WO2017036374A1 - 扩增dna的方法 - Google Patents

扩增dna的方法 Download PDF

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WO2017036374A1
WO2017036374A1 PCT/CN2016/097208 CN2016097208W WO2017036374A1 WO 2017036374 A1 WO2017036374 A1 WO 2017036374A1 CN 2016097208 W CN2016097208 W CN 2016097208W WO 2017036374 A1 WO2017036374 A1 WO 2017036374A1
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primer
sequence
reaction mixture
temperature
amplification
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PCT/CN2016/097208
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English (en)
French (fr)
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陆思嘉
殷广军
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上海序康医疗科技有限公司
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Priority to EP16840803.7A priority Critical patent/EP3346006B1/en
Priority to US15/756,987 priority patent/US11041192B2/en
Priority to ES16840803T priority patent/ES2894358T3/es
Publication of WO2017036374A1 publication Critical patent/WO2017036374A1/zh

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    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to methods of amplifying DNA, particularly in methods of amplifying a single cell whole genome.
  • 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.
  • the establishment of this technology requires two prerequisites: 1. High-quality whole genome amplification technology; 2. High-throughput low-cost sequencing technology.
  • PEP-PCR Primer Extension Preamplification-Polymerase Chain Reaction
  • DOP-PCR Polymerase chain reaction
  • MALBC Molecular Amplification-Coupled Device
  • it mainly consists of the following defects: 1. Generally, it is necessary to undergo a plurality of steps such as cell lysis, termination of lysis (warming/addition of neutralizing reagent), pre-amplification, amplification, etc. to obtain an amplification product, and the whole process involves Multiple reagent preparation, opening and adding liquid operation increases the risk of introducing environmental pollution; 2. The whole experimental process takes more than 4 hours, and the efficiency of personnel and instruments is low. It is impossible to give a sample that is urgently needed for clinical verification in a short time. Satisfactory results; 3. The entire experimental process requires a high level of proficiency for the operator, and the first contact person cannot quickly obtain satisfactory amplification results.
  • the present invention provides a method of amplifying cellular genomic DNA and a kit for amplifying genomic DNA.
  • a method of amplifying cellular genomic DNA comprising: (a) providing a reaction mixture, wherein the reaction mixture comprises the genomic DNA, a first type of primer, a second a primer, a nucleotide monomer mixture, and a nucleic acid polymerase, wherein the first type of primer comprises a universal sequence and a variable sequence from the 5' end to the 3' end, wherein the universal sequence is composed of G, A, C, and Three or two of the four bases of T, provided that the universal sequence does not include G and C at the same time, and the second type of primer comprises the universal sequence and does not comprise the variable sequence; b) placing the reaction mixture in a first temperature cycling sequence such that the variable sequence of the first type of primer is capable of pairing with the genomic DNA and amplifying the genomic DNA to obtain a genomic amplification product, wherein The 5' end of the genomic amplification product comprises the universal sequence, the 3' end comprises the complement of the universal sequence; (c) the reaction mixture
  • 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, Tay-Sax disease, Fragile X syndrome, spinal muscular atrophy Disease, 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 and fetal RHD.
  • the genomic DNA is contained in a cell, and the reaction mixture further comprises a surfactant and/or a lytic enzyme capable of cleaving the cell.
  • the reaction mixture prior to said step (b) and step (c), is further placed in a lysis temperature cycling sequence such that the cells lyse and release the genomic DNA.
  • the universal sequence is selected such that it does not substantially bind to genomic DNA to produce amplification. In some embodiments, the universal sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.
  • variable sequence comprises a random sequence.
  • variable sequence is 2-20 bases, 3-10 bases, 4-9 bases, or 5-8 bases in length.
  • three or more base positions in the variable sequence consist of one or several bases selected from G, A, and T, or by C, A, and T One or several bases. In some embodiments, the three or more base positions are located at or near the 3' end of the variable sequence.
  • variable sequence is selected from the group consisting of: (N) nGGG, (N) nTTT, (N) mTNTNG, (N) xGTGG(N)y, wherein N is any which can be carried out with a natural nucleic acid Base pairing Nucleotides, n is a positive integer selected from 3-17, m is a positive integer selected from 3-15, and x and y are each a positive integer selected from 3-13. In some embodiments, the variable sequences are selected such that they are respectively uniform on the genome and have a high degree of coverage.
  • the first class of primers comprises SEQ ID NO: 11 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNNNNNN], SEQ ID NO: 12 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNGGG], SEQ ID NO: 13 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNTTT], SEQ ID NO: 14 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNTNG] or SEQ ID NO: sequence of 15 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNGTGGNN], and the second type of primer has a sequence of SEQ ID NO: 1 [GTGAGTGATGGTTGAGGTAGTGT GGAG] from 5' to 3', wherein N is any base pairing with a natural nucleic acid. Nucleotide.
  • 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 (eg, Vent polymerase of Thermococcus litoralis, Deep Vent polymerase, Vent (-exo) polymerization.
  • Enzyme Deep Vent (-exo) polymerase, TOPOTaq DNA polymerase, 9°Nm polymerase, Klenow Fragment DNA polymerase I, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, T7 phase DNA polymerase variant (lack of 3'-5' exonuclease activity), Ultra-fidelity DNA polymerase, Taq polymerase, Bst DNA polymerase (full length), E. coli DNA polymerase, LongAmp Taq DNA polymerase, OneTaq DNA polymerase, and any combination thereof.
  • the reaction mixture further comprises a pH adjusting agent such that the pH of the reaction mixture is maintained between 7.0 and 9.0.
  • the reaction mixture further comprises one or more components selected from the group consisting of Mg 2+ , dTT, bovine serum albumin, DNase inhibitors, RNase, SO 4 2- , Cl ⁇ , K + , Ca 2+ , Na + , and (NH 4 ) + .
  • the first temperature cycling procedure comprises: (b1) placing the reaction mixture in a temperature program capable of opening a double strand of the genomic DNA; (b2) placing the reaction mixture in a a temperature program for binding the first type of primer to the DNA single strand template; (b3) placing the reaction mixture in a length that enables the first type of primer to bind to the DNA single strand template to extend under the action of the nucleic acid polymerase a temperature program to produce an amplification product; (b4) placing the reaction mixture in a temperature program capable of detaching the amplification product into a single strand; (b5) repeating steps (b2) through (b4) to a specified The number of first cycles. In some embodiments, the specified first number of cycles is greater than two.
  • the amplification product upon completion of the second cycle, comprises a genomic amplification product comprising the universal sequence at the 5' end and a complementary sequence of the universal sequence at the 3'
  • the method further comprises a step (b4') after step (b4) and before step (b5), wherein the reaction mixture is placed in an appropriate temperature program such that the genomic amplification product The 3' end is hybridized to the 5' end to form a circular structure, or the 3' end of the genomic amplification product is bound to a primer.
  • the method proceeds directly to step (b5) after step (b4).
  • the first number of cycles of step (b5) is greater than 3, greater than 4, greater than 5, greater than 6, and no greater than 10.
  • the step (c) comprises: (c1) placing the reaction mixture obtained in step (b) in a capable Opening a temperature program of the DNA duplex; (c2) placing the reaction mixture in a temperature program capable of binding the second type of primer to the single strand of the genomic amplification product obtained in the step (b); The reaction mixture is placed in a temperature program capable of extending the length of the second type of primer bound to the amplification product under the action of the nucleic acid polymerase; (c4) repeating steps (c1) to (c3) ) to the specified number of second cycles.
  • the second number of cycles in the step (c4) is greater than the first number of cycles described in the step (b5).
  • the temperature program described in step (b1) comprises reacting between temperatures of 90-95 ° C for 1-10 minutes.
  • the 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; in some embodiments
  • the more than one temperature program includes: a first temperature between 5-10 ° C, a second temperature between 25-30 ° C, and a third between 45-50 ° C temperature.
  • the step in the step (b2) comprises reacting at a first temperature for 3-50 seconds, reacting at a second temperature for 3-50 seconds, and reacting at a third temperature for 3-50 seconds.
  • the temperature program described in step (b3) comprises reacting between temperatures of 60-90 ° C for 1-15 minutes.
  • the temperature program described in step (b4) comprises reacting between temperatures of 90-95 ° C for 10-50 seconds.
  • the temperature program described in step (c1) comprises reacting between temperatures of 90-95 ° C for 10-30 seconds.
  • the temperature program described in step (c2) comprises reacting between temperatures of 45-65 ° C for 10-30 seconds.
  • the temperature program described in step (c3) comprises reacting between temperatures of 60-80 ° C for 1-15 minutes.
  • the genomic DNA in step (a) is released from the lysed cells, including pyrolysis, alkaline lysis, enzymatic cleavage or mechanical lysis.
  • the thermal cracking comprises cleavage for a period of 10-100 minutes between 20-100 °C. In some embodiments, the thermal cleavage is carried out in the presence of a lysis reagent.
  • the lysis reagent comprises one or more surfactants selected from the group consisting of NP-40, Tween, SDS, Triton X-100, EDTA, and guanidinium isothiocyanate, and/or Lyase.
  • a method of amplifying a cellular genome comprising: (a) providing a reaction mixture, wherein the reaction mixture comprises the genomic DNA, a first type of primer, a second a primer, a nucleotide monomer mixture, and a nucleic acid polymerase, wherein the first type of primer comprises a universal sequence and a variable sequence from the 5' end to the 3' end, wherein the universal sequence is composed of G, A, C, and Three or two of the four bases of T, provided that the universal sequence does not include G and C at the same time, and the second type of primer comprises the universal sequence and does not comprise the variable sequence; (b) The reaction mixture is placed in a first temperature cycling sequence such that the variable sequence of the first type of primer is capable of pairing with the genomic DNA and amplifying the genomic DNA to obtain a genomic amplification product, wherein the genome is expanded
  • the 5' end of the enhanced product comprises the universal sequence and the 3' end comprises the complement of the universal sequence; wherein the
  • the universal sequence comprises or consists of SEQ ID NO: 1; the variable sequence comprises or consists of NNNNNTTT or NNNNNGGG, and N is any nucleotide that can base pair with a natural nucleic acid.
  • a kit for amplifying genomic DNA comprising a mixture comprising a first type of primer and a second type of primer, wherein the first type of primer is from 5
  • the 'end to 3' end comprises a universal sequence and a variable sequence, wherein the universal sequence consists of three or two of the four bases G, A, C and T, provided that the universal sequence does not include G at the same time And C, and the second type of primer comprises the universal sequence and does not comprise the variable sequence.
  • the mixture further comprises a mixture of nucleotide monomers and Mg2+.
  • the mixture further comprises one or more components selected from the group: dTT, bovine serum albumin (BSA), pH adjusting agent (e.g. Tris HCl), DNase inhibitor, RNase, SO 4 2- , Cl - , K + , Ca 2+ , Na + , and / or (NH4) + .
  • BSA bovine serum albumin
  • pH adjusting agent e.g. Tris HCl
  • the mixture further comprises a nucleic acid polymerase.
  • the kit further comprises a surfactant and/or a lytic enzyme capable of lysing cells.
  • the surfactant is selected from one or more of the group consisting of NP-40, Tween, SDS, Triton X-100, EDTA, and guanidinium isothiocyanate.
  • the lytic enzyme is selected from one or more of the group consisting of proteinase K, pepsin, and papain.
  • the mixture further comprises a surfactant and/or a lytic enzyme capable of lysing cells.
  • a kit for amplifying genomic DNA comprising a first type of primer and a second type of primer, and further comprising instructions for use, the instruction manual describes The step of mixing the first type of primer and the second type of primer in the same container before starting the amplification, wherein the first type of primer comprises a universal sequence and a variable from the 5' end to the 3' end a sequence, wherein the universal sequence consists of three or two of four bases G, A, C, and T, provided that the universal sequence does not include G and C at the same time, and the second type of primer comprises The universal sequence is described and does not comprise the variable sequence.
  • Figure 1 shows the basic principle of the amplification method of the present application.
  • Figure 2 shows the amplification products obtained by the two methods of the genomic DNA of normal human epidermal fibroblasts (AFP) by the three-step method of Example 2 and the two-step method of Example 3, respectively.
  • the results of gel electrophoresis were respectively performed, wherein a is the two-step amplification result of Example 3, the first lane from left to right is the molecular weight marker, the lanes 2-11 are single cell amplification samples, and the lanes 12-14 are positive.
  • Figure 3 shows the amplification products obtained by the two methods of the genomic DNA of normal human epidermal fibroblasts (AFP) by the three-step method of Example 2 and the two-step method of Example 3, respectively. Seven samples were randomly selected, that is, a total of 14 samples were used as templates, and the primers shown in Table 7 were used to amplify the 20 pathogenic sites shown in Table 6, respectively, and the amplification products were subjected to gel electrophoresis. .
  • AFP normal human epidermal fibroblasts
  • ag represents a gel electrophoresis pattern of repeated single-cell genomic DNA amplification products
  • the upper strip is amplified by a two-step method and the lower strip is amplified by a three-step method: a : upper row corresponding sample 2_1 and lower row corresponding sample 3_1, b: upper row corresponding sample 2_2 and lower row corresponding sample 3_3, c: upper row corresponding sample 2_3 and lower row corresponding sample 3_4, d: upper row corresponding sample 2_4 and lower Row corresponding sample 3_5, e: upper row corresponding sample 2_5 and lower row corresponding sample 3_6, f: upper row corresponding sample 2_6 and lower row corresponding sample 3_7, g: upper row corresponding sample 2_7 and lower row corresponding sample 3_8; in each sheet
  • each lane from left to right sequentially represents the molecular weight marker, and the amplification results for the pathogenic sites 1-20 shown in Table 6 (1-16 in Fig. (a)
  • Figure 4 shows the amplification of the genomic DNA of normal human epidermal fibroblasts (AFP) by the three-step method of Example 2 and the two-step method of Example 3, respectively.
  • Three samples were randomly selected, that is, a total of six samples were used as templates, and the primers shown in Table 11 were used to amplify the 20 pathogenic sites shown in Table 10, and the amplification products were subjected to gel electrophoresis. .
  • ac represents a gel electrophoresis pattern of repeated single-cell genomic DNA amplification products
  • the upper strip is amplified by a two-step method and the lower strip is amplified by a three-step method: a : upper row corresponding sample 2_1 and lower row corresponding sample 3_1, b: upper row corresponding sample 2_2 and lower row corresponding sample 3_4, c: upper row corresponding sample 2_7 and lower row corresponding sample 3_8; in each electrophoresis picture, from left
  • the amplification results of the molecular weight markers and the pathogenic sites 1-20 shown in Table 6 are sequentially shown in each lane to the right.
  • Figure 5 shows the amplification products obtained by the two methods of the genomic DNA of normal human epidermal fibroblasts (AFP) by the three-step method of Example 2 and the two-step method of Example 3, respectively.
  • Four samples were randomly selected, that is, a total of eight samples were used as templates, and the results of qPCR amplification using the six pairs of quality test primers shown in Table 14 were used.
  • af denotes data for q-PCR detection of template DNA using a quality test primer for chromosomes CH1, CH2, CH3, CH4, CH5, CH6 and CH7, respectively.
  • C T represents the number of threshold cycles
  • DNA 1 and DNA 2 represent positive controls.
  • Fig. 6 shows the results of chromosomal copy number obtained by genomic DNA of normal human epidermal fibroblast (AFP) amplified by the two-step method of Example 3, and the obtained amplification product was constructed into a genomic library and sequenced.
  • the ordinate represents the copy number of the chromosome, and the normal person is 2; the abscissa represents the chromosome 1-22 and the sex chromosome of the chromosome.
  • a-j represent the results of chromosome copy numbers obtained by constructing genomic libraries of samples 2_1, 2_2, 2_3, 2_4, 2_5, 2_6, 2_7, 2_8, 2_9, and 2_10, respectively.
  • Fig. 7 shows the results of chromosomal copy number obtained by analysing genomic DNA of normal human epidermal fibroblasts (AFP) by the three-step method of Example 2, and structuring the obtained amplified product to construct a genomic library.
  • the ordinate represents the copy number of the chromosome, and the normal person is 2; the abscissa represents the chromosome 1-22 and the sex chromosome of the chromosome.
  • a-i represent the results of chromosome copy numbers obtained by constructing and sequencing the genomic libraries of samples 3_1, 3_3, 3_4, 3_5, 3_6, 3_7, 3_8, 3_9, and 3_10, respectively.
  • Figure 8 shows that genomic DNA of normal human epidermal fibroblasts (AFP) was amplified by the three-step method of Example 2 (i.e., samples 3_1, 3_3, 3_4, 3_5, 3_6, 3_7, 3_8, 3_9, and 3_10). And using the two-step amplification of Example 3 (ie, samples 2_1, 2_2, 2_3, 2_4, 2_5, 2_6, 2_7, 2_8, 2_9, and 2_10), and the amplification products obtained by the two methods are separately constructed into a genomic library. Generation sequencing, data statistics of the obtained sequencing results.
