a targeted manner according to a second or third generation sequencing platform.
  • the universal sequence is selected in a targeted manner according to Illumina's NGS sequencing platform.
  • the universal sequence is selected in a targeted manner according to the Ion torrent sequencing platform.
  • the universal sequence is selected from the group consisting of SEQ ID NO: 1 [TTGGTAGTGAGTG], SEQ ID NO: 2 [GAGGTGTGATGGA], SEQ ID NO: 3 [GTGATGGTTGAGGTA], SEQ ID NO: 4 [AGATGTGTATAAGAGACAG ], SEQ ID NO: 5 [GTGAGTGATGGTTGAGGTAGTGTGGAG] and SEQ ID NO: 6 [GCTCTTCCGATCT].
  • the first type of primer comprises a universal sequence and a variable sequence from the 5' end to the 3' end (eg, the first primer/third primer respectively comprise the first/third variable sequence), wherein the universal sequence of the first type of primer is The same, but the variable sequences may vary.
  • the first/third primers are a mixture of primers comprising the same universal sequence and different variable sequences, respectively.
  • a variable sequence refers to a stretch of base sequences that are not fixed, which may comprise a random sequence (eg, the first/third variable sequence comprises a first/third random sequence, respectively).
  • the variable sequence consists of a random sequence.
  • the variable sequence consists of a random sequence and a fixed sequence.
  • a random sequence means that the bases at each base position of the sequence are independently randomly selected from a specific set, and thus the above random sequence represents a collection of base sequences composed of different base combinations.
  • the first variable sequence may include a first random sequence, wherein the number of bases of the first random sequence is n, n is a positive integer selected from 3-20, and the first random sequence is from 5'
  • a selectable set of any of the above base positions can generally be represented by a degenerate identification method.
  • Random sequences can be selected in a completely random manner (ie, any base position in a random sequence), or certain qualifications can be further added on a random basis to exclude undesired conditions or increase matching to the target genomic DNA. degree.
  • any base position in the random sequence is selected from set D (ie, not C);
  • any base position in the random sequence is selected from the set H (ie, not G);
  • any base position in the random sequence is selected from the set B ( That is, not A); or when the universal sequence contains a large amount of A, any base position in the random sequence is selected from the set V (ie, not T).
  • the random sequence may have an appropriate length, such as 2-20 bases, 2-19 bases, 2-18 bases, 2-17 bases, 2-16 bases, 2-15 bases. 2-14 bases, 2-13 bases, 2-12 bases, 2-11 bases, 2-12 bases, 2-11 bases, 2-10 bases, 2-9 bases, 2-8 bases, 3-18 bases, 3-16 bases, 3-14 bases, 3-12 bases, 3-10 bases, 4 - 16 bases, 4-12 bases, 4-9 bases, or 5-8 bases.
  • the random sequence is 5 bases in length. In certain embodiments, the random sequence is 8 bases in length.
  • each of the first primers has the same universal sequence and the first random sequence described above, ie, the first primer in the particular first reactant is a group Primers, all of which have the same universal sequence and have the same or different random sequences consisting of bases selected from set B.
  • the third variable sequence in the third primer may comprise a third random sequence, wherein the third random sequence is from the 5' end to the 3' end X b1 X b2 . . .
  • first reaction mixture comprising a quantity of first primers, each having the same universal sequence and a first random sequence of length n, wherein each base of the first random sequence X ai All belong to the same set, and wherein the set is selected from B, D, H or V; and the first reaction mixture further comprises a certain amount of third primers, all of which have the same universal sequence and length a first random sequence of n, wherein each base X bi of the first random sequence belongs to the same set, and wherein the set is selected from B, D, H or V, and X bi and X ai belong to different sets .
  • the first random sequence and the third random sequence are the same length. In other embodiments, the first random sequence and the third random sequence are different in length.
  • the variable sequence may further comprise a fixed sequence at its 3' end, which may be selected from any combination of bases capable of increasing genomic coverage.
  • the fixed sequences described herein include, but are not limited to, those selected from the group consisting of CCC, AAA, TGGG, GTTT, GGG, TTT, TNTNG, or GTGG.
  • the N used in the description of the immobilization sequence in the present application means any one selected from the group consisting of A, T, C, and G, and does not represent a random sequence selected from N.
  • the first primer the 5' end to the 3' end may include the same universal sequence, a random sequence containing different sequence combinations, and the same fixed sequence (for example, all the first primers at the 3' end thereof.
  • the 5' end to the 3' end may include the same universal sequence, a random sequence containing different sequence combinations, and different fixed sequences (for example, the first primer is included in the first primer).
  • the 3' end includes a primer mixture of TGGG and a primer mixture including GTTT at its 3' end).
  • the first reaction mixture comprises a first primer and a third primer, wherein the first variable sequence in the first primer is selected from the group consisting of X a1 X a2 ... X an GGG, X a1 X a2 ... X an TTT, X a1 X a2 ...
  • the third variable sequence in the third primer is selected from X b1 X b2 ... X bn GGG, X b1 X b2 ... X bn TTT, X b1 X b2 ... X bn TGGG or X b1 X b2 ... X bn GTTT.
  • variable sequences with more uniform distribution and higher coverage on the genome can also be selected by statistical calculations, thereby increasing the chance of recognition of variable sequences and genomic DNA.
  • variable sequence is selected from the group consisting of: (B) n CCC, (B) n AAA, (B) n TGGG, (B) n GTTT, (B) n GGG, (B) n TTT , (B) n TNTNG, (B) n GTGGGGG, (D) n CCC, (D) n AAA, (D) n TGGG, (D) n GTTT, (D) n GGG, (D) n TTT, ( D) n TNTNG, (D) n GTGGGGG, (H) nCCC, (H) n AAA, (H) n TGGG, (H) n GTTT, (H) n GGG, (H) n TTT, (H) n TNTNG, (H) n GTGGGGG, (V) n CCC, (V) n AAA, (V) n TGGG, (V) n GTTT, (H) n GTGGGGG
  • the first variable sequence in the first primer may have (B) n CCC, (B) n AAA, (B) n TGGG, (B) n GTTT, (B) n GGG And (B) one or more sequences of n TTT, (B) n TNTNG, (B) n GTGGGGG.
  • the third variable sequence in the third primer may have (D) n CCC, (D) n AAA, (D) n TGGG, (D) n GTTT, (D) n GGG And one or more sequences of (D) n TTT, (D) n TNTNG, (D) n GTGGGGG.
  • the universal and variable sequences of the first class of primers may be directly contiguous or may have a spacer sequence of one or more bases.
  • the universal sequence and the variable sequence are joined by a spacer sequence of length m, wherein m is a positive integer selected from 1-3.
  • the random sequence in the variable sequence can be used in both universal and variable sequences. Introducing m bases randomly selected from A, T, G, and C (length m The spacer sequence) further increases the coverage of the first class of primers on the target genomic DNA without increasing the extent of primer dimer production.
  • the universal sequence and the third variable sequence in the third primer are joined by a third spacer sequence, wherein the first spacer sequence is Y b1 . . .
  • m is 1, ie, the universal sequence and the first variable sequence in the first primer are linked by a base selected from the set N, and the third primer is between the universal sequence and the third variable sequence. Connected by a base selected from the set N.
  • the first primer (and optionally the third primer) is designed such that its amplification product can be used directly in Illumina's NGS sequencing platform, where the first primer comprises GCTCTTCCGATCTY a1 X a1 X a2 X a3 X A4 X a5 TGGG, GCTCTTCCGATCTY a1 X a1 X a2 X a3 X a4 X a5
  • the first primer includes The sequence shown in SEQ ID NO: 7, SEQ ID NO: 11, or a mixture thereof
  • the third primer includes the sequence shown in SEQ ID NO: 8, SEQ ID NO: 12, or a mixture thereof.
  • the first class of primers comprises a primer consisting of the sequence set forth in SEQ ID NO: 7 and/or a primer consisting of the sequence set forth in SEQ ID NO: 11.
  • the first class of primers comprises a primer consisting of the sequence set forth in SEQ ID NO: 8 and a primer consisting of the sequence set forth in SEQ ID NO: 12.
  • the first class of primers comprises a primer consisting of the sequence set forth in SEQ ID NO: 7 or a primer consisting of the sequence set forth in SEQ ID NO: 11; And a primer consisting of the sequence shown in SEQ ID NO: 8 or a primer consisting of the sequence shown in SEQ ID NO: 12.
  • the first type of primer comprises a primer consisting of the sequence set forth in SEQ ID NO: 7, a primer consisting of the sequence set forth in SEQ ID NO: 11, and the sequence set forth in SEQ ID NO: A primer consisting of the primer consisting of the sequence shown in SEQ ID NO: 12.
  • the first class of primers comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 15-22, wherein the universal sequence of each of the first class of primers comprises or consists of SEQ ID NO: 1.
  • the first class of primers comprises a primer consisting of the sequence set forth in SEQ ID NO: 15 and/or a primer consisting of the sequence set forth in SEQ ID NO: 19.
  • the first class of primers comprises a primer consisting of the sequence set forth in SEQ ID NO: 16 and/or a primer consisting of the sequence set forth in SEQ ID NO: 20.
  • the first class of primers comprises a primer consisting of the sequence set forth in SEQ ID NO: 15 or a primer consisting of the sequence set forth in SEQ ID NO: 19; and the SEQ ID NO: 16 A primer consisting of a sequence or a primer consisting of the sequence shown in SEQ ID NO: 20.
  • the first class of primers comprises a primer consisting of the sequence set forth in SEQ ID NO: 15, a primer consisting of the sequence set forth in SEQ ID NO: 19, and the sequence set forth in SEQ ID NO: A primer consisting of the primer consisting of the sequence shown in SEQ ID NO: 20.
  • the first class of primers comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 23-30, wherein the universal sequence of each of the first class of primers comprises or consists of SEQ ID NO: 2.
  • the first class of primers comprises one or both of a primer consisting of the sequence set forth in SEQ ID NO: 23 and/or a primer consisting of the sequence set forth in SEQ ID NO: 27.
  • the first class of primers comprises one or both of a primer consisting of the sequence set forth in SEQ ID NO: 24 and/or a primer consisting of the sequence set forth in SEQ ID NO: 28.
  • the first class of primers comprises a primer consisting of the sequence set forth in SEQ ID NO: 23 or a primer consisting of the sequence set forth in SEQ ID NO: 27; and the SEQ ID NO: 24 A primer consisting of a sequence or a primer consisting of the sequence shown in SEQ ID NO: 28.
  • the first class of primers comprises a primer consisting of the sequence set forth in SEQ ID NO: 23, a primer consisting of the sequence set forth in SEQ ID NO: 27, and the sequence set forth in SEQ ID NO: A primer consisting of the primer consisting of the sequence shown in SEQ ID NO: 28.
  • the total concentration of the first and third primers in the first reaction mixture is from 10 to 150 ng/ ⁇ L. In some embodiments, the total concentration of the first and third primers in the first reaction mixture is 10-120 ng/ ⁇ L, 10-100 ng/ ⁇ L, 10-90 ng/ ⁇ L, 10-80 ng/ ⁇ L, 10-70 ng/ ⁇ L, 10-60 ng/ ⁇ L, 10-50 ng/ ⁇ L, 10-40 ng/ ⁇ L, 20-120 ng/ ⁇ L, 20-100 ng/ ⁇ L, 20-80 ng/ ⁇ L, 20-70 ng/ ⁇ L, 20-60 ng/ ⁇ L, 20-50 ng/ ⁇ L, 30-140 ng/ ⁇ L, 30-120 ng/ ⁇ L, 30-100 ng/ ⁇ L, 30-80 ng/ ⁇ L, 30-60 ng/ ⁇ L or 30-40 ng/ ⁇ L.
  • the concentrations of the first and third primers in the first reaction mixture are 10-140 ng/ ⁇ L, 10-120 ng/ ⁇ L, 10-100 ng/ ⁇ L, 10-80 ng/ ⁇ L, 10-60 ng/ ⁇ L, respectively. 10-30 ng/ ⁇ L, 10-20 ng/ ⁇ L, 20-120 ng/ ⁇ L, 20-100 ng/ ⁇ L, 20-80 ng/ ⁇ L, 20-60 ng/ ⁇ L, 20-40 ng/ ⁇ L or 20-30 ng/ ⁇ L. In some embodiments, the concentrations of the first and third primers in the first reaction mixture are 15 ng/ ⁇ L, 30 ng/ ⁇ L, or 60 ng/ ⁇ L, respectively.
  • the first primer and the third primer are at the same concentration in the first reaction mixture. In some embodiments, the first and third primers in the first reaction mixture are each 100-800 pmol. In some embodiments, the first and third primers in the first reaction mixture are collectively 400-600 pmol.
  • the first reaction mixture also includes other components required for DNA amplification, such as a nucleic acid polymerase, a mixture of nucleomonomers, and appropriate metal ion and buffer components required for enzymatic activity, and the like. At least one or more of these ingredients may use reagents known in the art.
  • a nucleic acid polymerase refers to an enzyme capable of synthesizing a new nucleic acid strand. Any nucleic acid polymerase suitable for use in the methods of the present application can be used. Preferably, a DNA polymerase is used.
  • the methods of the present application use thermostable nucleic acid polymerases, such as those that do not decrease or decrease by less than 1%, 3%, 5% at the temperature of PCR amplification (eg, 95 degrees Celsius). 7%, 10%, 20%, 30%, 40% or 50% of those nucleic acid polymerases.
  • the nucleic acid polymerases used in the methods of the present application have strand displacement activity.
  • strand displacement activity refers to an activity of a nucleic acid polymerase that is capable of separating a nucleic acid template from a complementary strand to which it is paired, and such separation is carried out in the direction from 5' to 3' with The creation of a new nucleic acid strand complementary to the template.
  • Nucleic acid polymerases having strand displacement capabilities and their use are known in the art, for example, see U.S. Patent No. 5,824,517, the disclosure of which is incorporated herein in its entirety by reference.
  • Suitable nucleic acid polymerases include, but are not limited to, Phi29 DNA polymerase, Bst DNA polymerase, Bst 2.0 DNA polymerase, Pyrophage 3137, Vent polymerase (eg, Ventpolymer of Thermococcus litoralis, Deep Vent polymerase, Vent (-exo) Polymerase, Deep Vent (-exo) polymerase, TOPOTaq DNA polymerase, 9°Nm polymerase, Klenow Fragment DNA polymerase I, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, T7phase DNA polymerase variant (lack of 3'-5' exonuclease activity), One of ultra-fidelity DNA polymerase, Taq polymerase, Psp GBD (exo-) DNA polymerase, Bst DNA polymerase (full length), E. coli DNA polymerase, LongAmp Taq DNA polymerase, OneTaq DNA polymerasekind or more.
  • a nucleomonomer mixture is referred to herein as a mixture of dATP, dTTP, dGTP, dCTP.
  • the first reaction mixture contains Vent polymerase of Thermococcus litoralis, One or more of Deep Vent polymerase, Vent(-exo) polymerase, or Deep Vent (-exo) polymerase.
  • the reaction mixture contains Vent polymerase of Thermococcus litoralis.
  • the Vent polymerase of Thermococcus litoralis refers to a natural polymerase isolated from Thermococcus litoralis.
  • the reaction mixture contains Deep Vent polymerase. Deep Vent polymerase refers to a natural polymerase isolated from Pyrococcus species GB-D.
  • the reaction mixture contains Vent(-exo) polymerase.
  • Vent (-exo) polymerase refers to an enzyme that has been genetically engineered with the D141A/E143A gene by the Vent polymerase of Thermococcus litoralis.
  • the reaction mixture contains Deep Vent (-exo) polymerase.
  • Deep Vent (-exo) polymerase is an enzyme that has been engineered with D141A/E143A for Deep Vent polymerase.
  • the various Vent polymerases described in this application are commercially available, for example, from New England Biolabs.
  • the first reaction mixture may also include a suitable metal ion required for the nucleic acid polymerase to perform an enzymatic activity (eg, a suitable concentration of Mg 2+ ions (eg, a final concentration may range from about 1.5 mM to about 8 mM), nucleomonomers Mixtures (e.g., dATP, dGTP, dTTP, and dCTP), bovine serum albumin (BSA), dTT (e.g., a final concentration of from about 2 mM to about 7 mM), purified water, and the like.
  • a suitable metal ion required for the nucleic acid polymerase to perform an enzymatic activity eg, a suitable concentration of Mg 2+ ions (eg, a final concentration may range from about 1.5 mM to about 8 mM)
  • nucleomonomers Mixtures e.g., dATP, dGTP, dTTP, and dCTP
  • BSA bovine
  • the pH adjusting agent may further be included in the first reaction mixture such that the pH of the mixture is maintained between 7.0 and 9.0.
  • Suitable pH adjusting agents can include, for example, Tris HCl and Tris SO 4 .
  • the first reaction mixture may further comprise one or more other ingredients, such as DNase inhibitors, RNase, SO 4 2- , Cl - , K + , Ca 2+ , Na + , and / or (NH 4 ) + and so on.
  • the method provided herein includes the step (b) of placing the first reaction mixture in a first temperature cycling sequence such that the first sequence of primers (first primer or first primer and third primer) has a variable sequence Genomic DNA can be replicated by the action of a nucleic acid polymerase by binding to the genomic DNA by base pairing.
  • Amplification refers to the addition of a nucleotide complementary to a nucleic acid template at the 3' end of a primer by the action of a nucleic acid polymerase to synthesize a new nucleic acid strand complementary to a nucleic acid template base.
  • Suitable methods for amplifying nucleic acids can be used, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), or other suitable amplification methods. These methods are known in the art and can be found, for example, in U.S. Patent Nos. 4,683,195 and 4,683,202, and to Innis et al. "PCR protocols: a guide to method and applications” Academic Press, Incorporated (1990) and Wu et al. (1989). Genomics 4: 560-569, the entire contents of which are incorporated herein by reference.
  • the reaction mixture is placed in an appropriate temperature cycle program, such that the DNA template double strand is unzipped into a single strand, the first/third primer hybridizes to the template single strand, and then under the action of DNA polymerase in the primer 3 'End to extend.
  • the temperature cycling program typically includes a denaturation or melting temperature at which the DNA template duplex is unzipped into a single strand; an annealing temperature at which the primer specifically hybridizes to the DNA template single strand; and an extension temperature at which temperature
  • the lower DNA polymerase adds a nucleotide complementary to the DNA template base at the 3' end of the primer, allowing the primer to be extended, resulting in a new DNA strand complementary to the DNA template.
  • the first reaction mixture is first placed in a temperature program capable of opening the double strand of the genomic DNA (step (b1)).
  • a temperature program capable of opening the double strand of the genomic DNA
  • higher reaction temperatures eg, 90 ° C - 95 ° C
  • longer reaction times can be maintained (For example, reacting at a temperature between 90-95 ° C for 1-20 minutes).
  • the double strand that needs to be unwrapped is the double strand generated during the amplification process, in which case, as long as the half amplicon or the full amplicon duplex to be amplified can be denatured into a single strand. Therefore, the melting time required is not required to be long (for example, a melting reaction between 3 and 50 seconds between temperatures of 90-95 ° C).
  • the first reaction mixture is placed in a temperature program capable of binding the first type of primers (the first primer or the first primer and the third primer) to the DNA single-strand template (step (b2)).
  • the variable sequences in the first type of primers are complementary to (ie, annealed) by complementary bases at different positions in the genomic DNA, and thereby the replication is initiated at different positions of the genomic DNA. Since the variable sequences in the first type of primers are different, and the base ratios and sequences are different, the optimal temperature for binding each variable sequence to the genomic DNA is also greatly different. Thus, at a particular annealing temperature, only a portion of the primers may bind well to the genomic DNA, and the binding of the other primer to the genomic DNA may not be ideal.
  • the step (b2) comprises the step of placing the reaction mixture at more than one temperature to cause the first type of primer to bind sufficiently to the DNA template.
  • the DNA denatured reaction mixture can be rapidly cooled to a low temperature, for example, about 10 ° C to 20 ° C, and then the reaction mixture is allowed to react at different annealing temperatures for a suitable time by gradient heating, thereby ensuring as much as possible. Primers are paired with genomic DNA.
  • step (b2) comprises reacting at a first annealing temperature (eg, 15 ° C) between 10-20 ° C for a suitable period of time (eg, 3-60 seconds) between 20-30 ° C
  • a first annealing temperature eg, 15 ° C
  • a suitable period of time eg, 3-60 seconds
  • the second annealing temperature for example, 25 ° C
  • a suitable time for example, 3 to 50 seconds
  • a third annealing temperature for example, 35 ° C between 30 and 50 ° C is reacted for an appropriate time (for example, 3-50 seconds).
  • the annealing temperature of the primer is generally not lower than the Tm value of the primer by more than 5 ° C, and the too low annealing temperature causes non-specific binding between the primer and the primer, resulting in primer assembly and non-specific amplification. Things. Therefore, a low temperature such as 10 ° C to 20 ° C is usually not used in the primer annealing temperature.
  • a low temperature such as 10 ° C to 20 ° C is usually not used in the primer annealing temperature.
  • the inventors of the present application unexpectedly found that even if the gradient is raised from a low temperature (for example, 10 ° C to 20 ° C), the pairing between the primer and the genomic DNA can maintain a good specificity, and the amplification result remains extremely low. The variability indicates that the amplification results are accurate and reliable.
  • the primer annealing temperature covers the low temperature, it can ensure a wider range of primer sequences and genomic DNA binding, thus providing better genomic coverage and depth of amplification.
  • the reaction mixture is placed in a temperature program capable of extending the length of the first type of primer bound to the DNA single-strand template under the action of the nucleic acid polymerase to generate an amplification product (step (b3) )).
  • the extension temperature is generally related to the optimum temperature of the DNA polymerase, and one skilled in the art can make a specific selection based on the particular reaction mixture.
  • the DNA polymerase in the reaction mixture can have strand displacement activity such that if the primer encounters a primer or amplicon that binds to the downstream template during extension, the strand displacement activity of the DNA polymerase These downstream bound primers can be separated from the template strand to ensure that the primers in the extension can continue to extend to obtain longer amplified sequences.
  • DNA polymerases having strand displacement activity include, but are not limited to, for example, phi29 DNA polymerase, T5 DNA polymerase, SEQUENASE 1.0, and SEQUENASE 2.0.
  • the DNA polymerase in the reaction mixture is a thermostable DNA polymerase.
  • Thermostable DNA polymerases include, but are not limited to, e.g., of Taq DNA polymerase, OmniBase TM Sequenase, of Pfu DNA polymerase, TaqBead TM hot start polymerase, Vent DNA polymerase (Thermococcus litoralis e.g. of Vent polymerase, Deep Vent Polymerase, Vent (-exo) polymerase, Deep Vent (-exo) polymerase, Tub DNA polymerase, TaqPlus DNA polymerase, Tfl DNA polymerase, Tli DNA polymerase, and Tth DNA polymerase.
  • the DNA polymerase in the reaction mixture can be a DNA polymerase that is thermostable and has strand displacement activity.
  • the DNA polymerase in the reaction mixture is selected from the group consisting of: Phi29 DNA polymerase, Bst DNA polymerase, Pyrophage 3137, Vent polymerase (eg, Vent polymerase of Thermococcus litoralis, Deep Vent polymerase, Vent (- Exo) polymerase, Deep Vent (-exo) polymerase, TOPOTaq DNA polymerase, 9°Nm polymerase, Klenow Fragment DNA polymerase I, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase , T7phase DNA polymerase variant (lack of 3'-5' exonuclease activity), One or more of ultra-fidelity DNA polymerase, Taq polymerase, Bst DNA polymerase (full length), E. coli DNA polymerase, LongAmp Taq DNA polymerase,
  • step (b3) comprises an extension temperature between 60-90 ° C (eg, at 65-90 ° C, 70-90 ° C, 75-90 ° C, 80-90 ° C, 60- 85 ° C, 60-80 ° C, 60-75 ° C, 70-80 ° C, or at 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 ° C) reaction for 10 seconds - 15 minutes (example For example, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-14, 3-14, 5-14, 6-14, 7-14, 8-14, 9-14, 10-14, 11-14, 12-14, 13-14 minutes, or 10-60, 10-50, 10-40, 10-30, 10-20, 20-60, 20-50, 20-40, 20-30, 30-60, 30-50, 30-40 seconds).
  • step (b3) comprises reacting at one or more temperatures between 60-80 ° C for 30 seconds to 2 minutes. In certain embodiments, step (b3) comprises reacting at 65 ° C for 40 seconds. In certain embodiments, step (b3) comprises reacting at 75 ° C for 40 seconds. In certain embodiments, step (b3) comprises reacting at 75 ° C for 40 seconds and then at 75 ° C for 40 seconds.
  • step (b1) in the cycle after the first cycle comprises reacting between temperatures of 90-95 °C for 10-50 seconds.
  • the first number of cycles described herein is at least two.
  • the sequence of the 3' end of the variable sequence of the first type of primer is extended, and the obtained amplified product is a universal sequence at the 5' end, and the 3' end is a sequence complementary to the single-stranded sequence of the genomic template.
  • Such amplification products are also referred to as semiamplifiers.
  • the previous half-amplifier itself can also be used as a DNA template to bind to the variable sequence in the first type of primer, and the primer extends to the 5' end of the amplified product by the action of the nucleic acid polymerase until The universal sequence of the 5' end of the amplified product is copied, thereby obtaining a genomic amplification product having a universal sequence at the 5' end and a complementary sequence at the 3' end, and the amplified product is also called a full amplicon.
  • the preamplification product described herein mainly refers to a full amplicon having a 5' end as a universal sequence and a 3' end being a complementary sequence of a universal sequence.
  • the DNA single strand in the reaction mixture contains not only the original genomic DNA single strand, but also the newly synthesized DNA single strand amplified, the original genomic DNA template and initial amplification.
  • the half amplicon produced in the same can be used as a new DNA template again, binds to the primer and initiates a new round of DNA synthesis; however, since the entire amplicon contains complementary sequences at both ends (the universal sequence contained at the 5' end and 3 The 'end sequence of the universal sequence contained in the end', thus forming a hairpin structure itself, so that it cannot be used as a new DNA template in the next reaction cycle for a new round of DNA synthesis.
  • the number of first cycles is controlled to an appropriate range to ensure that there are sufficient preamplification products for subsequent reactions without affecting the reaction time of the entire process due to excessive cycles.
  • the number of first cycles is 2-40 cycles (eg, 2-40, 4-40, 6-40, 8-40, 10-40, 12-40) , 14-40, 16-40, 18-40, 20-40, 15-40, 20-40, 25-40, 30-40, 5-35, 10-35 , 15-35, 20-35, 25-35, 30-35, 10-30, 15-30, 20-30, 25-30, 2-20, 2-18 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 4-20, 4-18, 4-16 4-14, 4-12, 4-10, 4-8, 4-6, 6-20, 6-18, 6-16, 6-14, 6-12, 6-10, 6-8, 8-20, 8-18, 8-16, 8-14, 8-12, 8-10, 10-20, 10-18, 10-16, 10-14, 10-12, 12-20, 12-18, 12-16, 12-14
  • the number of first cycles is at least 3, at least 4, at least 5, or at least 6, at least 7, at least 8, at least 9, or at least 10, at least 11, at least 12, at least Is at least 14, at least 15, or at least 16, at least 17, at least 18, at least 19, or at least 20, or preferably no more than 8, no more than 9, no more than 10, no more than 11.
  • step (b3') is further included after step (b3), wherein the reaction mixture is placed in an appropriate temperature program such that 3' of the entire amplicon in the genomic preamplification product The ends are hybridized to the 5' end to form a cyclic structure. It was previously thought that step (b3') can protect the ends of the entire amplicon, thereby avoiding the end-to-end polymerization between two or more full amplicons, thereby avoiding the two sequences that are not adjacent to the genome. integrate. This will help to improve the accuracy of the amplification results.
  • the method proceeds directly to the subsequent step (b1) or (c) without further steps (e.g., step (b3')) after step (b3).
  • step (b3') the whole amplicon does not undergo a specific step to avoid the first-to-tail polymerization, and therefore, theoretically, such amplification results should have certain defects in accuracy.
  • the method of the present application even if the whole amplicon is looped without a specific step after the step (b3), the final amplification result still has a relatively high degree of accuracy, and the use step
  • the method of (b3') is similar to the effect. This streamlines the reaction steps while still maintaining the specificity of the reaction.
  • the second reaction mixture comprises the preamplification product obtained in the step (b), the second primer, the nucleotide monomer mixture and the nucleic acid polymerase, and the second primer is from the 5' end to the 3' end.
  • the end contains a specific sequence and the universal sequence. Since the universal sequence is substantially non-complementary to the genomic sequence, if the other portions of the second type of primer are designed to be substantially non-complementary to the genomic sequence, then the second type of primer does not directly pair with the genomic DNA and initiates the multiplex of the genomic DNA.
  • the second reaction mixture can be obtained by adding a second primer directly to the reaction mixture obtained after the end of step (b).
  • the reaction mixture obtained after the end of step (b) is purified prior to step (c) to yield a purified preamplification product, which is then polymerized with a second primer, a mixture of nucleotide monomers, and a nucleic acid.
  • the enzyme and optionally any other reagents known in the art that can be used in the amplification reaction are combined to provide a second reaction mixture.
  • the "second primer” described herein pertains to the second type of primers described above.
  • the second type of primer comprises a universal sequence in the first type of primer, such that the second type of primer can bind to the complementary sequence of the universal sequence at the 3' end of the entire amplicon, thereby further replicating the full amplicon, thereby greatly increasing the number thereof. .
  • the second type of primer comprises or consists of a specific sequence and a universal sequence from 5' to 3'.
  • the second type of primers can be selected in a targeted manner according to different sequencing platforms.
  • a second type of primer is selected in accordance with a second generation sequencing platform.
  • the second type of primers are specifically selected according to Illumina's NGS sequencing platform (such as, but not limited to, Hiseq, Miseq, etc.) or Life Technologies' Ion torrent's NGS sequencing platform.
  • the second type of primer comprises a sequence that is complementary or identical to a portion or all of the primers for sequencing.
  • the sequence of the second type of primers that is complementary or identical to a portion or all of the primers for sequencing comprises or consists of the universal sequence.
  • the second primer described in the present application may be a pair of primer pairs having a second type of primer structure or a single primer having the same structure and sequence.
  • the particular sequence of the second primer includes at its 3' end a sequence that is complementary or identical to a portion or all of the primers for sequencing.
  • the sequence contained in a particular sequence of the second primer that is complementary or identical to a portion or all of the primers for sequencing comprises or consists of SEQ ID NO: 31 [ACACTCTTTCCCTACACGAC], or SEQ ID NO: 32 [GTGACTGGAGTTCAGACGTGT] composition.
  • the particular sequence in the second primer further includes at its 5' end a sequence that is complementary or identical to the capture sequence of the sequencing platform.
  • a capture sequence refers to a sequence contained on a sequencing plate in a sequencing platform for capturing a fragment to be sequenced.
  • the sequence contained in a particular sequence of the second primer that is partially or identical to the capture sequence of the sequencing platform comprises or consists of SEQ ID NO: 33 [AATGATACGGCGACCACCGAGATCT], or SEQ ID NO: 34 [CAAGCAGAAGACGGCATACGAGAT] composition.
  • the specific sequence of the second primer further comprises a stretch between the sequence that is partially or identical to the capture sequence of the sequencing platform and the sequence that is complementary or identical to the partial or total of the primer for sequencing.
  • Identification sequence barcode sequence
  • the identification sequence is Refers to the sequence used to identify a specific set of fragments to be sequenced. When the sequencing platform simultaneously sequences multiple sets of sequencing fragments, the sequencing data can be distinguished by screening the identification sequences carried in each set in the sequencing results.
  • the second primer is a primer pair comprising the same universal sequence and a different specific sequence, wherein the different specific sequences respectively comprise a complement or the same as part or all of a pair of capture sequences used in the same sequencing platform
  • the sequences, and/or the different specific sequences, respectively, comprise specific sequences that are complementary or identical to portions or all of the different primers in the pair of sequencing primers used in the same sequencing.
  • the second primer comprises a mixture of the sequences set forth in SEQ ID NO: 35 [AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT] and [CAAGCAGAAGACGGCATACGAGAT X...X GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT], wherein X...X is a marker sequence, which can be selected by a person skilled in the art according to actual needs. Identify the length of the sequence and its specific sequence.
  • the second primer comprises a mixture of the sequences set forth in SEQ ID NO: 35 [AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT], SEQ ID NO: 36 [CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT].
  • the second primer comprises a mixture of the sequences set forth in SEQ ID NO: 37 [CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATGCTCTTCCGATCT] and SEQ ID NO: 38 [CCATCTCATCCCTGCGTGTCTCCGACTCAGCTAAGGTAACGATGCTCTTCCGATCT].
  • the second primer comprises a mixture of the sequences set forth in SEQ ID NO: 39 [CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATTTGGTAGTGAGTG] and SEQ ID NO: 40 [CCATCTCATCCCTGCGTGTCTCCGACTCAGCTAAGGTAACGATTTGGTAGTGAGTG].
  • the concentration of the second primer in the second reaction mixture is from 1 to 15 ng/ ⁇ L. In some embodiments, the concentration of the second primer in the second reaction mixture is 1-12 ng/ ⁇ L, 1-10 ng/ ⁇ L, 1-8 ng/ ⁇ L, 1-7 ng/ ⁇ L, 1-6 ng/ ⁇ L, 1- 5 ng/ ⁇ L, 1-4 ng/ ⁇ L, 2-3 ng/ ⁇ L, 2-12 ng/ ⁇ L, 2-10 ng/ ⁇ L, 2-8 ng/ ⁇ L, 2-6 ng/ ⁇ L, 2-5 ng/ ⁇ L, 2-4 ng/ ⁇ L, 2-3 ng/ ⁇ L, 3-12 ng/ ⁇ L, 3-10 ng/ ⁇ L, 3-8 ng/ ⁇ L, 3-6 ng/ ⁇ L or 3-4 ng/ ⁇ L.
  • the second primer concentration in the second reaction mixture is 2-3 ng/ ⁇ L. In some embodiments, the second primer in the second reaction mixture is 5-50 pmol. In some embodiments, the second primer in the second reaction mixture is 10 pmol, 15 pmol or 20 pmol.
  • the nucleic acid polymerase contained in the second reaction mixture is selected from the group consisting of Vent polymerase, Deep Vent polymerase, Vent (-exo) polymerase, or Deep Vent (-exo) polymerase of Thermococcus litoralis.
  • the second reaction mixture contains Vent polymerase of Thermococcus litoralis.
  • the second reaction mixture contains Deep Vent polymerase.
  • the second reaction mixture contains Vent(-exo) polymerase.
  • the second reaction mixture contains Deep Vent (-exo) polymerase.
  • the various polymerases described in this application are commercially available, for example, from New England Biolabs.
  • the second reaction mixture may also include a suitable metal ion required for the nucleic acid polymerase to perform enzymatic activity (eg, a suitable concentration of Mg 2+ ions (eg, a final concentration may range from about 1.5 mM to about 8 mM).
  • a suitable metal ion required for the nucleic acid polymerase to perform enzymatic activity eg, a suitable concentration of Mg 2+ ions (eg, a final concentration may range from about 1.5 mM to about 8 mM).
  • nucleoside monomers eg, dATP, dGTP, dTTP, and dCTP
  • BSA bovine serum albumin
  • dTT eg, a final concentration of about 2 mM to about 7 mM
  • appropriate buffer components eg pH regulators such as TrisHCl and Tris SO 4
  • other ingredients commonly used in the art eg DNase inhibitors, RNase, SO 4 2- , Cl ⁇ , K + , Ca 2+ , Na) + , and / or (NH 4 ) +, etc.
  • the method provided by the present application further comprises the step (d): placing the second reaction mixture obtained in the step (c) in a second temperature cycle program such that the universal sequence of the second type of primer is capable of interacting with the genomic preamplification product The 3' end paires and amplifies the genomic preamplification product to obtain an amplified genomic amplification product.