  • AFP normal human epidermal fibroblasts
  • Figure 9 shows that the genomic DNA of normal human epidermal fibroblasts (AFP) was amplified by the three-step method of Example 2 (i.e., samples 3_1, 3_3, 3_4, 3_5, 3_6, 3_7, 3_8, 3_9, and 3_10). And using the two-step amplification of Example 3 (ie, samples 2_1, 2_2, 2_3, 2_4, 2_5, 2_6, 2_7, 2_8, 2_9, and 2_10), and the amplification products obtained by the two methods are separately constructed into a genomic library. Generation sequencing, and comparison of the copy number coefficient of variation.
  • AFP normal human epidermal fibroblasts
  • Figure 10 shows the amplification products obtained by the two methods of the genomic DNA of normal human epidermal fibroblasts (AFP) by the three-step method of Example 2 and the two-step method of Example 3, respectively.
  • the amplification products of the three-step method are shown as 3-1 and 3-2, the amplification products of the two-step method are shown as 2-1
  • the genomic DNA extracted by human epidermal fibroblast (AFP) cells shown as Gdna
  • Figure 11 shows that the genomic DNA of normal human epidermal fibroblasts (AFP) was amplified by the three-step method of Example 2 and the one-step amplification of Example 5, respectively, and the amplification products obtained by the two methods were respectively.
  • a is the one-step amplification result of Example 5, the first lane from left to right is the molecular weight marker, the lanes 2-11 are single cell amplification samples, and the lanes 12-14 are positive controls ( 40pg gDNA), Lane 15-17 is the negative control, Lane 18 is the molecular weight marker; b is the result of the three-step amplification of Example 2, the first lane from left to right is the molecular weight marker, and the lanes 2-11 are single cell expansion. Samples were added, lanes 12-14 were positive controls (40 pg gDNA), lanes 15-17 were negative controls, and lane 18 was molecular weight markers.
  • Figure 12 shows that the genomic DNA of normal human epidermal fibroblasts (AFP) was amplified by the three-step method of Example 2 and the one-step amplification of Example 5, respectively, and the amplification products obtained by the two methods were respectively Four samples were randomly selected, that is, a total of eight samples were used as templates, and the primers shown in Table 7 were used to amplify the 20 pathogenic sites shown in Table 6, respectively, and the amplification products were subjected to gel electrophoresis.
  • AFP normal human epidermal fibroblasts
  • ad represents a gel electrophoresis pattern of repeated single-cell genomic DNA amplification products
  • the upper strip is amplified by a two-step method and the lower strip is amplified by a three-step method: a : upper row corresponding sample 1_1 and lower row corresponding sample 3_1, b: upper row corresponding sample 1_2 and lower row corresponding sample 3_2, c: upper row corresponding sample 1_3 and lower row corresponding sample 3_3, d: upper row corresponding sample 1_4 and lower
  • the row corresponds to sample 3_4; in each electropherogram, each lane from left to right sequentially represents amplification results for the pathogenic sites 1-20 shown in Table 6.
  • Fig. 13 shows the results of chromosomal copy number obtained by sequencing the genomic DNA of normal human epidermal fibroblasts (AFP) by the one-step amplification of Example 5, and structuring the obtained amplified product into a genomic library.
  • the ordinate represents the copy number of the chromosome, and the normal person is 2; the abscissa represents the chromosome 1-22 and the sex chromosome of the chromosome.
  • a-j represent the results of chromosomal copy number obtained by constructing a genomic library of samples 1_1, 1_2, 1_3, 1_4, 1_5, 1_6, 1_7, 1_8, 1_9, and 1_10, respectively.
  • Fig. 14 shows the results of chromosomal copy number obtained by analysing genomic DNA of normal human epidermal fibroblasts (AFP) by the three-step method of Example 2, and structuring the obtained amplified product to construct a genomic library.
  • the ordinate represents the copy number of the chromosome, and the normal person is 2; the abscissa represents the chromosome 1-22 and the sex chromosome of the chromosome.
  • a-i represent the results of chromosome copy numbers obtained by constructing and sequencing the genomic libraries of samples 3_1, 3_2, 3_3, 3_4, 3_5, 3_6, 3_7, 3_8 and 3_10, respectively.
  • Figure 15 shows that the genomic DNA of normal human epidermal fibroblasts (AFP) was amplified by the three-step method of Example 2 and the one-step amplification of Example 5, respectively, and the amplification products obtained by the two methods were respectively.
  • the genomic library was constructed for second-generation sequencing, and the obtained sequencing results were statistically analyzed.
  • Figure 16 shows that the genomic DNA of normal human epidermal fibroblasts (AFP) was amplified by the three-step method of Example 2 and the one-step amplification of Example 5, respectively, and the amplification products obtained by the two methods were respectively.
  • the genomic library was constructed for second-generation sequencing and the comparison of the copy number variation coefficients was performed.
  • the present invention provides methods for amplifying genomic DNA, particularly methods for amplifying a single cell whole genome.
  • the invention is based, at least in part, on the discovery that prior to the amplification reaction of genomic DNA, all reagents required for amplification can be added to a single reaction mixture, and then the reaction mixture is placed under conditions of amplification reaction until completion Amplification.
  • This method eliminates the need to add reagents to the reaction mixture after the start of the amplification reaction, thus greatly reducing the extra handling due to the addition of reagents and the possible contamination, and greatly reducing the required reaction time.
  • the prior method of the present invention either adds only the primer required for the first step before the amplification reaction, and waits until the amplification reaction of the first step is completed, and then adds the second step to the reaction system.
  • the desired primers are then subjected to the amplification reaction of the second step (see, for example, WO2012/166425); or the first step can only be used at very low times to achieve the desired amplification efficiency.
  • the inventors of the present application have found that when primers originally thought to be likely to interfere with each other are placed in a single reaction mixture at a time and amplified under the reaction conditions of the present invention, it can be unexpectedly An amplification effect comparable to that when fractions were added to the primers was obtained. Therefore, the present invention greatly improves the reaction efficiency, shortens the reaction time, and reduces the risk of sample contamination and improves the reliability of the results.
  • the application provides a method of amplifying cellular genomic DNA, comprising: (a) providing a reaction mixture, wherein the reaction mixture comprises the genomic DNA, a first type of primer, a second type of primer, a nucleotide sequence a mixture, and a nucleic acid polymerase, wherein the first type of primer comprises a universal sequence and a variable sequence from the 5' end to the 3' end, wherein the universal sequence is comprised of four bases of G, A, C and T Three or two compositions, provided that the universal sequence does not include G and C at the same time, and the second type of primer comprises the universal sequence and does not comprise the variable sequence; (b) the reaction mixture is placed The first temperature cycling program 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 amplification product, wherein the 5' end of the genomic amplification product Including the universal sequence, the 3' end comprises the complementary sequence of the universal sequence; (c) placing the reaction mixture obtained in step
  • the methods of the present application are broadly applicable to the amplification of genomic DNA, particularly the rapid and precise 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.
  • 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 such as a cell expressing a particular biomarker, can be selected based on the different properties of the individual cells.
  • 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 (WO 2013153176 A1).
  • a mild lysis method can be used.
  • the 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 in PCR Buffer II (Applied Biosystems) heated at 70 ° C for 90 seconds (Kurimoto et al., Nucleic Acids Res 34 (5): e42 (2006), or using protease (such as proteinase K) or salt solution ( For example, guanidinium isothiocyanate is subjected to cleavage (U.S. Patent Application US 20070281313).
  • Thermal cracking includes heating and repeated freeze-thaw methods.
  • the thermal cracking comprises a temperature between 20-100 degrees Celsius and a cleavage of 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.
  • 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. Any time between -90, 30-80, 30-70, 30-60, 30-50 or 30-40 minutes. In some embodiments, the thermal cracking time is no less than 20, 30, 40, 50, 60, 70, 80 or 90 minutes. In some embodiments, the thermal cracking time is no more than 90, 80, 70, 60, 50, 40, 30 or 20 minutes. In some embodiments, 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 reaction mixture.
  • the biological sample can be subjected to a cleavage treatment in advance to obtain a cleavage product, and then the cleavage product is mixed with other components of the reaction mixture. If necessary, the cleavage product can be further processed to isolate the genomic DNA therein, and the isolated genomic DNA is mixed with other components of the reaction mixture to obtain a reaction mixture.
  • the lysed nucleic acid sample can be amplified without purification.
  • the present application also provides a more convenient method of directly mixing cells containing genomic DNA with other components required for amplification to obtain a reaction mixture, that is, genomic DNA in the reaction mixture exists inside the cell.
  • the reaction mixture may further comprise a surfactant capable of lysing the cells (such as, but not limited to, NP-40, Tween, SDS, Triton X-100, EDTA, guanidinium isothiocyanate).
  • a surfactant capable of lysing the cells
  • a surfactant capable of lysing the cells such as, but not limited to, NP-40, Tween, SDS, Triton X-100, EDTA, guanidinium isothiocyanate.
  • a lytic enzyme eg one or more of proteinase K, pepsin, 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 reaction mixture also contains two different types of 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 the universal sequence and does not comprise the variable sequence.
  • the variable sequence in the first type of primer can bind to the genomic DNA template, and a certain length of the genomic template can be replicated by the action of the nucleic acid polymerase to obtain an amplification product having a 5' end as a universal sequence and a 3' end as a genomic sequence.
  • a semiamplifier is also referred to in the present application.
  • variable sequences in the first type of primers can also be paired with a half-amplifier and replicated using a half-amplifier as a template to obtain amplification of the complementary sequence at the 5' end and the 3' end as the universal sequence.
  • the product also referred to herein as a full amplicon.
  • the second type of primers can bind to the complementary sequence of the universal sequence at the 3' end of the entire amplicon, thereby further replicating the full amplicon, and the number thereof is greatly increased.
  • a universal sequence in this application refers to a particular sequence located at the 5' end of a first type of primer.
  • the length of the universal sequence can be, for example, 10-30, 12-29, 15-28, 18-26 or 20-24 bases.
  • an appropriate universal sequence is selected such that it does not substantially bind to genomic DNA to produce amplification, and between the first type of primer and the first type of primer, or between the first type of primer and the second type of primer Aggregation.
  • 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, universal The 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 that is self-paired, or a sequence that results in a 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 is selected from the group consisting of SEQ ID NO: 1 (GTG AGT GAT GGT TGA GGT AGT GTG GAG), SEQ ID NO: 2 (GTGGAGTTAGTGAGTGTAATGGAT), SEQ ID NO: 3 (GGTTTGGTGTGGTGTGTGGTGGTG) SEQ ID NO: 4 (ACAACACTATCAATCCCTATCCTAC), SEQ ID NO: 5 (ATGGTAGTGGGTAGATGATTAGGT), SEQ ID NO: 6 (CATATCCCTATACCTAATACCATTAC).
  • the 5' end of the first type of primer is a universal sequence and the 3' end is a variable sequence.
  • the universal sequence and the variable sequence may be directly adjacent, or may have a spacer sequence of one or more bases.
  • a variable sequence in the present application refers to a sequence of bases whose sequence is not fixed, for example, may comprise a random sequence.
  • the random sequence may comprise any nucleotide that can base pair with a natural nucleic acid, such as four natural bases A, T, G, and C, as well as other nucleotide analogs, modified nucleosides known to those skilled in the art. An acid or the like can be paired with genomic DNA and an amplification reaction can be achieved. Nucleotide sequences in variable sequences can vary in a variety of ways.
  • a first type of primer may comprise a set of primers having different sequences, each of which has a universal sequence at the 5' end and a variable sequence at the 3' end, the universal sequences of these primers being identical, but the variable sequences May be different.
  • variable 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.
  • the variable sequence is 5 bases in length.
  • variable sequence is 8 bases in length. Theoretically, if each base position is randomly selected from four bases A, T, G, and C, then a variable sequence of 4 bases in length can combine 256 possible random sequences, length.
  • a variable sequence of 5 bases can combine 1024 possible random sequences, and so on.
  • variable sequence can be selected in a random manner, or some of the qualifications can be further added on a random basis to exclude some undesired conditions or increase the degree of matching with the target genomic DNA.
  • three or more base positions in the variable sequence consist of one or several bases selected from G, A, and T (ie, Not C), or consisting of one or several bases of C, A, and T (ie, not G) to avoid complementary pairing of the variable sequence with the universal sequence.
  • the universal sequence does not contain G but contains C
  • three or more base positions in the variable sequence consist of one or several bases of C, A, and T (ie, Not for G).
  • three or more base positions in the variable sequence consist of one or several bases of G, A, and T (ie, , not for C).
  • three or more base positions in the variable sequence are selected from one or more bases of G, A, and T. (ie, not C) or consist of one or several bases of C, A, and T (ie, not G).
  • the three or more bases may be located at the 3' end of the variable sequence or may be located in the middle of the variable sequence.
  • the three or more bases may be contiguous or may be discontinuous, for example, three adjacent bases at the 3' end of the variable sequence are not C, or the 3' end of the variable sequence
  • the three mutually spaced bases are not C, or one of the two consecutive bases at the 3' end of the variable sequence and the other base spaced therefrom is not C.
  • the three base positions are consecutive, they can be the following exemplary sequences: TTT, GGG, TTG, GAA or ATG.
  • variable sequence is selected from the group consisting of: (N) n GGG, (N) n TTT, (N) m TNTNG, and (N) x GTGG(N) y , wherein N is arbitrary A random nucleotide that can base pair with a natural nucleic acid, n is a positive integer selected from 3-17, m is a positive integer selected from 3-15, and x and y are positive integers selected from 3-13, respectively.
  • the variable sequence in the first type of primer may have one of (N) n GGG, (N) n TTT, (N) m TNTNG, (N) x GTGG(N) y One or more sequences. And X. .
  • 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.
  • the first class of primers may comprise SEQ ID NO: 11 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNNNNNN], SEQ ID NO: 12 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNGGG], SEQ ID NO: 13 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNTTT], SEQ ID NO: 14 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNTNG] or SEQ ID NO: 15 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNGTGGNN], wherein N is any nucleotide (for example, A, T, G, C) that can base pair with a natural nucleic acid.
  • N is any nucleotide (for example, A, T, G, C) that can base pair with a natural nucleic acid.
  • a second type of primer in the reaction mixture comprises the universal sequence and does not comprise the variable sequence.
  • the 5' and 3' ends of the second type of primer may or may not contain other additional sequences.
  • the sequence of the second type of primer consists of a universal sequence in the first class of primers.
  • the second class of primers comprises from 5' to 3' or consists of the sequence of SEQ ID NO: 1 [GTGAGTGATGGTTGAGGTAGTGTGGAG].
  • the concentration of the primer in the reaction mixture is from 300 ng to 1500 ng/ ⁇ L. In some embodiments, the concentration of the primer in the reaction mixture is from 300 ng to 1400 ng/ ⁇ L, from 300 ng to 1200 ng/ ⁇ L, from 300 ng to 1000 ng/ ⁇ L, 300 ng-800 ng/ ⁇ L, 300 ng-600 ng/ ⁇ L or 300 ng-400 ng/ ⁇ L.
  • the concentration of the primer in the reaction mixture is from 500 ng to 1400 ng/ ⁇ L, from 600 ng to 1400 ng/ ⁇ L, from 800 ng to 1400 ng/ ⁇ L, from 900 ng to 1400 ng/ ⁇ L, from 1000 ng to 1400 ng/ ⁇ L, or from 1200 ng to 1400 ng/ ⁇ L. In some embodiments, the concentration of the primer in the reaction mixture is from 400 ng to 1400 ng/ ⁇ L, from 500 ng to 1200 ng/ ⁇ L, from 600 ng to 1000 ng/ ⁇ L, or from 700 ng to 800 ng/ ⁇ L.
  • the 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, 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 , T7 phase 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.
  • the reaction mixture contains one or more of Vent polymerase, Deep Vent polymerase, Vent (-exo) polymerase, or Deep Vent (-exo) polymerase of Thermococcus litoralis.
  • 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 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), a mixture of nucleomonomers ( For example, dATP, dGTP, dTTP, and dCTP), bovine serum albumin (BSA), dTT (e.g., a final concentration of 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)
  • a suitable concentration of Mg 2+ ions eg, a final concentration may range from about 1.5 mM to about 8 mM
  • a pH adjusting agent may be further included in the reaction mixture such that the pH of the reaction mixture is maintained between 7.0 and 9.0.
  • Suitable pH adjusting agents can include, for example, Tris HCl and Tris SO 4 .
  • the 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.
  • reaction mixture is provided prior to the step (b) and the step (c). Since the configuration of the reaction mixture is completely completed before the temperature cycle program is performed, as long as the temperature cycle program is entered, the reaction mixture can be reacted according to a predetermined setting without opening the lid or adding any components, thereby avoiding contamination and improving the reaction efficiency. In some embodiments, it is no longer necessary to add reactants such as enzymes, primers and dNTPs to the reaction mixture after completion of step (a). . In some embodiments, providing the reaction mixture is completed prior to step (b). In some embodiments, no reactants, such as enzymes, primers, and dNTPs, are added to the reaction mixture after the start of step (b). In some embodiments, the step (a) precedes the step (b) and the step (c).