  • the genomic preamplification product obtained in the step (b), that is, the full amplicon has a complementary sequence of a universal sequence at the 3' end, it can be complementary to the universal sequence of the second type of primer, under the action of the nucleic acid polymerase,
  • the second type of primer extends to replicate the full length of the entire amplicon.
  • the reaction mixture is first placed in a temperature program capable of opening the DNA duplex (step (d1)).
  • the DNA duplex here mainly refers to the double strand of the genomic preamplification product (i.e., the full amplicon) obtained in the step (b) (including the single-stranded hairpin structure molecule of the whole amplicon).
  • the original genomic DNA may still be present in the second reaction mixture at this time, since the second type of primer does not substantially bind to the genomic DNA, the original genomic DNA is not the DNA to be amplified in step (d). template.
  • a higher reaction temperature (e.g., 90 ° C - 95 ° C) can be used to react for a suitable period of time such that the full amplicon duplex/hairpin structure to be amplified can be denatured into a linear single strand.
  • the temperature step in step (d1) places the reaction mixture in a temperature reaction capable of opening the DNA duplex for a sufficient time to ensure that the template DNA duplex or hairpin structure is completely denatured into a single strand, the temperature
  • the procedure involves reacting at a denaturation temperature between 90-95 ° C (eg, 95 ° C) for 5 seconds to 20 minutes (eg, 30 seconds or 3 minutes).
  • step (d1) the reaction mixture is placed in a temperature program capable of melting the double strand of the amplification product generated in the amplification of the xth round (x is an integer of ⁇ 1) contained therein into a single-stranded template (step ( D2)), ie a reaction at a melting temperature between 90-95 ° C (eg 95 ° C) for 3-50 seconds (eg 20 seconds).
  • step (d2) is not necessary in the first cycle, but since the temperatures used in the denaturation and melting procedures are similar and the melting time is short relative to the denaturation time, it can be considered as the first
  • the round is the delay of step (d1).
  • the reaction mixture is placed in a temperature program (step (d3)) capable of binding the second type of primer to the DNA single strand obtained in the step (d1) or (d2).
  • a temperature program (step (d3)) capable of binding the second type of primer to the DNA single strand obtained in the step (d1) or (d2).
  • the Tm value of the second type of primer can be calculated, and based on the Tm value, a suitable annealing temperature for the second type of primer can be found.
  • the temperature program in step (d3) comprises reacting at an annealing temperature (eg, 63 ° C) between 45-65 ° C for 3-50 seconds (eg, 40 seconds).
  • the second type of primer is a mixture of SEQ ID NO: 35, SEQ ID NO: 36, and the temperature program in step (d3) comprises reacting at 63 ° C for 3-50 seconds.
  • the annealing temperature in step (d3) is higher than the annealing temperature in step (b2).
  • the reaction mixture may still contain the first type of primers which are not reacted in the step (b), and the variable sequences in the first type of primers may be paired with the single-stranded template of the DNA obtained in the step (d3). Binding, resulting in an incomplete amplified sequence.
  • step (d3) When the annealing temperature in step (d3) is higher than the annealing temperature suitable for the first type of primer, binding of the first type of primer to the DNA single-strand template can be reduced or avoided, thereby selectively allowing amplification of the second type of primer.
  • the reaction mixture is placed in a temperature program capable of extending the length of the second type of primer that binds to the amplification product in a single strand under the action of the nucleic acid polymerase.
  • the temperature program described in step (d4) comprises reacting for an extension temperature (eg, 72 ° C) between 60-80 ° C for 10 seconds to 15 minutes (eg, 40 seconds or 3 minutes).
  • Steps (d2) through (d4) can be repeated to the second cycle number to obtain the desired expanded genomic amplification product.
  • the genomic amplification product obtained in step (b) is further replicated and amplified, and the amount is greatly increased to provide sufficient genomic DNA sequences for subsequent studies or operations.
  • the second number of cycles in step (d5) is greater than the first number of cycles in step (b4).
  • the number of second cycles is controlled to an appropriate range such that it is capable of providing a sufficient amount of DNA without affecting the accuracy of the amplification due to an excessive number of cycles.
  • the second number of cycles is 2-40 cycles (eg, 2-40, 4-40, 6-40, 8-40, 10-40, 12-40, 14-40, 16-40, 18-40, 20-40, 15-40, 20-40, 25-40, 30-40, 5-35, 10-35, 15-35, 20-35, 25-35, 30-35, 10-30, 15-30, 20-30, 25-30, 15-28, 15-26, 15-24, 15-22, 15-20, 15-18, 15-17, 16-30, 17-30, 18-30, 20-30, 22-30, 24-30, 26-30, 28-30, 32-40, 32-38, 32-36 or 32-34 cycles).
  • step (d) further comprises, after the second temperature cycling procedure, placing the reaction mixture in the same temperature program as step (d4) (eg, 72 ° C) for a suitable period of time (eg, 40 seconds). The reaction mixture was then placed at a temperature of 4 ° C to end the reaction. In certain embodiments, after the end of step (d), the reaction mixture is placed directly at a temperature of 4 ° C to terminate the reaction.
  • step (d4) eg, 72 ° C
  • a suitable period of time eg, 40 seconds
  • the present application also provides a method of amplifying a cellular genome, the method comprising:
  • B or D, or H, or V
  • X ai represents the ith nucleotide at the 5′ end of the first random sequence
  • n is a positive integer selected from 3-20
  • the first reaction mixture further comprises a third primer, wherein the third primer comprises the universal sequence and a third variable sequence from a 5' end to a 3' end, the third variable sequence A third random sequence is included, wherein the third random sequence is X b1 X b2 . . .
  • step (c) providing a second reaction mixture, the second reaction mixture comprising the genomic preamplification product, the second primer, the nucleomonomer mixture, and the nucleic acid polymerase obtained in the step (b), wherein
  • the second primer comprises a specific sequence and the universal sequence from the 5' end to the 3' end;
  • the genomic DNA in the reaction mixture of step (a) is present inside the cell, ie the reaction mixture contains cells and the genomic DNA to be amplified is contained in the cells.
  • the reaction mixture in step (a) contains cells, and further comprises components capable of lysing cells, such as surfactants and/or lytic enzymes and the like.
  • a suitable surfactant such as one or more of NP-40, Tween, SDS, Triton X-100, EDTA, and guanidinium isothiocyanate may be used.
  • the above method of amplifying a cellular genome further comprises, after step (a) and before step (b), placing the reaction mixture in a lysis temperature cycling sequence (eg, placing the reaction mixture at 50 ° C for 20 minutes). And then placed at 80 ° C for 10 minutes), allowing the cells to lyse and release the genomic DNA.
  • a lysis temperature cycling sequence eg, placing the reaction mixture at 50 ° C for 20 minutes.
  • 80 ° C for 10 minutes allowing the cells to lyse and release the genomic DNA.
  • the products amplified by the methods of the present application can be further used for sequencing, such as performing whole-genome sequencing.
  • various sequencing analysis platforms such as next-generation sequencing (NGS), microarray, and real-time PCR have higher requirements for the initial amount of samples to be analyzed (100 ng or more), therefore, from a single human cell ( A full-genome amplification is required to obtain a sufficient amount of nucleic acid material for analysis in a small amount of starting sample or about a small amount of starting sample.
  • the genomic DNA in a biological sample e.g., a single cell
  • the amplified product can be sequenced by an appropriate sequencing method in the art.
  • Exemplary sequencing methods include hybridization sequencing (SBH), ligase sequencing (SBL), quantitative incremental fluorescent nucleic acid increase sequencing (QIFNAS), stepwise ligation and cleavage, molecular beacon, pyrosequencing, and Fluorescence sequencing (FIS SEQ), fluorescence resonance energy transfer (FRET), multiplex sequencing (US Patent Application 12/027039; Porreca et al. (2007) NAT.
  • SBH hybridization sequencing
  • SBL ligase sequencing
  • QIFNAS quantitative incremental fluorescent nucleic acid increase sequencing
  • FRET fluorescence resonance energy transfer
  • sequencing of amplification products of the methods of the present invention can be accomplished in a high throughput manner.
  • High throughput methods typically fragment the nucleic acid molecules to be sequenced (eg, by enzymatic or mechanical cleavage, etc.) to form a large number of short fragments ranging from tens of bp to several hundred bp in length.
  • the throughput of sequencing can be greatly improved, and the sequencing needs can be shortened. time.
  • the sequence of the measured short fragments is processed by software and can be spliced into a complete sequence.
  • a variety of high throughput sequencing platforms are known in the art, such as Roche 454, Illumina Solexa, AB-SOLiD, Helicos, Polonator platform technology, and the like.
  • a variety of light-based sequencing techniques are also known in the art, for example, see Landegren et al. (1998) Genome Res. 8: 769-76, Kwok (2000) Pharmacogenomics 1: 95-100 and Shi (2001) Clin. Chem. : those described in 164-172.
  • the products amplified by the methods of the present application can also be used to analyze genotypes or genetic polymorphisms in genomic DNA, such as single nucleotide polymorphism (SNP) analysis, short tandem repeats.
  • SNP single nucleotide polymorphism
  • STR Sequence
  • RFLP restriction fragment length polymorphism
  • VNTRs variable number tandem repeat
  • CTR Complex Repetitive Sequence
  • microsatellite analysis can be referred to Krebs, JE, Goldstein, ES and Kilpatrick, ST (2009).
  • Lewin's Genes X Jones & Bartlett Publishers
  • the amplification products obtained by the methods of the present application can also be used in medical analysis and/or diagnostic analysis.
  • an individual biological sample can be amplified by the method of the present application, and an abnormality such as a mutation, a deletion, an insertion, or a fusion between chromosomes in the gene or DNA sequence of interest in the amplification product can be analyzed to evaluate The individual is at risk of developing a disease, the stage of progression of the disease, the genotyping of the disease, the severity of the disease, or the likelihood that the individual will respond to a particular therapy.
  • the gene or DNA sequence of interest can be analyzed using suitable methods known in the art, such as, but not limited to, by nucleic acid probe hybridization, primer-specific amplification, sequencing of sequences of interest, single-strand conformation polymorphism Sex (SSCP) and so on.
  • suitable methods known in the art such as, but not limited to, by nucleic acid probe hybridization, primer-specific amplification, sequencing of sequences of interest, single-strand conformation polymorphism Sex (SSCP) and so on.
  • the methods of the present application can be used to compare genomes derived from different single cells, particularly different single cells from the same individual. For example, when there is a difference between the genomes of different single cells of the same individual, such as between tumor cells and normal cells, the genomic DNA of different single cells can be separately amplified using the method of the present application, and the amplification products are further subjected to Analysis, for example, by sequencing analysis and comparison, or by comparative genomic hybridization (CGH) analysis.
  • CGH comparative genomic hybridization
  • the methods of the present application can be used to identify haploid structures or haploid genotypes in homologous chromosomes.
  • a haploid genotype refers to a combination of multiple alleles co-inherited on the chromosome of the same haploid.
  • a biological sample eg, a single cell from a diploid of an individual
  • each fraction is configured as a reaction mixture, and each reaction mixture is subjected to DNA amplification by the method of the present application, and then the amplified product is subjected to sequence analysis, and the reference genome sequence (for example, the published human standard genomic sequence, please See: International Human Genome Sequencing Consortium, Nature 431, 931-945 (2004)) for alignment to identify single nucleotide mutations therein. If there is no ready-made reference genomic sequence, a region of appropriate length can also be assembled from multiple fragment sequences of the genome for comparison by de-novo genome assembly.
  • the reference genome sequence for example, the published human standard genomic sequence, please See: International Human Genome Sequencing Consortium, Nature 431, 931-945 (2004)
  • the products amplified by the methods of the present application can be further used for analysis by gene cloning, real-time PCR, and the like.
  • the methods of the present application may further comprise analyzing the amplification product to identify sequence features associated with a disease or phenotype.
  • analyzing the amplification product comprises genotyping the DNA amplification product.
  • analyzing the amplification product comprises identifying a polymorphism of the DNA amplification product, such as a single nucleotide polymorphism analysis (SNP).
  • SNPs can be detected by well-known methods such as oligonucleotide ligation assay (OLA), single base extension, allele-specific primer extension, mismatch hybridization, and the like. The disease can be diagnosed by comparing the relationship of SNPs to known disease phenotypes.
  • sequence features associated with a disease or phenotype include chromosomal abnormalities, ectopic chromosomes, aneuploidy, deletions or duplications of some or all of the chromosomes, fetal HLA haplotypes, and paternal mutations .
  • the disease or phenotype can be beta-thalassemia, Down's syndrome, cystic fibrosis, sickle cell disease, Thai-saxophone disease, fragile X syndrome, spinal muscular atrophy, Hemoglobinopathy, alpha-thalassemia, X-linked disease (a disease dominated by genes on the X chromosome), spina bifida, no brain malformation, congenital heart disease, obesity, diabetes, cancer, fetal sex, or fetal RHD.
  • kits useful for genomic DNA amplification comprising a first primer.
  • the kit includes both a first primer and a third primer.
  • the kit further comprises a nucleic acid polymerase, wherein the nucleic acid polymerase is selected from the group consisting of: Phi29 DNA polymerase, Bst DNA polymerase, Pyrophage 3137, Vent polymerase, TOPOTaq DNA polymerase, 9° Nm polymerase , Klenow Fragment DNA Polymerase I, MMLV Reverse Transcriptase, AMV Reverse Transcriptase, HIV Reverse Transcriptase, T7phase DNA Polymerase Variant, Ultra-fidelity DNA polymerase, Taq polymerase, Bst DNA polymerase, E.coli DNA polymerase, LongAmp Taq DNA polymerase, OneTaq DNA polymerase, Deep Vent DNA polymerase, Vent (exo-) DNA polymerase, Deep Vent (exo-) DNA DNA polymerase, Deep Vent (exo-) DNA DNA sequence.
  • the kit further comprises one or more components selected from the group consisting of nucleotide monomer mixtures (eg, dATP, dGTP, dTTP, and dCTP, eg, total concentrations ranging from 1 mmol to 8 mmol/ ⁇ L) ), dTT (for example, concentration between 1 mmol and 7 mmol/ ⁇ L), Mg 2+ solution (for example, concentration between 2 mmol and 8 mmol/ ⁇ L), bovine serum albumin (BSA), pH adjuster (eg TrisHCl), DNase Inhibitor, RNase, SO 4 2- , Cl - , K + , Ca 2+ , Na + , and / or (NH 4 ) + .
  • nucleotide monomer mixtures eg, dATP, dGTP, dTTP, and dCTP, eg, total concentrations ranging from 1 mmol to 8 mmol/ ⁇ L
  • dTT for example, concentration between 1 m
  • the kit further comprises a component capable of lysing cells, such as one or more surfactants (eg, NP-40, Tween, SDS, Triton X-100, EDTA, guanidinium isothiocyanate) And/or one or more lytic enzymes (eg, proteinase K, pepsin, papain).
  • the kit further comprises a second type of primer (ie, a second primer). It will be appreciated that the first primer, the second primer and the third primer in the kit each have structural and sequence features as specifically described above.
  • all of the components in the kit are separately stored in separate containers. In some embodiments, all of the components in the kit are co-stored in the same container. In some embodiments, each primer in the kit is separately stored in a separate container, and all other components except the primer are stored in the same container.
  • the nucleic acid polymerase can be stored in a substantially pure form in a separate container, or alternatively can be combined with other ingredients.
  • the kit may comprise a mixture comprising all of the reactants other than genomic DNA required for a linear amplification reaction, when such a kit is used in the linear amplification reaction described herein,
  • the first reaction mixture in step (a) of the method of the present application can be obtained by directly mixing the sample containing the genomic DNA with the mixture in the kit, optionally by adding an appropriate amount of pure water to obtain the desired reaction volume.
  • the kit may comprise a mixture comprising all of the reactants other than the amplification template required for the exponential amplification reaction, when such a kit is used in the exponential amplification reaction described herein,
  • the DNA template sample containing the amplification product of step (b) can be directly mixed with the mixture in the kit, optionally by adding an appropriate amount of pure water to obtain a desired reaction volume, thereby obtaining the steps of the method of the present application.
  • the kit may comprise both a mixture of all reagents other than the amplification template required for the exponential amplification reaction and all but the amplification template required for the exponential amplification reaction.
  • a mixture of the reactants, the above mixture may be either a separate one or a mixed one.
  • kits useful for genomic DNA amplification comprising a first type of primer (eg, a first primer and/or a third primer) and a second type of primer (eg, The second primer), and also includes instructions for use, which describe the step of mixing the primers and other components to obtain the first/third reaction mixture before starting the amplification.
  • the instructions also document how to perform the amplifications described herein.
  • the first type of primers and the second type of primers in the kit may be placed in separate containers, respectively, but the instructions may include the step of mixing the two in the same container prior to initiating amplification.
  • Example 1 Preliminary validation of amplification effects using different linear amplification primer mixtures
  • the standard genomic DNA is genomic DNA of a human cell extracted in advance.
  • the standard genomic DNA was diluted to 50 ⁇ g/ ⁇ l of DNA solution with nuclease-free water, and 1 ⁇ l of the above solution (as a genomic DNA source) was added to the PCR tube, and each experimental group was added as shown in Table 1.
  • the primer mixture and other related reagents provide a first reaction mixture (containing Na + , Mg 2+ , Cl - , Tris-Cl, Triton X-100, dNTP, Vent polymerase, and a mixture of primers).
  • the primer mixtures used in each experimental group are shown in Table 1 below.
  • test group Primer used (or primer mixture) 1-2 Universal sequence + NNNNNTTT and universal sequence + NNNNNGGG 3-4 Universal sequence +NNNNNGTTT and universal sequence +NNNNNTGGG 5-6 SEQ ID NO: 7 and SEQ ID NO: 11 7-8 SEQ ID NO: 8 and SEQ ID NO: 12 9-10 SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11 and SEQ ID NO: 12 11-12 Universal sequence + KKKKK
  • the total amount of the first type of primers used in each of the experimental groups was 600 picomoles (if a plurality of primers were included, the total amount was 600 picomoles, and the contents of the various primers were the same).
  • Different universal sequences can be designed according to different sequencing platforms. SEQ ID NO: 6 was selected as the universal sequence according to the Illumina platform in 1-12 of the experiment.
  • the second primer -1 (SEQ ID NO: 35) : AAT GAT ACG GCG ACC ACC GAG ATC T AC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATCT
  • Second Primer-2 (SEQ ID NO: 36):
  • the bases indicated by the double underline in the second primer include a portion corresponding to the capture sequence of the sequencing platform, the base identified in italics is the portion corresponding to the sequencing sequence of the sequencing platform, and the portion identified by the dot is the portion of the identification sequence. , can be replaced with other identification sequences as needed, and the part identified by a single underscore is a general sequence part.
  • Each experimental group completed the above amplification temperature control program to obtain an amplification product.
  • Example 1 Five microliters of unpurified amplification products of each experimental group in Example 1 were separately taken, and 1 ⁇ l of 6x DNA loading buffer (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., product number CW0610A) was separately added for loading.
  • the gel was a 1% agarose gel and the marker was DM2000 (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., Cat. No. CW0632C). See Figure 3 for the electropherogram.
  • the first lane from left to right is the molecular weight marker
  • the lanes 2-13 are genomic DNA amplification samples
  • the 14 lanes are molecular weight markers.
  • the products of the experimental group 1-4 have obvious bands around 100 bp, but the product content is relatively low between 100-500 bp; the products of other experimental groups 5-12 are concentrated at about 500 bp, the experiment Groups 5-10 had unclear bands around 100 bp and were very low in concentration compared to experimental groups 1-4. It can be seen from Fig. 3 that there are many primer polymers in the experimental group 1-4, and the relative reaction efficiency is low, and the reaction sequence contained in the variable sequence of the first type of primers is TGGG or GTTT (experiment group 3-4).
  • the amplification efficiency of each experimental group was estimated at the concentration after purification: It can be seen from the concentration detection results shown in Table 2 that the experimental group 1-2 has the lowest amplification efficiency, and the experimental group 3-4 and the experiment The amplification efficiency of the group 5-12 was relatively low but higher than that of the experimental group 1-2. Except for the experimental groups 1-4, the total amount of amplification products of the other experimental groups was comparable, and there was no significant difference.
  • the amplified products of the above 12 experimental groups were taken and sequenced by Illumina's NGS sequencing platform hiseq2500 sequencer in a shallow sequencing manner, and the sequence obtained by sequencing was aligned to the human reference genome.
  • the unique ratio (unique_mapped_of_raw, the ratio of data that can be compared to the unique position of the human genome) in the original data is the most important measure.
  • the experiment The unique_mapped_of_raw in groups 5-12 is between 83% and 86%, with little difference between groups, but the unique_mapped_of_raw in experimental groups 1-4 is relatively low between 67 and 79%.
  • mapped_of_raw the ratio of data in the original data that can be compared to a location in the human genome.
  • Table 3 the mapped_of_raw in the experimental groups 5-12 was between 89% and 93%, and the difference between the groups was small, but the mapped_of_raw in the experimental groups 1-4 was relatively low at 73- Between 86%.
  • the data in Table 3 also shows that the reading data quality of experimental group 1-4 is also lower than other experimental groups.
  • the ratio of high-quality data in experimental data of experimental groups 3 and 4 is only 77.08%, 76.99%, and experimental group 5
  • the ratio of high quality data in -12 is between 94% and 96%.
  • the nucleotide readings for the starting position of the sequence reads in the sequencing library are shown in Figure 4.
  • the initial reading regions in experimental groups 1-4 and 9-10 include four bases A, T, C, and G.
  • the first few bases required for sequencing have higher randomness, and when the randomness of the first few bases of the whole sample is low, it is required.
  • a certain amount of positive control is added to each well to increase the randomness of the base when the whole plate is sampled, but this will inevitably waste a certain amount of data.
  • a certain amount of positive control is required due to the lack of A and C in the initial reading area, and it is generally necessary to add at least 20% according to experimental experience.
  • the positive control of the amount of sample can ensure the smooth progress of SBS sequencing.
  • the sample to be tested is AFP single cell.
  • Human epidermal fibroblasts (AFP) in good culture state were digested with trypsin, and the digested cells were collected into 1.5 ml EP tubes. The collected cells were centrifuged and rinsed with a 1x PBS solution. Flush After the wash was completed, 1 x PBS was added to suspend the cells. Pipette a portion of the suspension containing the cells, pick a single cell using a mouth pipette under a 10x microscope, pipette the PBS solution to a volume of no more than 1 ⁇ l, and transfer the picked single cells into a 5 ⁇ l lysis buffer.
  • AFP Human epidermal fibroblasts
  • the lysed reaction solution was replaced with the standard genomic DNA of Example 1a) as a genomic DNA source, and the other components and linear amplification, exponential amplification temperature control procedures were the same as in Example 1a).
  • the obtained amplification product was subjected to gel electrophoresis detection using the conditions described above.
  • gel electrophoresis please refer to Figure 5, wherein the first lane from left to right is the molecular weight marker, the lanes 2-13 are genomic DNA amplification samples, and the 14 lanes are molecular weight markers.
  • the experimental results are similar to those shown in Fig. 3.
  • the products of experimental group 1-4 showed obvious bands around 100 bp, but the product content was relatively low between 100-500 bp; the products of other experimental groups 5-12 were concentrated at about 500 bp, and the experimental groups 5-12 were not near 100 bp. Obvious strips. It can be seen from Fig. 5 that there are many primer polymers in the experimental group 1-4, and the relative reaction efficiency is low, and the reaction efficiency is high when the fixed sequence contained in the variable sequence of the first type of primer is TGGG or GTTT (experiment group 3-4).
  • the unique_mapped_of_raw in the experimental group 5-12 is between 78% and 85%, and the difference between the groups is not large, but the unique_mapped_of_raw in the experimental group 1-4 is relatively low between 63 and 70%.
  • the mapped_of_raw in the experimental group 5-12 is between 84% and 92%, and the difference between the groups is not large, but the mapped_of_raw in the experimental group 1-4 is relatively low at 73-86%. between.
  • the data in Table 3 also indicates that the quality of the reading data of the experimental group 1-4 is also lower than that of the other experimental groups.
  • the ratio of the high-quality data in the original data of the experimental group 3 and 4 is only 68.97%, 72.29%, and the experimental group 5
  • the ratio of high quality data in -12 is between 94% and 96%.
  • the linear amplification primer mixture used in the experimental groups 1-4 produced more primer polymers, so that the amplification efficiency was greatly reduced, and accordingly the amount of data was lower under the same amplification conditions.
  • the A, T, C, and G distributions of the experimental group 1-4 were uniform in the starting sequencing region, the ratio of the data in the comparison was especially low, which resulted in the use of the sequencing data for subsequent processing. difficult.
  • Example 2 Further verification of amplification effects using different linear amplification primer mixtures
  • Human epidermal fibroblasts were isolated and lysed according to the method described in Example 1b) to obtain single-cell genomic DNA, and the primer mixtures used in the experimental group 11/12 of Table 1 and the experimental group 9/10 were used, respectively.
  • the primer mixture was amplified, and 10 parallel experiments were performed for each primer mixture (represented by 1_1, 1_2...1_10 and 2_1, 2_2...2_10, respectively).
  • Amplification was carried out according to the procedure described in Example 1a) and amplification products were obtained, and the amplification products were subjected to gel electrophoresis detection, and the results of electrophoresis detection are shown in Fig. 6.
  • the concentration of the amplified product in the experimental group 2_1, 2_2...2_10 is slightly lower than the concentration of the amplified product in the experimental group 1_1, 1_2...1_10
  • Table 6 Main quality indicators of high-throughput sequencing results of experimental groups 1_1, 1_2...1_10
  • Table 7 Main quality indicators of high-throughput sequencing results of experimental group 2_1, 2_2...2-10
  • the data in Tables 6 and 7 shows that the unique_mapped_of_raw in the experimental group 2_1, 2_2...2_10 is around 83%-84%, the mapped_of_raw is around 90%-91%, and the unique_mapped_of_raw in the experimental group 1_1, 1_2...1_10 is at 84%- About 85%, the mapped_of_raw is around 91%-92%, and there is little difference between the two groups.
  • Experimental group 1_1, 1_2...1_10 except for the abnormal experimental group 1_1 (possibly due to errors in the recovery process, the data volume is extremely low)
  • Other experimental groups The amount of data in the middle is between 1.5-2M, and the average amount of data on the experimental group 1_2, 1_3...1_10 is about 1.7M.
  • the data volume is between 1.5-2.5M, and the average data volume of the experimental group 2_1, 2_2...2_10 is about 1.8M, and the data volume in the experimental group 2_1, 2_2...2_10 Slightly higher.
  • the copy number variation coefficient CV in the experimental group data is summarized as shown in Fig. 8: After the experimental group 1_1 excluding the obvious abnormality, the average copy number variation coefficient CV of the experimental group 1_2, 1_3...1_10 is about 0.046, the experimental group The average copy number variation coefficient CV of 2_1, 2_2...2_10 was about 0.049, and there was no significant difference in the copy number coefficient of variation between the two experimental groups.
  • the copy number variation map of each experimental group is separately listed in Fig. 9, in which the ordinate represents the copy number of the chromosome, the normal person is 2; the abscissa represents the chromosome 1-22 and the sex chromosome of the chromosome. As shown, chromosomes 1-22 in each experimental group were approximately two copies except for individual data points, while sex chromosomes X and Y were approximately 1 copy, respectively.
  • the amplification products in the experimental groups of 1_1, 1_2, 2_1, 2_2 according to Example 2 were randomly selected as template DNA, respectively.
  • the template DNA was subjected to PCR detection using 2x Goldstar Master Mix (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., product number CW0960).
  • the composition of the amplification system is shown in Table 10, and the amplification procedure is shown in Table 11.
  • the results of the amplification are shown in the gel electrophoresis pattern in FIG.
  • the amplification results showed that the pathogenic sites 4 and 13 in the samples 1_1 and 1_2 were not amplified in both samples, and the pathogenic site 21 was not amplified in the sample 1_1, and the pathogenic site was not found. 20, 29, 31 were not amplified in sample 1_2.
  • the pathogenic site 31 was not amplified in both samples, and the pathogenic sites 18, 21, 32, 35 were not amplified in the sample 2_1, and the pathogenic site 8 was 22 was not amplified in sample 1_2.
  • the results showed that the two sets of primer set samples (1_1, 1_2, and 2_1, 2_2) did not differ significantly in the accuracy of amplification and the amount of amplification products.
  • the amplification products in the experimental group of 1_1, 1_2, 2_1, 2_2 in the above Example 2, the positive control (gDNA of the same concentration), and the negative control (no template) were used as template DNA, respectively.
  • the template DNA was subjected to q-PCR detection using DNA sets of primers shown in Table 12 for DNA sequences on different chromosomes.
  • 2xFastSYBR Mixture purchased from Beijing Kangwei Century Biotechnology Co., Ltd., Cat. No. CW0955 was used in the real-time PCR.
  • the composition of the amplification system is shown in Table 13, and the amplification procedure is shown in Table 14.
  • Table 12 6 pairs of random primer information used for quality inspection
  • Component Volume (microliter) 2xFastSYBR mixture 25 Forward primer (10uM) 1 Reverse primer (10uM) 1 DNA template (10ng/ul) 1 RNase-free water twenty two Total amount 50
  • the results of the amplification are shown in Table 15, in which the q-PCR detection data of the primer pairs CH1, CH2, CH4, CH5, CH6 and CH7 for each set of template DNA are respectively listed. Among them, the larger the Ct value, the lower the number of templates corresponding to the primer, and the worse the amplification efficiency in gDNA amplification.
  • the amplification results showed that the amplification efficiency of CH1, CH2, CH4, CH5, CH6 and CH7 in sample 2_1 was high.
  • the amplification efficiencies of CH1, CH2, CH4, CH5, CH6 and CH7 in sample 2_2 were all high. There is no essential difference from the amplification of samples 1-1 and 1-2.
  • Table 15 Amplification efficiency of 4 samples in Figure 6 using qPCR detection using 6 pairs of primers in Table 12
  • Example 4 The amplification method of the present application is used in the Ion torrent sequencing platform
  • Fresh blood is drawn and the lymphocytes are separated using a lymphocyte separation solution.
  • a part of the suspension containing the cells is extracted with a pipette, and about 3 white blood cells are picked up using a mouth pipette under a 10x microscope, and the volume of the absorbed PBS solution is not more than 1 micrometer.
  • Second Primer-1 (SEQ ID NO: 37): CCA CTA CGC CTC CGC TTT CCT CTC TAT GGG CAG TCG GTG AT G CTC TTC CGA TCT;
  • Second Primer-2 (SEQ ID NO: 38): (wherein, the bases indicated by the double underline in the second primer include a portion corresponding to the capture sequence of the sequencing platform, and the portion identified by the dot is the portion of the identification sequence, which can be replaced with other identification sequences as needed, and the single underlined portion is a universal sequence.
  • an amplification product was obtained by exponential amplification, and four sets of experiments were performed in parallel. All other reaction conditions were consistent with those described in Example 1 b). The amplification effect is shown by gel electrophoresis in Figure 11.
  • sample 1 and sample 2 shown in Fig. 11 Two samples (shown as sample 1 and sample 2 shown in Fig. 11) amplified according to Example 4 were then randomly selected as template DNA, respectively.
  • the template DNA was subjected to PCR detection using 2x Goldstar Master Mix (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., Cat. No. CW0960), and the 35 disease-causing sites as shown in Table 8 were amplified using primers as shown in Table 9.
  • the composition of the amplification system is shown in Table 10, and the amplification procedure is shown in Table 11.
  • FIG 11 shows four samples taken after an equal volume were purified using Life Technologies' Ion torrent sequencing platforms PGM TM sequencer for sequencing, and sequenced to obtain the sequence alignment of the human genome reference. The sequencing results are shown in Table 16 below and Figure 13 below.
  • the data in Table 16 shows that the unique_mapped_of_raw in samples 1, 2, 3, and 4 is around 68%, the mapped_of_raw is in the range of 72%-73%, and the data volume is between 0.38 and 0.53M.
  • the copy number variation in the data on the machine. Department The number CV is approximately 0.06.
  • the copy number variation map of the sequencing reads is shown in Fig. 13. In each experimental group, chromosomes 1-22 are roughly two copies except for individual data points, and the sex chromosomes X and Y are each approximately one copy.
  • Example 5 Amplification method of the present application for preimplantation chromosome detection of blastocyst trophoblast
  • the fertilized eggs were cultured in vitro, and a plurality of cells (about 3 cells) of the ectotrophoblast layer were taken for detection of abnormal chromosome copy number in the blastocyst stage (day 5 of in vitro culture).
  • the method of collecting blastocyst ectotrophoblast cells may be any method known to those skilled in the art, such as, but not limited to, Wang L, Cram DS et al. Validation of copy number variation sequencing for detecting chromosome imbalances in human preimplantation embryos. Biol Reprod , 2014, 91 (2): 37 method.
  • the collected blastocyst ectotrophoblast cells were transferred into a PCR tube containing 5 ⁇ l of lysis buffer, lyase was added, lysed according to the procedure described in Example 1b), and the experimental group 9/10 in Table 1 was used.
  • the primer mixture used in the amplification of genomic DNA (four sets of experiments in parallel).
  • the amplified product was purified and sequenced as described in Example 1. The sequencing results are shown in Table 17 below.
  • Table 17 shows that the unique_mapped_of_raw in samples 1, 2, and 3 is around 66-73%, the mapped_of_raw is around 71%-78%, and the data volume is between 1.4-2.6M.
  • the copy number variation map of the sequencing reads is shown in Fig. 14. In each experimental group, chromosomes 1-22 are roughly two copies except for individual data points, and the sex chromosomes X and Y are each approximately one copy.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

一种扩增基因组DNA的方法，包括：(a)提供第一反应混合物，包括包含基因组DNA的样本、第一引物、核苷酸单体混合物和核酸聚合酶，其中所述第一引物从5'端到3'端包含通用序列和包括第一随机序列的第一可变序列；(b)将第一反应混合物置于第一温度循环程序获得预扩增产物；(c)提供第二反应混合物，包括预扩增产物、第二引物、核苷酸单体混合物和核酸聚合酶，其中所述第二引物从5'端到3'端包含或由特定序列及所述通用序列组成；(d)将第二反应混合物置于第二温度循环程序进行扩增，获得扩增产物。还涉及一种用于扩增基因组DNA的试剂盒。

Description

扩增DNA的方法 技术领域
本发明涉及扩增DNA的方法，特别涉及扩增单细胞全基因组DNA并对其进行测序的方法。
背景技术
单细胞全基因组测序技术是在单细胞水平对全基因组进行扩增与测序的一项新技术。其原理是将分离的单个细胞的微量全基因组DNA进行扩增，获得高覆盖率的完整基因组后进行高通量测序。
目前主要的全基因组扩增技术主要有四类：扩增前引物延伸聚合酶链式反(Primer Extension Preamplification-Polymerase Chain Reaction，简称为PEP-PCR，具体方法参见Zhang L,Cui X,Schmitt K,Hubert R,Navidi W,Arnheim N.1992.Whole genome amplification from a single cell:implications for genetic analysis.Proc Natl Acad Sci U S A.89(13):5847-51.)、退变寡核苷酸引物聚合酶链式反应(Degenerate Oligonucleotide–Primed Polymerase Chain Reaction，简称为DOP-PCR，具体方法参见Telenius H,Carter NP,Bebb CE,Nordenskjo M,Ponder BA,Tunnacliffe A.1992.Degenerate oligonucleotide-primed PCR:general amplification of target DNA by a single degenerate primer.Genomics13:718–25)、多重置换扩增(Multiple Displacement Amplification，简称为MDA，具体方法参见Dean FB,Nelson JR,Giesler TL,LaskenRS.2001.Rapid amplification of plasmid and phageDNA using phi29 DNA polymerase and multiply-primed rolling circle amplification.Genome Res.11:1095–99)和多次退火环状循环扩(Multiple Annealing and Looping Based Amplification Cycles，简称为MALBAC，具体方法参见PCT专利申请WO2012166425)。
基因测序技术经过了三个发展阶段：第一代DNA测序技术包括化学降解法、双脱氧链终止法以及在它们基础上发展起来的各种测序技术，其中最具代表性的是1975年由桑格(Sanger)和考尔森(Coulson)提出的链终止法。第一代技术准确率高，读取长，是至今唯一可以进行“从头至尾”测序的方法，但存在成本高、速度慢等方面的不足，并不是最理想的测序方法。随后的二、三代测序技术以高通量为共同特征，也被称为“新一代测序技术(NGS)”。其中第二代测序技术以焦磷酸测序技术、边合成边测序(SBS)技术、以及连接测序技术为代表，经过几年的发展，焦磷酸测序技术以及连接测序技术已很少使用，现在主流的二代测序技术为边合成边测序技术、半导体测序技术以及CG测序技术。第三代测序 技术大体分为两类，一类为单分子荧光测序，具有代表性的技术为TSMS技术和SMRT技术，另一类为纳米孔单分子技术。与前两代技术相比，第三代测序技术最大的特点是单分子测序。虽然第三代测序技术取得了一定的进展，但现阶段主流的测序技术仍是第二代测序技术。
目前的全基因组扩增技术扩增出的全基因序列无法直接用于二代测序技术。因此，无论是将上述全基因组序列应用于二代测序技术中的边合成边测序技术、半导体测序技术或者CG测序技术，在上机测序之前都需要进行文库制备过程。每种测序技术都具有各自对应的文库制备方法，其中边合成边测序平台的文库制备主要分为两类，一类为片段化DNA经过末端修复后添加Y形接头技术或颈环接头技术，另一类为transpson技术。半导体测序平台的文库制备同样分为两类，一类为片段化DNA经过末端修复后添加接头技术，另一类为transpson技术。CG平台文库制备过程比较复杂，片段化DNA经过末端修复后需要酶切、以及两次环化过程，操作繁琐，耗时较长。
当将目前的主流扩增方法扩增出的产物用于上述测序技术时，要么需要另行进行建库，要么测序的效果不佳。因此，目前急需一种能够克服主流扩增方法的一个、多个或全部缺陷的改进的扩增方法。
发明内容
本发明提供了一种扩增细胞基因组DNA的方法和一种用于扩增基因组DNA的试剂盒。
在本申请的一个方面中，提供了一种扩增基因组DNA的方法，所述方法包括：(a)提供第一反应混合物，其中所述第一反应混合物包括包含所述基因组DNA的样本、第一引物、核苷酸单体混合物和核酸聚合酶，其中所述第一引物从5’端到3’端包含通用序列和第一可变序列，所述第一可变序列包括第一随机序列，其中所述第一随机序列从5’端到3’端依次为Xa1Xa2……Xan，所述第一随机序列的Xai(i＝1-n)均属于同一个集合，所述集合选自B、或D、或H、或V，其中B＝{T、G、C}，D＝{A、T、G}，H＝{T、A、C}，V＝{A、C、G}，其中Xai表示第一随机序列5’端的第i个核苷酸，n是选自3-20的正整数，可选地，所述第一反应混合物进一步包括第三引物，其中所述第三引物从5’端到3’端包含所述通用序列和第三可变序列，所述第三可变序列包括第三随机序列，其中所述第三随机序列从5’端到3’端依次为Xb1Xb2……Xbn，所述第三随机序列的Xbi(i＝1-n)均属于同一个集合，所述集合选自B、或D、或H、或V，其中B＝{T、G、C}，D＝{A、T、G}，H＝{T、A、C}，V＝{A、C、G}，并且Xbi(i＝1-n)和Xai(i＝1-n)属于不同的集合，其中Xbi表示第三随机序列 5’端的第i个核苷酸，n是选自3-20的正整数；(b)将所述第一反应混合物置于第一温度循环程序进行预扩增，获得预扩增产物；(c)提供第二反应混合物，所述第二反应混合物包括步骤(b)中得到的预扩增产物、第二引物、核苷酸单体混合物和核酸聚合酶，其中所述第二引物从5’端到3’端包含或由特定序列及所述通用序列组成；(d)将所述第二反应混合物置于第二温度循环程序进行扩增，获得扩增产物。
在一些实施方式中，第一随机序列的Xai(i＝1-n)均属于集合B，第三随机序列的Xbi(i＝1-n)均属于集合D。
在一些实施方式中，所述第一可变序列和所述第三可变序列进一步在其3’端包括固定序列，所述固定序列能够提高基因组覆盖度的碱基组合。在一些实施方式中，所述固定序列选自CCC、AAA、TGGG、GTTT、GGG、TTT、TNTNG或GTGG。
在一些实施方式中，所述第一可变序列选自Xa1Xa2……XanTGGG或Xa1Xa2……XanGTTT，所述第三可变序列选自Xb1Xb2……XbnTGGG或Xb1Xb2……XbnGTTT。
在一些实施方式中，选择所述通用序列以使得其基本上不会与基因组DNA结合产生扩增，所述通用序列长度为6-60bp。在一些实施方式中，选择所述通用序列使得扩增产物能够直接进行测序。在一些实施方式中，所述通用序列选自SEQ ID NO:1[TTGGTAGTGAGTG]、SEQ ID NO:2[GAGGTGTGATGGA]、SEQ ID NO:3[GTGATGGTTGAGGTA]、SEQ ID NO:4[AGATGTGTATAAGAGACAG]、SEQ ID NO:5[GTGAGTGATGGTTGAGGTAGTGTGGAG]或SEQ ID NO:6[GCTCTTCCGATCT]。
在一些实施方式中，所述通用序列和所述第一可变序列直接相连，或者所述通用序列和所述第一可变序列通过第一间隔序列相连，所述第一间隔序列为Ya1……Yam，其中Yaj(j＝1-m)∈{A、T、G、C}，其中Yaj表示间隔序列5’端的第j个核苷酸，m是选自1-3的正整数。
在一些实施方式中，所述通用序列和所述第三可变序列直接相连，或者所述通用序列和所述第三可变序列通过第三间隔序列相连，所述第三间隔序列为Yb1……Ybm，其中Ybj(j＝1-m)∈{A、T、G、C}，其中Ybj表示间隔序列5’端的第j个核苷酸，m是选自1-3的正整数。
在一些实施方式中，所述m＝1。
在一些实施方式中，所述第一引物包括GCTCTTCCGATCTYa1Xa1Xa2Xa3Xa4Xa5TGGG、 GCTCTTCCGATCTYa1Xa1Xa2Xa3Xa4Xa5GTTT或其混合物，所述第三引物包括GCTCTTCCGATCTYb1Xb1Xb2Xb3Xb4Xb5TGGG、GCTCTTCCGATCTYb1Xb1Xb2Xb3Xb4Xb5GTTT或其混合物，其中Ya1∈{A、T、G、C}，Yb1∈{A、T、G、C}，所述Xai(i＝1-5)∈{T、G、C}，所述Xbi(i＝1-5)∈{A、T、G}。
在一些实施方式中，所述方法进一步包括对步骤(d)中获得的扩增产物进行测序的步骤，其中所述第二引物包括与测序用引物的部分或全部互补或者相同的序列。
在一些实施方式中，所述通用序列包括与测序用引物的部分或全部互补或者相同的序列。
在一些实施方式中，所述第二引物的特定序列包括与测序用引物的部分或全部互补或者相同的序列。
在一些实施方式中，所述第二引物的特定序列进一步包括与测序平台的捕捉序列部分或全部互补或者相同的序列。
在一些实施方式中，所述第二引物的特定序列中包含的与测序用引物的部分或全部互补或相同的序列包含或由SEQ ID NO:31[ACACTCTTTCCCTACACGAC]、或SEQ ID NO:32[GTGACTGGAGTTCAGACGTGT]组成。
在一些实施方式中，所述第二引物的特定序列中包含的与测序平台的捕捉序列部分或全部互补或相同的序列包含或由SEQ ID NO:33[AATGATACGGCGACCACCGAGATCT]、或SEQ ID NO:34[CAAGCAGAAGACGGCATACGAGAT]组成。
在一些实施方式中，所述第二引物的特定序列进一步包括标识序列，所述标识序列位于所述与测序平台的捕捉序列部分或全部互补或相同的序列和所述与测序用引物的部分或全部互补或相同的序列之间。
在一些实施方式中，所述第二引物包括具有相同通用序列和不同特定序列的引物混合物，所述不同特定序列分别与同一测序中用到的测序引物对中不同引物的部分或全部互补或相同。
在一些实施方式中，所述第二引物包括SEQ ID NO:35[AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT]和SEQ ID NO:36[CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCT TCCGATCT]所示的序列的混合物。
在一些实施方式中，所述核酸聚合酶具有热稳定和/或链置换活性。在一些实施方式中，所述核酸聚合酶选自：Phi29DNA聚合酶、Bst DNA聚合酶、Pyrophage 3137、Vent聚合酶、TOPOTaq DNA聚合酶、9°Nm聚合酶、Klenow Fragment DNA聚合酶I、MMLV反转录酶、AMV反转录酶、HIV反转录酶、T7phase DNA聚合酶变种、
Figure PCTCN2017081977-appb-000001
超保真DNA聚合酶、Taq聚合酶、Bst DNA聚合酶、E.coli DNA聚合酶、LongAmp Taq DNA聚合酶、OneTaq DNA聚合酶、Deep Vent DNA聚合酶、Vent(exo-)DNA聚合酶、Deep Vent(exo-)DNA聚合酶，及其任意组合。
在一些实施方式中，步骤(b)使得所述第一类引物的可变序列能够与所述基因组DNA配对并扩增所述基因组DNA以得到基因组预扩增产物，其中所述基因组预扩增产物的5’端包含所述通用序列，3’端包含所述通用序列的互补序列。
在一些实施方式中，所述第一温度循环程序包括：(b1)能够打开所述DNA双链以获得DNA单链模板的温度程序；(b2)能够使所述第一引物以及可选的第三引物与所述DNA单链模板结合的温度程序；(b3)在所述核酸聚合酶的作用下能够使与所述DNA单链模板结合的第一类引物延伸长度以产生预扩增产物的温度程序；(b4)重复步骤(b1)到(b3)至指定的第一循环次数，其中所述指定的第一循环次数大于1。
在一些实施方式中，在进行第一次循环时，步骤(b1)中所述DNA双链为基因组DNA双链，所述温度程序包括在90-95℃的温度之间变性反应1-20分钟。在一些实施方式中，在进行第一次循环后，步骤(b1)中所述的温度程序包括在90-95℃的温度之间解链反应3-50秒。
在一些实施方式中，当进行到第二次循环后，所述预扩增产物包含在5’端包含所述通用序列，3’端包含所述通用序列的互补序列的基因组预扩增产物。
在一些实施方式中，在步骤(b1)后并且在步骤(b2)之前不包括额外的将所述第一反应混合物置于适当的温度程序，使得所述基因组预扩增产物的3’端与5’端杂交结合以形成发卡结构的步骤(b2’)。在一些实施方式中，所述步骤(b2)包括将所述反应混合物置于多于一种的温度程序，以促使所述第一类引物充分与所述DNA模板有效结合。在一些实施方式中，所述多于一种的温度程序包括：介于10-20℃之间的第一温度，介于20-30℃之间的第二温度，和介于30-50℃之间的第三温度。在一些实施方式中，所述步骤(b2)中所述步骤包 括在第一温度退火反应3-60秒、在第二温度退火反应3-50秒和在第三温度退火反应3-50秒。在一些实施方式中，所述步骤(b3)中所述的温度程序包括在60-80℃的温度之间延伸反应10秒-15分钟。在一些实施方式中，所述步骤(b4)的所述第一循环次数为2-40。
在一些实施方式中，所述步骤(d)使得所述第二引物的所述通用序列能够与所述基因组预扩增产物的3’端配对并扩增所述基因组预扩增产物以得到扩大的基因组扩增产物。
在一些实施方式中，所述步骤(d)包括：(d1)能够打开DNA双链的温度程序；(d2)进一步能打开DNA双链的温度程序；(d3)能够使所述第二引物与所述经步骤(b)获得的基因组预扩增产物的单链结合的温度程序；(d4)能够使与所述基因组预扩增产物单链结合的第二引物在所述核酸聚合酶的作用下延伸长度的温度程序；(d5)重复步骤(d2)到(d4)至指定的第二循环次数，其中所述指定的第二循环次数大于1。
在一些实施方式中，步骤(d1)中所述DNA双链为所述基因组预扩增产物，并且所述DNA双链包括DNA发卡结构中包含的双链，所述温度程序包括90-95℃的温度之间变性反应5秒-20分钟。
在一些实施方式中，步骤(d2)中所述的温度程序包括在90-95℃的温度之间解链反应3-50秒。在一些实施方式中，所述步骤(d3)中所述的温度程序包括在45-65℃的温度之间退火反应3-50秒。在一些实施方式中，所述步骤(d4)中所述的温度程序包括在60-80℃的温度之间延伸反应10秒-15分钟。
在一些实施方式中，所述方法进一步包括分析所述扩增产物以识别与疾病或表型相关的序列特征。在一些实施方式中，所述与疾病或表型相关的序列特征包括染色体水平异常、染色体的异位、非整倍体、部分或全部染色体的缺失或重复、胎儿HLA单倍型和父源突变，或者所述疾病或表型选自下组：β-地中海贫血、唐氏综合征、囊性纤维化、镰状细胞病、泰-萨克斯病、脆性X综合征、脊髓性肌萎缩症、血红蛋白病、α-地中海贫血、X连锁疾病(由在X染色体上基因主导的疾病)、脊柱裂、无脑畸形、先天性心脏病、肥胖、糖尿病、癌症、胎儿性别、胎儿RHD。在一些实施方式中，所述基因组DNA来源于卵裂球、囊胚滋养层、培养的细胞、提取后的gDNA或囊胚培养液。
本申请的一方面提供了一种扩增基因组DNA的方法，所述方法包括：(a)提供第一反应混合物，其中所述第一反应混合物包括包含所述基因组DNA的样本、第一引物、核苷酸单体混合物、和核酸聚合酶，其中所述第一引物从5’端到3’端包含通用序列和可变序列， 其中所述第一引物从5’端到3’端包含通用序列和第一可变序列，所述第一可变序列包括第一随机序列，其中所述第一随机序列从5’端到3’端依次为Xa1Xa2……Xan，所述第一随机序列的Xai(i＝1-n)均属于同一个集合，所述集合选自B、或D、或H、或V，其中B＝{T、G、C}，D＝{A、T、G}，H＝{T、A、C}，V＝{A、C、G}，其中Xai表示第一随机序列5’端的第i个核苷酸，n是选自3-20的正整数，其中所述通用序列和所述第一可变序列直接相连、或所述通用序列和所述第一可变序列通过第一间隔序列相连，所述第一间隔序列为Ya1……Yam，其中Yaj(j＝1-m)∈{A、T、G、C}，其中Yaj表示间隔序列5’端的第j个核苷酸，可选地，其中所述第一反应混合物进一步包括第三引物，其中所述第三引物从5’端到3’端包含所述通用序列和第三可变序列，所述第三可变序列包括第三随机序列，其中所述第三随机序列从5’端到3’端依次为Xb1Xb2……Xbn，所述第三随机序列的Xbi(i＝1-n)均属于同一个集合，所述集合选自B、或D、或H、或V，其中B＝{T、G、C}，D＝{A、T、G}，H＝{T、A、C}，V＝{A、C、G}，并且Xbi(i＝1-n)和Xai(i＝1-n)属于不同的集合，其中Xbi表示第一随机序列5’端的第i个核苷酸，n是选自3-20的正整数，其中所述通用序列和所述第三可变序列直接相连，或者所述通用序列和所述第三可变序列通过第三间隔序列相连，所述第三间隔序列为Yb1……Ybm，其中Ybj(j＝1-m)∈{A、T、G、C}，其中Ybj表示间隔序列5’端的第j个核苷酸，m是选自1-3的正整数；(b)将所述第一反应混合物置于第一温度循环程序，使得所述第一引物的第一可变序列以及可选的第三引物的第三可变序列能够与所述基因组DNA配对并扩增所述基因组DNA以得到基因组预扩增产物，其中所述基因组预扩增产物的5’端包含所述通用序列，3’端包含所述通用序列的互补序列；其中所述第一温度循环程序包括：(b1)第一个循环为在介于90-95℃的温度之间的第一变性温度反应1-20分钟，第一个循环之后为在介于90-95℃的温度之间的第二解链温度反应3-50秒；(b2)在介于10-20℃之间的第一退火温度反应3-60秒，介于20-30℃之间的第二退火温度反应3-50秒，和介于30-50℃之间的第三退火温度反应3-50秒；(b3)在介于60-80℃之间的第一延伸温度反应10秒-15分钟；(b4)重复步骤(b1)到(b3)至2-40个循环；(c)提供第二反应混合物，所述第二反应混合物包括步骤(b)中得到的所述基因组预扩增产物、第二引物、核苷酸单体混合物、和核酸聚合酶，其中所述第二引物的从5’端到3’端包含或由特定序列及所述通用序列组成；(d)将所述第二反应混合物置于第二温度循环程序，使得所述第二引物的所述通用序列能够与所述基因组预扩增产物的3’端配对并扩增所述基因组预扩增产物以得到扩大的基因组扩增产物，其中所述第二温度循环程序包括：(d1)在介于90-95℃之间的第二变性温度反应5秒-20分钟；(d2)在介于90-95℃之间的第二解链温度反应3-50 秒；(d3)在介于45-65℃之间的第四退火温度反应3-50秒；(d4)在介于60-80℃之间的第二延伸温度反应10秒-15分钟；(d5)重复步骤(d2)到(d4)2-40个循环。
在一些实施方式中，所述通用序列包含或由SEQ ID NO:6组成；所述第一随机序列的Xai(i＝1-n)均属于D，所述第三随机序列的Xbi(i＝1-n)均属于B。
在一些实施方式中，步骤(d)得到的扩增产物已完成了文库构建。
在本申请的再一个方面中，提供了一种用于扩增基因组DNA的试剂盒，所述试剂盒包括第一引物，其中所述第一引物从5’端到3’端包含通用序列和第一可变序列，所述第一可变序列包括第一随机序列，其中所述第一随机序列从5’端到3’端依次为Xa1Xa2……Xan，所述第一随机序列的Xai(i＝1-n)均属于同一个集合，所述集合选自B、或D、或H、或V，其中B＝{T、G、C}，D＝{A、T、G}，H＝{T、A、C}，V＝{A、C、G}，其中Xai表示第一随机序列5’端的第i个核苷酸，n是选自3-20的正整数，其中所述通用序列和所述第一可变序列直接相连、或所述通用序列和所述第一可变序列通过第一间隔序列相连，所述第一间隔序列为Ya1……Yam，其中Yaj(j＝1-m)∈{A、T、G、C}，其中Yaj表示间隔序列5’端的第j个核苷酸，m是选自1-3的正整数，可选地，其中所述第一反应混合物进一步包括第三引物，其中所述第三引物从5’端到3’端包含所述通用序列和第三可变序列，所述第三可变序列包括第三随机序列，其中所述第三随机序列从5’端到3’端依次为Xb1Xb2……Xbn，所述第三随机序列的Xbi(i＝1-n)均属于同一个集合，所述集合选自B、或D、或H、或V，其中B＝{T、G、C}，D＝{A、T、G}，H＝{T、A、C}，V＝{A、C、G}，并且Xbi(i＝1-n)和Xai(i＝1-n)属于不同的集合，其中Xbi表示第一随机序列5’端的第i个核苷酸，n是选自3-20的正整数，其中所述通用序列和所述第三可变序列直接相连，或者所述通用序列和所述第三可变序列通过第三间隔序列相连，所述第三间隔序列为Yb1……Ybm，其中Ybj(j＝1-m)∈{A、T、G、C}，其中Ybj表示间隔序列5’端的第j个核苷酸，m是选自1-3的正整数。
在一些实施方式中，所述通用序列包含或由SEQ ID NO:6组成；所述第一随机序列的Xai(i＝1-n)均属于D，所述第三随机序列的Xbi(i＝1-n)均属于B。在一些实施方式中，所述通用序列包含或由SEQ ID NO:1组成；所述第一随机序列的Xai(i＝1-n)均属于D，所述第三随机序列的Xbi(i＝1-n)均属于B。在一些实施方式中，所述通用序列包含或由SEQ ID NO:2组成；所述第一随机序列的Xai(i＝1-n)均属于D，所述第三随机序列的Xbi(i＝1-n)均属于B。
在一些实施方式中，所述试剂盒用于构建全基因组DNA文库。
在一些实施方式中，所述试剂盒进一步包括核酸聚合酶，其中所述核酸聚合酶选自：Phi29DNA聚合酶、Bst DNA聚合酶、Pyrophage 3137、Vent聚合酶、TOPOTaq DNA聚合酶、9°Nm聚合酶、Klenow Fragment DNA聚合酶I、MMLV反转录酶、AMV反转录酶、HIV反转录酶、T7phase DNA聚合酶变种、
Figure PCTCN2017081977-appb-000002
超保真DNA聚合酶、Taq聚合酶、Bst DNA聚合酶、E.coli DNA聚合酶、LongAmp Taq DNA聚合酶、OneTaq DNA聚合酶、Deep Vent DNA聚合酶、Vent(exo-)DNA聚合酶、Deep Vent(exo-)DNA聚合酶、及其任意组合。
在一些实施方式中，所述试剂盒进一步包括包含一种或多种选自下组的成分：核苷酸单体混合物、Mg2+、dTT、牛血清白蛋白、pH调节剂、DNase抑制剂、RNase、SO4 2-、Cl-、K+、Ca2+、Na+、(NH4)+的一种或多种试剂。
在一些实施方式中，所述混合物进一步包括细胞裂解剂，所述细胞裂解剂选自：蛋白酶K、胃蛋白酶、木瓜蛋白酶、NP-40、吐温、SDS、TritonX-100、EDTA和异硫氰酸胍中的一种或多种。
附图说明
通过下面说明书和所附的权利要求书并与附图结合，将会更加充分地描述本申请内容的上述和其他特征。可以理解，这些附图仅描绘了本申请内容的若干实施方式，因此不应认为是对本申请内容范围的限定。通过采用附图，本申请内容将会得到更加明确和详细地说明。
图1示出了本申请扩增方法的基本原理。
图2示出了本申请扩增方法中使用的第一类引物(线性扩增引物)的结构示意图。
图3示出了使用不同的第一类引物混合物对50pg人基因组DNA进行扩增，并将得到的扩增产物分别进行凝胶电泳的结果，其中自左向右第1泳道为分子量标记(M)，第2-13泳道为使用实验组1-12的引物混合物(具体见表1)对gDNA进行扩增获得的扩增样品，第14泳道为分子量标记。
图4示出了实验组1-12中获得的扩增产物在SBS测序中每个读数位置的A、T、C、G分布。
图5示出了使用表1中所示的1-12个实验组的引物混合物以正常人表皮成纤维细胞(AFP细胞)为起始样本进行扩增的扩增结果，自左向右第1泳道为分子量标记，第2-11泳道为单细胞扩增样品，第12泳道为分子量标记。
图6示出了使用表1中所示的实验组9/10和实验组11/12的引物混合物以正常人表皮成纤维细胞(AFP细胞)为起始样本进行扩增，并将得到的扩增产物分别进行凝胶电泳的结果。自左向右第1泳道为分子量标记，第2-11泳道为使用实验组11/12的引物混合物对单细胞进行扩增获得的扩增样品，第12泳道为分子量标记，第13-22泳道为使用实验组9/10的引物混合物对单细胞进行扩增获得的扩增样品，第23泳道为分子量标记。
图7示出了图6中的每个样本1_1、1_2…1_10及2_1、2_2…2_10在SBS测序中的数据量(以等体积扩增产物进行测序)。
图8示出了图6中的每个样本1_1、1_2…1_10及2_1、2_2…2_10在SBS测序中的拷贝数变异系数。
图9示出了图6中的每个样本1_1、1_2…1_10及2_1、2_2…2_10在SBS测序中每个染色体的拷贝数。
图10示出了对图6中的扩增样本1_1、1_2及2_1、2_2分别进一步针对表8中所列的35个致病位点基因进行PCR扩增，将扩增产物进行凝胶电泳的结果。从左向右每个泳道依次表示分子量标记物、针对表8所示的致病位点1-23进行的扩增结果、分子量标记物、针对表8所示的致病位点24-35进行的扩增结果、分子量标记物。
图11示出了使用表1中所示的实验组9/10的引物混合物以正常人表皮成纤维细胞(AFP细胞)为起始样本进行扩增，并将得到的扩增产物分别进行凝胶电泳的结果。从左向右每个泳道依次表示分子量标记物、使用实验组9/10的引物混合物对单细胞进行扩增获得的扩增样品(4个平行实验孔)、分子量标记物。
图12示出了对图11中的扩增样本1和2别进一步针对表8中所列的35个致病位点基因进行PCR扩增，将扩增产物进行凝胶电泳的结果。从左向右每个泳道依次表示分子量标记物、针对表8所示的致病位点1-23进行的扩增结果、分子量标记物、针对表6所示的致病位点24-35进行的扩增结果、分子量标记物。
图13示出了对图11中的扩增样本在半导体测序中每个染色体的拷贝数。
图14示出了使用表1中所示的实验组9/10的引物混合物对以囊胚培养液中DNA为起始样本进行扩增，并将扩增样本进行SBS测序得到的染色体拷贝数。
具体实施方式
本发明提供了扩增基因组DNA的方法，特别是扩增单细胞全基因组DNA的方法。
在本发明之前，通常是在基因扩增完成之后进行建库，在建库完成后再对其进行测序，这种方法流程复杂，耗时时间长。而本申请发明人通过设计特殊结构的引物并且优化扩增的过程，使得在单细胞扩增之后能够直接成库从而大幅减少单细胞全基因组DNA文库构建所需的时间。虽然在某些文献中报道了对引物的某些设计，但是这些设计均存在这样或那样的缺陷。例如在WO2012/166425中进行单细胞全基因组预扩增步骤时，引物的随机序列选自四种碱基(即，A、T、C和G)，但是使用这种方法进行直接扩增建库时会不可避免的自体或相互之间成环或形成二聚体，从而显著降低了扩增的效率。再例如，在US8,206,913中报道了引物中随机序列选自两种碱基(即，G和T、G和A、A和C、C和T)以避免自体或相互之间成环，但是由于使用这类引物扩增出的序列中目标序列前的碱基随机性很差，所以在整板上机进行SBS测序时必须添加阳性对照品来校正碱基随机性，否则无法进行检测，因此这种方法势必会浪费一定的数据量。而与上述现有技术不同，本发明中涉及的引物虽然包含较高的碱基随机性，但引物自身或引物之间基本不形成或与四碱基随机引物相比形成非常少的成环或二聚体，并且本发明构建出的文库中目标序列前具有较高的碱基随机性，因此根据本发明的方法进行扩增获得的扩增产物二聚体少、可以直接成库、可用于整版上机并且测序结果良好。
在一方面，本申请提供了扩增基因组DNA的方法，所述方法包括：(a)提供第一反应混合物，其中所述第一反应混合物包括包含所述基因组DNA的样本、第一引物、核苷酸单体混合物和核酸聚合酶，其中所述第一引物从5’端到3’端包含通用序列和第一可变序列，所述第一可变序列包括第一随机序列，其中所述第一随机序列从5’端到3’端依次为Xa1Xa2……Xan，所述第一随机序列的Xai(i＝1-n)均属于同一个集合，所述集合选自B、或D、或H、或V，其中B＝{T、G、C}，D＝{A、T、G}，H＝{T、A、C}，V＝{A、C、G}，其中Xai表示第一随机序列5’端的第i个核苷酸，n是选自3-20的正整数，可选地，所述第一反应混合物进一步包括第三引物，其中所述第三引物从5’端到3’端包含所述通用序列和第三可变序列，所述第三可变序列包括第三随机序列，其中所述第三随机序列从5’端到3’端依 次为Xb1Xb2……Xbn，所述第三随机序列的Xbi(i＝1-n)均属于同一个集合，所述集合选自B、或D、或H、或V，其中B＝{T、G、C}，D＝{A、T、G}，H＝{T、A、C}，V＝{A、C、G}，并且Xbi(i＝1-n)和Xai(i＝1-n)属于不同的集合，其中Xbi表示第三随机序列5’端的第i个核苷酸，n是选自3-20的正整数；(b)将所述第一反应混合物置于第一温度循环程序进行预扩增，获得预扩增产物；(c)提供第二反应混合物，所述第二反应混合物包括步骤(b)中得到的预扩增产物、第二引物、核苷酸单体混合物和核酸聚合酶，其中所述第二引物从5’端到3’端包含或由特定序列及所述通用序列组成；(d)将所述第二反应混合物置于第二温度循环程序进行扩增，获得扩增产物。本申请提供的方法的一种实施方式的图示请见图1。
步骤(a)：提供第一反应混合物
本申请的方法广泛适用于基因组DNA的扩增，特别是痕量的基因组DNA的扩增。
i.基因组DNA
本申请的方法优选适用于基因组DNA。在某些实施方式中，反应混合物中包含的基因组DNA的起始量不超过10ng、不超过5ng、不超过1ng、不超过500pg、不超过200pg、不超过100pg、不超过50pg、不超过20pg、或者不超过10pg。
基因组DNA可以来自生物样品，例如生物组织或含有细胞或游离DNA的体液。含有基因组DNA的样品可以通过已知的方法获取，例如通过口腔粘膜样本、鼻腔样本、头发、漱口水、脐带血、血浆、羊水、胚胎组织、内皮细胞、指甲样本、蹄样本等获取。生物样品可以是任何适当的形式提供，例如可以是石蜡包埋的形式，新鲜分离的形式等。基因组DNA可以来自任何物种或生物种类，例如但不限于，人类、哺乳动物、牛、猪、羊、马、啮齿动物、禽类、鱼类、斑马鱼、虾、植物、酵母、病毒或细菌。
在某些实施方式中，基因组DNA是来自于单个细胞的基因组DNA，或者来自两个或多个同类细胞的基因组DNA。单个细胞或同类细胞可以来自，例如，植入前的胚胎、孕妇外周血中的胚胎细胞、单精子、卵细胞、受精卵、癌细胞、细菌细胞、肿瘤循环细胞、肿瘤组织细胞、或者从任意组织获得的单个或多个同类细胞。本申请的方法可以用于扩增一些宝贵的样本或起始量低样本中的DNA，如人类的卵细胞、生殖细胞、肿瘤循环细胞、肿瘤组织细胞等。
在一些实施方式中，基因组DNA来源于卵裂球、囊胚滋养层、培养的细胞、提取后的gDNA或囊胚培养液。
获得单细胞的方法在本领域也是公知的，例如，可以通过流式细胞分选的方法(Herzenberg等人Proc Natl Acad Sci USA 76:1453-55,1979；lverson等人Prenatal Diagnosis 1:61-73,1981；Bianchi等人Prenatal Diagnosis 11:523-28,1991)、荧光激活细胞分选法、通过磁珠分离的方法(MACS,Ganshirt-Ahlert等人Am J Obstet Gynecol 166:1350,1992)、使用半自动细胞挑取仪(例如Stoelting公司生产的细胞转移系统QuixellTM)或者上述多种方法的结合。在一些实施方式中，可以使用梯度离心和流式细胞技术来提高分离和分选的效率。在一些实施方式中，可以根据单个细胞不同的性质来挑选特定类型的细胞，例如表达某种特定的生物标记的细胞。