  • the method provided herein includes the step (b) of placing the reaction mixture in a first temperature cycling sequence such that the variable sequence of the first type of primer is capable of pairing with the genomic DNA and amplifying the genomic DNA
  • a genomic amplification product is obtained, wherein the 5' end of the genomic amplification product comprises the universal sequence and the 3' end comprises the complement of the universal sequence.
  • 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 cycling program such that the DNA template duplex is unzipped into a single strand, the primer hybridizes to the template single strand, and the primer is extended.
  • 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 DNA polymerase adds a nucleus complementary to the DNA template base at the 3' end of the primer
  • the nucleotides allow the primers to be extended, resulting in a new DNA strand complementary to the DNA template.
  • the newly synthesized DNA strand can be used as a new DNA template in the next reaction cycle for a new round of DNA synthesis.
  • step (b) of the method of the present application the reaction mixture is placed in a first temperature cycling sequence such that the variable sequence of the first type of primer in the reaction mixture is capable of binding to the genomic DNA by base pairing, in the polymerization of the nucleic acid Genomic DNA is replicated by the action of an enzyme.
  • the 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 such as 90 °C - 95 °C can be used and longer reaction times can be maintained.
  • the temperature program in step (b1) comprises reacting at a temperature between 90-95 ° C for 1-10 minutes.
  • the reaction mixture is placed in a temperature program capable of binding the first type of 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 5 ° C to 10 ° C, and then the reaction mixture is allowed to react at different annealing temperatures for a suitable time by a gradient heating method, thereby ensuring as much as possible.
  • Primers are paired with genomic DNA.
  • step (b2) comprises reacting at a first annealing temperature (eg, 10 ° C) between 5-10 ° C for a suitable period of time (eg, 3-50 seconds), between 25-30 ° C
  • a suitable period of time eg, 3-50 seconds
  • the second annealing temperature for example, 30 ° C
  • a suitable time for example, 3 to 50 seconds
  • a third annealing temperature for example, 50 ° C
  • 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 the occurrence of the primer polymer and the non-specific amplification product. . Therefore, a low temperature such as 5 ° C to 10 ° C is usually not used in the primer annealing temperature.
  • a low temperature such as 5 ° C to 10 ° 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, 5 ° C to 10 ° C), the pairing between the primer and the genomic DNA can maintain a good specificity, and the amplification result remains very 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, 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 transcription Enzyme, T7 phase 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.
  • Vent polymerase eg, Ventpolymer of Thermococcus litoralis
  • Deep Vent polymerase Vent ( -exo) polymerase, Deep Vent (-exo) polyme
  • step (b3) comprises an extension temperature between 60-90 ° C (eg, 65-90 ° C, 70-90 ° C, 75-90 ° C, 80-90 ° C, 60-85 °C, 60-80 ° C, 60-75 ° C, or 60-70 ° C) reaction for 1-15 minutes (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, or 13-14 minutes). In certain embodiments, step (b3) comprises reacting at 70 ° C for 2 minutes.
  • 60-90 ° C eg, 65-90 ° C, 70-90 ° C, 75-90 ° C, 80-90 ° C, 60-85 °C, 60-80 ° C, 60-75 ° C, or 60-70 ° C
  • step (b3)
  • step (b4) After the primer extension program, the reaction mixture is placed in a temperature program capable of detaching the amplification product into a single strand (step (b4)).
  • the temperature in this step may be similar to the temperature in the step (b1), but the reaction time is slightly shorter.
  • step (b4) comprises reacting between temperatures of 90-95 °C for 10-50 seconds.
  • 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, both of which can be used as DNA templates in the next cycle, combined with primers and turned on. The next round of replication.
  • Steps (b2) through (b4) are then repeated to the designated first cycle number to obtain a genomic amplification product.
  • the number of first cycles should be at least 2.
  • the sequence at the 3' end of the variable sequence of the first type of primer is extended and the resulting amplification is obtained.
  • the product is a universal sequence at the 5' end and a 3' end is a sequence complementary to the genomic template single-stranded sequence.
  • Such an amplification product is also referred to as a semiamplifier.
  • the previous amplification product 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 amplification product under the action of the nucleic acid polymerase until replication
  • the universal sequence at the 5' end of the amplified product is completed, thereby obtaining a genomic amplification product which is a universal sequence at the 5' end and a complementary sequence of the universal sequence at the 3' end, and such an amplification product is also referred to as a full amplicon.
  • the number of first cycles is controlled to an appropriate range to ensure that there are sufficient amplification products for subsequent reactions without affecting the accuracy of the amplification products due to excessive cycles.
  • the number of first cycles is at least 3, at least 4, at least 5, or at least 6, and preferably no more than 8, no more than 9, no more than 10, no more than 11, or no more than 12. If the number of cycles is too low, the obtained amplification product is small. In order to obtain sufficient amplification product, it is necessary to increase the number of cycles in the next amplification (ie, step (c)), which reduces the accuracy of the amplification result. If the number of cycles is too high, it will lead to a sequence of mutations during the amplification of genomic DNA, causing deviations in the template of the next amplification (ie, step (c)), resulting in inaccurate final amplification results.
  • step (b) after step (b4), further comprising the step (b4'), wherein the reaction mixture is placed in an appropriate temperature program such that the genomic amplification product The 3' end is hybridized with the 5' end to form a circular structure, or the 3' end of the genomic amplification product is bound to a primer. It was previously thought that step (b4') could protect the 3' end of the entire amplicon, thereby avoiding the end-to-end polymerization between the entire amplicon, thereby avoiding the two sequences that were not adjacent to the genome. . This will help to improve the accuracy of the amplification results.
  • the method proceeds directly to step (b5) without further steps (e.g., step (b4')) after step (b4).
  • step (b4') 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.
  • step (b4) even after the step (b4), the whole amplicon is cyclized or the 3' end is bound to the primer without a specific step, and the final amplification result is still quite high.
  • the accuracy is similar to the method using step (b4'). This streamlines the reaction steps while still maintaining the specificity of the reaction.
  • the second type of primers comprise a universal sequence in the first class of primers. Since the universal sequence is substantially non-complementary to the genomic sequence, if the specificity of the amplification reaction is sufficiently high, then in step (b) the second type of primer does not directly pair with the genomic DNA and initiates replication of the genomic DNA. However, when step (b) is carried out for two cycles, a full amplicon having a universal sequence complementary sequence at the 3' end begins to appear in the reaction mixture, and the 3' end of such a full amplicon can be compared with the second class.
  • the primers pass base pairing, thus potentially causing the second type of primer to replicate the entire amplicon in step (b) (eg, starting from the third cycle). This may interfere with the replication of genomic DNA by the first type of primers, such that the amplification of genomic DNA by the first type of primers is insufficient to achieve the desired coverage of genomic DNA.
  • the first type of primer and the second type of primer are simultaneously present in the reaction system, it is possible that the first type of primer and the second type of primer generate a primer-primer amplification reaction independent of the template, resulting in generation of the primer multimer. .
  • step (b) the inventors of the present application unexpectedly discovered that both the first type of primer and the second type of primer are present in the reaction mixture and both In the case of amplification reaction, the first type of primer does not appear to be interfered by the second type of primer, and the amplification of genomic DNA still maintains high specificity and wide coverage, and in step (b) The results obtained are generally comparable in comparison to the method of using the first type of primers alone and then adding the second type of primers in step (c).
  • the method provided herein further comprises the step (c) of placing the reaction mixture obtained in the step (b) in a second temperature cycling sequence such that the universal sequence of the second type of primer is capable of interacting with the genomic amplification product The 3' end pairs and amplifies the genomic amplification product to obtain an amplified genomic amplification product.
  • the genomic amplification product obtained in the step (b), that is, the whole 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, and 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 (c1)).
  • the DNA double strand herein mainly refers to the double strand of the genomic amplification product (i.e., the full amplicon) obtained in the step (b).
  • the second type of primer does not substantially bind to the genomic DNA, the original genomic DNA is not the DNA template to be amplified in the step (c).
  • a higher reaction temperature for example, 90 ° C - 95 ° C can be used for a suitable period of time, as long as the full amplicon duplex to be amplified can be denatured into a single strand.
  • the temperature program in step (c1) comprises reacting at a melting temperature between 90-95 ° C (eg, 94 ° C) for 10-30 seconds (eg, 20 seconds).
  • a melting temperature between 90-95 ° C (eg, 94 ° C) for 10-30 seconds (eg, 20 seconds).
  • the reaction mixture is placed in a temperature program capable of binding the second type of primer to the single strand of the genomic amplification product obtained in the step (b) (step (c2)).
  • 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 (c2) comprises reacting at an annealing temperature (eg, 58 ° C) between 45-65 ° C for 10-30 seconds (eg, 15 seconds).
  • the second type of primer is SEQ ID NO: 1
  • the temperature program in step (c2) comprises reacting at 58 ° C for 10-30 seconds.
  • the annealing temperature in step (c2) 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 (c1). Binding, resulting in an incomplete amplified sequence.
  • step (c2) When the annealing temperature in step (c2) 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 second class capable of binding a single strand to the amplification product.
  • the temperature program described in step (c3) comprises reacting for an extension temperature (eg, 72 ° C) between 60-80 ° C for 1-15 minutes (eg, 2 minutes).
  • Steps (c1) through (c3) 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 (c4) is greater than the first number of cycles in step (b5).
  • 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 15-30 cycles (eg, 15-30, 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 cycles ).
  • step (c) further comprises, after the second temperature cycling procedure, placing the reaction mixture in the same temperature sequence as step (c3) (eg, 72 ° C) for a suitable period of time (eg, 5 minutes). The reaction mixture was then placed at a temperature of 4 ° C to end the reaction. In certain embodiments, the reaction mixture is placed at a temperature of 4 ° C directly after the end of step (c) to terminate the reaction.
  • the present application also provides a method of amplifying a cellular genome, the method comprising:
  • reaction mixture comprising the genomic DNA, a first type of primer, a second type of primer, a mixture of nucleomonomers, and a nucleic acid polymerase, wherein the first type of primer is from 5'
  • the terminal to the 3' end comprises a universal sequence and a variable sequence, wherein the universal sequence consists of three or two of the four bases G, A, C and T, provided that the universal sequence does not include G and C, the second type of primer comprises the universal sequence and does not comprise the variable sequence;
  • step (c) placing the reaction mixture obtained in step (b) in a second temperature cycling sequence such that the universal sequence of the second type of primer is capable of pairing with the 3' end of the genomic amplification product and amplifying the Genomically amplifying the product to obtain an expanded genomic amplification product;
  • the second temperature cycling program comprises:
  • reaction mixture is provided prior to the step (b) and the step (c).
  • 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.
  • a suitable lytic enzyme such as one or more of proteinase K, pepsin, papain may also be selected.
  • 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 first class of primers comprises or consists of SEQ ID NOs: 11, 12, 13, 14, and/or 15, wherein the universal sequence comprises or consists of SEQ ID NO: 1.
  • the invention combines the steps of pre-amplification, amplification, and the like of nucleic acids into one reaction under thermal cycling conditions.
  • This counter The human operation should be reduced, and the whole genome amplification of the nucleic acid can be realized by putting the nucleic acid into the PCR tube and performing a specific procedure, and the amplification product genome coverage is high, and the amplification deviation is small. It eliminates the need for reagent preparation, opening and adding liquid, which reduces the risk of contamination caused by the experimental environment and operators, and shortens the overall experimental time period.
  • the overall amplification time takes only 2.5 hours.
  • the present invention can also combine steps of cell lysis, pre-amplification of nucleic acids, amplification, and the like into one reaction under thermal cycling conditions. This further reduces human intervention and eliminates the need for separate cell lysis steps, further reducing the time of experimentation and reducing the risk of contamination.
  • the method of the present application also maintains high accuracy and wide coverage of amplification based on the simplified operation.
  • the method of the present application uses quasi-linear amplification techniques to reduce sequence-dependent amplification bias.
  • pre-amplification the focus is on amplification from the original sample DNA template, and the genome coverage is high and the amplification bias is small.
  • the full amplicon produced during the preamplification phase is extensively amplified during the amplification phase.
  • the yield of this technology is high, the minimum starting template is a few picograms, and the results are reliable and repeatable.
  • 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, etc.
  • 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 for example, refer 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 for genomic DNA amplification comprising a mixture comprising a first type of primer and a second type of primer, wherein the first type of primer is from the 5' end to the 3' end
  • a universal sequence and a variable sequence are included, wherein the universal sequence is composed of three or two of four bases G, A, C, and T, provided that the universal sequence does not include G and C at the same time, and
  • a second type of primer comprises the universal sequence and does not comprise the variable sequence.
  • the mixture further comprises a mixture of nucleomonomers (eg, dATP, dGTP, dTTP, and dCTP), dTT, and Mg 2+ .
  • the mixture has a Mg2+ concentration of from 2 mmol to 8 mmol/ ⁇ L, a dNTP concentration of from 1 mmol to 8 mmol/ ⁇ L, and a dTT concentration of from 2 mmol to 7 mmol/ ⁇ L.
  • the mixture further comprises one or more components selected from the group consisting of bovine serum albumin (BSA), pH adjusting agents (eg, Tris HCl), DNase inhibitors, RNase, SO 4 2 - , Cl - , K + , Ca 2+ , Na+, and/or (NH 4 ) + and the like.
  • BSA bovine serum albumin
  • pH adjusting agents eg, Tris HCl
  • DNase inhibitors e.g, RNase, SO 4 2 - , Cl - , K + , Ca 2+ , Na+, and/or (NH 4 ) + and the like.
  • the pH of the mixture ranges between 7.0 and 9.0.
  • the kit further comprises a nucleic acid polymerase, and the nucleic acid polymerase is not included in the mixture of the first type of primer and the second type of primer.
  • the nucleic acid polymerase can be stored in a separate container, optionally in a mixture with other ingredients, or it can be in substantially pure form.
  • the nucleic acid polymerase is further included in the mixture of the first type of primer and the second type of primer.
  • the mixture comprises: the first primer, the second primer, a mixture of nucleotide monomers, Mg 2+, dTT, Tris HCl , and nucleic acid polymerase, and one or more selected from Components from the following groups: BSA, DNase inhibitor, RNase, SO 4 2- , Cl - , K + , Ca 2+ , Na+, and (NH 4 ) + .
  • the mixture may contain all of the reactants other than genomic DNA required for the amplification reaction.
  • the reaction containing the genomic DNA can be directly mixed with the mixture in the kit, optionally an appropriate amount of pure water can be added to obtain the desired reaction volume, ie The reaction mixture in step (a) of the process of the invention can be obtained.
  • the kit further comprises a component capable of lysing cells, such as one or more surfactants, and/or one or more lytic enzymes.
  • exemplary surfactants include, but are not limited to, one or more of NP-40, Tween, SDS, Triton X-100, EDTA, guanidinium isothiocyanate.
  • An exemplary lytic enzyme can be one or more of proteinase K, pepsin, papain.
  • the components of the lysed cells can be stored separately in separate containers or mixed with some other ingredients.
  • the kit includes a surfactant and a lytic enzyme, each placed in a separate container or placed in the same container.
  • the first class of primers, the second class of primers, and the mixture of nucleic acid polymerases further comprise a surfactant and/or a lytic enzyme.
  • the kit includes a container containing all of the reactants.
  • the kit includes two containers, one of which stores the components required for the amplification reaction, including the nucleic acid polymerase, and the other of which stores the cell lysis, including the lytic enzyme.
  • the kit includes four containers, wherein the first container stores the nucleic acid polymerase, the second container stores the components required for the amplification reaction other than the nucleic acid polymerase, and the third container stores the lyase The fourth container stores the components required for cell lysis other than the lytic enzyme.
  • kits for genomic DNA amplification comprising a first type of primer and a second type of primer, and further comprising instructions for use, which are described at the beginning
  • the first primer and the second primer in the kit may each be placed in separate containers, but the instructions may include the step of mixing the two in the same container prior to initiating amplification.
  • Example 1 Acquisition of single cell genome, positive control and negative control
  • Single cell genomic DNA 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. After the completion of the rinsing, 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, draw a volume of PBS solution no more than 1 microliter, and transfer the picked single cells into the bag.
  • AFP Human epidermal fibroblasts
  • lysis buffer containing Tris-Cl, KCl, EDTA, Triton X-100 and Qiagen Protease was contained in a PCR tube. After a brief centrifugation, the PCR tube was placed on a PCR machine to perform a lysis procedure. The specific procedure is shown in Table 1.
  • Standard genomic DNA was diluted to 30 pg/ ⁇ l of DNA solution with nuclease-free water, and 1 ⁇ l of the above solution was added to a PCR tube containing 4 ⁇ l of cell lysis buffer.