获得基因组DNA的方法也是本领域公知的。在某些实施方式中，可以从生物样品中或单个细胞中裂解细胞并释放获得基因组DNA。可以使用本领域公知的任何适当的方法进行裂解，例如可以通过热裂解、碱裂解、酶裂解、机械裂解，或其任意组合的方式进行裂解(具体可参见，例如，U.S.7,521,246、Thermo Scientific Pierce Cell Lysis Technical Handbook v2和Current Protocols in Molecular Biology(1995).John Wiley和Sons,Inc.(supplement 29)pp.9.7.1-9.7.2.)。
机械裂解包括使用超声、高速搅拌、均质、加压(例如法式滤压壶)、减压和研磨等使用机械力破坏细胞的方法。最常用的机械裂解法是液体均质法，其迫使细胞悬浮液通过一个很狭窄的空间，从而对细胞膜施加剪切力(例如，如WO2013153176A1中所描述的)。
在某些实施方式中，可以使用温和的裂解方法。例如，可以将细胞在含有Tween-20的溶液中72℃加热2分钟、在水中65℃加热10分钟(Esumi等人，Neurosci Res 60(4):439-51(2008)、在含有0.5％NP-40的PCR缓冲液II(Applied Biosystems)中70℃加热90秒(Kurimoto等人，Nucleic Acids Res 34(5):e42(2006)、或者使用蛋白酶(例如蛋白酶K)或者离盐液(例如异硫氰酸胍)进行裂解(例如，如美国专利申请US 20070281313中所描述的)。
热裂解包括加热法和反复冻融法。在一些实施方式中，所述热裂解包括温度在20-100℃之间，裂解10-100分钟。在一些实施方式中，热裂解的温度可以是介于在20-90、30-90、40-90、50-90、60-90、70-90、80-90、30-80、40-80、50-80、60-80或70-80℃之间的任意温度。在一些实施方式中，热裂解的温度不低于20、30、40或50℃。在一些实施方式中，热裂解的温度不高于100、90或80℃。在一些实施方式中，热裂解时间可以是介于20-100、20-90、20-80、20-70、20-60、20-50、20-40、20-30、30-100、30-90、30-80、 30-70、30-60、30-50或30-40分钟之间的任意时间。在一些实施方式中，热裂解的时间不少于20、30、40、50、60、70、80或90分钟。在一些实施方式中，热裂解的时间不多于90、80、70、60、50、40、30或20分钟。在一些实施方式中，热裂解温度是随时间进行变化的。在一些实施方式中，热裂解是温度在30-60℃保持10-30分钟，之后在70-90℃保持5-20分钟。
在一些实施方式中，所述热裂解是在裂解试剂存在的条件下进行的。当裂解试剂存在时，可以降低裂解所需的时间或降低裂解所需的温度。裂解试剂可以破坏蛋白-蛋白、脂质-脂质和/或蛋白-脂质相互作用，从而促进细胞释放基因组DNA。
在一些实施方式中，所述裂解试剂包括表面活性剂和/或裂解酶。表面活性剂可以分为离子型、两性和非离子型表面活性剂。一般情况下，两性和非离子型表面活性剂的裂解效能弱于离子型表面活性剂。示例性的表面活性剂包括，但不限于，NP-40、吐温、SDS、GHAPS、TritonX-100、TritonX-114、EDTA、脱氧胆酸钠、胆酸钠、异硫氰酸胍中的一种或多种。本领域技术人员可以根据实际的需要选择表面活性剂的种类和浓度。在一些实施方式中，表面活性剂的工作浓度为0.01％-5％、0.1％-3％、0.3％-2％或0.5-1％。
示例性的裂解酶可以是蛋白酶K、胃蛋白酶、木瓜蛋白酶等，或其任意组合。在一些实施方式中，裂解酶的工作浓度为0.01％-1％、0.02％-0.5％、0.03％-0.2％或0.4-0.1％。
在本申请提供的方法中，可以在第一反应混合物中直接使用含有基因组DNA的裂解产物，例如，可以将生物样品预先进行裂解处理，得到裂解产物，然后将裂解产物与第一反应混合物的其他成分混合。如有需要，可以对裂解产物经过进一步的处理，以分离得到其中的基因组DNA，再将该分离的基因组DNA与第一反应混合物的其他成分混合得到第一反应混合物。
在一些实施方式中，裂解后的核酸样品无需进行纯化即可进行扩增。在一些实施方式中，裂解后的核酸样品在进行纯化后再进行扩增。在一些实施方式中，裂解过程中DNA已经发生了不同程度的断裂，而无需特殊打断步骤即可用于扩增。在一些实施方式中，裂解后的核酸样品在经过打断处理后再进行扩增。
本申请还提供了一种更为简便的方法，即，直接将包含基因组DNA的细胞与扩增所需的其他成分混合得到第一反应混合物，也就是说，在第一反应混合物中的基因组DNA存在于细胞内部。在这样的情况下，第一反应混合物中还可以进一步含有能够裂解所述细 胞的表面活性剂(例如但不限于，NP-40、吐温、SDS、TritonX-100、EDTA、异硫氰酸胍中的一种或多种)和/或裂解酶(例如蛋白酶K、胃蛋白酶、木瓜蛋白酶中的一种或多种)。这样，细胞的裂解和基因组DNA的预扩增都在同一个反应混合物中进行，能够提高了反应效率和缩短反应时间。
在某些实施方式中，本申请提供的方法在步骤(a)完成以后并且在进行步骤(b)之前还可以进一步包括将所述反应混合物置于裂解温度循环程序，使得所述细胞裂解并释放出所述基因组DNA。本领域技术人员根据反应混合物中含有的裂解成分、细胞的种类等可以选择适当的裂解温度循环程序。示例的裂解温度循环程序包括，将反应混合物置于50℃3分钟到8小时(例如，在3分钟到7小时、3分钟到6小时、3分钟到5小时、3分钟到4小时、3分钟到3小时、3分钟到2小时、3分钟到1小时、3分钟到40分钟、3分钟到20分钟之间的任意时间，例如10分钟、20分钟、30分钟等)，然后置于80℃2分钟到8小时(例如，在2分钟到7小时、2分钟到6小时、2分钟到5小时、2分钟到4小时、2分钟到3小时、2分钟到2小时、2分钟到1小时、2分钟到40分钟、2分钟到20分钟之间的任意时间，例如10分钟、20分钟、30分钟等)。裂解温度程序可以进行1个循环，如有需要，也可以进行两个或更多个循环，取决于具体的裂解条件。
ii.第一类引物
本申请所述的方法中涉及两大类不同的引物，其中第一类引物从5’端到3’端包含通用序列和可变序列，所述第二类引物包含特定序列和通用序列，但是不包含任何可变序列。本文中所述的“第一引物”和“第三引物”均属于上述第一类引物。在第一反应混合物中包括的第一引物从5’端到3’端包含通用序列和第一可变序列；而在第一反应混合物中可选地包括的第三引物从5’端到3’端包括通用序列和第三可变序列。在一些实施方式中，第一类引物由通用序列和可变序列组成。在另一些实施方式中，第一类引物由通用序列、可变序列和间隔序列组成。
通用序列
通用序列在本申请中是指第一类引物和第二类引物在其5’端均具有的核苷酸序列。通用序列的长度可以是例如，6-60、8-50、9-40、10-30、10-15或25-30个碱基。在本申请中，选择适当的通用序列，使得基本上不会与基因组DNA结合而产生扩增，并且避免第一类引物与第一类引物之间的聚合(例如，第一引物与第一引物之间、第三引物与第三引物之间或第一引物和第三引物之间)以及第一类引物自身的成环(例如，第一引物5’端的部分 序列与3’端的部分序列互补而第一引物自身形成发卡结构、或第三引物5’端的部分序列与3’端的部分序列互补而第三引物自身形成发卡结构)，以及第一类引物与第二类引物之间的聚合或成环的情况。
在某些实施方式中，通用序列中包含全部4类碱基A、T、C、G。在某些实施方式中，通用序列中仅包含三类或两类自身互补配对能力较弱的碱基，而不含有另一种或两种碱基。在某些实施方式中，通用序列由G、A和T三种碱基组成，即通用序列中不含有C碱基。在某些实施方式中，通用序列由C、A和T三种碱基组成，即通用序列中不含有G碱基。在某些实施方式中，通用序列由A和T、A和C、A和G、T和C或T和G两种碱基组成，即通用序列中不同时含有G和C碱基。不希望受理论限制，但认为通用序列中如果含有C或G碱基可能会导致引物与引物之间的相互聚合，产生多聚体，从而削弱对基因组DNA的扩增能力。优选地，通用序列中不具有能够自身配对的序列、会导致引物与引物之间配对的序列，或者连续多个同种的碱基。
在某些实施方式中，可以选择适当的通用序列的碱基序列以及其中各碱基的比例，以确保通用序列本身不与基因组DNA模板序列发生碱基配对或产生扩增。
在某些实施方式中，可以选择所述通用序列使得扩增产物能够直接进行测序。不希望受到理论的约束，可以将通用序列设计成包括与测序用引物的部分或全部互补或者相同的序列(例如，与测序用引物的部分相同、全部相同、部分互补、或全部互补的序列)。在某些实施方式中，根据不同的测序平台针对性地选择通用序列。在某些实施方式中，根据第二代或第三代测序平台针对性地选择通用序列。在某些实施方式中，根据Illumina的NGS测序平台针对性地选择通用序列。在某些实施方式中，根据Ion torrent测序平台针对性地选择通用序列。
在某些实施方式中，所述通用序列选自下组：SEQ ID NO:1[TTGGTAGTGAGTG]、SEQ ID NO:2[GAGGTGTGATGGA]、SEQ ID NO:3[GTGATGGTTGAGGTA]、SEQ ID NO:4[AGATGTGTATAAGAGACAG]、SEQ ID NO:5[GTGAGTGATGGTTGAGGTAGTGTGGAG]和SEQ ID NO:6[GCTCTTCCGATCT]。
可变序列
第一类引物从5’端到3’端包含通用序列和可变序列(例如第一引物/第三引物分别包含第一/第三可变序列)，其中第一类引物中的通用序列都相同，但是可变序列可能各不相同。 例如，在一些实施方式中，第一/第三引物分别为包括相同的通用序列和不同的可变序列的引物混合物。可变序列在本申请中是指序列不固定的一段碱基序列，其可以包含随机序列(例如第一/第三可变序列分别包含第一/第三随机序列)。在一些实施方式中，可变序列由随机序列组成。在另一些实施方式中，可变序列由随机序列和固定序列组成。
a)随机序列
随机序列是指该序列每个碱基位置上的碱基均从某个特定集合中各自独立地随机选出，因此上述随机序列代表了由不同碱基组合构成的碱基序列的集合。
具体而言，例如，第一可变序列可包括第一随机序列，其中所述第一随机序列的碱基数为n，n是选自3-20的正整数，第一随机序列从5’端到3’端的序列可以表示为Xa1Xa2……Xan，而其中任意碱基位置i上的碱基(即，第一随机序列5’端的第i个核苷酸，i＝1-n)可用Xai来代表，其中每个Xai均是从一个特定的集合中随机选择，例如，由A、T、G、C中的特定两种或三种核苷酸组成的集合。通常可以通过简并标识的方法表示上述任意碱基位置上可选择的集合，例如，可将仅包含A、G两种核苷酸的集合表示为R(即R＝{A、G})，其他的可以简并标识方式表示的集合还包括：Y＝{C、T}、M＝{A、C}、K＝{G、T}、S＝{C、G}、W＝{A、T}、H＝{A、C、T}、B＝{C、G、T}、V＝{A、C、G}、D＝{A、G、T}、N＝{A、C、G、T}。
可以通过完全随机的方式选择随机序列(即随机序列中的任意碱基位置)，也可以在随机的基础上进一步增加某些限定条件，从而排除一些不希望的情况或者增加与目标基因组DNA的匹配程度。在某些实施方式中，为避免可变序列与通用序列产生互补配对，当通用序列含有大量G时，随机序列中的任意碱基位置均选自集合D(即，不为C)；或者当通用序列含有大量C时，随机序列中的任意碱基位置均选自集合H(即，不为G)；当通用序列含有大量T时，随机序列中的任意碱基位置均选自集合B(即，不为A)；或者当通用序列含有大量A时，随机序列中的任意碱基位置均选自集合V(即，不为T)。
随机序列可以具有适当的长度，例如2-20个碱基、2-19个碱基、2-18个碱基、2-17个碱基、2-16个碱基、2-15个碱基、2-14个碱基、2-13个碱基、2-12个碱基、2-11个碱基、2-12个碱基、2-11个碱基、2-10个碱基、2-9个碱基、2-8个碱基、3-18个碱基、3-16个碱基、3-14个碱基、3-12个碱基、3-10个碱基，4-16个碱基、4-12个碱基、4-9个碱基、或5-8个碱基。在某些实施方式中，随机序列的长度为5个碱基。在某些实施方式中，随机序列的长度为8个碱基。理论上来说，如果随机序列的每个碱基位置都从A、T、G三 种碱基中随机选择的话，那么长度为4个碱基的可变序列可以组合出34＝81种可能的随机序列，长度为5个碱基的随机序列可以组合出35＝243种可能的随机序列，以此类推。这些随机序列可以与基因组DNA上的不同位置的对应序列互补配对，从而在基因组DNA的不同位置开始复制。
在一个实施方式中，第一随机序列中每个任意碱基位置i上的碱基Xai(i＝1-n)均属于同一个集合，并且其中所述集合选自B、D、H或V中的一个。作为一个非限制性的例子，第一引物以具有通用序列和第一随机序列，其中n＝5，随机序列的每个任意Xai(i＝1-5)均属于同一集合B，即，该随机序列可表示为BBBBB或者(B)5，随机序列可以选自{TTTTT,TGTTT,TCTTT,TTGTT,TTCTT……}，共35＝243种序列组合。在包括这种第一引物的特定第一反应混合物中，这些第一引物均具有相同的通用序列及上述的第一随机序列，即，在这个特定第一反应物中的第一引物是一组引物，这些引物均具有相同的通用序列，并且具有由选自集合B的碱基组成的相同或不同的随机序列。
除非另有明确的说明，本文中所有对于第一引物及其各个部分的描述均适用与第三引物及其相应部分。相似地，在第一反应混合物中进一步包含第三引物的情况下，第三引物中的第三可变序列可包括第三随机序列，其中所述第三随机序列从5’端到3’端依次为Xb1Xb2……Xbn，优选地所述第三随机序列的Xbi(i＝1-n)均属于同一个集合，所述集合选自B、或D、或H、或V，其中B＝{T、G、C}，D＝{A、T、G}，H＝{T、A、C}，V＝{A、C、G}，并且Xbi(i＝1-n)和Xai(i＝1-n)属于不同的集合，其中Xbi表示第三随机序列5’端的第i个核苷酸，n是选自3-20的正整数。在一个特定的第一反应混合物中，包括一定量的第一引物，这些第一引物均具有相同的通用序列及长度为n的第一随机序列，其中第一随机序列的每个碱基Xai均属于同一个集合，并且其中所述集合选自B、D、H或V；同时上述第一反应混合物中进一步包括一定量的第三引物，这些第三引物均具有相同的通用序列及长度为n的第一随机序列，其中第一随机序列的每个碱基Xbi均属于同一个集合，并且其中所述集合选自B、D、H或V，并且Xbi和Xai属于不同的集合。在一些实施方式中，第一随机序列和第三随机序列的长度相同。在另一些实施方式中，第一随机序列和第三随机序列的长度不同。
b)固定序列
可变序列在其3’端还可以进一步包括固定序列，所述固定序列可以选自任何能够提高基因组覆盖度的碱基组合。本申请所述的固定序列包括但不限于选自CCC、AAA、TGGG、 GTTT、GGG、TTT、TNTNG或GTGG的序列。在本申请中描述固定序列时使用的N表示选自A、T、C、G中的任一种单核苷酸，而并非表示选自N的随机序列。同一引物组中，例如第一引物中，从5’端到3’端可以依次包括相同的通用序列、含有不同序列组合的随机序列和相同的固定序列(例如所有第一引物在其3’端均包括TGGG或GTTT中的任一种)。或者，同一引物组中，例如第一引物中，从5’端到3’端可以依次包括相同的通用序列、含有不同序列组合的随机序列和不同的固定序列(例如第一引物中包含在其3’端均包括TGGG的引物混合物以及或在其3’端均包括GTTT的引物混合物)。在一些实施方式中，第一反应混合物包括第一引物和第三引物，其中第一引物中的第一可变序列选自Xa1Xa2……XanGGG、Xa1Xa2……XanTTT、Xa1Xa2……XanTGGG或Xa1Xa2……XanGTTT，第三引物中的第三可变序列选自Xb1Xb2……XbnGGG、Xb1Xb2……XbnTTT、Xb1Xb2……XbnTGGG或Xb1Xb2……XbnGTTT。
在某些实施方式中，还可以通过统计计算，选择在基因组上分布更加均匀，覆盖度更高的可变序列，从而增加可变序列与基因组DNA的识别机会。
在某些实施方式中，可变序列选自下组：(B)nCCC、(B)nAAA、(B)nTGGG、(B)nGTTT、(B)nGGG、(B)nTTT、(B)nTNTNG、(B)nGTGGGGG、(D)nCCC、(D)nAAA、(D)nTGGG、(D)nGTTT、(D)nGGG、(D)nTTT、(D)nTNTNG、(D)nGTGGGGG、(H)nCCC、(H)nAAA、(H)nTGGG、(H)nGTTT、(H)nGGG、(H)nTTT、(H)nTNTNG、(H)nGTGGGGG、(V)nCCC、(V)nAAA、(V)nTGGG、(V)nGTTT、(V)nGGG、(V)nTTT、(V)nTNTNG、(V)nGTGGGGG，其中n是选自3-17的正整数。在某些实施方式中，所述第一引物中的第一可变序列可以具有(B)nCCC、(B)nAAA、(B)nTGGG、(B)nGTTT、(B)nGGG、(B)nTTT、(B)nTNTNG、(B)nGTGGGGG中的一种或多种序列。在某些实施方式中，所述第三引物中的第三可变序列可以具有(D)nCCC、(D)nAAA、(D)nTGGG、(D)nGTTT、(D)nGGG、(D)nTTT、(D)nTNTNG、(D)nGTGGGGG中的一种或多种序列。
间隔序列
第一类引物的通用序列和可变序列可以是直接相邻的，或者也可以具有一个或多个碱基的间隔序列。在某些实施方式中，通用序列和可变序列通过长度为m的间隔序列相连，其中m是选自1-3的正整数。在为了排除一些不希望的情况(例如引物二聚体等)或者为了增加与目标基因组DNA的匹配程度而对可变序列中的随机序列进行一定程度的限制时，可以在通用序列和可变序列中引入m个完全随机地选自A、T、G、C的碱基(长度为m 的间隔序列)，以在不增加引物二聚体产生程度的情况下进一步增加第一类引物在目标基因组DNA上的覆盖率。
在一些实施方式中，第一引物中的通用序列和第一可变序列之间通过第一间隔序列相连，所述第一间隔序列为Ya1……Yam，其中Yaj(j＝1-m)∈{A、T、G、C}，其中Yaj表示第一间隔序列5’端的第j个核苷酸，m是选自1-3的正整数。在一些实施方式中，第三引物中的通用序列和第三可变序列之间通过第三间隔序列相连，所述第一间隔序列为Yb1……Ybm，其中Ybj(j＝1-m)∈{A、T、G、C}，其中Ybj表示第三间隔序列5’端的第j个核苷酸，m是选自1-3的正整数。在一些实施方式中，m为1，即第一引物中通用序列和第一可变序列之间通过一个选自集合N的碱基相连，第三引物中通用序列和第三可变序列之间通过一个选自集合N的碱基相连。
在某些实施方式中，设计第一引物(以及可选的第三引物)以使得其扩增产物可直接用于Illumina的NGS测序平台，其中第一引物包括GCTCTTCCGATCTYa1Xa1Xa2Xa3Xa4Xa5TGGG、GCTCTTCCGATCTYa1Xa1Xa2Xa3Xa4Xa5GTTT所示的序列或其混合物，第三引物包括GCTCTTCCGATCTYb1Xb1Xb2Xb3Xb4Xb5TGGG、GCTCTTCCGATCTYb1Xb1Xb2Xb3Xb4Xb5GTTT或其混合物，其中每个任意碱基位置i上的碱基Xai(i＝1-n)均属于同一个集合，其中所述集合选自B、D、H或V中的一个，以及每个任意碱基位置i上的碱基Xbi(i＝1-n)均属于同一个集合，其中所述集合选自B、D、H或V中的一个，并且Xbi(i＝1-n)和Xai(i＝1-n)属于不同的集合；其中Ya1∈{A、T、G、C}，Yb1∈{A、T、G、C}。在某些特定实施方式中，上述Xai(i＝1-5)∈{T、G、C}，Xbi(i＝1-5)∈{A、T、G}，即第一引物包括SEQ ID NO:7、SEQ ID NO:11所示的序列或其混合物，第三引物包括SEQ ID NO:8、SEQ ID NO:12所示的序列或其混合物。
在某些实施方式中，第一类引物包含或者由选自SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:14所示的序列组成，其中各第一类引物的通用序列包含或由SEQ ID NO:6组成。在某些实施方式中，第一类引物包括由SEQ ID NO:7所示的序列组成的引物和/或由SEQ ID NO:11所示的序列组成的引物。在某些实施方式中，第一类引物包括由SEQ ID NO:8所示的序列组成的引物和由SEQ ID NO:12所示的序列组成的引物。在某些实施方式中，第一类引物包括由SEQ ID NO:7所示的序列组成的引物或由SEQ ID NO:11所示的序列组成的引物； 以及由SEQ ID NO:8所示的序列组成的引物或由SEQ ID NO:12所示的序列组成的引物。在某些实施方式中，第一类引物包含由SEQ ID NO:7所示的序列组成的引物、由SEQ ID NO:11所示的序列组成的引物、由SEQ ID NO:8所示的序列组成的引物和由SEQ ID NO:12所示的序列组成的引物。
在某些实施方式中，第一类引物包含或者由选自SEQ ID NO:15-22所示的序列组成，其中各第一类引物的通用序列包含或由SEQ ID NO:1组成。在某些实施方式中，第一类引物包括由SEQ ID NO:15所示的序列组成的引物和/或由SEQ ID NO:19所示的序列组成的引物。在某些实施方式中，第一类引物包括由SEQ ID NO:16所示的序列组成的引物和/或由SEQ ID NO:20所示的序列组成的引物。在某些实施方式中，第一类引物括由SEQ ID NO:15所示的序列组成的引物或由SEQ ID NO:19所示的序列组成的引物；以及由SEQ ID NO:16所示的序列组成的引物或由SEQ ID NO:20所示的序列组成的引物。在某些实施方式中，第一类引物包含由SEQ ID NO:15所示的序列组成的引物、由SEQ ID NO:19所示的序列组成的引物、由SEQ ID NO:16所示的序列组成的引物和由SEQ ID NO:20所示的序列组成的引物。
在某些实施方式中，第一类引物包含或者由选自SEQ ID NO:23-30所示的序列组成，其中各第一类引物的的通用序列包含或由SEQ ID NO:2组成。在某些实施方式中，第一类引物包括由SEQ ID NO:23所示的序列组成的引物和/或由SEQ ID NO:27所示的序列组成的引物中的一种或两种。在某些实施方式中，第一类引物包括由SEQ ID NO:24所示的序列组成的引物和/或由SEQ ID NO:28所示的序列组成的引物中的一种或两种。在某些实施方式中，第一类引物括由SEQ ID NO:23所示的序列组成的引物或由SEQ ID NO:27所示的序列组成的引物；以及由SEQ ID NO:24所示的序列组成的引物或由SEQ ID NO:28所示的序列组成的引物。在某些实施方式中，第一类引物包含由SEQ ID NO:23所示的序列组成的引物、由SEQ ID NO:27所示的序列组成的引物、由SEQ ID NO:24所示的序列组成的引物和由SEQ ID NO:28所示的序列组成的引物。
在一些实施方式中，第一和第三引物在第一反应混合物中的总浓度为10-150ng/μL。在一些实施方式中，第一和第三引物在第一反应混合物中的总浓度为10-120ng/μL、10-100ng/μL、10-90ng/μL、10-80ng/μL、10-70ng/μL、10-60ng/μL、10-50ng/μL、10-40ng/μL、20-120ng/μL、20-100ng/μL、20-80ng/μL、20-70ng/μL、20-60ng/μL、20-50ng/μL、30-140ng/μL、30-120ng/μL、30-100ng/μL、30-80ng/μL、30-60ng/μL或30-40ng/μL。在一些实 施方式中，第一和第三引物在第一反应混合物中的浓度分别为10-140ng/μL、10-120ng/μL、10-100ng/μL、10-80ng/μL、10-60ng/μL、10-30ng/μL、10-20ng/μL、20-120ng/μL、20-100ng/μL、20-80ng/μL、20-60ng/μL、20-40ng/μL或20-30ng/μL。在一些实施方式中，第一和第三引物在第一反应混合物中的浓度分别为15ng/μL、30ng/μL或60ng/μL。在一些实施方式中，第一引物和第三引物在第一反应混合物中的浓度相同。在一些实施方式中，在第一反应混合物中的第一和第三引物分别为100-800pmol。在一些实施方式中，在第一反应混合物中的第一和第三引物一共为400-600pmol。
iii.其他成分
第一反应混合物还包括DNA扩增所需的其他组分，例如核酸聚合酶、核苷酸单体混合物、以及酶活性所需的适当的金属离子和缓冲液成分等。至少一种或多种这些成分可以使用本领域已知的试剂。
核酸聚合酶在本申请中是指能够合成新的核酸链的酶。任何适用于本申请方法的核酸聚合酶都可以使用。优选使用DNA聚合酶。在某些实施方式中，本申请的方法使用热稳定的核酸聚合酶，例如那些在PCR扩增的温度下(例如95摄氏度)聚合酶活性不会下降或者下降小于1％、3％、5％、7％、10％、20％、30％、40％或者50％的那些核酸聚合酶。在某些实施方式中，本申请的方法使用的核酸聚合酶具有链置换活性。本申请所述的“链置换活性”是指核酸聚合酶的一种活性，其能够使得核酸模板和与其配对结合的互补链分离，并且这种分离以从5’到3’的方向进行并伴随着新的与模板互补的核酸链的生成。具有链置换能力的核酸聚合酶及其应用是本领域已知的，例如可以参见美国专利U.S.5824517，该专利的全部内容通过引用并入本申请。适合的核酸聚合酶包括，但不限于：Phi29DNA聚合酶、Bst DNA聚合酶、Bst 2.0DNA聚合酶、Pyrophage 3137、Vent聚合酶(例如Thermococcus litoralis的Vent聚合酶、Deep Vent聚合酶、Vent(-exo)聚合酶、Deep Vent(-exo)聚合酶)、TOPOTaq DNA聚合酶、9°Nm聚合酶、Klenow Fragment DNA聚合酶I、MMLV反转录酶、AMV反转录酶、HIV反转录酶、T7phase DNA聚合酶变种(缺少3’-5’外切酶活性)、
Figure PCTCN2017081977-appb-000003
超保真DNA聚合酶、Taq聚合酶、Psp GBD(exo-)DNA聚合酶、Bst DNA聚合酶(全长)、E.coli DNA聚合酶、LongAmp Taq DNA聚合酶、OneTaq DNA聚合酶中的一种或多种。
核苷酸单体混合物在本申请中是指dATP、dTTP、dGTP、dCTP的混合物。
在某些实施方式中，第一反应混合物中含有Thermococcus litoralis的Vent聚合酶、 Deep Vent聚合酶、Vent(-exo)聚合酶、或Deep Vent(-exo)聚合酶中的一种或多种。在某些实施方式中，反应混合物中含有Thermococcus litoralis的Vent聚合酶。Thermococcus litoralis的Vent聚合酶是指分离自Thermococcus litoralis的天然的聚合酶。在某些实施方式中，反应混合物中含有Deep Vent聚合酶。Deep Vent聚合酶是指分离自Pyrococcus species GB-D的天然的聚合酶。在某些实施方式中，反应混合物中含有Vent(-exo)聚合酶。Vent(-exo)聚合酶是指将Thermococcus litoralis的Vent聚合酶进行过D141A/E143A基因改造的酶。在某些实施方式中，反应混合物中含有Deep Vent(-exo)聚合酶。Deep Vent(-exo)聚合酶是指对Deep Vent聚合酶进行过D141A/E143A基因改造的酶。本申请中所述的各种Vent聚合酶可以从商业途径获得，例如从New England Biolabs公司获得。
第一反应混合物中还可以包括核酸聚合酶发挥酶活性所需的适当的金属离子(例如，适当浓度的Mg2+离子(例如终浓度可以为约1.5mM到约8mM)，核苷酸单体混合物(例如dATP、dGTP、dTTP和dCTP)、牛血清白蛋白(BSA)、dTT(例如终浓度可以为约2mM到约7mM)、纯水等。
在某些实施方式中，第一反应混合物中还可以进一步包括pH调节剂，使得混合物的pH值维持在7.0-9.0之间。适当的pH调节剂可以包括，例如Tris HCl和Tris SO4。在某些实施方式中，第一反应混合物中还可以进一步包括一种或多种其他成分，例如DNase抑制剂、RNase、SO4 2-、Cl-、K+、Ca2+、Na+、和/或(NH4)+等。
步骤(b)：置于第一温度循环程序
本申请提供的方法包括步骤(b)：将所述第一反应混合物置于第一温度循环程序，使得所述第一类引物(第一引物或第一引物和第三引物)的可变序列能够与所述基因组DNA通过碱基配对结合，在核酸聚合酶的作用下复制基因组DNA。
“扩增”在本申请中是指，在核酸聚合酶的作用下，在引物的3’端添加与核酸模板互补的核苷酸，从而合成得到与核酸模板碱基互补的新的核酸链。可以使用适合的扩增核酸的方法，例如聚合酶链式反应(PCR)、连接酶链式反应(LCR)，或其他适合的扩增方法。这些方法都是本领域已知的，可以参见例如美国专利U.S.4,683,195和U.S.4,683,202，以及Innis等人"PCR protocols:a guide to method and applications"Academic Press,Incorporated(1990)和Wu等人(1989)Genomics 4:560-569，这些文献和专利的全部内容通过引用并入本申请。
在扩增过程中，将反应混合物置于适当的温度循环程序，使得DNA模板双链解开成单链，第一/第三引物与模板单链杂交，然后在DNA聚合酶的作用下在引物的3’端进行延伸。因此，温度循环程序通常包括：变性或解链温度，在该温度下DNA模板双链解开成单链；退火温度，在该温度下引物与DNA模板单链特异性杂交；以及延伸温度，在该温度下DNA聚合酶在引物的3’端添加与DNA模板碱基互补的核苷酸，使得引物得以延长，得到与DNA模板互补的新的DNA链。
在第一温度循环程序中的第一次循环，首先将第一反应混合物置于能够打开所述基因组DNA的双链的温度程序(步骤(b1))。在第一轮循环中，为确保基因组DNA双链完全解开成单链(即，变性/解链)，可以使用较高的反应温度(例如90℃-95℃)，并且可以保持较长的反应时间(例如在介于90-95℃之间的温度反应1-20分钟)。而在后续循环中，需要解开的双链为扩增过程中生成的双链，在此情况下，只要待扩增的半扩增子或全扩增子双链能够变性成为单链即可，因此需要的解链时间无需很长(例如在介于90-95℃的温度之间解链反应3-50秒)。
然后，将第一反应混合物置于能够使所述第一类引物(第一引物或第一引物和第三引物)与DNA单链模板结合的温度程序(步骤(b2))。在这个温度程序中，第一类引物中的可变序列与基因组DNA中的不同位置的互补序列通过碱基互补结合(即，退火)，并由此在基因组DNA的不同位置开启复制。由于第一类引物中的可变序列各不相同，其中的碱基比例、序列都存在差异，因此每个可变序列与基因组DNA结合的最佳温度也存在较大的差别。这样，在某个特定的退火温度下，可能只有一部分的引物能够很好地与基因组DNA结合，而另一部分引物与基因组DNA的结合可能并不理想。在某些实施方式中，所述步骤(b2)包括将所述反应混合物置于多于一种温度的程序，以促使所述第一类引物充分与所述DNA模板有效结合。例如，可以将DNA变性的反应混合物快速降温至低温，例如约10℃-20℃，再通过梯度升温的方式，使得反应混合物分别在不同的退火温度下反应适当的时间，从而确保尽可能多的引物与基因组DNA配对结合。在某些实施方式中，步骤(b2)包括在介于10-20℃之间的第一退火温度(例如15℃)反应适当的时间(例如3-60秒)，在介于20-30℃之间的第二退火温度(例如25℃)反应适当的时间(例如3-50秒)，以及在介于30-50℃之间的第三退火温度(例如35℃)反应适当的时间(例如3-50秒)。
本领域公知，引物的退火温度通常不会比引物Tm值低5℃以上，而过低的退火温度会导致引物与引物之间发生非特异性结合，从而导致出现引物聚合体以及非特异性扩增产 物。因此，通常在引物退火温度中不会使用如10℃-20℃这样的低温。但是，本申请的发明人意想不到地发现，即使从低温(例如10℃-20℃)开始梯度升温，引物与基因组DNA之间的配对仍然能够保持很好的特异性，扩增结果仍然保持非常低的变异性，表明扩增的结果准确可靠。同时，由于引物退火温度覆盖了低温的情况，因此可以确保更广范围的引物序列与基因组DNA的结合，从而能够提供更好的基因组覆盖率和扩增深度。在退火温度程序后，将所述反应混合物置于能够使与DNA单链模板结合的第一类引物在所述核酸聚合酶的作用下延伸长度的温度程序，以产生扩增产物(步骤(b3))。
延伸温度通常与DNA聚合酶的最适温度相关，本领域技术人员可以根据具体的反应混合物进行具体的选择。在某些实施方式中，在反应混合物中的DNA聚合酶可以具有链置换活性，这样，如果引物在延伸的过程中遇到与下游模板结合的引物或扩增子，DNA聚合酶的链置换活性可以使这些下游结合的引物与模板链分开，从而确保延伸中的引物可以继续延伸，以得到较长的扩增序列。具有链置换活性的DNA聚合酶包括但不限于，例如，phi29DNA聚合酶、T5DNA聚合酶、SEQUENASE 1.0和SEQUENASE 2.0。在某些实施方式中，在反应混合物中的DNA聚合酶是热稳定的DNA聚合酶。热稳定的DNA聚合酶包括但不限于，例如，Taq DNA聚合酶、OmniBaseTM序列酶、Pfu DNA聚合酶、TaqBeadTM热启动聚合酶、Vent DNA聚合酶(例如Thermococcus litoralis的Vent聚合酶、Deep Vent聚合酶、Vent(-exo)聚合酶、Deep Vent(-exo)聚合酶)、Tub DNA聚合酶、TaqPlus DNA聚合酶、Tfl DNA聚合酶、Tli DNA聚合酶和Tth DNA聚合酶。在某些实施方式中，反应混合物中的DNA聚合酶可以是热稳定并且具有链置换活性的DNA聚合酶。在某些实施方式中，在反应混合物中的DNA聚合酶选自：Phi29DNA聚合酶、Bst DNA聚合酶、Pyrophage 3137、Vent聚合酶(例如Thermococcus litoralis的Vent聚合酶、Deep Vent聚合酶、Vent(-exo)聚合酶、Deep Vent(-exo)聚合酶)、TOPOTaq DNA聚合酶、9°Nm聚合酶、Klenow Fragment DNA聚合酶I、MMLV反转录酶、AMV反转录酶、HIV反转录酶、T7phase DNA聚合酶变种(缺少3’-5’外切酶活性)、
Figure PCTCN2017081977-appb-000004
超保真DNA聚合酶、Taq聚合酶、Bst DNA聚合酶(全长)、E.coli DNA聚合酶、LongAmp Taq DNA聚合酶、OneTaq DNA聚合酶中的一种或多种。
在某些实施方式中，步骤(b3)包括在介于60-90℃之间的延伸温度(例如，在65-90℃、70-90℃、75-90℃、80-90℃、60-85℃、60-80℃、60-75℃、70-80℃之间，或在60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75℃下)反应10秒-15分钟(例 如，1-14、1-13、1-12、1-11、1-10、1-9、1-8、1-7、1-6、1-5、1-4、1-3、1-2、2-14、3-14、5-14、6-14、7-14、8-14、9-14、10-14、11-14、12-14、13-14分钟，或者10-60、10-50、10-40、10-30、10-20、20-60、20-50、20-40、20-30、30-60、30-50、30-40秒)。在某些实施方式中，步骤(b3)包括在60-80℃之间的一个或多个温度下反应30秒-2分钟。在某些实施方式中，步骤(b3)包括在65℃反应40秒。在某些实施方式中，步骤(b3)包括在75℃反应40秒。在某些实施方式中，步骤(b3)包括在65℃反应40秒之后再在75℃反应40秒。
引物延伸程序后，重复步骤(b1)到(b3)至指定的第一循环次数，如上所述在后续循环中，步骤(b1)中的解链温度与第一轮循环中的解链温度相近，但反应时间可以略短。在某些实施方式中，在第一轮循环后的循环中步骤(b1)包括在90-95℃的温度之间反应10-50秒。
本申请所述的第一循环次数至少为2。在第一次循环时，第一类引物的可变序列的3’端的序列得以延长，得到的扩增产物在5’端为通用序列，3’端为与基因组模板单链序列互补的序列，这样的扩增产物也称为半扩增子。在第二次循环时，之前的半扩增子本身也可以作为DNA模板与第一类引物中的可变序列结合，引物在核酸聚合酶的作用下向扩增产物的5’端延伸，直到复制完扩增产物5’末端的通用序列，由此得到在5’端为通用序列，3’端为通用序列的互补序列的基因组扩增产物，这样的扩增产物也称为全扩增子。本申请所述的预扩增产物主要是指5’端为通用序列而3’端为通用序列的互补序列的全扩增子。
在第一次循环后的后续扩增中，反应混合物中的DNA单链不仅包含原始的基因组DNA单链，也包含扩增得到的新合成的DNA单链，其中原始基因组DNA模板以及初始扩增中产生的半扩增子均可再次作为新的DNA模板，与引物结合并开启新一轮的DNA合成；但由于全扩增子两端包含互补的序列(5’端包含的通用序列和3’端包含的通用序列的互补序列)，因此会自身形成发卡结构，从而不能在下一个反应循环中再次作为新的DNA模板，进行新一轮的DNA合成。
在某些实施方式中，将第一循环的次数控制在适当的范围内，以确保既有足够的预扩增产物用于后续的反应，又不会因为循环次数过多影响整个流程的反应时间。在某些实施方式中，第一循环的次数为2-40个循环(例如，2-40个、4-40个、6-40个、8-40个、10-40个、12-40个、14-40个、16-40个、18-40个、20-40个、15-40个、20-40个、25-40个、30-40个、5-35个、10-35个、15-35个、20-35个、25-35个、30-35个、10-30个、15-30个、20-30个、25-30个、2-20个、2-18个、2-16个、2-14个、2-12个、2-10个、2-8个、2-6个、2-4个、4-20个、4-18个、4-16个、4-14个、4-12个、4-10个、4-8个、4-6个、 6-20个、6-18个、6-16个、6-14个、6-12个、6-10个、6-8个、8-20个、8-18个、8-16个、8-14个、8-12个、8-10个、10-20个、10-18个、10-16个、10-14个、10-12个、12-20个、12-18个、12-16个、12-14个、14-20个、1-18个、14-16个、16-20个、16-18个和18-20个循环)。例如，第一循环次数次数至少为3、至少为4、至少为5、或至少为6、至少为7、至少为8、至少为9、或至少为10、至少为11、至少为12、至少为13、至少为14、至少为15、或至少为16、至少为17、至少为18、至少为19或至少为20，或者最好不超过8、不超过9、不超过10、不超过11、不超过12、不超过13、不超过14、不超过15、不超过16、不超过17、或不超过18、不超过19、不超过20、不超过21、不超过22、不超过23、不超过24、或不超过25、不超过26、不超过27、不超过28、不超过29、不超过30、不超过31、不超过32、不超过33、不超过34、不超过35、不超过36、不超过37、不超过38、不超过39或不超过40。如果第一循环次数过低，则得到的预扩增产物少，为获得足够的扩增产物，就需要在扩增步骤(d)中增加循环次数，这样会降低扩增结果的准确性。而如果第一循环次数过高，则会由于耗时较长而导致整个流程反应时间过长。
在某些实施方式中，在步骤(b3)后进一步包括步骤(b3’)，其中将所述反应混合物置于适当的温度程序，使得所述基因组预扩增产物中全扩增子的3’端与5’端杂交结合以形成环状结构。此前认为，步骤(b3’)能够将全扩增子的末端保护起来，从而避免两条或多条全扩增子之间发生首尾聚合，从而避免将两个原本在基因组上不相邻的序列结合在一起。这将有助于提高扩增结果的准确性。
在某些实施方式中，所述方法在步骤(b3)后不经其他步骤(例如步骤(b3’))而直接到后续步骤(b1)或(c)。这样，全扩增子并未经过特定的步骤以避免首尾聚合的情况，因此，理论上，这样的扩增结果应该在准确性上存在一定的缺陷。但是，意想不到的是，在本申请的方法中，即使在步骤(b3)后不经特定的步骤使全扩增子成环，最终的扩增结果仍然具有相当高的准确度，与使用步骤(b3’)的方法相比效果差不多。这精简了反应步骤，同时仍然保持了反应的特异性。
步骤(c)：提供第二反应混合物
在步骤(c)中，第二反应混合物中包含步骤(b)中得到的预扩增产物、第二引物、核苷酸单体混合物和核酸聚合酶，第二引物从5’端到3’端包含特定序列及所述通用序列。由于通用序列基本上不与基因组序列互补，因此如果第二类引物的其他部分被设计为基本不与基因组序列互补，那么第二类引物不会直接与基因组DNA发生配对并开启基因组DNA的复 制，因而在某些特定的实施方式中可以通过直接在步骤(b)结束后获得的反应混合物中加入第二引物而获得第二反应混合物。在另一些实施方式中，在步骤(c)之前对步骤(b)结束后获得的反应混合物进行纯化，得到纯化的预扩增产物，然后与第二引物、核苷酸单体混合物和核酸聚合酶以及可选地与任何其他本领域公知的可以用于扩增反应的试剂混合得到第二反应混合物。
i.第二类引物
本文中所述的“第二引物”属于上文所述的第二类引物。第二类引物包含第一类引物中的通用序列，从而第二类引物可以结合全扩增子中的3’端的通用序列的互补序列，从而进一步复制该全扩增子，使其数量大大增加。
在某些实施方式中，第二类引物从5’到3’包含或由特定序列和通用序列组成。可以根据不同的测序平台针对性地选择第二类引物。在某些实施方式中，根据第二代测序平台针对性地选择第二类引物。在某些实施方式中，根据Illumina的NGS测序平台(例如但不限于Hiseq、Miseq等)或Life technologies的Ion torrent的NGS测序平台针对性地选择第二类引物。在某些实施方式中，第二类引物包括与测序用引物的部分或全部互补或者相同的序列。在某些实施方式中，上述第二类引物中的与测序用引物的部分或全部互补或者相同的序列包含或由所述的通用序列组成。
本申请中所述的第二引物可以是具有第二类引物结构特征的一对引物对或者是具有相同结构和序列的单一引物。在一些实施方式中，第二引物的特定序列在其3’端包括与测序用引物的部分或全部互补或者相同的序列。在一些实施方式中所述第二引物的特定序列中包含的与测序用引物的部分或全部互补或相同的序列包含或由SEQ ID NO:31[ACACTCTTTCCCTACACGAC]、或SEQ ID NO:32[GTGACTGGAGTTCAGACGTGT]组成。在一些实施方式中，第二引物中的特定序列在其5’端进一步包括与测序平台的捕捉序列部分或全部互补或者相同的序列。捕捉序列是指在测序平台中测序板上包含的用于捕捉待测序片段的序列。在一些实施方式中，第二引物的特定序列中包含的与测序平台的捕捉序列部分或全部互补或相同的序列包含或由SEQ ID NO:33[AATGATACGGCGACCACCGAGATCT]、或SEQ ID NO:34[CAAGCAGAAGACGGCATACGAGAT]组成。在一些实施方式中，第二引物的特定序列进一步在所述与测序平台的捕捉序列部分或全部互补或相同的序列和所述与测序用引物的部分或全部互补或相同的序列之间包括一段标识序列(barcode序列)，所述标识序列是 指用于标识特定的待测序片段集合的序列，当测序平台同时对多个测序片段集合进行测序时，可以通过在测序结果中筛选每个集合带有的标识序列来区分测序数据。
在一些实施方式中，第二引物是包括具有相同通用序列和不同特定序列的引物对，其中所述不同特定序列分别包含与同一测序平台中用到的一对捕捉序列的部分或全部互补或者相同的序列，和/或所述不同特定序列分别包含与同一测序中用到的测序引物对中不同引物的部分或全部互补或相同的特定序列。在一些实施方式中，第二引物包括SEQ ID NO:35[AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT]和[CAAGCAGAAGACGGCATACGAGATX…XGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT]所示的序列的混合物，其中X…X为标识序列，本领域技术人员可以根据实际需要选择标识序列的长度和其具体序列。在一些实施方式中，第二引物包括SEQ ID NO:35[AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT]、SEQ ID NO:36[CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT]所示的序列的混合物。在一些实施方式中，第二引物包括SEQ ID NO:37[CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATGCTCTTCCGATCT]和SEQ ID NO:38[CCATCTCATCCCTGCGTGTCTCCGACTCAGCTAAGGTAACGATGCTCTTCCGATCT]所示的序列的混合物。在一些实施方式中，第二引物包括SEQ ID NO：39[CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATTTGGTAGTGAGTG]和SEQ ID NO：40[CCATCTCATCCCTGCGTGTCTCCGACTCAGCTAAGGTAACGATTTGGTAGTGAGTG]所示的序列的混合物。
在一些实施方式中，第二引物在第二反应混合物中的浓度为1-15ng/μL。在一些实施方式中，第二引物在第二反应混合物中的浓度为1-12ng/μL、1-10ng/μL、1-8ng/μL、1-7ng/μL、1-6ng/μL、1-5ng/μL、1-4ng/μL、2-3ng/μL、2-12ng/μL、2-10ng/μL、2-8ng/μL、2-6ng/μL、2-5ng/μL、2-4ng/μL、2-3ng/μL、3-12ng/μL、3-10ng/μL、3-8ng/μL、3-6ng/μL或3-4ng/μL。在一些实施方式中，在第二反应混合物中的第二引物浓度为2-3ng/μL。在一些实施方式中，在第二反应混合物中的第二引物为5-50pmol。在一些实施方式中，在第二反应混合物中的第二引物为10pmol、15pmol或20pmol。
ii.其它成分
在某些实施方式中，第二反应混合物中含有的核酸聚合酶为选自Thermococcus litoralis的Vent聚合酶、Deep Vent聚合酶、Vent(-exo)聚合酶、或Deep Vent(-exo)聚合酶中的一种或多种。在某些实施方式中，第二反应混合物中含有Thermococcus litoralis的Vent聚合酶。在某些实施方式中，第二反应混合物中含有Deep Vent聚合酶。在某些实施方式中，第二反应混合物中含有Vent(-exo)聚合酶。在某些实施方式中，第二反应混合物中含有Deep Vent(-exo)聚合酶。本申请中所述的各种聚合酶均可以从商业途径获得，例如从New England Biolabs公司获得。
在某些实施方式中，第二反应混合物中还可以包括核酸聚合酶发挥酶活性所需的适当的金属离子(例如，适当浓度的Mg2+离子(例如终浓度可以为约1.5mM到约8mM)；核苷酸单体混合物(例如dATP、dGTP、dTTP、和dCTP)、牛血清白蛋白(BSA)、dTT(例如终浓度可以为约2mM到约7mM)、纯水、适当的缓冲液成分(例如pH调节剂，如TrisHCl和Tris SO4)或其他本领域通用的一种或多种其他成分(例如DNase抑制剂、RNase、SO4 2-、Cl-、K+、Ca2+、Na+、和/或(NH4)+等)等。
步骤(d)：置于第二温度循环程序
本申请提供的方法还包括步骤(d)：将步骤(c)得到的第二反应混合物置于第二温度循环程序，使得所述第二类引物的通用序列能够与所述基因组预扩增产物的3’端配对并扩增所述基因组预扩增产物以得到扩大的基因组扩增产物。
由于步骤(b)得到的基因组预扩增产物，即全扩增子，在3’端具有通用序列的互补序列，因此可以与第二类引物的通用序列互补，在核酸聚合酶的作用下，第二类引物延伸，复制全扩增子的全长。
在第二温度循环程序中，首先将反应混合物置于能够打开DNA双链的温度程序(步骤(d1))。这里的DNA双链主要是指在步骤(b)中得到的基因组预扩增产物(即全扩增子)的双链(包括全扩增子的单链发卡结构分子)。虽然此时的第二反应混合物中仍然可能存在原始的基因组DNA，但由于第二类引物基本上不与基因组DNA配对结合，因此原始的基因组DNA并不是步骤(d)中的待扩增的DNA模板。可以使用较高的反应温度(例如90℃-95℃)反应适当的时间使得待扩增的全扩增子双链/发卡结构能够变性成为线性单链。 在某些实施方式中，步骤(d1)中的温度程序中将反应混合物置于能够打开DNA双链的温度反应足够的时间，以确保模板DNA双链或发卡结构全部变性成单链，该温度程序包括在介于90-95℃之间(例如95℃)的变性温度反应5秒-20分钟(例如30秒或3分钟)。在步骤(d1)以后，将反应混合物置于能够使其中包含的第x轮(x为≥1的整数)扩增中生成的扩增产物双链解链为单链模板的温度程序(步骤(d2))，即在介于90-95℃之间(例如95℃)的解链温度反应3-50秒(例如20秒)。应当理解的是，在第一轮循环中步骤(d2)并非必须，但由于变性和解链程序中使用的温度相近，且相对于变性时间来说解链时间很短，所以可以认为其在第一轮中为步骤(d1)的延时。
在步骤(d2)以后，将反应混合物置于能够使所述第二类引物与步骤(d1)或(d2)中获得的DNA单链结合的温度程序(步骤(d3))。根据第二类引物中的碱基组成，可以计算出第二类引物的Tm值，并基于该Tm值找出对于第二类引物的适合的退火温度。在某些实施方式中，步骤(d3)中的温度程序包括在介于45-65℃之间的退火温度(例如63℃)反应3-50秒(例如40秒)。在某些实施方式中，第二类引物为SEQ ID NO:35、SEQ ID NO:36的混合物，且步骤(d3)中的温度程序包括在63℃反应3-50秒。在某些实施方式中，步骤(d3)中的退火温度高于在步骤(b2)中的退火温度。在步骤(d3)时，反应混合物可能仍然含有在步骤(b)中未反应的第一类引物，这些第一类引物中的可变序列可能与步骤(d3)中得到的DNA单链模板配对结合，从而产生不完整的扩增序列。当步骤(d3)中的退火温度高于第一类引物适合的退火温度时，可以减少或避免第一类引物与DNA单链模板结合，从而选择性地允许第二类引物进行扩增。
在引物退火完成以后，将所述反应混合物置于能够使与所述扩增产物单链结合的第二类引物在所述核酸聚合酶的作用下延伸长度的温度程序。在某些实施方式中，步骤(d4)中所述的温度程序包括在介于60-80℃之间的延伸温度(例如72℃)反应10秒-15分钟(例如40秒或3分钟)。
可以重复步骤(d2)到(d4)至第二循环次数，以获得所需的扩大的基因组扩增产物。在这个过程中，步骤(b)中得到的基因组扩增产物被进一步复制扩增，数量大大增加，以提供足够的基因组DNA序列用于后续的研究或操作。在某些实施方式中，步骤(d5)中的所述第二循环次数大于所述步骤(b4)中的第一循环次数。在某些实施方式中，将第二循环的次数控制在适当的范围内，使得其既能够提供足够量的DNA，又不会因为过多的循环数而影响扩增的准确度。在某些实施方式中，第二循环次数为2-40个循环(例如，2-40个、4-40个、 6-40个、8-40个、10-40个、12-40个、14-40个、16-40个、18-40个、20-40个、15-40个、20-40个、25-40个、30-40个、5-35个、10-35个、15-35个、20-35个、25-35个、30-35个、10-30个、15-30个、20-30个、25-30个、15-28个、15-26个、15-24个、15-22个、15-20个、15-18个、15-17个、16-30个、17-30个、18-30个、20-30个、22-30个、24-30个、26-30个、28-30个、32-40个、32-38个、32-36个或32-34个循环)。
在某些实施方式中，步骤(d)进一步包括在第二温度循环程序以后，将反应混合物置于与步骤(d4)相同的温度程序(例如72℃)反应适当的时间(例如40秒)。然后将反应混合物置于4℃的温度下以结束反应。在某些实施方式中，步骤(d)反应结束后直接将反应混合物置于4℃的温度下以结束反应。
在某些特定的实施方式中，本申请还提供了一种扩增细胞基因组的方法，所述方法包括：
(a)提供第一反应混合物，其中所述第一反应混合物包括所述基因组DNA、第一引物、核苷酸单体混合物、和核酸聚合酶，其中所述第一引物从5’端到3’端包含通用序列和可变序列，其中所述第一引物从5’端到3’端包含通用序列和第一可变序列，所述第一可变序列包括第一随机序列，其中所述第一随机序列从5’端到3’端依次为Xa1Xa2……Xan，所述第一随机序列的Xai(i＝1-n)均属于同一个集合，所述集合选自B、或D、或H、或V，其中B＝{T、G、C}，D＝{A、T、G}，H＝{T、A、C}，V＝{A、C、G}，其中Xai表示第一随机序列5’端的第i个核苷酸，n是选自3-20的正整数，其中所述通用序列和所述第一可变序列直接相连、或所述通用序列和所述第一可变序列通过第一间隔序列相连，所述第一间隔序列为Ya1……Yam，其中Yaj(j＝1-m)∈{A、T、G、C}，其中Yaj表示间隔序列5’端的第j个核苷酸，
可选地，其中所述第一反应混合物进一步包括第三引物，其中所述第三引物从5’端到3’端包含所述通用序列和第三可变序列，所述第三可变序列包括第三随机序列，其中所述第三随机序列从5’端到3’端依次为Xb1Xb2……Xbn，所述第三随机序列的Xbi(i＝1-n)均属于同一个集合，所述集合选自B、或D、或H、或V，其中B＝{T、G、C}，D＝{A、T、G}，H＝{T、A、C}，V＝{A、C、G}，并且Xbi(i＝1-n)和Xai(i＝1-n)属于不同的集合，其中Xbi表示第一随机序列5’端的第i个核苷酸，n是选自3-20的正整数，其中所述通用序列和所述第三可变序列直接相连，或者所述通用序列和所述第三可变序列通过第三间隔序列相连，所述第三间隔序列为Yb1……Ybm，其中Ybj(j＝1-m)∈{A、T、G、C}，其中Ybj表示间隔序 列5’端的第j个核苷酸，m是选自1-3的正整数；
(b)将所述第一反应混合物置于第一温度循环程序，使得所述第一引物和第三引物的可变序列能够与所述基因组DNA配对并扩增所述基因组DNA以得到基因组扩增产物，其中所述基因组扩增产物的5’端包含所述通用序列，3’端包含所述通用序列的互补序列；其中所述第一温度循环程序包括：
(b1)在介于90-95℃的温度之间的第一变性温度反应1-10分钟(第一轮循环中)或者10-50秒(后续循环中)；
(b2)介于5-15℃之间的第一退火温度反应3-50秒，介于15-25℃之间的第二退火温度反应3-50秒，和介于30-50℃之间的第三退火温度反应3-50秒；
(b3)在介于60-80℃之间的(一个或多个)第一延伸温度反应10秒-15分钟；
(b4)重复步骤(b1)到(b3)至2-40个循环；
(c)提供第二反应混合物，所述第二反应混合物包括步骤(b)中得到的所述基因组预扩增产物、第二引物、核苷酸单体混合物、和核酸聚合酶，其中所述第二引物的从5’端到3’端包含特定序列及所述通用序列；
(d)将所述第二反应混合物置于第二温度循环程序，使得所述第二引物的所述通用序列能够与所述基因组预扩增产物的3’端配对并扩增所述基因组预扩增产物以得到扩大的基因组扩增产物，其中所述第二温度循环程序包括：
(d1)在介于90-95℃之间的第二变性温度反应5秒-20分钟；
(d2)在介于90-95℃之间的第二解链温度反应3-50秒；
(d3)在介于45-65℃之间的第四退火温度反应3-50秒；
(d4)在介于60-80℃之间的第二延伸温度反应10秒-15分钟；
(d5)重复步骤(d2)到(d4)2-40个循环，获得基因组扩增产物。
在某些实施方式中，在步骤(a)的反应混合物中的基因组DNA存在于细胞内部，即：反应混合物含有细胞，而在细胞中包含了待扩增的基因组DNA。在某些实施方式中，在步骤(a)的反应混合物含有细胞，而且还进一步包含能够裂解细胞的成分，例如表面活性剂和/或裂解酶等。可以使用适当的表面活性剂，例如NP-40、吐温、SDS、TritonX-100、EDTA、异硫氰酸胍中的一种或多种。也可以选择适当的裂解酶，例如蛋白酶K、胃蛋白酶、木瓜 蛋白酶中的一种或多种。在这样的实施方式中，上述扩增细胞基因组的方法在步骤(a)之后以及步骤(b)之前进一步包括将所述反应混合物置于裂解温度循环程序(例如将反应混合物置于50℃20分钟，然后置于80℃10分钟)，使得所述细胞裂解并释放出所述基因组DNA。
应用
在某些实施方式中，本申请方法扩增得到的产物可以进一步用于进行测序，如进行全基因测序。由于各种测序分析平台如新一代测序(NGS)，基因芯片(Microarray)，荧光定量PCR等均对待分析样本的起始量有较高的要求(100ng以上)，因此如需要从单个人类细胞(6pg左右)或者少量起始量的样本中得到足量用于分析的核酸物质，则需要进行全基因组扩增。可以通过本申请的方法对生物样品(例如单细胞)中的基因组DNA进行扩增，再通过本领域适当的测序方法对扩增得到的产物进行测序。示例的测序方法包括，杂交测序法(SBH)、连接酶测序法(SBL)、定量增量荧光核酸增加测序法(QIFNAS)、逐步连接和切割法、分子信标法、焦磷酸测序法、原位荧光测序法(FISSEQ)、荧光共振能量转移法(FRET)、多重测序法(美国专利申请12/027039；porreca等人(2007)NAT.Methods4:931)、聚合群体(POLONY)测序法(U.S.6,432,360、U.S.6,485,944和PCT/US05/06425)、摆动测序法(PCT US05/27695)、TaqMan报告分子探针消化法、微粒滚动循环测序法(ROLONY)(美国专利申请12/120541)、FISSEQ小珠法(U.S.7,425,431)和等位基因特异的寡核苷酸连接分析法等。
在某些实施方式中，可以以高通量的方法实现对本申请方法的扩增产物的测序。高通量的方法通常将待测序的核酸分子片段化(例如通过酶解或机械剪切等方式)，以形成大量的长度为几十bp到几百bp的短片段。通过在一次测序反应中平行地对几万个、几十万个、几百万个、几千万个、甚至上亿个这样的短片段测序，可以大大提高测序的通量、缩短测序所需的时间。将测得的短片段的序列通过软件进行数据处理，可以拼接成完整的序列。本领域已知多种高通量测序平台，例如Roche 454、Illumina Solexa、AB-SOLiD、Helicos、Polonator平台技术等。本领域还已知多种基于光的测序技术，例如可以参见Landegren等人(1998)Genome Res.8:769-76、Kwok(2000)Pharmacogenomics 1:95-100和Shi(2001)Clin.Chem.47:164-172中描述的那些。
在某些实施方式中，本申请方法扩增得到的产物还可以用于对基因组DNA中的基因型或遗传多态性进行分析，例如单核苷酸多态性(SNP)分析、短串联重复序列(STR)分析、限制性片段长度多态性(RFLP)分析、可变数目串联重复序列(VNTRs)分析、 复杂重复序列(CTR)分析或微卫星分析等，例如可以参考Krebs,J.E.,Goldstein,E.S.和Kilpatrick,S.T.(2009).Lewin’s Genes X(Jones & Bartlett Publishers)，其公开内容通过引用整体并入本申请。
在某些实施方式中，本申请的方法得到的扩增产物还可以用于医学分析和/或诊断分析。例如，可以对个体的生物样品用本申请的方法进行扩增，分析扩增产物中在感兴趣的基因或DNA序列中是否存在突变、缺失、插入或染色体之间的融合等异常情况，从而评估该个体患上某种疾病的风险、疾病的进展阶段、疾病的基因分型、疾病的严重程度、或者该个体对某种疗法反应的可能性。可以使用本领域已知的适当的方法对感兴趣的基因或DNA序列进行分析，例如但不限于，通过核酸探针杂交、引物特异性扩增、对感兴趣的序列测序、单链构象多态性(SSCP)等。
在某些实施方式中，本申请的方法可以用于比较来源于不同单细胞的基因组，特别是来自于同一个体的不同单细胞。例如，当同一个体的不同单细胞的基因组之间存在差异时，例如肿瘤细胞和正常细胞之间，可以使用本申请的方法分别扩增不同单细胞的基因组DNA，并对扩增产物进行进一步的分析，例如，通过测序分析和比较，或者进行比较基因组杂交(CGH)分析。可以参考Fan,H.C.,Wang,J.,Potanina,A.和Quake,S.R.(2011).Whole-genome molecular haplotyping of single cells.Nature Biotechnology 29,51–57.以及Navin,N.,Kendall,J.,Troge,J.,Andrews,P.,Rodgers,L.,McIndoo,J.,Cook,K.,Stepansky,A.,Levy,D.,Esposito,D.等人(2011).Tumour evolution inferred by single-cell sequencing.Nature 472,90–94，其公开内容通过引用整体并入本申请。
在某些实施方式中，本申请的方法可以用于识别在同源染色体中的单倍体结构或单倍体基因型。单倍体基因型是指同一单倍体的染色体上共同遗传的多个基因座上等位基因的组合。可以将生物样品(例如来自个体的二倍体的单细胞)分成足够多的部分，以使得同源的两个单倍体上的DNA序列在统计学意义上被分隔到不同的部分中。每一个部分配置成一个反应混合物，对每一个反应混合物通过本申请的方法进行DNA扩增，然后将扩增产物进行序列分析，并与参照的基因组序列(例如公开的人的标准基因组序列，请参见：International Human Genome Sequencing Consortium,Nature 431,931-945(2004))进行比对，以识别其中的单核苷酸突变情况。如果没有现成的参照基因组序列，也可以通过从头基因组组装(de-novo genome assembly)的方法从基因组的多个片段序列组装得到适当长度的一段区域以供比较。
在某些实施方式中，本申请的方法扩增得到的产物可以进一步用于基因克隆、荧光定量PCR等分析。
在某些实施方式中，本申请的方法还可以进一步包括分析所述扩增产物以识别与疾病或表型相关的序列特征。在一些实施方式中，分析所述扩增产物包括对DNA扩增物的基因型分析。在另一些实施方式中，分析所述扩增产物包括识别DNA扩增物的多态性，如单核苷酸多态性分析(SNP)。SNP可以通过一些众所周知的方法进行检测，例如寡核苷酸连接测定法(OLA)、单碱基延生法、等位基因特异性引物延伸法、错配杂交法等。可以通过比对SNP与已知疾病表型的关系来诊断疾病。
在一些实施方式中，所述与疾病或表型相关的序列特征包括染色体水平异常、染色体的异位、非整倍体、部分或全部染色体的缺失或重复、胎儿HLA单倍型和父源突变。
在一些实施方式中，所述疾病或表型可以是β-地中海贫血、唐氏综合征、囊性纤维化、镰状细胞病、泰-萨克斯病、脆性X综合征、脊髓性肌萎缩症、血红蛋白病、α-地中海贫血、X连锁疾病(由在X染色体上基因主导的疾病)、脊柱裂、无脑畸形、先天性心脏病、肥胖、糖尿病、癌症、胎儿性别、或胎儿RHD。
试剂盒
在本申请的另一方面还提供了可用于基因组DNA扩增的试剂盒，其中包括第一引物。在某些实施方式中，所述试剂盒同时包括第一引物和第三引物。在某些实施方式中，试剂盒进一步包括核酸聚合酶，其中所述核酸聚合酶选自：Phi29DNA聚合酶、Bst DNA聚合酶、Pyrophage 3137、Vent聚合酶、TOPOTaq DNA聚合酶、9°Nm聚合酶、Klenow Fragment DNA聚合酶I、MMLV反转录酶、AMV反转录酶、HIV反转录酶、T7phase DNA聚合酶变种、