  • the standard genomic DNA is genomic DNA of a human cell extracted in advance.
  • Negative control 5 microliters of cell lysis buffer was added to the PCR tube.
  • Example 2 Genomic amplification using multiple annealing circular cycle amplification (MALBAC) (referred to as a three-step method)
  • MALBAC multiple annealing circular cycle amplification
  • the method of this example is also referred to herein as a three-step process because it basically comprises three steps, namely, lysing cells, pre-amplification, and exponential amplification.
  • Human epidermal fibroblasts were isolated and lysed according to the method described in Example 1 to obtain single-cell genomic DNA.
  • the single-cell whole genome amplification kit (Cat. No. YK001A/B) of Jiangsu Yikang Gene Technology Co., Ltd. was used for amplification according to its product specification. Specifically, the preamplification buffer and the preamplification enzyme mixture were mixed in a ratio of 30:1 to form a preamplified mixture. Thirty microliters of the preamplification mixture was separately added to the PCR tubes respectively including the samples to be amplified (genomic DNA obtained according to Example 1, positive control, and negative control). The PCR tube was placed in a PCR machine for pre-amplification, and the procedure for pre-amplification was as shown in Table 2.
  • the amplification buffer and the amplification enzyme mixture were mixed in a ratio of 30:0.8 to form an amplification mixture.
  • 30 microliters of the amplification mixture was added to the PCR tube after completion of the pre-amplification, followed by exponential amplification.
  • the procedure of the exponential amplification is shown in Table 3.
  • Example 3 Genomic amplification using mixed primers (referred to as a two-step method)
  • the method of this example also referred to herein as a two-step process, consists essentially of two steps, namely lysis of cells and amplification reactions.
  • Human epidermal fibroblasts were isolated and lysed according to the method described in Example 1 to obtain single-cell genomic DNA.
  • the primers used in this example were designed according to the following principles:
  • the genomic recognition site is statistically calculated, and sequences that meet the above conditions and are more evenly distributed and covered in the genome are selected to increase the recognition opportunities of variable base sequences and genomic DNA. .
  • the proportion and composition of the three types of bases in the universal sequence of primers are specially designed to ensure that the universal sequence does not bind to genomic DNA for amplification.
  • 60 microliters of the amplification mixture was separately added to the PCR tubes of each sample to be amplified (genomic DNA, positive control, and negative control obtained according to Example 1).
  • the PCR tube was placed in a PCR machine for amplification, and the amplification procedure is shown in Table 4.
  • Example 4 Comparison of the two-step amplification product of the present application with the MALBAC three-step amplification product
  • Example 2 Five microliters of the unpurified three-step amplification product of Example 2 and the two-step amplification product of Example 3 were separately taken, and one microliter of 6x DNA loading buffer was separately added (purchased from Beijing Kangwei Century Biotechnology). Ltd., item number CW0610A) ready to load.
  • the gel was a 1% agarose gel and the marker was DM2000 (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., Cat. No. CW0632C).
  • Electropherograms are shown in Figure 2, where a is the two-step amplification result of Example 3, the first lane from left to right is the molecular weight marker, the lanes 2-11 are single-cell amplification samples, and the lanes 12-14 are positive.
  • Control 40 pg gDNA
  • lanes 15-17 were negative controls (without genomic DNA)
  • lane 18 was molecular weight marker
  • b was the result of three-step amplification of example 2
  • lane 1 from left to right was molecular weight marker
  • Lanes 2-11 were single-cell amplified samples
  • lanes 12-14 were positive controls (40 pg gDNA)
  • lanes 15-17 were negative controls (without genomic DNA)
  • lane 18 was molecular weight markers.
  • the electropherogram shows that the band and brightness of the two-step amplification product of Example 3 are comparable to those of the three-step amplification product of Example 2, and the brightness is equivalent, and there is no significant difference, indicating that the two-step amplification is performed.
  • the accuracy and yield of the resulting product are comparable to those of the three-step process.
  • Example 5 50 ⁇ l of the unpurified three-step amplification product of Example 2 and the two-step amplification product of Example 3 were used, and a universal column purification kit (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., article number CW2301) was used.
  • the amplification product is subjected to purification treatment, and the purification step is carried out in accordance with the kit instructions. Elution was performed using 50 microliters of EB. After the purification was completed, 2 ⁇ l of the purified product was taken and detected using Nanodrop (AOSHENG, NANO-100). The concentration detection results are shown in Table 5.
  • concentration test results showed that the amplification products obtained by the two amplification methods were comparable in concentration after purification, and there was no significant difference.
  • the 7 amplification products amplified according to Example 2 and the 7 amplification products amplified according to Example 3 were randomly selected as template DNA, respectively.
  • Use dye-containing 2xTaq MasterMix purchased from Beijing Kangwei Century Biotechnology Co., Ltd., The product number CW0682 was subjected to PCR to amplify the above 20 pathogenic sites.
  • the composition of the amplification system is shown in Table 8, and the amplification procedure is shown in Table 9.
  • PKDH1-3681, PKDH1-1713, DMD-13exe, DMD-19exe annealing temperature is 50 degrees Celsius, and the remaining 16 primers have an annealing temperature of 55 degrees Celsius.
  • Example 2 Three samples amplified according to Example 2 (shown as 2_1, 2_3, and 2-7 in Figure 4) and three samples amplified according to Example 3 (shown as 3_1, 3_4, and 3_8 in Figure 4) were randomly selected. ) used as template DNA, respectively.
  • the dye-containing 2xTaq MasterMix purchased from Beijing Kangwei Century Biotechnology Co., Ltd., product number CW0682 was subjected to PCR amplification to amplify the above 20 pathogenic sites.
  • the amplification system and amplification procedure are shown in Tables 8 and 9, respectively, except that the annealing temperature is selected to be 55 degrees Celsius.
  • the results of the amplification are shown in Figure 4.
  • the amplification results showed that 20 pathogenic sites were well amplified in the amplification products of the above two amplification methods, and the two methods did not have the accuracy of amplification and the amount of amplification products. Significant difference.
  • Example 2 Four samples amplified according to the three-step method of Example 2 (shown as 3-5, 3-6, 3-9, 3-10 in FIG. 5) and a two-step method according to Example 3 were randomly selected. The four additional samples (shown as 2-1, 2-3, 2-7, 2-10 in Figure 5) were used as template DNA, respectively.
  • the template DNA was subjected to q-PCR detection using the six sets of quality test primers as shown in Table 14, respectively, 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 12, and the amplification procedure is shown in Table 13.
  • a-f represents the data for q-PCR detection of the template DNA for the DNA sequences on chromosomes CH1, CH2, CH3, CH4, CH5, CH6 and CH7, respectively.
  • the amplification results showed that when the two-step and three-step amplification products were used as a template for q-PCR, the Cr values obtained by q-PCR were comparable, and there was no significant difference, indicating that the number of starting templates of q-PCR was not significant. The difference, that is, the amount of amplification products of the two-step method and the three-step method, was not significantly different.
  • 6 pairs of quality test primers verified different sequences on chromosomes 1, 2, 4, 5, 6, and 7, respectively, and the results consistently showed that there was no significant difference between the two initial template amounts.
  • the purified products of the two-step method of Example 3 after purification were randomly selected (shown as 2-1, 2-2, 2-3, 2-4, 2- in Figures 6, 8 and 9). 5, 2-6, 2-7, 2-8, 2-9, 2-10) and 10 purified samples of the three-step method of Example 2 (in Figures 7, 8, and 9) Shown as 3-1, 3-2, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10), using the interrupt method to construct the genome
  • the library was sequenced using a hiseq2500 sequencer in a shallow sequencing manner. Each sample was assayed for 1.5 Mb of data and the sequenced sequence was aligned to the human reference genome (hg19).
  • the results of the two-step method of Example 3 are shown in Fig. 6, and the results of the three-step method of Example 2 are shown in Fig. 7.
  • the ordinate represents the copy number of the chromosome, and the normal person is 2; the abscissa represents the chromosome 1-22 and the sex chromosome of the chromosome.
  • the above results indicate that the three-step method of Example 2 and the two-step method of Example 3 are consistent with the chromosome detection results of the cells.
  • the copy number variation coefficient can be used to compare the degree of dispersion of the sample copy number after amplification of the sample by the two types of amplification methods.
  • the two-step method of Example 3 had an average copy number coefficient of variation of 0.1200 for all amplified samples, while the average copy number coefficient of variation for all amplified samples of the three-step method of Example 2 was 0.1205. There is no significant difference in the copy number coefficient of variation between the two types of amplification methods. See Figure 9 for specific data.
  • SNP loci were randomly selected and the corresponding primers were designed. The selected loci and corresponding primers are shown in Table 15.
  • the genomic library was constructed by interrupting the construction of the amplified products, and sequenced using a hiseq2500 sequencer.
  • the average sequencing depth was 5 Mb. See Figure 10 for statistical results.
  • the multiplex PCR data showed that there was no significant difference in the GC content, high quality data (high_quality_of_raw), raw data specific map ratio (unique_mapped_of_raw), and average coverage (average depth) of the amplification products of the above three samples.
  • the results of the second-generation sequencing analysis showed that 23 homozygous sites were detected in the multiplex PCR product starting from gDNA, and the two homozygous sites were the two amplification products in the three-step method of Example 2. 23 and 22 were detected respectively; 21 of the amplification products of the two-step method of Example 3 were detected, and the ADO ratio of the amplification product of Example 2 and the homozygous site of the amplification product of Example 3 was not significant. difference. See Table 18 for specific data.
  • the results of the second-generation sequencing analysis showed that 62 heterozygous sites were detected in the multiplex PCR product starting from gDNA, and the 62 heterozygous sites detected 59 and 56 in the two samples of Example 2, respectively. There were 51 detected in one sample of Example 3, and there was no significant difference in the ADO ratio of the amplification product of Example 2 and the amplification product of the amplification product of Example 3. See Table 19 for specific data.
  • Hybrid site 2_1 3_1 3_2 62 (gDNA) 4/51 1/59 4/56 ADO 7.84314 1.69492 7.14286 Amplification efficiency 82.2581 95.1613 90.3226
  • Example 5 Genomic amplification using a one-step method of cleavage and amplification (referred to as one-step method)
  • the method of this example is also referred to herein as a one-step method because it is accomplished by combining the lysed cells, pre-amplification, and exponential amplification in one step.
  • Human epidermal fibroblasts were isolated and lysed according to the method described in Example 1 to obtain single-cell genomic DNA.
  • Example 6 Comparison of the one-step amplification product of Example 5 with the three-step amplification product of Example 2.
  • Example 2 Five microliters of the unpurified three-step amplification product of Example 2 and the one-step amplification product of Example 5 were separately taken, and one microliter of 6x DNA loading buffer was separately added (purchased from Beijing Kangwei Century Biotechnology Co., Ltd.). Company, item number CW0610A) ready to load.
  • the gel was a 1% agarose gel and the marker was DM2000 (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., Cat. No. CW0632C).
  • the first row is the one-step amplification result of Example 5.
  • the first lane from left to right is the molecular weight marker
  • the lanes 2-11 are single-cell amplification samples
  • the lanes 12-14 are Positive control (40 pg gDNA)
  • lanes 15-17 were negative controls (without genomic DNA)
  • lane 18 was molecular weight markers.
  • the electropherogram shows that the band and brightness of the one-step amplification product of Example 5 were comparable to those of the three-step amplification product of Example 2, and there was no significant difference.
  • Example 2 50 ⁇ l of the unpurified three-step amplification product of Example 2 and the one-step amplification product of Example 5 were used, using a universal column purification kit (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., article number CW2301)
  • the amplified product is subjected to purification treatment, and the purification step is carried out in accordance with the kit instructions. Elution was performed using 50 microliters of EB. After the purification was completed, 2 ⁇ l of the purified product was taken and detected using Nanodrop (AOSHENG, NANO-100). See Table 21 for the concentration test results.
  • concentration test results showed that the amplification products obtained by the two amplification methods were comparable in concentration after purification, and there was no significant difference.
  • Example 2 The four samples amplified according to Example 2 and the four samples amplified according to Example 5 were randomly selected as template DNA, respectively, and the amplification system composition and amplification procedure were as shown in Tables 8 and 9 of Example 4, respectively. As shown, only the number of cycles in Figures 12(a) and 12(b) is 30 rounds. PCR was carried out using a dye-containing 2xTaq MasterMix (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., Cat. No. CW0682) to amplify the above 20 pathogenic sites.
  • a dye-containing 2xTaq MasterMix purchased from Beijing Kangwei Century Biotechnology Co., Ltd., Cat. No. CW0682
  • Example 2 Ten purified products amplified by the three-step method of Example 2 were randomly selected (shown as 2-1, 2-2, 2-3, 2-4, 2-5, 2-6 in Figure 14). 2-7, 2-8, 2-9, 2-10) and 10 purified products of the one-step amplification of Example 5 (shown as 1-1, 1-2, 1- in Figure 13) 3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10), using the interrupt method to construct a genomic library, and using a shallow sequencing method using the hiseq2500 sequencer Sequencing, 1.5 Mb of data was determined for each sample, and the sequence obtained by sequencing was aligned to the human reference genome (hg19).
  • the results of the one-step method of Example 5 are shown in Fig. 13, and the results of the three-step method of Example 2 are shown in Fig. 14.
  • the ordinate represents the copy number of the chromosome, and the normal person is 2; the abscissa represents the chromosome 1-22 and the sex chromosome of the chromosome.
  • the above results indicate that the three-step method of Example 2 and the one-step method of Example 5 are consistent with the results of chromosome detection of cells.
  • the copy number variation coefficient can be used to compare the degree of dispersion of the sample copy number after amplification of the sample by the two types of amplification methods.
  • the one-step method of Example 5 The average copy number variation coefficient of all amplified samples was similar to the average copy number variation coefficient of all amplified samples of the three-step method of Example 2. See Figure 16 for specific data.