Figure PCTCN2017081977-appb-000005
超保真DNA聚合酶、Taq聚合酶、Bst DNA聚合酶、E.coli DNA聚合酶、LongAmp Taq DNA聚合酶、OneTaq DNA聚合酶、Deep Vent DNA聚合酶、Vent(exo-)DNA聚合酶、Deep Vent(exo-)DNA聚合酶，及其任意组合。在某些实施方式中，试剂盒进一步包括一种或多种选自下组的成分：核苷酸单体混合物(例如dATP、dGTP、dTTP和dCTP，例如，总浓度介于1mmol-8mmol/μL)、dTT(例如，浓度介于1mmol-7mmol/μL)、Mg2+溶液(例如，浓度介于2mmol-8mmol/μL)、牛血清白蛋白(BSA)、pH调节剂(例如TrisHCl)、DNase抑制剂、RNase、SO4 2-、Cl-、K+、Ca2+、Na+、和/或(NH4)+。在某些实施方式中，试剂盒进一步包括能够裂解细胞的成分，例如一种或多种表面活性剂(例如，NP-40、吐温、SDS、TritonX-100、EDTA、异硫氰酸胍)，和/或一种或多种裂解酶(例如，蛋白酶 K、胃蛋白酶、木瓜蛋白酶)。在一些实施方式中，所述试剂盒进一步包括第二类引物(即，第二引物)。应当理解，试剂盒中的第一引物、第二引物及第三引物均具有如上文中具体描述的结构和序列特征。
在一些实施方式中，试剂盒中的所有组分均分别存放于单独的容器中。在一些实施方式中，试剂盒中的所有组分均共同存放在同一容器中。在一些实施方式中，试剂盒中的每种引物均分别各自存放在单独的容器中，而除引物以外的所有其他组分均存放在同一容器中。当试剂盒中包括核酸聚合酶时，核酸聚合酶可以以基本上纯的形式存放于单独的容器中，或者可选地可以与其他成分组成混合物。
在一些实施方式中，所述试剂盒可以包含含有线性扩增反应所需的除基因组DNA以外的全部反应物的混合物，当这样的试剂盒用于本申请所述的线性扩增反应时，可以将含有基因组DNA的样本与试剂盒中的混合物直接混合，可选地可以加入适量的纯水以获得需要的反应体积，即可获得本申请方法的步骤(a)中的第一反应混合物。在一些实施方式中，所述试剂盒可以包含含有指数扩增反应所需的除扩增模板以外的全部反应物的混合物，当这样的试剂盒用于本申请所述的指数扩增反应时，可以将含有步骤(b)中的扩增产物的DNA模板样本与试剂盒中的该混合物直接混合，可选地可以加入适量的纯水以获得需要的反应体积，即可获得本申请方法的步骤(c)中的第二反应混合物。在另一些实施方式中，所述试剂盒可以既包含含有指数扩增反应所需的除扩增模板以外的全部反应物的混合物又包含含有指数扩增反应所需的除扩增模板以外的全部反应物的混合物，上述混合物可以是分开的两种，也可以是混合的一种。
在本申请的另一方面还提供了可用于基因组DNA扩增的试剂盒，所述试剂盒包括第一类引物(例如，第一引物和/或第三引物)和第二类引物(例如，第二引物)，并且还包括使用说明书，所述使用说明书记载了在开始进行所述扩增之前混合引物和其他组分得到第一/第三反应混合物的步骤。在另一些实施方式中，所述说明书还记载了如何进行本申请所述的扩增。试剂盒中的第一类引物和第二类引物可以分别置于不同的容器中，但说明书中可以包括在开始扩增前将两者混合在同一容器中的步骤。
具体实施例
实施例1：初步验证使用不同线性扩增引物混合物的扩增效果
a)使用标准基因组DNA样本进行验证
标准基因组DNA为事先提取好的人类细胞的基因组DNA。用无核酸酶水将标准基因组DNA稀释为50皮克/微升的DNA溶液，取1微升上述溶液(作为基因组DNA源)加入到PCR管中，在各实验组中加入如表1所示的引物混合物及其他相关试剂，得到第一反应混合物(其中含有Na+、Mg2+、Cl-、Tris-Cl、TritonX-100、dNTP、Vent聚合酶和引物混合物)。
线性扩增
每组引物组合/混合物均使用两个实验组进行平行实验以保证其准确性，并将各实验组的反应混合液置于如下第一温控程序进行反应：
Figure PCTCN2017081977-appb-000006
各实验组中使用的引物混合物如下表1中所示。
表1：各实验组中使用的引物
实验组 使用的引物(或引物混合物)
1-2 通用序列+NNNNNTTT和通用序列+NNNNNGGG
3-4 通用序列+NNNNNGTTT和通用序列+NNNNNTGGG
5-6 SEQ ID NO:7和SEQ ID NO:11
7-8 SEQ ID NO:8和SEQ ID NO:12
9-10 SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:11和SEQ ID NO:12
11-12 通用序列+KKKKK
其中每个实验组中使用的第一类引物的总量为600皮摩尔(如果其中包括多种引物，则总量为600皮摩尔，各种引物含量均相同)。可根据测序平台的不同设计不同的通用序列。在本实验的1-12中根据Illumina平台选用SEQ ID NO：6作为通用序列。
线性扩增程序后，在各反应体系中加入1微升10皮摩/微升的第二引物，获得12组第二反应混合物，并将各实验组的反应混合液置于如下第二温控程序进行反应：
Figure PCTCN2017081977-appb-000007
可根据测序平台的不同设计不同的第二引物序列。在本实验的1-12中根据Illumina平台均使用如下第二引物的混合物：
第二引物-1(SEQ ID NO：35)：AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATCT
第二引物-2(SEQ ID NO：36)：
Figure PCTCN2017081977-appb-000008
Figure PCTCN2017081977-appb-000009
其中，第二引物中以双下划线标识的碱基包括与测序平台的捕捉序列对应的部分，以斜体标识的碱基为与测序平台的测序序列对应的部分，以点标识的部分为标识序列部分，可根据需要替换为其他标识序列，以单下划线标识的部分为通用序列部分。
各实验组结束上述扩增温控程序后得到扩增产物。
凝胶电泳(定性)
分别取5微升未纯化的实施例1中各实验组的扩增产物，并分别添加1微升6xDNA加样缓冲液(购自北京康为世纪生物科技有限公司，货号CW0610A)准备上样。凝胶使用1％琼脂糖凝胶，标记物使用DM2000(购自北京康为世纪生物科技有限公司，货号CW0632C)。电泳图请参见附图3，其中自左向右第1泳道为分子量标记，2-13泳道为基因组DNA扩增样品，14泳道为分子量标记。
如图3电泳图所示，实验组1-4的产物在100bp附近有明显条带，但在100-500bp之间产物含量相对较低；其他实验组5-12的产物集中在500bp左右，实验组5-10在100bp附近有不清晰条带且与实验组1-4相比浓度非常低。由图3可知实验组1-4中引物聚合物较多，相对反应效率较低，而第一类引物可变序列内包含的固定序列为TGGG或者GTTT(实验组3-4)时反应效率高于固定序列为GGG或者TTT的引物(实验组1-2)；实验组 5-12组间的扩增产物区别不大，但与实验组1-4相比显著更高，说明引物聚合物产生的程度比第1-4组低。
纯化产物(定量)
取50微升未纯化的扩增产物，使用磁珠法DNA纯化回收试剂盒(购自北京康为世纪生物科技有限公司，货号CW2508)对扩增产物进行纯化处理，纯化步骤按照试剂盒说明书操作。使用20微升EB洗脱。纯化完成后取2微升纯化产物使用Nanodrop(AOSHENG，NANO-100)检测浓度。浓度检测结果如表2所示。
表2：图3中样本回收后浓度
实验组 浓度(纳克/微升) 体积(微升)
实验组1 16.647 20
实验组2 20.158 20
实验组3 82.085 20
实验组4 79.712 20
实验组5 130.52 20
实验组6 130.024 20
实验组7 144.456 20
实验组8 128.041 20
实验组9 118.145 20
实验组10 101.04 20
实验组11 126.059 20
实验组12 130.079 20
根据获得的扩增产物在纯化后的浓度推测各实验组的扩增效率：由表2所示的浓度检测结果可以看出，实验组1-2扩增效率最低，实验组3-4与实验组5-12相比扩增效率相对较低但比实验组1-2扩增效率高。除实验组1-4以外，其他实验组扩增产物总量相当，没有显著差别。
测序(定性)
取上述纯化后的12实验组的扩增产物，采取浅测序的方式使用Illumina的NGS测序平台hiseq2500测序仪进行测序，并将测序得到的序列比对到人类参考基因组上。
在表3中提供了高通量测序结果的各个指标参数。
表3的各指标参数中，原始数据中唯一比对比例(unique_mapped_of_raw，即能够比对到人类基因组的唯一位置的数据比例)是最重要的衡量指标。如表3中数据所示，实验 组5-12中的unique_mapped_of_raw在83％-86％之间，各组间差别不大，但实验组1-4中的unique_mapped_of_raw相对较低在67-79％之间。
另一个重要指标参数为原始数据中比对比例(mapped_of_raw，即原始数据中能够比对到人类基因组的某个位置的数据比例)。类似地，如表3中数据所示，实验组5-12中的mapped_of_raw在89％-93％之间，各组间差别不大，但实验组1-4中的mapped_of_raw相对较低在73-86％之间。
另外，表3中的数据还表明实验组1-4的读数数据质量也低于其他实验组，例如实验组3、4的原始数据中高质量数据比例仅为77.08％、76.99％，而实验组5-12中高质量数据比例在94％-96％之间。
图4中显示了测序文库中序列读数起始位置的各核苷酸读数情况。由图4可见，实验组1-4和9-10中起始读数区域包括A、T、C、G四种碱基。实验组5-7中的起始读数区域缺乏A或者C，实验组11-12中的起始读数区域缺乏A和C。本领域技术人员应知，在测序时，特别是使用SBS测序时要求测序的前几个碱基有较高的随机性，当测序样本整体的前几个碱基随机性较低时，则需要在整板上样时在每个上样孔中添加一定量的阳性质控品以增加碱基随机性，但这势必会浪费一定的数据量。例如，当使用实验组11-12中制备的文库进行整板上样测序时，由于起始读数区域缺乏A、C则需要加入一定数量的阳性质控品，根据实验经验一般需要加入至少20％上样量的阳性质控才能保证SBS测序的顺利进行。
表3：实验组1-12的高通量测序结果主要质量指标
Figure PCTCN2017081977-appb-000010
b)使用AFP单细胞样本进行验证
待测样本为AFP单细胞。使用胰蛋白酶消化培养状态良好的人表皮成纤维细胞(AFP)，将消化后的细胞收集进入1.5ml EP管内。将收集的细胞离心并用1x的PBS溶液冲洗。冲 洗完成后加入1x的PBS使细胞悬浮。用移液器抽取一部分包含细胞的悬浮液，在10x显微镜下使用口吸管挑取单细胞，吸取的PBS溶液体积不超过1微升，并将挑取的单细胞转移进入包含5微升裂解缓冲液(含有Tris-Cl、KCl、EDTA、Triton X-100和Qiagen Protease)的PCR管内。短暂离心后将PCR管置于PCR仪上执行裂解程序，具体程序如表4所示。
表4：裂解程序
Figure PCTCN2017081977-appb-000011
将裂解后的反应液作为基因组DNA源替换实施例1a)中的标准基因组DNA，其它成分和线性扩增、指数扩增温控程序与实施例1a)中相同。对获得的扩增产物采用上文所述条件进行凝胶电泳检测。凝胶电泳电泳图请参见附图5，其中自左向右第1泳道为分子量标记，2-13泳道为基因组DNA扩增样品，14泳道为分子量标记。
如图5电泳图所示，实验结果与图3中所示的相似。实验组1-4的产物在100bp附近有明显条带，但在100-500bp之间产物含量相对较低；其他实验组5-12的产物集中在500bp左右，实验组5-12在100bp附近无明显条带。由图5可知实验组1-4中引物聚合物较多，相对反应效率较低，而第一类引物可变序列内包含的固定序列为TGGG或者GTTT(实验组3-4)时反应效率高于固定序列为GGG或者TTT的引物(实验组1-2)；实验组5-10组间的扩增产物得率区别不大，但与实验组1-4相比更高，说明引物聚合物产生的程度比第1-4组低，但与实验组11-12相比略低。
根据上文所述的操作对扩增产物进行纯化并且测序，测序结果的各项指标如下表5中所示。其中实验组5-12中的unique_mapped_of_raw在78％-85％之间，各组间差别不大，但实验组1-4中的unique_mapped_of_raw相对较低在63-70％之间。如表5中数据所示，实验组5-12中的mapped_of_raw在84％-92％之间，各组间差别不大，但实验组1-4中的mapped_of_raw相对较低在73-86％之间。另外，表3中的数据还表明实验组1-4的读数数据质量也低于其他实验组，例如实验组3、4的原始数据中高质量数据比例仅为68.97％、72.29％，而实验组5-12中高质量数据比例在94％-96％之间。
表5：实验组1-12的高通量测序结果主要质量指标
Figure PCTCN2017081977-appb-000012
Figure PCTCN2017081977-appb-000013
由上述结果可见，实验组1-4所使用的线性扩增引物混合物会产生较多的引物聚合物，从而使得扩增效率大大降低，相应地在同等扩增条件下数据量较低。虽然实验组1-4在起始测序区域的A、T、C、G分布均匀，但是数据中的比对比例尤其是唯一比对比例较低，从而导致使用其测序数据在后续处理上更为困难。
实施例2：进一步验证使用不同线性扩增引物混合物的扩增效果
按照实施1b)所述的方法对人表皮成纤维细胞进行分离和裂解，以获得单细胞基因组DNA，并且分别使用表1实验组11/12中使用的引物混合物以及实验组9/10中使用的引物混合物进行扩增，对每种引物混合物均进行10个平行试验(分别以1_1、1_2…1_10和2_1、2_2…2_10表示)。根据实施例1a)中所述的程序进行扩增并获得扩增产物，并对扩增产物进行凝胶电泳检测，电泳检测结果如图6所示。其中实验组2_1、2_2…2_10中扩增产物的浓度略低于实验组1_1、1_2…1_10中扩增产物的浓度
根据实施例1a)中所述的程序对上述扩增产物进行纯化并取相同体积的纯化扩增产物进行测序，测序结果的相关指标参数如下表6-7所示。
表6：实验组1_1、1_2…1_10的高通量测序结果主要质量指标
Figure PCTCN2017081977-appb-000014
表7：实验组2_1、2_2…2-10高通量测序结果主要质量指标
Figure PCTCN2017081977-appb-000015
表6和7中的数据显示，实验组2_1、2_2…2_10中的unique_mapped_of_raw在83％-84％左右，mapped_of_raw在90％-91％左右，实验组1_1、1_2…1_10中的unique_mapped_of_raw在84％-85％左右，mapped_of_raw在91％-92％左右，两组间差别不大。每组中各样品的上机数据量统计结果显示如图7：实验组1_1、1_2…1_10中，除异常的实验组1_1以外(可能由于回收过程中的失误导致数据量极低)其他实验组中数据量均在1.5-2M之间，实验组1_2、1_3…1_10的上机数据量平均值为约1.7M左右。实验组2_1、2_2…2_10中，数据量均在1.5-2.5M之间，实验组2_1、2_2…2_10的上机数据量平均值为约1.8M左右，实验组2_1、2_2…2_10中数据量略高。此外，将实验组上机数据中的拷贝数变异系数CV总结如图8：在排除明显异常的实验组1_1后，实验组1_2、1_3…1_10的平均拷贝数变异系数CV约为0.046，实验组2_1、2_2…2_10的平均拷贝数变异系数CV约为0.049，两个实验组中拷贝数变异系数相比无明显差别。图9中单独列出了每个实验组的拷贝数变异图，其中纵坐标代表染色体的拷贝数，正常人为2；横坐标代表染色体的1-22号染色体及性染色体。如图所示，每个实验组中染色体1-22除个别数据点以外均大致为两个拷贝，而性染色体X和Y均分别为大致1个拷贝。
实施例3：致病位点和质检引物检测
致病位点检测
随机选取35个致病位点(选择的位点参见下表8)，并设计引物。选取的致病位点及其相应的引物分别如表8和表9所示。
表8：随机选取的35个致病位点
  致病位点名称 染色体定位
1 SMN1-1 chr5
2 SMN1-2 chr5
3 SMN1-3 chr5
4 SMN1-4 chr5
5 SMN1-1R chr5
6 SMN1-2R chr5
7 SMN1-3R chr5
8 SMN1-4R chr5
9 PDS-IV15 chr7
10 PDS-EXON5 chr7
11 PDS-EXON7+8 chr7
12 PDS-EXON10 chr7
13 PDS-EXON17 chr7
14 PDS-EXON19 chr7
15 HBB3 chr11
16 HBB chr11
17 MMACHC chr1
18 HBA2 chr16
19 GJB2 chr13
20 GJB2-C796 chr13
21 ATP7B-8 chr13
22 PKHD1-3681 chr6
23 PKHD1-1713 chr6
24 WASP-C21 chrX
25 WASP-C12 chrX
26 DMD-13exe chrX
27 GJB2 chr13
28 GJB2-c79 chr13
29 PDS-7+8 chr7
30 PDS-10 chr7
31 CFTR-IVS13 chr17
32 IL2RG chrX
33 IL2RGIVS4 chrX
34 FLG-c3319 chr1
35 IDS chrX
表9：与表8中的致病位点相应的引物
致病位点名称 引物序列
SMN1-1+ AAAATGTCTTGTGAAACAAAATGC
SMN1-1- TTTTACAAAAGTAAGATTCACTTTCATAAT
SMN1-2+ AGGGTTTCAGACAAAATCAAAAAGAAG
SMN1-2- CTAATAGTTTTGGCATCAAAATTCTTTAAT
SMN1-3+ CTTTATGGTTTGTGGAAAACAAATG
SMN1-3- GTCTGCCTACTAGTGATATAAAATGG
SMN1-4+ CTGGAATGTGAAGCGTTATAG
SMN1-4- CAAAATCTAATCCACATTCAAATTTT
SMN1-1R+ TGTGGGATTGTAGGCATGAG
SMN1-1R- GCTGGCAGACTTACTCCTTAAT
SMN1-2R+ AAGTCTGCCAGCATTATGAAAG
SMN1-2R- CCACATAACCAACCAGTTAAG
SMN1-3R+ GTTCAGATGTTAAAAAGTTGAAAG
SMN1-3R- TGGTCTGCCTACTAGTGATATAAA
SMN1-4R+ GGAAGTGGAATGGGTAACTCTT
SMN1-4R- CCACATACGCCTCACATACAT
PDS-IV15+ CCAAAGGTTGGATTTGATGCC
PDS-IV15- GAATAGCTCAGTTGTTCTTTGATACG
PDS-EXON5+ CCGACGAACACTTTCTCGTATC
PDS-EXON5- GGGTTCCAGGAAATTACTTTGTTT
PDS-EXON7+8+ AAGTCTCCCTGTTCTGTCCTA
PDS-EXON7+8- AGGGTGTTGCAGACAAAGT
PDS-EXON10+ TTCACTGCTGGATTGCTCAC
PDS-EXON10- CCCCTTGGGATGGATTTAAC
PDS-EXON17+ GGAGGAACTTGATATCCCAACC
PDS-EXON17- ATACTGGACAACCCACATCATT
PDS-EXON19+ GAGCAATGCGGGTTCTTTG
PDS-EXON19- GCTAGACTAGACTTGTGTAATGTTTG
HBB3+ TCATGCCTCTTTGCACCATT
HBB3- AATCCAGCCTTATCCCAACCA
HBB+ GGTTGGCCAATCTACTCCCA
HBB- AAGGTGCCCTTGAGGTTGTC
MMACHC+ GGAGTCGAAGCTGACTCA
MMACHC- CAGTTGCAACGAAGCCAATC
HBA2+ CTTCTCTGCACAGCTCCTAAG
HBA2- GCTGCCCACTCAGACTTTAT
GJB2+ GACGCCAAGTTTGAAGGAAC
GJB2- CTACTGCTAGAAACAGCCTACTC
GJB2-C79+ TCGCATTATGATCCTCGTTG
GJB2-C79- GGACACAAAGCAGTCCACAG
ATP7B-8+ AAAAGCTGAGAAGTTCAGAAAAC
ATP7B-8- AAATTTGTATTTAACAAGTGCTTGTC
PKHD1-3681+ AGTGATTGTCATTGAAATTGGTGATTC
PKHD1-3681- AGCCAATGACTCCCTTTGAC
PKHD1-1713+ CAGAGCGATGACATCTTAACCT
PKHD1-1713- GTGAACACCAGGGCAGATGAG
WASP-C21+ TGTCCCTTGTGGTTTTTTGCATTTC
WASP-C21- TTTCGTCCAAGCATCTCAAAGAGTC
WASP-C12+ CTCTTCTTACCCTGCACCCAGAG
WASP-C12- GCATTTTCGTCCAAGCATCTCAAAGAG
DMD-13exe+ AAGAACAAGTCAGGGTCAAT
DMD-13exe- TTAAAATACTTTTCAAGTTATAGTTCTTTT
GJB2+ GACGCCAAGTTTGAAGGAAC
GJB2- CTACTGCTAGAAACAGCCTACTC
GJB2-c79+ TCGCATTATGATCCTCGTTG
GJB2-c79- GGACACAAAGCAGTCCACAG
PDS-7+8+ AAGTCTCCCTGTTCTGTCCTA
PDS-7+8- AGGGTGTTGCAGACAAAGT
PDS-10+ TTCACTGCTGGATTGCTCAC
PDS-10- CCCCTTGGGATGGATTTAAC
CFTR-IVS13+ TTTGCAGAGAATGGGATAGAGAG
CFTR-IVS13- CACCTATTCACCAGATTTCGTAGT
IL2RG+ TGACCAGGAAATAGAGAGGAAATG
IL2RG- CATTCTGCCATACCAACAATGG
IL2RGIVS4+ ATTGGAAGCCGTGGTTATCTC
IL2RGIVS4- CTTCCATCACCAAACCCTCTT
FLG-c3319+ CTGAGTGAATCCCAGCTAGAAC
FLG-c3319- GCAGAGAACAGGAGCTTGAT
IDS+ CTCCAGACACTCAGGCATTC
IDS- GTGCTCACCTGGTAGATGAAA
随机选择根据实施例2中的1_1、1_2、2_1、2_2实验组中的扩增产物分别作为模板DNA。使用2xGoldstarMasterMix(购自北京康为世纪生物科技有限公司，货号CW0960)对模板DNA进行PCR检测。扩增体系组成如表10所示，扩增程序如表11所示。
表10：用于致病位点检测的PCR反应体
Figure PCTCN2017081977-appb-000016
表11：用于致病位点检测的PCR扩增程序
Figure PCTCN2017081977-appb-000017
扩增的结果如图10中的凝胶电泳图所示。扩增结果显示：在样品1_1、1_2中致病位点4、13在两个样本中均未被扩增出，而致病位点21在样品1_1中未被扩增出，致病位点20、29、31在样品1_2中未被扩增出。在样品2_1、2_2中致病位点31在两个样本中均未被扩增出，而致病位点18、21、32、35在样品2_1中未被扩增出，致病位点8、22在样品1_2中未被扩增出。结果显示两组引物组样品(1_1、1_2和2_1、2_2)在扩增的准确性和扩增产物的量上没有显著差别。
质检引物q-PCR检测
使用上述实施例2中的1_1、1_2、2_1、2_2实验组中的扩增产物、阳性对照(相同浓度的gDNA)、阴性对照(无模板)分别作为模板DNA。使用如表12所示的6组质检引物，分别针对不同染色体上的DNA序列，对模板DNA进行q-PCR检测。在荧光定量PCR中使用2xFastSYBR Mixture(购自北京康为世纪生物科技有限公司，货号CW0955)。扩增体系组成如表表13所示，扩增程序如表14所示。
表12：质检使用的6对随机引物信息
Figure PCTCN2017081977-appb-000018
表13：用于qPCR检测的反应体系
组分 体积(微升)
2xFastSYBR混合液 25
前向引物(10uM) 1
反向引物(10uM) 1
DNA模板(10ng/ul) 1
无RNA酶的水 22
总量 50
表14：用于qPCR检测的扩增程序
Figure PCTCN2017081977-appb-000019
扩增的结果如表15中所示，其中分别列出了引物对CH1、CH2、CH4、CH5、CH6和CH7对各组模板DNA的q-PCR检测数据。其中当Ct值越大表明，该引物对应的模板数越低，对应在gDNA扩增中扩增效率越差。扩增结果显示：样品2_1中CH1、CH2、CH4、CH5、CH6和CH7扩增效率均较高。样品2_2中CH1、CH2、CH4、CH5、CH6和CH7的扩增效率均很高。与样本1-1与1-2的扩增没有本质区别。
表15：使用表12中的6对引物使用qPCR检测对图6中4个样本的扩增效率
Figure PCTCN2017081977-appb-000020
Figure PCTCN2017081977-appb-000021
实施例4：本申请的扩增方法用于Ion torrent测序平台
抽取新鲜的血液并使用淋巴细胞分离液分离淋巴细胞，其中用移液器抽取一部分包含细胞的悬浮液，在10x显微镜下使用口吸管挑取约3个白细胞，吸取的PBS溶液体积不超过1微升，并将挑取的3个左右白细胞转移进入包含4微升裂解缓冲液含有Tris-Cl、KCl、EDTA、Triton X-100和Qiagen Protease的PCR管内，并根据实施例1b)中所描述的步骤进 行裂解，并使用表1中的实验组9/10中所使用的引物混合物对基因组DNA进行线性扩增，并且使用如下第二引物混合物：第二引物-1(SEQ ID NO：37)：CCA CTA CGC CTC CGC TTT CCT CTC TAT GGG CAG TCG GTG ATG CTC TTC CGA TCT；
第二引物-2(SEQ ID NO：38)：
Figure PCTCN2017081977-appb-000022
Figure PCTCN2017081977-appb-000023
(其中，第二引物中以双下划线标识的碱基包括与测序平台的捕捉序列对应的部分，以点标识的部分为标识序列部分，可根据需要替换为其他标识序列，单下划线部分为通用序列部分)进行指数扩增得到扩增产物，平行进行4组实验。所有其他反应条件均与实施例1b)中所描述的一致。扩增效果如图11中的凝胶电泳所示。
均一性检测
之后随机选择根据实施例4扩增出的2个样品(在图11中显示为样本1和样本2)分别作为模板DNA。使用2xGoldstarMasterMix(购自北京康为世纪生物科技有限公司，货号CW0960)对模板DNA进行PCR检测，其中使用如表9中所示的引物扩增如表8中所示的35个致病位点。扩增体系组成如表10所示，扩增程序如表11所示。
扩增的结果如图12所示。扩增结果显示：35个致病位点在上述两个扩增产物样本中均能得到了很好地扩增，两个样本在扩增的准确性和扩增产物的量上没有显著差别。
基因测序
图11中显示的4个样本在经纯化后分别取等体积，使用Life Technologies的Ion torrent测序平台PGMTM测序仪进行测序，并将测序得到的序列比对到人类参考基因组上。测序结果如下表16及附图13所示。
表16：图11中样本半导体测序(PGM平台)测序结果
Figure PCTCN2017081977-appb-000024
表16中的数据显示，样本1、2、3、4中的unique_mapped_of_raw在68％左右，mapped_of_raw在72％-73％左右，数据量在0.38-0.53M之间，上机数据中的拷贝数变异系 数CV约为0.06。此外测序读数的拷贝数变异图如图13所示，每个实验组中染色体1-22除个别数据点以外均大致为两个拷贝，而性染色体X和Y均分别为大致1个拷贝。
值得注意的是，在Ion Torrent测序平台中并不需要针对测序平台设计特定的通用序列，原则上任何基本上不会与基因组DNA结合产生扩增的6-60bp长度范围内的序列均可选作通用序列。我们在Ion Torrent测序平台中还使用了另外两种引物组合进行检测：1)使用与表1中的实验组9/10中所使用的第一类引物混合物类似的第一类引物混合物：SEQ ID NO：15、16、19和20(但其中通用序列为SEQ ID NO:1)，对应的指数扩增引物混合物为SEQ ID NO：39CCA CTA CGC CTC CGC TTT CCT CTC TAT GGG CAG TCG GTG ATT TGG TAG TGA GTG和SEQ ID NO：40 
Figure PCTCN2017081977-appb-000025
Figure PCTCN2017081977-appb-000026
2)使用与表1中的实验组9/10中所使用的第一类引物混合物类似的第一类引物混合物：SEQ ID NO：23、24、27和28(但其中通用序列为SEQ ID NO:2)，对应的指数扩增引物混合物为SEQ ID NO：41CCA CTA CGC CTC CGC TTT CCT CTC TAT GGG CAG TCG GTG ATG AGG TGT GAT GGA和SEQ ID NO：42 
Figure PCTCN2017081977-appb-000027
Figure PCTCN2017081977-appb-000028
扩增与测序结果与图11及表16中所示的结果无明显差别(此处未提供具体数据)。
实施例5：本申请的扩增方法用于囊胚滋养层的胚胎植入前染色体检测
受精卵在体外培养，在囊胚期(体外培养第5天)取外胚滋养层的多个细胞(约3个细胞)进行染色体拷贝数异常的检测。采集囊胚外胚滋养层细胞的方法可以为任何本领域技术人员公知的方法，例如但不限于Wang L,Cram DS et al.Validation of copy number variation sequencing for detecting chromosome imbalances in human preimplantation embryos.Biol Reprod,2014,91(2):37中所述的方法。采集的囊胚外胚滋养层细胞转移进入包含5微升裂解缓冲液的PCR管内，加入裂解酶，根据实施例1b)中所描述的步骤进行裂解，并使用表1中的实验组9/10中所使用的引物混合物对基因组DNA进行扩增(平行进行4组实验)。如实施例1中所述的步骤对扩增产物进行纯化及测序。测序结果如下表17中所示。
表17：囊胚滋养样本测序结果
Figure PCTCN2017081977-appb-000029
Figure PCTCN2017081977-appb-000030
表17中的数据显示，样本1、2、3中的unique_mapped_of_raw在66-73％左右，mapped_of_raw在71％-78％左右，数据量在1.4-2.6M之间。此外测序读数的拷贝数变异图如图14所示，每个实验组中染色体1-22除个别数据点以外均大致为两个拷贝，而性染色体X和Y均分别为大致1个拷贝。
尽管本发明已公开了多个方面和实施方式，但是其它方面和实施方式对本领域技术人员而言将是显而易见的。本发明公开的多个方面和实施方式仅用于举例说明，其并非旨在限制本发明，本发明的实际保护范围以权利要求为准。

Claims (52)

  1. 一种扩增基因组DNA的方法，所述方法包括：
    (a)提供第一反应混合物，其中所述第一反应混合物包括包含所述基因组DNA的样本、第一引物、核苷酸单体混合物和核酸聚合酶，其中所述第一引物从5’端到3’端包含通用序列和第一可变序列，所述第一可变序列包括第一随机序列，其中所述第一随机序列从5’端到3’端依次为Xa1Xa2……Xan，所述第一随机序列的Xai(i＝1-n)均属于同一个集合，所述集合选自B、或D、或H、或V，其中B＝{T、G、C}，D＝{A、T、G}，H＝{T、A、C}，V＝{A、C、G}，其中Xai表示第一随机序列5’端的第i个核苷酸，n是选自3-20的正整数，
    可选地，所述第一反应混合物进一步包括第三引物，其中所述第三引物从5’端到3’端包含所述通用序列和第三可变序列，所述第三可变序列包括第三随机序列，其中所述第三随机序列从5’端到3’端依次为Xb1Xb2……Xbn，所述第三随机序列的Xbi(i＝1-n)均属于同一个集合，所述集合选自B、或D、或H、或V，其中B＝{T、G、C}，D＝{A、T、G}，H＝{T、A、C}，V＝{A、C、G}，并且Xbi(i＝1-n)和Xai(i＝1-n)属于不同的集合，其中Xbi表示第三随机序列5’端的第i个核苷酸，n是选自3-20的正整数；
    (b)将所述第一反应混合物置于第一温度循环程序进行预扩增，获得预扩增产物；
    (c)提供第二反应混合物，所述第二反应混合物包括步骤(b)中得到的预扩增产物、第二引物、核苷酸单体混合物和核酸聚合酶，其中所述第二引物从5’端到3’端包含或由特定序列及所述通用序列组成；
    (d)将所述第二反应混合物置于第二温度循环程序进行扩增，获得扩增产物。
  2. 根据权利要求1所述的方法，其中第一随机序列的Xai(i＝1-n)均属于集合B，第三随机序列的Xbi(i＝1-n)均属于集合D。
  3. 根据权利要求1所述的方法，其中所述第一可变序列和所述第三可变序列进一步在其3’端包括固定序列，所述固定序列能够提高基因组覆盖度的碱基组合。
  4. 根据权利要求3所述的方法，其中所述固定序列选自CCC、AAA、TGGG、GTTT、GGG、TTT、TNTNG或GTGG。
  5. 根据权利要求1所述的方法，其中所述第一可变序列选自Xa1Xa2……XanTGGG或Xa1Xa2……XanGTTT，所述第三可变序列选自Xb1Xb2……XbnTGGG或Xb1Xb2……XbnGTTT。
  6. 根据权利要求1所述的方法，其中选择所述通用序列以使得其基本上不会与基因组DNA结合产生扩增，所述通用序列长度为6-60bp。
  7. 根据权利要求6所述的方法，其中选择所述通用序列使得扩增产物能够直接进行测序。
  8. 根据权利要求1所述的方法，其中所述通用序列选自SEQ ID NO:1[TTGGTAGTGAGTG]、SEQ ID NO:2[GAGGTGTGATGGA]、SEQ ID NO:3[GTGATGGTTGAGGTA]、SEQ ID NO:4[AGATGTGTATAAGAGACAG]、SEQ ID NO:5[GTGAGTGATGGTTGAGGTAGTGTGGAG]或SEQ ID NO:6[GCTCTTCCGATCT]。
  9. 根据权利要求1所述的方法，其中所述通用序列和所述第一可变序列直接相连，或者所述通用序列和所述第一可变序列通过第一间隔序列相连，所述第一间隔序列为Ya1……Yam，其中Yaj(j＝1-m)∈{A、T、G、C}，其中Yaj表示间隔序列5’端的第j个核苷酸，m是选自1-3的正整数。
  10. 根据权利要求1所述的方法，其中所述通用序列和所述第三可变序列直接相连，或者所述通用序列和所述第三可变序列通过第三间隔序列相连，所述第三间隔序列为Yb1……Ybm，其中Ybj(j＝1-m)∈{A、T、G、C}，其中Ybj表示间隔序列5’端的第j个核苷酸，m是选自1-3的正整数。
  11. 根据权利要求9或10所述的方法，其中所述m＝1。
  12. 根据权利要求11所述的方法，其中所述第一引物包括GCTCTTCCGATCTYa1Xa1Xa2Xa3Xa4Xa5TGGG、GCTCTTCCGATCTYa1Xa1Xa2Xa3Xa4Xa5GTTT或其混合物，所述第三引物包括GCTCTTCCGATCTYb1Xb1Xb2Xb3Xb4Xb5TGGG、GCTCTTCCGATCTYb1Xb1Xb2Xb3Xb4Xb5GTTT或其混合物，其中Ya1∈{A、T、G、C}，Yb1∈{A、T、G、C}，所述Xai(i＝1-5)∈{T、G、C}，所述Xbi(i＝1-5)∈{A、T、G}。
  13. 根据权利要求1所述的方法，其中所述方法进一步包括对步骤(d)中获得的扩增产物进行测序的步骤，其中所述第二引物包括与测序用引物的部分或全部互补或者相同的序列。
  14. 根据权利要求13所述的方法，其中所述通用序列包括与测序用引物的部分或全部 互补或者相同的序列。
  15. 根据权利要求13所述的方法，其中所述第二引物的特定序列包括与测序用引物的部分或全部互补或者相同的序列。
  16. 根据权利要求15所述的方法，其中所述第二引物的特定序列进一步包括与测序平台的捕捉序列部分或全部互补或者相同的序列。
  17. 根据权利要求15所述的方法，其中所述第二引物的特定序列中包含的与测序用引物的部分或全部互补或相同的序列包含或由SEQ ID NO:31[ACACTCTTTCCCTACACGAC]、或SEQ ID NO:32[GTGACTGGAGTTCAGACGTGT]组成。
  18. 根据权利要求16所述的方法，其中所述第二引物的特定序列中包含的与测序平台的捕捉序列部分或全部互补或相同的序列包含或由SEQ ID NO:33[AATGATACGGCGACCACCGAGATCT]、或SEQ ID NO:34[CAAGCAGAAGACGGCATACGAGAT]组成。
  19. 根据权利要求16所述的方法，其中所述第二引物的特定序列进一步包括标识序列，所述标识序列位于所述与测序平台的捕捉序列部分或全部互补或相同的序列和所述与测序用引物的部分或全部互补或相同的序列之间。
  20. 根据权利要求1所述的方法，其中所述第二引物包括具有相同通用序列和不同特定序列的引物混合物，所述不同特定序列分别与同一测序中用到的测序引物对中不同引物的部分或全部互补或相同。
  21. 根据权利要求1所述的方法，其中所述第二引物包括SEQ ID NO:35[AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT]和SEQ ID NO:36[CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT]所示的序列的混合物。
  22. 根据权利要求1所述的方法，其中所述核酸聚合酶具有热稳定和/或链置换活性。
  23. 根据权利要求1所述的方法，其中所述核酸聚合酶选自：Phi29 DNA聚合酶、Bst  DNA 聚合酶、Pyrophage 3137、Vent聚合酶、TOPOTaq DNA聚合酶、9°Nm聚合酶、Klenow Fragment DNA聚合酶I、MMLV反转录酶、AMV反转录酶、HIV反转录酶、T7 phase DNA聚合酶变种、
    Figure PCTCN2017081977-appb-100001
    超保真DNA聚合酶、Taq聚合酶、Bst DNA聚合酶、E.coli DNA聚合酶、LongAmp Taq DNA聚合酶、OneTaq DNA聚合酶、Deep Vent DNA聚合酶、Vent(exo-)DNA聚合酶、Deep Vent(exo-)DNA聚合酶，及其任意组合。
  24. 根据权利要求1所述的方法，其中步骤(b)使得所述第一类引物的可变序列能够与所述基因组DNA配对并扩增所述基因组DNA以得到基因组预扩增产物，其中所述基因组预扩增产物的5’端包含所述通用序列，3’端包含所述通用序列的互补序列。
  25. 根据权利要求1所述的方法，其中所述第一温度循环程序包括：
    (b1)能够打开所述DNA双链以获得DNA单链模板的温度程序；
    (b2)能够使所述第一引物以及可选的第三引物与所述DNA单链模板结合的温度程序；
    (b3)在所述核酸聚合酶的作用下能够使与所述DNA单链模板结合的第一类引物延伸长度以产生预扩增产物的温度程序；
    (b4)重复步骤(b1)到(b3)至指定的第一循环次数，其中所述指定的第一循环次数大于1。
  26. 根据权利要求25所述的方法，其中在进行第一次循环时，步骤(b1)中所述DNA双链为基因组DNA双链，所述温度程序包括在90-95℃的温度之间变性反应1-20分钟。
  27. 根据权利要求26所述的方法，其中在进行第一次循环后，步骤(b1)中所述的温度程序包括在90-95℃的温度之间解链反应3-50秒。
  28. 根据权利要求26所述的方法，当进行到第二次循环后，所述预扩增产物包含在5’端包含所述通用序列，3’端包含所述通用序列的互补序列的基因组预扩增产物。
  29. 根据权利要求25所述的方法，其中在步骤(b1)后并且在步骤(b2)之前不包括将所述第一反应混合物置于适当的温度程序使得所述基因组预扩增产物的3’端与5’端杂交结合以形成发卡结构的额外步骤。
  30. 根据权利要求25所述的方法，其中所述步骤(b2)包括将所述反应混合物置于多于一种的温度程序，以促使所述第一类引物充分与所述DNA模板有效结合。
  31. 根据权利要求30所述的方法，其中所述多于一种的温度程序包括：介于10-20℃之间的第一温度，介于20-30℃之间的第二温度，和介于30-50℃之间的第三温度。
  32. 根据权利要求31所述的方法，其中所述步骤(b2)中所述步骤包括在第一温度退火反应3-60秒、在第二温度退火反应3-50秒和在第三温度退火反应3-50秒。
  33. 根据权利要求25所述的方法，其中所述步骤(b3)中所述的温度程序包括在60-80℃的温度之间延伸反应10秒-15分钟。
  34. 根据权利要求25所述的方法，其中所述步骤(b4)的所述第一循环次数为2-40。
  35. 根据权利要求1所述的方法，其中所述步骤(d)使得所述第二引物的所述通用序列能够与所述基因组预扩增产物的3’端配对并扩增所述基因组预扩增产物以得到扩大的基因组扩增产物。
  36. 根据权利要求1所述的方法，其中所述步骤(d)包括：
    (d1)能够打开DNA双链的温度程序；
    (d2)进一步能打开DNA双链的温度程序；
    (d3)能够使所述第二引物与所述经步骤(b)获得的基因组预扩增产物的单链结合的温度程序；
    (d4)能够使与所述基因组预扩增产物单链结合的第二引物在所述核酸聚合酶的作用下延伸长度的温度程序；
    (d5)重复步骤(d2)到(d4)至指定的第二循环次数，其中所述指定的第二循环次数大于1。
  37. 根据权利要求36所述的方法，其中步骤(d1)中所述DNA双链为所述基因组预扩增产物，并且所述DNA双链包括DNA发卡结构中包含的双链，所述温度程序包括90-95℃的温度之间变性反应5秒-20分钟。
  38. 根据权利要求36所述的方法，其中步骤(d2)中所述的温度程序包括在90-95℃的温度之间解链反应3-50秒。
  39. 根据权利要求36所述的方法，其中所述步骤(d3)中所述的温度程序包括在45-65℃的温度之间退火反应3-50秒。
  40. 根据权利要求36所述的方法，其中所述步骤(d4)中所述的温度程序包括在60-80℃的温度之间延伸反应10秒-15分钟。
  41. 根据权利要求1所述的方法，进一步包括分析所述扩增产物以识别与疾病或表型相关的序列特征。
  42. 根据权利要求41所述的方法，其中所述与疾病或表型相关的序列特征包括染色体水平异常、染色体的异位、非整倍体、部分或全部染色体的缺失或重复、胎儿HLA单倍型和父源突变，或者所述疾病或表型选自下组：β-地中海贫血、唐氏综合征、囊性纤维化、镰状细胞病、泰-萨克斯病、脆性X综合征、脊髓性肌萎缩症、血红蛋白病、α-地中海贫血、X连锁疾病(由在X染色体上基因主导的疾病)、脊柱裂、无脑畸形、先天性心脏病、肥胖、糖尿病、癌症、胎儿性别、胎儿RHD。
  43. 根据权利要求41所述的方法，其中所述基因组DNA来源于卵裂球、囊胚滋养层、培养的细胞、提取后的gDNA或囊胚培养液。
  44. 一种扩增基因组DNA的方法，所述方法包括：
    (a)提供第一反应混合物，其中所述第一反应混合物包括包含所述基因组DNA的样本、第一引物、核苷酸单体混合物、和核酸聚合酶，其中所述第一引物从5’端到3’端包含通用序列和可变序列，其中所述第一引物从5’端到3’端包含通用序列和第一可变序列，所述第一可变序列包括第一随机序列，其中所述第一随机序列从5’端到3’端依次为Xa1Xa2……Xan，所述第一随机序列的Xai(i＝1-n)均属于同一个集合，所述集合选自B、或D、或H、或V，其中B＝{T、G、C}，D＝{A、T、G}，H＝{T、A、C}，V＝{A、C、G}，其中Xai表示第一随机序列5’端的第i个核苷酸，n是选自3-20的正整数，其中所述通用序列和所述第一可变序列直接相连、或所述通用序列和所述第一可变序列通过第一间隔序列相连，所述第一间隔序列为Ya1……Yam，其中Yaj(j＝1-m)∈{A、T、G、C}，其中Yaj表示间隔序列5’端的第j个核苷酸，
    可选地，其中所述第一反应混合物进一步包括第三引物，其中所述第三引物从5’端到3’端包含所述通用序列和第三可变序列，所述第三可变序列包括第三随机序列，其中所述第三随机序列从5’端到3’端依次为Xb1Xb2……Xbn，所述第三随机序列的Xbi(i＝1-n)均属于同一个集合，所述集合选自B、或D、或H、或V，其中B＝{T、G、C}，D＝{A、T、G}， H＝{T、A、C}，V＝{A、C、G}，并且Xbi(i＝1-n)和Xai(i＝1-n)属于不同的集合，其中Xbi表示第一随机序列5’端的第i个核苷酸，n是选自3-20的正整数，其中所述通用序列和所述第三可变序列直接相连，或者所述通用序列和所述第三可变序列通过第三间隔序列相连，所述第三间隔序列为Yb1……Ybm，其中Ybj(j＝1-m)∈{A、T、G、C}，其中Ybj表示间隔序列5’端的第j个核苷酸，m是选自1-3的正整数；
    (b)将所述第一反应混合物置于第一温度循环程序，使得所述第一引物的第一可变序列以及可选的第三引物的第三可变序列能够与所述基因组DNA配对并扩增所述基因组DNA以得到基因组预扩增产物，其中所述基因组预扩增产物的5’端包含所述通用序列，3’端包含所述通用序列的互补序列；其中所述第一温度循环程序包括：
    (b1)第一个循环为在介于90-95℃的温度之间的第一变性温度反应1-20分钟，第一个循环之后为在介于90-95℃的温度之间的第一变性温度反应3-50秒；
    (b2)在介于10-20℃之间的第一退火温度反应3-60秒，介于20-30℃之间的第二退火温度反应3-50秒，和介于30-50℃之间的第三退火温度反应3-50秒；
    (b3)在介于60-80℃之间的第一延伸温度反应10秒-15分钟；
    (b4)重复步骤(b1)到(b3)至2-40个循环；
    (c)提供第二反应混合物，所述第二反应混合物包括步骤(b)中得到的所述基因组预扩增产物、第二引物、核苷酸单体混合物、和核酸聚合酶，其中所述第二引物的从5’端到3’端包含或由特定序列及所述通用序列组成；
    (d)将所述第二反应混合物置于第二温度循环程序，使得所述第二引物的所述通用序列能够与所述基因组预扩增产物的3’端配对并扩增所述基因组预扩增产物以得到扩大的基因组扩增产物，其中所述第二温度循环程序包括：
    (d1)在介于90-95℃之间的第二变性温度反应5秒-20分钟；
    (d2)在介于90-95℃之间的第二解链温度反应3-50秒；
    (d3)在介于45-65℃之间的第四退火温度反应3-50秒；
    (d4)在介于60-80℃之间的第二延伸温度反应10秒-15分钟；
    (d5)重复步骤(d2)到(d4)2-40个循环。
  45. 如权利要求44所述的方法，其中所述通用序列包含或由SEQ ID NO:6组成；所述第一随机序列的Xai(i＝1-n)均属于D，所述第三随机序列的Xbi(i＝1-n)均属于B。
  46. 如权利要求1所述的方法，其中步骤(d)得到的扩增产物已完成了文库构建。
  47. 一种用于扩增基因组DNA的试剂盒，所述试剂盒包括第一引物，其中所述第一引物从5’端到3’端包含通用序列和第一可变序列，所述第一可变序列包括第一随机序列，其中所述第一随机序列从5’端到3’端依次为Xa1Xa2……Xan，所述第一随机序列的Xai(i＝1-n)均属于同一个集合，所述集合选自B、或D、或H、或V，其中B＝{T、G、C}，D＝{A、T、G}，H＝{T、A、C}，V＝{A、C、G}，其中Xai表示第一随机序列5’端的第i个核苷酸，n是选自3-20的正整数，其中所述通用序列和所述第一可变序列直接相连、或所述通用序列和所述第一可变序列通过第一间隔序列相连，所述第一间隔序列为Ya1……Yam，其中Yaj(j＝1-m)∈{A、T、G、C}，其中Yaj表示间隔序列5’端的第j个核苷酸，m是选自1-3的正整数，
    可选地，其中所述第一反应混合物进一步包括第三引物，其中所述第三引物从5’端到3’端包含所述通用序列和第三可变序列，所述第三可变序列包括第三随机序列，其中所述第三随机序列从5’端到3’端依次为Xb1Xb2……Xbn，所述第三随机序列的Xbi(i＝1-n)均属于同一个集合，所述集合选自B、或D、或H、或V，其中B＝{T、G、C}，D＝{A、T、G}，H＝{T、A、C}，V＝{A、C、G}，并且Xbi(i＝1-n)和Xai(i＝1-n)属于不同的集合，其中Xbi表示第一随机序列5’端的第i个核苷酸，n是选自3-20的正整数，其中所述通用序列和所述第三可变序列直接相连，或者所述通用序列和所述第三可变序列通过第三间隔序列相连，所述第三间隔序列为Yb1……Ybm，其中Ybj(j＝1-m)∈{A、T、G、C}，其中Ybj表示间隔序列5’端的第j个核苷酸，m是选自1-3的正整数。
  48. 如权利要求47所述试剂盒，其中所述通用序列包含或由SEQ ID NO:6组成；所述第一随机序列的Xai(i＝1-n)均属于D，所述第二随机序列的Xbi(i＝1-n)均属于B。
  49. 如权利要求47所述试剂盒，其中所述试剂盒用于构建全基因组DNA文库。
  50. 如权利要求47所述试剂盒，所述试剂盒进一步包括核酸聚合酶，其中所述核酸聚合酶选自：Phi29 DNA聚合酶、Bst DNA聚合酶、Pyrophage 3137、Vent聚合酶、TOPOTaq  DNA聚合酶、9°Nm聚合酶、Klenow Fragment DNA聚合酶I、MMLV反转录酶、AMV反转录酶、HIV反转录酶、T7 phase DNA聚合酶变种、
    Figure PCTCN2017081977-appb-100002
    超保真DNA聚合酶、Taq聚合酶、Bst DNA聚合酶、E.coli DNA聚合酶、LongAmp Taq DNA聚合酶、OneTaq DNA聚合酶、Deep Vent DNA聚合酶、Vent(exo-)DNA聚合酶、Deep Vent(exo-)DNA聚合酶，及其任意组合。
  51. 如权利要求47-50任一项所述试剂盒，其中所述试剂盒进一步包括包含一种或多种选自下组的成分：核苷酸单体混合物、Mg2+、dTT、牛血清白蛋白、pH调节剂、DNase抑制剂、RNase、SO4 2-、Cl-、K+、Ca2+、Na+、(NH4)+的一种或多种试剂。
  52. 如权利要求47所述试剂盒，其中所述混合物进一步包括细胞裂解剂，所述细胞裂解剂选自：蛋白酶K、胃蛋白酶、木瓜蛋白酶、NP-40、吐温、SDS、TritonX-100、EDTA和异硫氰酸胍中的一种或多种。
PCT/CN2017/081977 2016-04-26 2017-04-26 扩增dna的方法 Ceased WO2017186117A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP17788769.2A EP3450569B1 (en) 2016-04-26 2017-04-26 Dna amplification method
US16/093,975 US20190106738A1 (en) 2016-04-26 2017-04-26 Dna amplification method