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Abstract

提供一种扩增细胞基因组DNA的方法，所述方法包括：（a）提供反应混合物，其中所述反应混合物包括所述基因组DNA、第一类引物、第二类引物、核苷酸单体混合物和核酸聚合酶，其中所述第一类引物从5'端到3'端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C，并且所述第二类引物包含所述通用序列且不包含所述可变序列；（b）将所述反应混合物置于第一温度循环程序，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；（c）将步骤（b）得到的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所述通用序列能够与所述基因组扩增产物的3'端配对并扩增所述基因组扩增产物以得到扩大的基因组扩增产物，其中，在所述步骤（b）和所述步骤（c）之前提供所述反应混合物。还提供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，特别是在扩增单细胞全基因组的方法。
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。该技术的建立需要两个必备条件：1.高质量的全基因组扩增技术；2.高通量低成本的测序技术。
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)。但是现有的主流扩增方法中的一些虽然操作简单但最终的扩增效果不甚理想，而另一些扩增效果好但操作过程较为繁琐。以MALBAC为例，其主要由如下缺陷：1.一般需经过细胞裂解、终止裂解(升温/添加中和试剂)、预扩增、扩增等若干个步骤才可得到扩增产物，整个过程涉及多次试剂配制，开盖加液操作，增加了引入环境污染的风险；2.整个实验过程需4小时以上，人员及仪器效率较低，无法对临床上急需验证的样本在短时间内给出令人满意的结果；3.整个实验过程对操作人员的熟练程度要求较高，初次接触者无法很快的获得满意的扩增结果。
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，提供了一种扩增细胞基因组DNA的方法，所述方法包括：(a)提供反应混合物，其中所述反应混合物包括所述基因组DNA、第一类引物、第二类引物、核苷酸单体混合物、和核酸聚合酶，其中所述第一类引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C，，并且所述第二类引物包含所述通用序列且不包含所述可变序列；(b)将所述反应混合物置于第一温度循环程序，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；(c)将步骤(b)得到的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所述通用序列能够与所述基因组扩增产物的3’端配对并扩增所述基因组扩增产物以得到扩大的基因组扩增产物，其中，在所述步骤(b)和所述步骤(c)之前提供所述反应混合物。
在一些实施方式中，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。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、胎儿性别和胎儿RHD。
在一些实施方式中，所述基因组DNA包含在细胞中，并且所述反应混合物进一步包含能够裂解所述细胞的表面活性剂和/或裂解酶。
在一些实施方式中，在所述步骤(b)和步骤(c)之前还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。
在一些实施方式中，所述通用序列被选择以使得其基本上不会与基因组DNA结合产生扩增。在一些实施方式中，所述通用序列选自下组：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、和SEQ ID NO:6。
在一些实施方式中，所述可变序列包含随机序列。在一些实施方式中，所述可变序列的长度为2-20个碱基、3-10个碱基，4-9个碱基或5-8个碱基。在一些实施方式中，所述可变序列中的三个或三个以上的碱基位置由选自G、A、和T的一种或几种碱基组成，或者由C、A和T的一种或几种碱基组成。在一些实施方式中，所述三个或三个以上的碱基位置位于所述可变序列的3’端或者中间。在一些实施方式中，所述可变序列选自下组：(N)nGGG、(N)nTTT，(N)mTNTNG，(N)xGTGG(N)y，其中N为任意的可与天然核酸进行碱基配对的 核苷酸，n是选自3-17的正整数，m是选自3-15的正整数、x和y分别是选自3-13的正整数。在一些实施方式中，所述可变序列被选择以使得在基因组上分别均匀并且覆盖度高。
在一些实施方式中，所述第一类引物包括SEQ ID NO:11[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNNNN]、SEQ ID NO:12[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNGGG]、SEQ ID NO:13[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNTTT]、SEQ ID NO:14[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNTNTNG]或SEQ ID NO:15[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNGTGGNN]的序列，并且所述第二类引物从5’到3’具有SEQ ID NO:1[GTGAGTGATGGTTGAGGTAGTGT GGAG]的序列，其中N为任意的可与天然核酸进行碱基配对的核苷酸。
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：Phi29 DNA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、T7 phase DNA聚合酶变种(缺少3’-5’外切酶活性)、
Figure PCTCN2016097208-appb-000001
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，及其任意组合。
在一些实施方式中，反应混合物进一步包含pH值调节剂，使得所述反应混合物的pH值维持在7.0-9.0之间。
在一些实施方式中，所述反应混合物进一步包含一种或多种选自下组的成分：Mg2+、dTT、牛血清白蛋白、DNase抑制剂、RNase、SO4 2-、Cl-、K+、Ca2+、Na+、和(NH4)+。
在一些实施方式中，所述第一温度循环程序包括：(b1)将所述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；(b2)将所述反应混合物置于能够使所述第一类引物与DNA单链模板结合的温度程序；(b3)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；(b4)将所述反应混合物置于能够使所述扩增产物脱落成单链的温度程序；(b5)重复步骤(b2)到(b4)至指定的第一循环次数。在一些实施方式中，所述指定的第一循环次数大于2。在一些实施方式中，当进行到第二次循环后，所述扩增产物包含在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扩增产物。在一些实施方式中，所述方法在步骤(b4)后并且在步骤(b5)之前进一步包括步骤(b4’)，其中将所述反应混合物置于适当的温度程序，使得所述基因组扩增产物的3’端与5’端杂交结合以形成环状结构，或者使所述基因组扩增产物的3’端与引物结合。在一些实施方式中，所述方法在步骤(b4)后直接到步骤(b5)。在一些实施方式中，所述步骤(b5)的所述第一循环次数大于3、大于4、大于5、大于6，并且不超过10。
在一些实施方式中，所述步骤(c)包括：(c1)将经步骤(b)获得的所述反应混合物置于能够 打开DNA双链的温度程序；(c2)将所述反应混合物置于能够使所述第二类引物与所述经步骤(b)获得的基因组扩增产物的单链结合的温度程序；(c3)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；(c4)重复步骤(c1)到(c3)至指定的第二循环次数。在一些实施方式中，所述步骤(c4)中的所述第二循环次数大于所述步骤(b5)中所述的第一循环次数。在一些实施方式中，所述步骤(b1)中所述的温度程序包括在90-95℃的温度之间反应1-10分钟。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5-10℃之间的第一温度，介于25-30℃之间的第二温度，和介于45-50℃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反应3-50秒、在第二温度反应3-50秒、和在第三温度反应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60-90℃的温度之间反应1-15分钟。在一些实施方式中，所述步骤(b4)中所述的温度程序包括在90-95℃的温度之间反应10-50秒。在一些实施方式中，所述步骤(c1)中所述的温度程序包括在90-95℃的温度之间反应10-30秒。在一些实施方式中，所述步骤(c2)中所述的温度程序包括在45-65℃的温度之间反应10-30秒。在一些实施方式中，所述步骤(c3)中所述的温度程序包括在60-80℃的温度之间反应1-15分钟。在一些实施方式中，所述步骤(a)中的基因组DNA从被裂解的细胞释放，所述裂解包括热裂解、碱裂解、酶裂解或机械裂解。
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，所述热裂解是在裂解试剂存在的条件下进行的。在一些实施方式中，所述裂解试剂包括一种或多种选自下组的表面活性剂：NP-40、吐温、SDS、TritonX-100、EDTA、和异硫氰酸胍，和/或裂解酶。
在本申请的另一个方面中，提供了一种扩增细胞基因组的方法，所述方法包括：(a)提供反应混合物，其中所述反应混合物包括所述基因组DNA、第一类引物、第二类引物、核苷酸单体混合物、和核酸聚合酶，其中所述第一类引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C，所述第二类引物包含所述通用序列且不包含所述可变序列；(b)将所述反应混合物置于第一温度循环程序，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；(b2)介于5-10℃之间的第一退火温度反应3-50秒，介于25-30℃之间的第二退火温度反应3-50秒，和介于45-50℃之间的第三退火温度反应3-50秒；(b3)在介于60-90℃之间的第一延伸温度反应1-15分钟；(b4)在介于90-95℃的第一解链温度之间反应10-50秒；(b5)重复步骤(b2)到(b4)至6-9个循环；(c)将步骤(b)得到的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所述 通用序列能够与所述基因组扩增产物的3’端配对并扩增所述基因组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：(c1)在介于90-95℃之间的第二变性温度反应1-10分钟；(c2)在介于90-95℃之间的第二解链温度反应10-30秒；(c3)在介于45-65℃之间的第四退火温度反应10-30秒；(c4)在介于60-80℃之间的第二延伸温度反应1-15分钟；(c5)重复步骤(c2)到(c4)5-30个循环；(d)获得所述步骤(c)得到的扩增产物；其中，在所述步骤(b)和所述步骤(c)之前提供所述反应混合物。
在一些实施方式中，所述通用序列包含或由SEQ ID NO:1组成；所述可变序列包含或由NNNNNTTT或NNNNNGGG组成，N为任意的可与天然核酸进行碱基配对的核苷酸。
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，所述试剂盒包括含有第一类引物和第二类引物的混合物，其中所述第一类引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C，并且所述第二类引物包含所述通用序列且不包含所述可变序列。
在一些实施方式中，所述混合物进一步包括核苷酸单体混合物和Mg2+。
在一些实施方式中，所述混合物进一步包括一种或多种选自下组的成分：dTT、牛血清白蛋白(BSA)、pH调节剂(例如Tris HCl)、DNase抑制剂、RNase、SO4 2-、Cl-、K+、Ca2+、Na+、和/或(NH4)+。
在一些实施方式中，所述混合物进一步包括核酸聚合酶。
在一些实施方式中，所述试剂盒进一步包括能够裂解细胞的表面活性剂和/或裂解酶。在一些实施方式中，所述表面活性剂选自NP-40、吐温、SDS、TritonX-100、EDTA、和异硫氰酸胍中的一种或多种。在一些实施方式中，所述裂解酶选自蛋白酶K、胃蛋白酶、和木瓜蛋白酶中的一种或多种。
在一些实施方式中，所述混合物进一步包括能够裂解细胞的表面活性剂和/或裂解酶。
在本申请的又一个方面中，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类引物和第二类引物，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在同一容器中混合所述第一类引物和所述第二类引物的步骤，其中所述第一类引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C，并且所述第二类引物包含所述通用序列且不包含所述可变序列。
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示出了对正常人表皮成纤维细胞(AFP)的基因组DNA分别用实施例2的三步法扩增以及用实施例3的两步法扩增，将两种方法得到的扩增产物分别进行凝胶电泳的结果，其中a为实施例3的两步法扩增结果，自左向右第1泳道为分子量标记，2-11泳道为单细胞扩增样品，12-14泳道为阳性对照(40pg gDNA)，15-17泳道为阴性对照，第18泳道为分子量标记；b为实施例2的三步法扩增结果，自左向右第1泳道为分子量标记，2-11泳道为单细胞扩增样品，12-14泳道为阳性对照(40pg gDNA)，15-17泳道为阴性对照，第18泳道为分子量标记。
图3示出了对正常人表皮成纤维细胞(AFP)的基因组DNA分别用实施例2的三步法扩增以及用实施例3的两步法扩增，将两种方法得到的扩增产物分别随机选取7个样品，即总共14个样品分别作为模板，使用表7所示的引物分别针对表6所示的20个致病位点进行扩增，将扩增产物进行凝胶电泳的结果。其中a-g分别表示重复进行的单细胞基因组DNA扩增产物的凝胶电泳图，其中上排条带是用两步法扩增的结果，下排条带是用三步法扩增的结果：a：上排对应样品2_1和下排对应样品3_1、b：上排对应样品2_2和下排对应样品3_3、c：上排对应样品2_3和下排对应样品3_4、d：上排对应样品2_4和下排对应样品3_5、e：上排对应样品2_5和下排对应样品3_6、f：上排对应样品2_6和下排对应样品3_7、g：上排对应样品2_7和下排对应样品3_8；在每张电泳图片中，从左向右每个泳道依次表示分子量标记物、针对表6所示的致病位点1-20进行的扩增结果(在图(a)中是1-16)。
图4示出了对正常人表皮成纤维细胞(AFP)的基因组DNA分别用实施例2的三步法扩增以及用实施例3的两步法扩增，将两种方法得到的扩增产物分别随机选取3个样品，即总共6个样品分别作为模板，使用表11所示的引物分别针对表10所示的20个致病位点进行扩增，将扩增产物进行凝胶电泳的结果。其中a-c分别表示重复进行的单细胞基因组DNA扩增产物的凝胶电泳图，其中上排条带是用两步法扩增的结果，下排条带是用三步法扩增的结果：a：上排对应样品2_1和下排对应样品3_1、b：上排对应样品2_2和下排对应样品3_4、c：上排对应样品2_7和下排对应样品3_8；在每张电泳图片中，从左向右每个泳道依次表示分子量标记物、针对表6所示的致病位点1-20进行的扩增结果。
图5示出了对正常人表皮成纤维细胞(AFP)的基因组DNA分别用实施例2的三步法扩增以及用实施例3的两步法扩增，将两种方法得到的扩增产物分别随机选取4个样品，即总共8个样品分别作为模板，使用表14所示的6对质检引物进行qPCR扩增的结果。其中a-f分别表示使用针对染色体CH1、CH2、CH3、CH4、CH5、CH6和CH7的质检引物，对模板DNA进行的q-PCR检测的数据。其中的CT表示阈值循环数，DNA1和DNA2代表阳性对照。
图6示出了对正常人表皮成纤维细胞(AFP)的基因组DNA用实施例3的两步法扩增，将得到的扩增产物构建基因组文库并进行测序得到的染色体拷贝数的结果。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。其中a-j分别表示将样品2_1、2_2、2_3、2_4、2_5、2_6、2_7、2_8、2_9和2_10构建基因组文库并进行测序得到的染色体拷贝数的结果。
图7示出了对正常人表皮成纤维细胞(AFP)的基因组DNA用实施例2的三步法扩增，将得到的扩增产物构建基因组文库进行测序得到的染色体拷贝数的结果。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。其中a-i分别表示将样品3_1、3_3、3_4、3_5、3_6、3_7、3_8、3_9和3_10构建基因组文库并进行测序得到的染色体拷贝数的结果。
图8示出了对正常人表皮成纤维细胞(AFP)的基因组DNA分别用实施例2的三步法扩增(即样品3_1、3_3、3_4、3_5、3_6、3_7、3_8、3_9和3_10)以及用实施例3的两步法扩增(即样品2_1、2_2、2_3、2_4、2_5、2_6、2_7、2_8、2_9和2_10)，将两种方法得到的扩增产物分别构建基因组文库进行二代测序，得到的测序结果的数据统计。
图9示出了对正常人表皮成纤维细胞(AFP)的基因组DNA分别用实施例2的三步法扩增(即样品3_1、3_3、3_4、3_5、3_6、3_7、3_8、3_9和3_10)以及用实施例3的两步法扩增(即样品2_1、2_2、2_3、2_4、2_5、2_6、2_7、2_8、2_9和2_10)，将两种方法得到的扩增产物分别构建基因组文库进行二代测序，并对其拷贝数变异系数的比较结果。
图10示出了对正常人表皮成纤维细胞(AFP)的基因组DNA分别用实施例2的三步法扩增以及用实施例3的两步法扩增，将两种方法得到的扩增产物(其中三步法的扩增产物示为3-1和3-2，两步法的扩增产物示为2-1)以及人类表皮成纤维细胞(AFP)细胞提取的基因组DNA(示为Gdna)分别进行多重PCR，并将多重PCR扩增产物进行高通量测序得到的结果。
图11示出了对正常人表皮成纤维细胞(AFP)的基因组DNA分别用实施例2的三步法扩增以及用实施例5的一步法扩增，将两种方法得到的扩增产物分别进行凝胶电泳的结果，其中a为实施例5的一步法扩增结果，自左向右第1泳道为分子量标记，2-11泳道为单细胞扩增样品，12-14泳道为阳性对照(40pg gDNA)，15-17泳道为阴性对照，第18泳道为分子量标记；b为实施例2三步法扩增结果，自左向右第1泳道为分子量标记，2-11泳道为单细胞扩增样品，12-14泳道为阳性对照(40pg gDNA)，15-17泳道为阴性对照，第18泳道为分子量标记。
图12示出了对正常人表皮成纤维细胞(AFP)的基因组DNA分别用实施例2的三步法扩增以及用实施例5的一步法扩增，将两种方法得到的扩增产物分别随机选取4个样品，即总共8个样品分别作为模板，使用表7所示的引物分别针对表6所示的20个致病位点进行扩增，将扩增产物进行凝胶电泳的结果。其中a-d分别表示重复进行的单细胞基因组DNA扩增产物的凝胶电泳图，其中上排条带是用两步法扩增的结果，下排条带是用三步法扩增的结果：a：上排对应样品1_1和下排对应样品3_1、b：上排对应样品1_2和下排对应样品3_2、c：上排对应样品1_3和下排对应样品3_3、d：上排对应样品1_4和下排对应样品3_4；在每张电泳图中，从左向右每个泳道依次表示针对表6所示的致病位点1-20进行的扩增结果。
图13示出了对正常人表皮成纤维细胞(AFP)的基因组DNA用实施例5的一步法扩增，将得到的扩增产物构建基因组文库进行测序得到的染色体拷贝数的结果。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。其中a-j分别表示将样品1_1、1_2、1_3、1_4、1_5、1_6、1_7、1_8、1_9和1_10构建基因组文库并进行测序得到的染色体拷贝数的结果。
图14示出了对正常人表皮成纤维细胞(AFP)的基因组DNA用实施例2的三步法扩增，将得到的扩增产物构建基因组文库进行测序得到的染色体拷贝数的结果。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。其中a-i分别表示将样品3_1、3_2、3_3、3_4、3_5、3_6、3_7、3_8和3_10构建基因组文库并进行测序得到的染色体拷贝数的结果。
图15示出了对正常人表皮成纤维细胞(AFP)的基因组DNA分别用实施例2的三步法扩增以及用实施例5的一步法扩增，将两种方法得到的扩增产物分别构建基因组文库进行二代测序，得到的测序结果的数据统计。
图16示出了对正常人表皮成纤维细胞(AFP)的基因组DNA分别用实施例2的三步法扩增以及用实施例5的一步法扩增，将两种方法得到的扩增产物分别构建基因组文库进行二代测序，并对其拷贝数变异系数的比较结果。
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的方法。
本发明至少部分基于以下发现，即，在基因组DNA的扩增反应之前，可以在单个反应混合物中加入扩增所需的全部试剂，然后将该反应混合物置于扩增反应的条件下，直到完成扩增。该方法在扩增反应开始后无需再向反应混合物中添加试剂，因此大大降低了因添加试剂而带来的额外操作以及可能导致的污染，并且将所需的反应时间大大缩短。