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610264059.0A CN105925675B (zh) 2016-04-26 2016-04-26 扩增dna的方法
CN201610264059.0 2016-04-26

Publications (1)

Publication Number Publication Date
WO2017186117A1 true WO2017186117A1 (zh) 2017-11-02

Family

ID=56836189

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/081977 Ceased WO2017186117A1 (zh) 2016-04-26 2017-04-26 扩增dna的方法

Country Status (5)

Country Link
US (1) US20190106738A1 (zh)
EP (1) EP3450569B1 (zh)
CN (2) CN111621548A (zh)
TW (1) TWI742059B (zh)
WO (1) WO2017186117A1 (zh)

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111621548A (zh) * 2016-04-26 2020-09-04 序康医疗科技（苏州）有限公司 扩增dna的方法
CN108070586A (zh) * 2016-11-18 2018-05-25 杭州拓宏生物科技有限公司 Pcr扩增引物及其应用
CN106755506A (zh) * 2016-12-27 2017-05-31 安诺优达基因科技(北京)有限公司 用于检测肿瘤ffpe样本中基因变异的试剂盒
WO2018148903A1 (zh) * 2017-02-16 2018-08-23 上海亿康医学检验所有限公司 泌尿系统肿瘤的辅助诊断方法
CN108504651B (zh) * 2017-02-27 2020-09-08 深圳乐土生物科技有限公司 基于高通量测序的pcr产物大样本量混合建库的文库构建方法和试剂
AU2018231240A1 (en) * 2017-03-08 2019-10-31 President And Fellows Of Harvard College Methods of amplifying DNA to maintain methylation status
CN111406114A (zh) * 2017-05-29 2020-07-10 哈佛学院董事及会员团体 扩增单个细胞转录组的方法
CN107254541B (zh) * 2017-08-03 2020-05-08 广州万德基因医学科技有限公司 用于扩增cfDNA样品中多个目标的NGS建库引物池及应用
CN109593757B (zh) * 2017-09-30 2021-08-03 厦门艾德生物医药科技股份有限公司 一种探针及其适用于高通量测序的对目标区域进行富集的方法
CN108456713A (zh) * 2017-11-27 2018-08-28 天津诺禾致源生物信息科技有限公司 接头封闭序列、文库构建试剂盒及测序文库的构建方法
CN107937582A (zh) * 2017-12-29 2018-04-20 苏州普瑞森基因科技有限公司 一种用于分析肠道微生物的引物组及其应用
CN108959851B (zh) * 2018-06-12 2022-03-18 哈尔滨工程大学 一种Illumina高通量测序数据误差校正方法
CN109629009B (zh) * 2019-01-10 2022-02-22 北京中科遗传与生殖医学研究院有限责任公司 一种基于RAD-seq对胚胎进行无创PGS的方法
CN111575346A (zh) * 2020-05-19 2020-08-25 泰州亿康医学检验有限公司 一种针对细胞分选后的单细胞全基因组扩增方法
CN111826421B (zh) * 2020-07-09 2021-09-21 广州迈景基因医学科技有限公司 一种pcr随机引物和使用其构建靶向测序文库的方法
CN113832147B (zh) * 2021-09-08 2024-06-14 华南农业大学 一种高效的大片段dna合成与扩增的pcr引物、方法及应用
CN114015751A (zh) * 2021-10-26 2022-02-08 江苏海伯基因科技有限公司 扩增基因组dna的方法、试剂盒及其获得扩增引物的方法
CN116656787A (zh) * 2023-04-04 2023-08-29 墨卓生物科技(浙江)有限公司 一种核酸序列扩增和标记的方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030100006A1 (en) * 1999-04-21 2003-05-29 Periannan Senapathy Shot-gun sequencing and amplification without cloning
US20090099040A1 (en) * 2007-10-15 2009-04-16 Sigma Aldrich Company Degenerate oligonucleotides and their uses
CN103890191A (zh) * 2011-05-27 2014-06-25 哈佛大学校长及研究员协会 单细胞全基因组扩增方法
CN104630202A (zh) * 2013-11-13 2015-05-20 北京大学 一种能够减小微量核酸物质整体扩增时产生偏倚的扩增方法
WO2015171656A1 (en) * 2014-05-06 2015-11-12 Baylor College Of Medicine Methods of linearly amplifying whole genome of a single cell
CN105368936A (zh) * 2015-11-05 2016-03-02 上海序康医疗科技有限公司 一种利用囊胚培养液检测胚胎染色体异常的方法
CN105925675A (zh) * 2016-04-26 2016-09-07 序康医疗科技（苏州）有限公司 扩增dna的方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7993839B2 (en) * 2001-01-19 2011-08-09 General Electric Company Methods and kits for reducing non-specific nucleic acid amplification
EP2209912A1 (en) * 2007-10-26 2010-07-28 Rosetta Inpharmatics LLC Cdna synthesis using non-random primers
KR102299305B1 (ko) * 2013-06-21 2021-09-06 시쿼넘, 인코포레이티드 유전적 변이의 비침습 평가를 위한 방법 및 프로세스
US9587263B2 (en) * 2014-03-26 2017-03-07 General Electric Company Isothermal amplification under low salt condition

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030100006A1 (en) * 1999-04-21 2003-05-29 Periannan Senapathy Shot-gun sequencing and amplification without cloning
US20090099040A1 (en) * 2007-10-15 2009-04-16 Sigma Aldrich Company Degenerate oligonucleotides and their uses
CN103890191A (zh) * 2011-05-27 2014-06-25 哈佛大学校长及研究员协会 单细胞全基因组扩增方法
CN104630202A (zh) * 2013-11-13 2015-05-20 北京大学 一种能够减小微量核酸物质整体扩增时产生偏倚的扩增方法
WO2015171656A1 (en) * 2014-05-06 2015-11-12 Baylor College Of Medicine Methods of linearly amplifying whole genome of a single cell
CN105368936A (zh) * 2015-11-05 2016-03-02 上海序康医疗科技有限公司 一种利用囊胚培养液检测胚胎染色体异常的方法
CN105925675A (zh) * 2016-04-26 2016-09-07 序康医疗科技（苏州）有限公司 扩增dna的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHAPMAN, A.R. ET AL.: "Single Cell Transcriptome Amplification with MALBAC", PLOS ONE, vol. 10, no. 3, 30 March 2015 (2015-03-30), pages e0120889, XP055433842 *

Also Published As

Publication number Publication date
CN105925675A (zh) 2016-09-07
EP3450569B1 (en) 2026-05-13
CN105925675B (zh) 2020-06-05
TWI742059B (zh) 2021-10-11
EP3450569A4 (en) 2020-03-11
EP3450569A1 (en) 2019-03-06
CN111621548A (zh) 2020-09-04
TW201738383A (zh) 2017-11-01
US20190106738A1 (en) 2019-04-11

Similar Documents

Publication Publication Date Title
CN105925675B (zh) 扩增dna的方法
AU2022202625B2 (en) Methods and compositions for rapid nucleic acid library preparation
US20220389408A1 (en) Methods and compositions for phased sequencing
US11041192B2 (en) Method for amplifying DNA
WO2021052310A1 (zh) 一种dna文库构建方法
US20250230485A1 (en) Barcoding of nucleic acids
US20250084403A1 (en) Compositions and methods for making controls for sequence-based genetic testing
CN114015751A (zh) 扩增基因组dna的方法、试剂盒及其获得扩增引物的方法
WO2014129894A1 (en) Sequencing strategies for genomic regions of interest
HK1228461B (zh) 扩增dna的方法
HK1228461A1 (zh) 扩增dna的方法
HK1228461A (zh) 擴增dna的方法
HK40114910A (zh) 用於定相测序的方法和组合物
HK40064558A (zh) 用於制备快速核酸库的组合物
US20180282799A1 (en) Targeted locus amplification using cloning strategies
HK40013260B (zh) 用於定相测序的方法和组合物
HK40013260A (zh) 用於定相测序的方法和组合物
NZ749719A (en) Single cell whole genome libraries and combinatorial indexing methods of making thereof
HK1237376A1 (zh) 用於多重捕获反应的通用阻断寡聚物系统和改进的杂交捕获的方法

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2017788769

Country of ref document: EP

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17788769

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2017788769

Country of ref document: EP

Effective date: 20181126

WWG Wipo information: grant in national office

Ref document number: 2017788769

Country of ref document: EP