在本发明之前，当使用两种及以上引物进行扩增基因组DNA时，需要将扩增反应分成至少两步，每步中使用不同的引物，由此实现不同的扩增目的。此前一直认为，只有在第一步完成后才能在反应混合物中加入第二步所需的引物或者第一步仅使用非常低的循环次数(例如，1次)，这样才能避免引物之间发生相互干扰进而影响扩增效果，因此，本发明以前的方法要么在扩增反应前只加入第一步所需的引物，等到第一步的扩增反应结束后，再向反应体系中加入第二步所需的引物，然后再进行第二步的扩增反应(例如，请参见，WO2012/166425)；要么第一步仅能使用非常低的次数，无法达到理想的扩增效率。但是，出乎意料的是，本申请的发明人发现，当把原本认为可能相互产生干扰的引物一次性地放入单个反应混合物，并且在本发明的反应条件下进行扩增时，能够意外地得到与分次加入引物时相当的扩增效果。因此，本发明大大提高了反应效率，缩短了反应时间，而且降低了样品污染的风险，提高了结果的可靠性。
在一方面，本申请提供了扩增细胞基因组DNA的方法，包括：(a)提供反应混合物，其中所述反应混合物包括所述基因组DNA、第一类引物、第二类引物、核苷酸单体混合物、和核酸聚合酶，其中所述第一类引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C，并且所述第二类引物包含所述通用序列且不包含所述可变序列；(b)将所述反应混合物置于第一温度循环程序，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；(c)将步骤(b)得到的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所述通用序列能够与所述基因组扩增产物的3’端配对并扩增所述基因组扩增产物以得到扩大的基因组扩增产物。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的快速、精确扩增。
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。
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进行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(WO2013153176 A1)。
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 et al.,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(美国专利申请US 20070281313)。
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摄氏度之间，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与反应混合物的其他成分混合得到反应混合物。
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。
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反应混合物，也就是说，在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，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的扩增都在同一个反应混合物中进行，不仅提高了反应效率，缩短了反应时间，而且仍然保持了相当好的扩增效果。
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.引物
反应混合物中还含有两类不同的引物，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所述通用序列且不包含所述可变序列。第一类引物中的可变序列可以与基因组DNA模板结合，并且在核酸聚合酶的作用下复制一定长度的基因组模板以得到5’端为通用序列而3’端为基因组序列的扩增产物，在本申请中也称为半扩增子。第一类引物中的可变序列还可以与半扩增子配对结合，并以半扩增子为模板进行复制，得到5’端为通用序列而3’端为通用序列的互补序列的扩增产物，在本申请中也称为全扩增子。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。
通用序列在本申请中是指位于第一类引物5’端的特定序列。通用序列的长度可以是例如，10-30、12-29、15-28、18-26或20-24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结合产生扩增，并且避免第一类引物与第一类引物之间，或者第一类引物与第二类引物之间的聚合。
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，通用序列中不具有自身配对的序列，或者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，所述通用序列选自下组：SEQ ID NO:1(GTG AGT GAT GGT TGA GGT AGT GTG GAG)、SEQ ID NO:2(GTGGAGTTAGTGAGTGTAATGGAT)、SEQ ID NO:3(GGTTTGGTGTGGTGTGTGGTGGTG)、SEQ ID NO:4(ACAACACTATCAATCCCTATCCTAC)、SEQ ID NO:5(ATGGTAGTGGGTAGATGATTAGGT)、SEQ ID NO:6(CATATCCCTATACCTAATACCATTAC)。
在第一类引物的5’端为通用序列，3’端为可变序列。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。可变序列在本申请中是指序列不固定的一段碱基序列，例如可以包含随机序列。随机序列可以包含任意的可与天然核酸进行碱基配对的核苷酸，例如A、T、G、和C四种天然碱基以及本领域技术人员公知的其他核苷酸类似物、修饰核苷酸等，只要能够与基因组DNA配对并实现扩增反应即可。可变序列中的核苷酸序列可以有多种的变化可能。因此，第一类引物可以包含一组序列不同的引物，其中每一个引物都具有位于5’端的通用序列，以及位于3’端的可变序列，这些引物中的通用序列都相同，但是可变序列可能各不相同。
可变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，可变序列的长度为5个碱基。在某些实施方式中，可变序列的长度为8个碱基。理论上来说，如果每个碱基位置都从A、T、G、C四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256种可能的随机序列，长度为5个碱基的可变序列可以组合出1024种可能的随机序列，以此类推。这些可变序列可以与基因组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。
可以通过随机的方式选择可变序列，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，可变序列中的三个或三个以上的碱基位置由选自G、A、和T的一种或几种碱基组成(即， 不为C)，或者由C、A和T的一种或几种碱基组成(即，不为G)，以避免可变序列与通用序列产生互补配对。在一些实施方式中，当通用序列不含有G但含有C时，可变序列中的三个或三个以上的碱基位置由C、A和T的一种或几种碱基组成(即，不为G)。在一些实施方式中，当通用序列不含有C但含有G时，可变序列中的三个或三个以上的碱基位置由G、A、和T的一种或几种碱基组成(即，不为C)。在一些实施方式中，当通用序列不含有C也不含有G时，可变序列中的三个或三个以上的碱基位置选自G、A、和T的一种或几种碱基组成(即，不为C)或者由C、A和T的一种或几种碱基组成(即，不为G)。所述三个或三个以上的碱基可以位于可变序列的3’端，或者也可以位于可变序列的中间部分。所述三个或三个以上的碱基可以是连续的或者也可以是不连续的，例如，可变序列的3’端的三个相邻碱基均不为C，或者可变序列3’端的三个相互间隔的碱基不为C，或者可变序列3’端的某两个连续碱基以及另一个与之间隔的碱基不为C。当所述三个碱基位置连续时，其可以为以下的示例性的序列：TTT、GGG、TTG、GAA或ATG。
在某些实施方式中，可变序列选自下组：(N)nGGG、(N)nTTT，(N)mTNTNG，和(N)xGTGG(N)y，其中其中N为任意的可与天然核酸进行碱基配对的随机的核苷酸，n是选自3-17的正整数，m是选自3-15的正整数、x和y分别是选自3-13的正整数。在某些实施方式中，所述第一类引物中的可变序列可以具有(N)nGGG、(N)nTTT，(N)mTNTNG，(N)xGTGG(N)y中的一种或多种序列。在某些实施方式中，可变序列选自下组：SEQ ID NO:7(NNNNNGGG)，SEQ ID NO:8(NNNNNTTT)，SEQ ID NO:9(NNNTNTNG)，SEQ ID NO:10(NNNGTGGNN)。
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，第一类引物可以包含SEQ ID NO:11[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNNNN]、SEQ ID NO:12[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNGGG]、SEQ ID NO:13[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNTTT]、SEQ ID NO:14[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNTNTNG]或SEQ ID NO:15[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNGTGGNN]，其中N为任意的可与天然核酸进行碱基配对的核苷酸(例如，A、T、G、C)。
反应混合物中的第二类引物包含所述通用序列且不包含所述可变序列。第二类引物的5’端和3’端可以含有或不含有其他额外的序列。在某些实施方式中，第二类引物的序列由第一类引物中的通用序列组成。在某些实施方式中，第二类引物从5’到3’包含或者由SEQ ID NO:1[GTGAGTGATGGTTGAGGTAGTGTGGAG]的序列组成。
在一些实施方式中，引物在反应混合物中的浓度为300ng-1500ng/μL。在一些实施方式中，引物在反应混合物中的浓度为300ng-1400ng/μL、300ng-1200ng/μL、300ng-1000ng/μL、 300ng-800ng/μL、300ng-600ng/μL或300ng-400ng/μL。在一些实施方式中，引物在反应混合物中的浓度为500ng-1400ng/μL、600ng-1400ng/μL、800ng-1400ng/μL、900ng-1400ng/μL、1000ng-1400ng/μL或1200ng-1400ng/μL。在一些实施方式中，引物在反应混合物中的浓度为400ng-1400ng/μL、500ng-1200ng/μL、600ng-1000ng/μL或700ng-800ng/μL。
iii.其他成分
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：Phi29 DNA聚合酶、Bst DNA聚合酶、Bst 2.0 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、T7 phase DNA聚合酶变种(缺少3’-5’外切酶活性)、
Figure PCTCN2016097208-appb-000002
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。
在某些实施方式中，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。
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，反应混合物中还可以进一步包括pH调节剂，使得反应混合物的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，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。
本申请提供的方法的特点之一在于，在所述步骤(b)和所述步骤(c)之前提供所述反应混合物。由于反应混合物的配置在进行温度循环程序之前全部完成，只要进入温度循环程序，反应混合物就可以按照预定的设置进行反应，无需再开盖或者增加任何组分，从而避免污染而且提高反应效率。在一些实施方式中，在所述步骤(a)完成之后无需再向所述反应混合物中添加反应物，例如酶，引物和dNTP。。在一些实施方式中，在所述步骤(b)之前完成提供所述反应混合物。在一些实施方式中，在所述步骤(b)开始之后不再向所述反应混合物中添加反应物，例如酶，引物和dNTP。在一些实施方式中，所述步骤(a)在所述步骤(b)和所述步骤(c)之前。
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)：将所述反应混合物置于第一温度循环程序，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。
“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、引物与模板单链杂交、和引物延伸。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。新合成的DNA链在下一个反应循环中又可以作为新的DNA模板，进行新一轮的DNA合成。
在本申请方法的步骤(b)中，将反应混合物置于第一温度循环程序，使得反应混合物中的第一类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。
在第一温度循环程序中，首先将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))。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。在某些实施方式中，步骤(b1)中的温度程序包括在介于90-95℃之间的温度反应1-10分钟。
然后，将反应混合物置于能够使所述第一类引物与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5℃-10℃，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5-10℃之间的第一退火温度(例如10℃)反应适当的时间(例如3-50秒)，在介于25-30℃之间的第二退火温度(例如30℃)反应适当的时间(例如3-50秒)，以及在介于45-50℃之间的第三退火温度(例如50℃)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5℃-10℃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5℃-10℃)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。
在引物退火的温度程序后，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，phi29 DNA聚合酶、T5 DNA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、TflDNA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：Phi29 DNA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、T7 phase DNA聚合酶变种(缺少3’-5’外切酶活性)、
Figure PCTCN2016097208-appb-000003
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，65-90℃、70-90℃、75-90℃、80-90℃、60-85℃、60-80℃、60-75℃、或60-70℃)反应1-15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、或13-14分钟)。在某些实施方式中，步骤(b3)包括在70℃反应2分钟。
引物延伸程序后，将所述反应混合物置于能够使所述扩增产物脱落成单链的温度程序(步骤(b4))。该步骤中的温度可以与步骤(b1)中的温度相近，但反应时间略短。在某些实施方式中，步骤(b4)包括在90-95℃的温度之间反应10-50秒。这时的反应混合物中的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，两者都可以作为下一个循环中的DNA模板，与引物结合并开启下一轮的复制。
然后重复步骤(b2)到(b4)至指定的第一循环次数，以获得基因组扩增产物。第一循环次数应至少为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，之前的扩增产物本身也可以作为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。
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，以确保既有足够的扩增产物用于后续的反应，又不会因为循环次数过多影响扩增产物的准确度。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，并且最好不超过8、不超过9、不超过10、不超过11、或不超过12。如果循环次数过低，则得到的扩增产物少，为获得足够的扩增产物，就需要在下一次扩增(即步骤(c))中增加循环次数，这样会降低扩增结果的准确性。而如果循环次数过高，则会导致在基因组DNA的扩增过程中出现变异的序列，使得下一次扩增(即步骤(c))的模板中出现偏差，导致最终的扩增结果不准确。
在某些实施方式中，在步骤(b)中，在步骤(b4)后进一步包括包括步骤(b4’)，其中将所述反应混合物置于适当的温度程序，使得所述基因组扩增产物的3’端与5’端杂交结合以形成环状结构，或者使所述基因组扩增产物的3’端与引物结合。此前认为，步骤(b4’)能够将全扩增子的3’末端保护起来，从而避免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(b4)后不经其他步骤(例如步骤(b4’))而直接到步骤(b5)。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(b4)后不经特定的步骤使全扩增子成环或者3’端与引物结合，最终的扩增结果仍然具有相当高的准确度，与使用步骤(b4’)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。
在步骤(b)中，反应体系中不仅存在第一类引物，而且还存在第二类引物。第二类引物包含第一类引物中的通用序列。由于通用序列基本上不与基因组序列互补，因此如果扩增反应的特异性足够高，那么在步骤(b)中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。但是，当步骤(b)进行了两个循环时，反应混合物中开始出现在3’端具有通用序列互补序列的全扩增子，而这样的全扩增子的3’端能够与第二类引物通过碱基配对，因此有可能导致第二类引物在步骤(b)中(例如从第三个循环开始)就开始对全扩增子进行复制。这有可能会干扰第一类引物对基因组DNA复制，使得第一类引物对基因组DNA的扩增不够充分，达不到期望的对基因组DNA的覆盖范围。此外，当第一类引物和第二类引物同时存在于反应体系中时，有可能第一类引物与第二类引物发生与模板无关的引物-引物扩增反应，导致引物多聚体的产生。但是出乎意料的是，即使存在上述这些不确定的因素，本申请的发明人意想不到地发现，在第一类引物与第二类引物同时存在于反应混合物中并且都能 进行扩增反应的情况下，第一类引物似乎并不受第二类引物的干扰，对基因组DNA的扩增仍然保持了较高的特异性以及较广的覆盖度，并且与在步骤(b)中单独使用第一类引物然后在步骤(c)中额外添加第二类引物的方法相比，得到的结果在总体上相当。
步骤(c)：置于第二温度循环程序
本申请提供的方法还包括步骤(c)：将步骤(b)得到的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所述通用序列能够与所述基因组扩增产物的3’端配对并扩增所述基因组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(c1))。这里的DNA双链主要是指在步骤(b)中得到的基因组扩增产物(即全扩增子)的双链。虽然此时的反应混合物中仍然存在原始的基因组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(c)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，只要待扩增的全扩增子双链能够变性成为单链即可。在某些实施方式中，步骤(c1)中的温度程序包括在介于90-95℃之间(例如94℃)的解链温度反应10-30秒(例如20秒)。在某些实施方式中，在第一温度循环程序结束以后但是在第二温度循环程序开始之前，进一步包括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。这将有助于确保模板DNA双链全部变性成单链。
在步骤(c1)以后，将反应混合物置于能够使所述第二类引物与步骤(b)获得的基因组扩增产物的单链结合的温度程序(步骤(c2))。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(c2)中的温度程序包括在介于45-65℃之间的退火温度(例如58℃)反应10-30秒(例如15秒)。在某些实施方式中，第二类引物为SEQ ID NO:1，且步骤(c2)中的温度程序包括在58℃反应10-30秒。在某些实施方式中，步骤(c2)中的退火温度高于在步骤(b2)中的退火温度。在步骤(c2)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(c1)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(c2)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(c3)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1-15分钟(例如2分钟)。
可以重复步骤(c1)到(c3)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(c4)中的所述第二循环次数大于所述步骤(b5)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15-30个循环(例如，15-30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个循环)。
在某些实施方式中，步骤(c)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(c3)相同的温度程序(例如72℃)反应适当的时间(例如5分钟)。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(c)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)提供反应混合物，其中所述反应混合物包括所述基因组DNA、第一类引物、第二类引物、核苷酸单体混合物、和核酸聚合酶，其中所述第一类引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C，所述第二类引物包含所述通用序列且不包含所述可变序列；
(b)将所述反应混合物置于第一温度循环程序，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；
(b2)介于5-10℃之间的第一退火温度反应3-50秒，介于25-30℃之间的第二退火温度反应3-50秒，和介于45-50℃之间的第三退火温度反应3-50秒；
(b3)在介于60-90℃之间的第一延伸温度反应1-15分钟；
(b4)在介于90-95℃的第一解链温度之间反应10-50秒；
(b5)重复步骤(b2)到(b4)至6-9个循环；
(c)将步骤(b)得到的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所述通用序列能够与所述基因组扩增产物的3’端配对并扩增所述基因组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：
(c1)在介于90-95℃之间的第二变性温度反应1-10分钟；
(c2)在介于90-95℃之间的第二解链温度反应10-30秒；
(c3)在介于45-65℃之间的第四退火温度反应10-30秒；
(c4)在介于60-80℃之间的第二延伸温度反应1-15分钟；
(c5)重复步骤(c2)到(c4)5-30个循环；
(d)获得所述步骤(c)得到的扩增产物；
其中，在所述步骤(b)和所述步骤(c)之前提供所述反应混合物。
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。这样，整个扩增反应实际上是将裂解细胞和扩增基因组都发生在同一个反应混合物中，通过将该反应混合物置于不同的温度循环程序中完成。这大大简化了操作步骤，避免了多次操作样品而带来的污染的风险，而且在扩增结果上同样能够实现很好的扩增特异性和低变异性。
在某些实施方式中，第一类引物包含或者由SEQ ID NO:11、12、13、14、和/或15组成，其中的通用序列包含或由SEQ ID NO:1组成。
优势
本申请提供的方法与现有技术中的方法相比具有多种优势。
一方面，本发明在热循环条件下将核酸的预扩增、扩增等步骤组合成一个反应。这一反 应减少了人为操作，将核酸放入PCR管内并执行特定的程序即可实现核酸的全基因组扩增，扩增产物基因组覆盖程度高，扩增偏差小。省去了试剂配制，开盖加液等操作，减少了实验环境和操作人员带来的污染风险，同时缩短整体实验时间周期，整体扩增时间仅需2.5小时。
另一方面，本发明还可以在热循环条件下将细胞裂解、核酸的预扩增、扩增等步骤组合成一个反应。这又进一步减少了人为操作，省去了单独进行细胞裂解的步骤，进一步缩短实验时间和降低污染的风险。
本申请的方法在简化操作的基础上还保持了扩增的高准确性和广覆盖程度。本申请的方法使用准线性扩增技术，减少序列依赖的扩增偏倚性。在预扩增中，侧重于从原始的样品DNA模板扩增，并且基因组覆盖度高，扩增偏差小。预扩增阶段产生的全扩增子在扩增阶段被大量的扩增。该技术扩增过程产量高，最低起始模板几个皮克，结果可靠，可重复。
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.Methods 4:931)、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.,and 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.,et al.(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，其中包括含有第一类引物和第二类引物的混合物，其中所述第一类引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C，并且所述第二类引物包含所述通用序列且不包含所述可变序列。
在某些实施方式中，其中所述混合物进一步包括核苷酸单体混合物(例如dATP、dGTP、dTTP、和dCTP)、dTT和Mg2+。在某些实施方式中，所述混合物的Mg2+浓度介于2mmol-8mmol/μL，dNTP浓度介于1mmol-8mmol/μL，dTT浓度介于2mmol-7mmol/μL。在某些实施方式中，所述混合物进一步包括一种或多种选自下组的成分：牛血清白蛋白(BSA)、pH调节剂(例如Tris HCl)、DNase抑制剂、RNase、SO4 2-、Cl-、K+、Ca2+、Na+、和/或(NH4)+等。在某些实施方式中，所述混合物的pH值范围介于7.0—9.0之间。
在某些实施方式中，试剂盒进一步包括核酸聚合酶，且所述核酸聚合酶不包含在第一类引物和第二类引物的混合物中。在这样的实施方式中，核酸聚合酶可以存放于单独的容器中，可选地可以与其他成分组成混合物，或者也可以是以基本上纯的形式存在。
在某些实施方式中，所述第一类引物和第二类引物的混合物中进一步包括核酸聚合酶。在某些实施方式中，所述混合物包括：第一类引物、第二类引物、核苷酸单体混合物、Mg2+、dTT、Tris HCl、和核酸聚合酶，以及一种或多种选自下组的成分：BSA、DNase抑制剂、RNase、SO4 2-、Cl-、K+、Ca2+、Na+、和(NH4)+等。在这样的实施方式中，所述混合物可以含有扩增反应所需的除基因组DNA以外的全部反应物。当这样的混合物用于本申请所述的扩增反应 时，可以将含有基因组DNA的反应物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)中的反应混合物。
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，和/或一种或多种裂解酶。示例性的表面活性剂包括，但不限于，NP-40、吐温、SDS、TritonX-100、EDTA、异硫氰酸胍中的一种或多种。示例性的裂解酶可以是蛋白酶K、胃蛋白酶、木瓜蛋白酶中的一种或多种。裂解细胞的成分可以单独存放于独立的容器中，也可以与其他某些成分混合在一起。在某些实施方式中，试剂盒包括表面活性剂和裂解酶，两者分别置于不同的容器中，或者置于同一个容器中。
在某些实施方式中，所述第一类引物、第二类引物以及核酸聚合酶的混合物中进一步包含表面活性剂和/或裂解酶。
在某些实施方式中，所述试剂盒中包括一个容器，其中包含了所有的反应物。在某些实施方式中，试剂盒中包括两个容器，其中一个容器存放包括核酸聚合酶在内的在扩增反应中所需的成分，另一个容器存放包括裂解酶在内的在细胞裂解中所需的成分。在某些实施方式中，试剂盒中包括四个容器，其中第一容器存放核酸聚合酶，第二容器存放除核酸聚合酶以外的在扩增反应中所需的成分，第三容器存放裂解酶，第四容器存放除裂解酶以外的在细胞裂解中所需的成分。
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和第二类引物，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在同一容器中混合所述第一类引物和所述第二类引物的步骤，其中所述第一类引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C，并且所述第二类引物包含所述通用序列且不包含所述可变序列。试剂盒中的第一引物和第二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：单细胞基因组、阳性对照和阴性对照的获得
单细胞基因组DNA：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4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1所示。
表1：裂解程序
Figure PCTCN2016097208-appb-000004
阳性对照：用无核酸酶水将标准基因组DNA稀释为30皮克/微升的DNA溶液，取1微升上述溶液加入到含有4微升细胞裂解缓冲液的PCR管中。标准基因组DNA为事先提取好的人类细胞的基因组DNA。
阴性对照：将5微升细胞裂解缓冲液加入到PCR管中。
实施例2：使用多次退火环状循环扩增(MALBAC)(简称为三步法)进行基因组扩增
本实施例的方法在这里也称为三步法，因为基本上包括三个步骤，即：裂解细胞、预扩增和指数扩增。
按照实施1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。使用江苏亿康基因科技有限公司的单细胞全基因组扩增试剂盒(货号YK001A/B)并按照其产品说明书进行扩增。具体而言，按照30:1的比例混合预扩增缓冲液和预扩增酶混合物以形成预扩增混合液。向分别包括待扩增样品(根据实施例1获得的基因组DNA、阳性对照和阴性对照)的PCR管内分别加入30微升预扩增混合液。将PCR管放入PCR仪进行预扩增，预扩增的程序如表2所示。
表2：MALBAC三步法预扩增程序
Figure PCTCN2016097208-appb-000005
按照30:0.8的比例混合扩增缓冲液和扩增酶混合物以形成扩增混合液。向完成预扩增后的PCR管中加入30微升扩增混合液，之后进行指数扩增，指数扩增的程序如表3所示。
表3：MALBAC三步法指数扩增程序
Figure PCTCN2016097208-appb-000006
实施例3：使用混合的引物(简称为两步法)进行基因组扩增
本实施例的方法在这里也称为两步法，基本上包括两个步骤，即：裂解细胞和扩增反应。
按照实施1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。
配制扩增混合液，其中含有Na+、Mg2+、Cl-、Tris-Cl、TritonX-100、dNTP、Vent聚合酶、SEQ ID NO:1所示的引物、SEQ ID NO:12所示的引物和SEQ ID NO:13所示的引物。
本实施例中使用的引物根据如下原则设计：
1.在引物的通用序列中仅包含三类自身互补配对能力较弱的碱基，如G,A,T.
2.在引物可变碱基序列的3’端(连续或不连续三个及以上碱基)使用1中提到的三类碱基中的一种或几种，保证引物3’端与自身或不同引物的5’端不会产生互补配对的现象。
3.引物可变碱基序列在基因组上的识别位点经过统计计算，选择符合上述条件且在基因组上分布更加均匀，覆盖度更高的序列，增加可变碱基序列与基因组DNA的识别机会。
4.引物通用序列中三类碱基的使用比例及组成方式经过特殊设计，保证通用序列不会与基因组DNA结合产生扩增。分别向每个待扩增样品(根据实施例1获得的基因组DNA、阳性对照和阴性对照)的PCR管内加入60微升扩增混合液。将PCR管放入PCR仪进行扩增，扩增的程序如表4所示。
表4：本申请两步法扩增程序
Figure PCTCN2016097208-appb-000007
实施例4：本申请两步法扩增产物与MALBAC三步法扩增产物的比较
凝胶电泳
分别取5微升未纯化的实施例2的三步法扩增产物和实施例3的两步法扩增产物，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2，其中a为实施例3的两步法扩增结果，自左向右第1泳道为分子量标记，2-11泳道为单细胞扩增样品，12-14泳道为阳性对照(40pg gDNA)，15-17泳道为阴性对照(不含基因组DNA)，第18泳道为分子量标记；b为实施例2的三步法扩增结果，自左向右第1泳道为分子量标记，2-11泳道为单细胞扩增样品，12-14泳道为阳性对照(40pg gDNA)，15-17泳道为阴性对照(不含基因组DNA)，第18泳道为分子量标记。电泳图显示：实施例3的两步法扩增产物的条带和亮度与实施例2的三步法扩增产物的条带位置相当、亮度相当，没有显著的差别，表明两步法扩增的准确性和得到的产物的产量与三步法的结果相当。
纯化产物
取50微升未纯化的实施例2的三步法扩增产物和实施例3的两步法扩增产物，使用通用柱式纯化试剂盒(购自北京康为世纪生物科技有限公司，货号CW2301)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50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5所示。
表5：纯化后扩增产物的浓度
两步法 浓度(纳克/微升) 三步法 浓度(纳克/微升)
2-1 69.668 3-1 60.146
2-2 57.332 3-2 67.512
2-3 44.119 3-3 71.704
2-4 57.859 3-4 75.275
2-5 73.391 3-5 67.615
2-6 70.341 3-6 79.219
2-7 71.845 3-7 86.552
2-8 44.211 3-8 79.712
2-9 60.947 3-9 65.838
2-10 90.09 3-10 64.263
平均值 63.98 平均值 71.78
浓度检测结果显示：两种扩增方法获得的扩增产物在纯化后的浓度相当，没有显著差别。
致病位点检测
随机选取20个致病位点(选择的位点参见下表)，并设计引物。选取的致病位点及其相应的引物分别如表6和表7所示。
表6：第一批致病位点
Figure PCTCN2016097208-appb-000008
表7：第一批致病位点相应的引物
致病位点名称 引物序列
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
DMD-19exe+ GTGAAACATCTTAAGGCTTGAAAG
DMD-19exe- TAACAAGTGCTTGTCTGATATAAT
ATP7B-8+ AAAAGCTGAGAAGTTCAGAAAAC
ATP7B-8- AAATTTGTATTTAACAAGTGCTTGTC
ATP7B-13+ GTTTATTCTCTGGTCATCCTGGT
ATP7B-13- GGTGTTCAGAGGAAGTGAGATT
ERCC6-C643+ GAACTCTCAACCTGCCTCTG
ERCC6-C643- CTTGATGAGGATGCCGTTCT
ERCC6-C3776+ CCATTCAAGGAACAACAGCTAAA
ERCC6-C3776- ACCCAGGCAAAGACTAAAGAG
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-PL88+ AAATCCCAGTCCCTATTCCTAT
CFTR-PL88- CTAAGAGGAACACCACACTCAC
CFTR-IVS13+ TTTGCAGAGAATGGGATAGAGAG
CFTR-IVS13- CACCTATTCACCAGATTTCGTAGT
IL2RG+ TGACCAGGAAATAGAGAGGAAATG
IL2RG- CATTCTGCCATACCAACAATGG
IL2RG IVS4+ ATTGGAAGCCGTGGTTATCTC
IL2RG IVS4- CTTCCATCACCAAACCCTCTT
FLG-c3319+ CTGAGTGAATCCCAGCTAGAAC
FLG-c3319- GCAGAGAACAGGAGCTTGAT
IDS+ CTCCAGACACTCAGGCATTC
IDS- GTGCTCACCTGGTAGATGAAA
随机选择根据实施例2扩增出的7个扩增产物以及根据实施例3扩增出的7个扩增产物分别作为模板DNA。使用含染料的2xTaq MasterMix(购自北京康为世纪生物科技有限公司， 货号CW0682)进行PCR，分别扩增上述20个致病位点。扩增体系组成如表8所示，扩增程序如表9所示。
表8：用于致病位点检测的PCR反应体系
2×Taq MasterMix 25微升
正向引物，10微摩 2微升
负向引物，10微摩 2微升
模板DNA 40纳克
无RNA酶水 定容至50微升
表9：用于致病位点检测的PCR扩增程序
Figure PCTCN2016097208-appb-000009
*PKDH1-3681、PKDH1-1713、DMD-13exe、DMD-19exe退火温度为50摄氏度，其余16个引物退火温度为55摄氏度。
扩增的结果如图3所示。扩增结果显示：两种方法在扩增的准确性和扩增产物的量上没有显著差别。
再次随机选取另外20个致病位点(选择的位点参见下表)，并设计引物。选取的致病位点及其相应的引物分别如表10和表11所示。
表10：第二批致病位点
Figure PCTCN2016097208-appb-000010
表11：第二批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
HBB+ GGTTGGCCAATCTACTCCCA
HBB- AAGGTGCCCTTGAGGTTGTC
HBB3+ TCATGCCTCTTTGCACCATT
HBB3- AATCCAGCCTTATCCCAACCA
MMACHC+ GGAGTCGAAGCTGACTCA
MMACHC- CAGTTGCAACGAAGCCAATC
HBA2+ CTTCTCTGCACAGCTCCTAAG
HBA2- GCTGCCCACTCAGACTTTAT
GJB2+ GACGCCAAGTTTGAAGGAAC
GJB2- CTACTGCTAGAAACAGCCTACTC
GJB2-C79+ TCGCATTATGATCCTCGTTG
GJB2-C79- GGACACAAAGCAGTCCACAG
随机选择根据实施例2扩增出的3个样品(在图4中显示为2_1、2_3和2_7)以及根据实施例3扩增出的3个样品(在图4中显示为3_1、3_4和3_8)分别作为模板DNA，使用 含染料的2xTaq MasterMix(购自北京康为世纪生物科技有限公司，货号CW0682)进行PCR扩增，分别扩增上述20个致病位点。扩增体系和扩增程序分别如表8和表9所示，只是退火温度均选择55摄氏度。
扩增的结果如图4所示。扩增结果显示：20个致病位点在上述两个扩增方法的扩增产物中均能得到了很好地扩增，两种方法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
随机选择根据实施例2的三步法扩增出的4个样品(在图5中示为3-5、3-6、3-9、3-10)以及根据实施例3的两步法扩增出的4个样品(在图5中示为2-1、2-3、2-7、2-10)分别作为模板DNA。使用如表14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12所示，扩增程序如表13所示。
表12：q-PCR扩增体系
2×FastSYBR Mixture 25微升
正向引物，10微摩 2微升
负向引物，10微摩 2微升
模板DNA 40纳克
无RNA酶水 定容至50微升
表13：q-PCR扩增程序
Figure PCTCN2016097208-appb-000011
表14：质检引物对
Figure PCTCN2016097208-appb-000012
扩增的结果如图5所示，其中a-f分别表示针对染色体CH1、CH2、CH3、CH4、CH5、CH6和CH7上的DNA序列，对模板DNA进行的q-PCR检测的数据。扩增结果显示：当用两步法和三步法的扩增产物作为q-PCR的模板进行q-PCR时得到的Cr值相当，没有显著差别，表明q-PCR的起始模板数没有显著差别，即两步法和三步法的扩增产物的量没有显著差别。而且6对质检引物分别验证了染色体1、2、4、5、6、7上的不同序列，结果都一致显示两种起始模板量没有显著差别。
基因测序
随机选取10个纯化后的实施例3的两步法扩增出的产物(在图6、图8和图9中示为2-1、2-2、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10)和10个纯化后的实施例2的三步法扩增出的样品(在图7、图8和图9中示为3-1、3-2、3-3、3-4、3-5、3-6、3-7、3-8、3-9、3-10)，使用打断方法构建基因组文库，并采取浅测序的方式使用hiseq2500测序仪进行测序，每个样品测定1.5 Mb的数据量，并将测序得到的序列比对到人类参考基因组(hg19)上。
实施例3的两步法的结果如图6所示，实施例2的三步法的结果如图7所示。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。上述结果表明：实施例2的三步法和实施例3的两步法对细胞的染色体检测结果一致。
在测序结果中还提供了高通量测序结果的各个指标参数，如图8所示。其中原始数据中“唯一比对到人类基因组的数据比例”(即，unique_mapped_of_raw)是最重要的衡量指标，实施例3的两步法所有样品的平均unique_mapped_of_raw为74.15％，而实施例2的三步法所有样品的平均unique_mapped_of_raw为68.5％，这表明实施例3的两步法扩增样品的unique_mapped_of_raw的比例显著高于MALBAC三步法扩增样品。
拷贝数变异系数可以用来比较两类扩增方法扩增样品后样品拷贝数的离散程度的大小。实施例3的两步法所有扩增样品的平均拷贝数变异系数为0.1200，而实施例2的三步法所有扩增样品的平均拷贝数变异系数为0.1205。两类扩增方法拷贝数变异系数相比无明显差别。具体数据参见图9。
ADO评价
随机挑选150个SNP位点并设计相应的引物，挑选的位点以及相应引物参见表15。
表15：150个SNP位点及其相应引物
Figure PCTCN2016097208-appb-000013
Figure PCTCN2016097208-appb-000014
Figure PCTCN2016097208-appb-000015
Figure PCTCN2016097208-appb-000016
Figure PCTCN2016097208-appb-000017
Figure PCTCN2016097208-appb-000018
为了衡量扩增给定位点的效率和等位基因脱扣率(ADO)，以确定扩增效率和扩增是否成功，随机选取实施例2的三步法的两个扩增产物(3-1和3-2)，实施例3的两步法的一个扩增产物(2-1)和人类表皮成纤维细胞(AFP)细胞提取的120ng DNA分别进行多重PCR，引物参见表15。扩增体系的组成如表16所示，扩增程序如表17所示。
表16：多重PCR扩增混合物
2×Taq MasterMix 25微升
正向引物，10微摩 1微升
负向引物，10微摩 1微升
模板DNA 120纳克
无RNA酶水 定容至50微升
表17：多重PCR扩增程序
Figure PCTCN2016097208-appb-000019
对扩增产物采用打断建库方式构建基因组文库，使用hiseq2500测序仪测序，平均测序深度为5Mb，统计结果参见图10。多重PCR数据表明：上述三种样本的扩增产物的GC含量、高质量数据(high_quality_of_raw)、原始数据特定map比率(unique_mapped_of_raw)，以及平均覆盖度(average depth)等各项指标参数无明显差距。
此外，二代测序分析结果显示，以gDNA为起始材料的多重PCR产物中共检测到纯合位点23个，这23个纯合位点在实施例2的三步法的两个扩增产物中分别检测到23和22个；实施例3的两步法的扩增产物中检测到21个，实施例2的扩增产物和实施例3的扩增产物纯合位点的ADO比例无显著差别。具体数据参见表18。
表18：gDNA纯合位点中实施例3扩增产物与实施例2扩增产物的ADO比较
纯合位点 2_1 3_1 3_2
23(gDNA) 1/21 1/23 0/22
ADO 4.7619 4.34783 0
扩增效率 91.30％ 100％ 95.65％
二代测序分析结果显示，以gDNA为起始材料的多重PCR产物中共检测到杂合位点62个，这62个杂合位点在实施例2的两个样品中分别检测到59和56个；实施例3的一个样品中检测到51个，实施例2的扩增产物和实施例3的扩增产物杂合位点的ADO比例无显著差别。具体数据参见表19。
表19：gDNA杂合位点中实施例3扩增产物与实施例2扩增产物的ADO比较
杂合位点 2_1 3_1 3_2
62(gDNA) 4/51 1/59 4/56
ADO 7.84314 1.69492 7.14286
扩增效率 82.2581 95.1613 90.3226
实施例5：使用裂解和扩增一步完成的方法(简称为一步法)进行基因组扩增
本实施例的方法在这里也称为一步法，因为其中将裂解细胞、预扩增和指数扩增合并在一个步骤中完成。
按照实施1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。
配制扩增混合液，其中含有Na+、Mg2+、Cl-、Tris-Cl、dNTP、TritonX-100、Vent聚合酶、SEQ ID NO:1所示的引物、SEQ ID NO:12所示的引物和SEQ ID NO:13所示的引物。
向每个待扩增样品(根据实施例1制备的阳性对照和阴性对照，以及未裂解的单细胞)的PCR管内加入50微升扩增混合液。将PCR管放入PCR仪进行扩增，扩增的程序如表20所示。
表20：本申请一步法扩增程序
Figure PCTCN2016097208-appb-000020
Figure PCTCN2016097208-appb-000021
实施例6：实施例5的一步法扩增产物与实施例2的三步法扩增产物的比较
凝胶电泳
分别取5微升未纯化的实施例2的三步法扩增产物和实施例5的一步法扩增产物，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11，其中第一排为实施例5的一步法扩增结果，自左向右第1泳道为分子量标记，2-11泳道为单细胞扩增样品，12-14泳道为阳性对照(40pg gDNA)，15-17泳道为阴性对照(不含基因组DNA)，第18泳道为分子量标记。电泳图显示：实施例5的一步法扩增产物的条带和亮度与实施例2的三步法扩增产物的条带位置和亮度相当，没有显著差异。
纯化产物
取50微升未纯化的实施例2的三步法扩增产物和实施例5的一步法扩增产物，使用通用柱式纯化试剂盒(购自北京康为世纪生物科技有限公司，货号CW2301)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50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。浓度检测结果参见表21。
表21：纯化后扩增产物的浓度
一步法 浓度(纳克/微升) 三步法 浓度(纳克/微升)
1-1 49.481 3-1 47.044
1-2 51.956 3-2 46.268
1-3 40.976 3-3 71.316
1-4 45.041 3-4 73.675
1-5 57.89 3-5 68.86
1-6 49.494 3-6 64.411
1-7 47.94 3-7 52.018
1-8 65.17 3-8 49.532
1-9 36.519 3-9 无扩增产物
1-10 39.538 3-10 13.157
平均值 48.401 平均值 54.031
浓度检测结果显示：两种扩增方法获得的扩增产物在纯化后的浓度相当，没有显著差异。
致病位点检测
随机选取20个致病位点，并设计引物。选取的致病位点及其相应的引物分别如实施例4中的表6和表7所示。
随机选择根据实施例2扩增出的4个样品以及根据实施例5扩增出的4个样品分别作为模板DNA，扩增体系组成和扩增程序分别如实施例4中的表8和表9所示，只是图12(a)和图12(b)中的循环次数为30轮。使用含染料的2xTaq MasterMix(购自北京康为世纪生物科技有限公司，货号CW0682)进行PCR，分别扩增上述20个致病位点。
扩增的结果如图12所示。扩增结果显示：两种方法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
随机选取10个纯化后的实施例2的三步法扩增出的产物(在图14中示为2-1、2-2、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10)和10个纯化后的实施例5的一步法扩增出的产物(在图13中示为1-1、1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10)，使用打断方法构建基因组文库，并采取浅测序的方式使用hiseq2500测序仪进行测序，每个样品测定1.5Mb的数据量，并将测序得到的序列比对到人类参考基因组(hg19)上。
实施例5的一步法的结果如图13所示，实施例2的三步法的结果如图14所示。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。上述结果表明：实施例2的三步法和实施例5的一步法对细胞的染色体检测结果一致。
在测序结果中还提供了高通量测序结果的各个指标参数，如图15所示。其中原始数据中“唯一比对到人类基因组的数据比例”(即，unique_mapped_of_raw)是最重要的衡量指标，实施例5的一步法所有样品的平均unique_mapped_of_raw为79.13％，而实施例2的三步法所有样品的平均unique_mapped_of_raw为74.25％，这表明实施例5的一步法扩增样品的unique_mapped_of_raw的比例显著高于实施例2的三步法扩增样品。
拷贝数变异系数可以用来比较两类扩增方法扩增样品后样品拷贝数的离散程度的大小。实施例5的一步法所有扩增样品的平均拷贝数变异系数为与实施例2的三步法所有扩增样品的平均拷贝数变异系数相近。具体数据可参见图16。
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 (49)

  1. 一种扩增细胞基因组DNA的方法，所述方法包括：
    (a)提供反应混合物，其中所述反应混合物包括所述基因组DNA、第一类引物、第二类引物、核苷酸单体混合物、和核酸聚合酶，其中所述第一类引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C，并且所述第二类引物包含所述通用序列且不包含所述可变序列；
    (b)将所述反应混合物置于第一温度循环程序，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；
    (c)将步骤(b)得到的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所述通用序列能够与所述基因组扩增产物的3’端配对并扩增所述基因组扩增产物以得到扩大的基因组扩增产物，
    其中，在所述步骤(b)和所述步骤(c)之前提供所述反应混合物。
  2. 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。
  3. 根据权利要求2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。
  4. 根据权利要求1-3中任一项所述的方法，其中所述基因组DNA包含在细胞中，并且所述反应混合物进一步包含能够裂解所述细胞的表面活性剂和/或裂解酶。
  5. 根据权利要求1-4中任一项所述的方法，其中在所述步骤(b)和步骤(c)之前还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。
  6. 根据权利要求1-5中任一项所述的方法，其中所述通用序列被选择以使得其基本上不会与基因组DNA结合产生扩增。
  7. 根据权利要求1-6中任一项所述的方法，其中所述通用序列选自下组：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、和SEQ ID NO:6。
  8. 根据权利要求1-7中任一项所述的方法，其中所述可变序列包含随机序列。
  9. 根据权利要求1-8中任一项所述的方法，其中所述可变序列的长度为2-20个碱基、3-10 个碱基，4-9个碱基或5-8个碱基。
  10. 根据权利要求1-9中任一项所述的方法，其中所述可变序列中的三个或三个以上的碱基位置由选自G、A和T的一种或几种碱基组成，或者由C、A和T的一种或几种碱基组成。
  11. 根据权利要求10所述的方法，其中所述三个或三个以上的碱基位置位于所述可变序列的3’端或者中间。
  12. 根据权利要求1-11中任一项所述的方法，其中所述可变序列选自下组：(N)nGGG、(N)nTTT，(N)mTNTNG，(N)xGTGG(N)y，其中N为任意的可与天然核酸进行碱基配对的核苷酸，n是选自3-17的正整数，m是选自3-15的正整数、x和y分别是选自3-13的正整数。
  13. 根据权利要求1-12中任一项所述的方法，其中所述可变序列被选择以使得在基因组上分别均匀并且覆盖度高。
  14. 根据权利要求1-13中任一项所述的方法，其中所述第一类引物包括SEQ ID NO:11[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNNNN]、SEQ ID NO:12[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNGGG]、SEQ ID NO:13[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNTTT]、SEQ ID NO:14[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNTNTNG]或SEQ ID NO:15[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNGTGGNN]的序列，并且所述第二类引物从5’到3’具有SEQ ID NO:1[GTGAGTGATGGTTGAGGTAGTGTGGAG]的序列，其中N为任意的可与天然核酸进行碱基配对的核苷酸。
  15. 根据权利要求1-14中任一项所述的方法，其中所述核酸聚合酶具有热稳定和/或链置换活性。
  16. 根据权利要求1-15中任一项所述的方法，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(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、T7 phase DNA聚合酶变种(缺少3’-5’外切酶活性)、
    Figure PCTCN2016097208-appb-100001
    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，及其任意组合。
  17. 根据权利要求1-16中任一项所述的方法，其中所述反应混合物进一步包含pH值调节剂，使得所述反应混合物的pH值维持在7.0-9.0之间。
  18. 根据权利要求1-17中任一项所述的方法，其中所述反应混合物进一步包含一种或多种选自下组的成分：Mg2+、dTT、牛血清白蛋白、DNase抑制剂、RNase、SO4 2-、Cl-、K+、Ca2+、Na+、和(NH4)+。
  19. 根据权利要求1-18中任一项所述的方法，其中所述第一温度循环程序包括：
    (b1)将所述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；
    (b2)将所述反应混合物置于能够使所述第一类引物与DNA单链模板结合的温度程序；
    (b3)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；
    (b4)将所述反应混合物置于能够使所述扩增产物脱落成单链的温度程序；
    (b5)重复步骤(b2)到(b4)至指定的第一循环次数。
  20. 根据权利要求19所述的方法，其中所述指定的第一循环次数大于2。
  21. 根据权利要求19或20所述的方法，当进行到第二次循环后，所述扩增产物包含在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扩增产物。
  22. 根据权利要求19-21中任一项所述的方法，所述方法在步骤(b4)后并且在步骤(b5)之前进一步包括步骤(b4’)，其中将所述反应混合物置于适当的温度程序，使得所述基因组扩增产物的3’端与5’端杂交结合以形成环状结构，或者使所述基因组扩增产物的3’端与引物结合。
  23. 根据权利要求19-22中任一项所述的方法，所述方法在步骤(b4)后直接到步骤(b5)。
  24. 根据权利要求19-23中任一项所述的方法，其中所述步骤(b5)的所述第一循环次数大于3、大于4、大于5、大于6，并且不超过10。
  25. 根据权利要求19-24中任一项所述的方法，其中所述步骤(c)包括：
    (c1)将经步骤(b)获得的所述反应混合物置于能够打开DNA双链的温度程序；
    (c2)将所述反应混合物置于能够使所述第二类引物与所述经步骤(b)获得的基因组扩增产物的单链结合的温度程序；
    (c3)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；
    (c4)重复步骤(c1)到(c3)至指定的第二循环次数。
  26. 根据权利要求25所述的方法，其中所述步骤(c4)中的所述第二循环次数大于所述步骤(b5)中所述的第一循环次数。
  27. 根据权利要求19-26中任一项所述的方法，其中所述步骤(b1)中所述的温度程序包括在90-95℃的温度之间反应1-10分钟。
  28. 根据权利要求19-27中任一项所述的方法，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。
  29. 根据权利要求28所述的方法，其中所述多于一种的温度程序包括：介于5-10℃之间的第一温度，介于25-30℃之间的第二温度，和介于45-50℃之间的第三温度。
  30. 根据权利要求19-29中任一项所述的方法，其中所述步骤(b2)中所述步骤包括在第一温度反应3-50秒、在第二温度反应3-50秒、和在第三温度反应3-50秒。
  31. 根据权利要求19-30中任一项所述的方法，其中所述步骤(b3)中所述的温度程序包括在60-90℃的温度之间反应1-15分钟。
  32. 根据权利要求19-31中任一项所述的方法，其中所述步骤(b4)中所述的温度程序包括在90-95℃的温度之间反应10-50秒。
  33. 根据权利要求25-32中任一项所述的方法，其中所述步骤(c1)中所述的温度程序包括在90-95℃的温度之间反应10-30秒。
  34. 根据权利要求25-33中任一项所述的方法，其中所述步骤(c2)中所述的温度程序包括在45-65℃的温度之间反应10-30秒。
  35. 根据权利要求25-34中任一项所述的方法，其中所述步骤(c3)中所述的温度程序包括在60-80℃的温度之间反应1-15分钟。
  36. 根据权利要求1-35中任一项所述的方法，其中所述步骤(a)中的基因组DNA从被裂解的细胞释放，所述裂解包括热裂解、碱裂解、酶裂解或机械裂解。
  37. 根据权利要求36所述的方法，其中所述热裂解包括温度在20-100℃之间裂解10-100分钟。
  38. 根据权利要求36或37所述的方法，其中所述热裂解是在裂解试剂存在的条件下进行的。
  39. 根据权利要求38所述的方法，其中所述裂解试剂包括一种或多种选自下组的表面活性剂：NP-40、吐温、SDS、TritonX-100、EDTA、和异硫氰酸胍，和/或裂解酶。
  40. 一种扩增细胞基因组的方法，所述方法包括：
    (a)提供反应混合物，其中所述反应混合物包括所述基因组DNA、第一类引物、第二类引物、核苷酸单体混合物、和核酸聚合酶，其中所述第一类引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C，所述第二类引物包含所述通用序列且不包含所述可变序列；
    (b)将所述反应混合物置于第一温度循环程序，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；
    (b2)介于5-10℃之间的第一退火温度反应3-50秒，介于25-30℃之间的第二退火温度反应3-50秒，和介于45-50℃之间的第三退火温度反应3-50秒；
    (b3)在介于60-90℃之间的第一延伸温度反应1-15分钟；
    (b4)在介于90-95℃的第一解链温度之间反应10-50秒；
    (b5)重复步骤(b2)到(b4)至6-9个循环；
    (c)将步骤(b)得到的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所述通用序列能够与所述基因组扩增产物的3’端配对并扩增所述基因组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：
    (c1)在介于90-95℃之间的第二变性温度反应1-10分钟；
    (c2)在介于90-95℃之间的第二解链温度反应10-30秒；
    (c3)在介于45-65℃之间的第四退火温度反应10-30秒；
    (c4)在介于60-80℃之间的第二延伸温度反应1-15分钟；
    (c5)重复步骤(c2)到(c4)5-30个循环；
    (d)获得所述步骤(c)得到的扩增产物；
    其中，在所述步骤(b)和所述步骤(c)之前提供所述反应混合物。
  41. 如权利要求40所述的方法，其中所述通用序列包含或由SEQ ID NO:1组成；所述可变序列包含或者由NNNNNGGG或NNNNNTTT组成，N为任意的可与天然核酸进行碱基配对的核苷酸。
  42. 一种用于扩增基因组DNA的试剂盒，所述试剂盒包括含有第一类引物和第二类引物的混合物，其中所述第一类引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C，并且所述第二类引物包含所述通用序列且不包含所述可变序列。
  43. 如权利要求42所述试剂盒，其中所述混合物进一步包括核苷酸单体混合物和Mg2+。
  44. 如权利要求42或43所述试剂盒，其中所述混合物进一步包括一种或多种选自下组的成分：dTT、牛血清白蛋白(BSA)、pH调节剂(例如Tris HCl)、DNase抑制剂、RNase、SO4 2-、Cl-、K+、Ca2+、Na+、和/或(NH4)+。
  45. 如权利要求42-44中任一项所述试剂盒，其中所述混合物进一步包括核酸聚合酶。
  46. 如权利要求42-45中任一项所述试剂盒，其中所述试剂盒进一步包括能够裂解细胞的表面活性剂和/或裂解酶。
  47. 如权利要求46所述试剂盒，其中所述表面活性剂选自NP-40、吐温、SDS、TritonX-100、EDTA、和异硫氰酸胍中的一种或多种，所述裂解酶选自蛋白酶K、胃蛋白酶、和木瓜蛋白酶中的一种或多种。
  48. 如权利要求42-47中任一项所述试剂盒，其中所述混合物进一步包括能够裂解细胞的表面活性剂和/或裂解酶。
  49. 一种用于扩增基因组DNA的试剂盒，所述试剂盒包括第一类引物和第二类引物，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在同一容器中混合所述第一类引物和所述第二类引物的步骤，其中所述第一类引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C，并且所述第二类引物包含所述通用序列且不包含所述可变序列。
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