HK1228461B - A method for dna amplification - Google Patents

Description

Methods for amplifying DNA

Technical Field

The present invention relates to a method for amplifying DNA, and in particular to a method for amplifying the whole genome DNA of a single cell and sequencing the same.

Background Art

Single-cell whole-genome sequencing is a new technology that amplifies and sequences the entire genome at the single-cell level. Its principle is to amplify the trace amount of whole-genome DNA from isolated single cells, obtain a complete genome with high coverage, and then perform high-throughput sequencing.

Currently, there are four main types of whole genome amplification technologies: Primer Extension Preamplification-Polymerase Chain Reaction (PEP-PCR, for specific methods, see 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.), Degenerate Oligonucleotide–Primed Polymerase Chain Reaction (DOP-PCR, for specific methods, see 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), Multiple Displacement Amplification (MDA, for specific methods, see Dean FB, Nelson JR, Giesler TL, Lasken RS.2001. Rapid amplification of plasmid and phage DNA using phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res.11:1095–99) and Multiple Annealing and Looping Based Amplification Cycles (MALBAC, for specific methods, see PCT patent application WO2012166425).

Gene sequencing technology has evolved through three stages. First-generation DNA sequencing technologies include chemical degradation, dideoxy chain termination, and various sequencing technologies developed from these. The most representative of these is the chain termination method proposed by Sanger and Coulson in 1975. First-generation technologies offer high accuracy and long read times, making them the only method capable of "end-to-end" sequencing to date. However, they suffer from high costs and slow speeds, making them less than ideal. Subsequent second- and third-generation sequencing technologies, characterized by high throughput, are also known as "next-generation sequencing (NGS)." Second-generation sequencing technologies are typified by pyrosequencing, sequencing by synthesis (SBS), and sequencing by ligation. After several years of development, pyrosequencing and sequencing by ligation have fallen into obscurity. Sequencing by synthesis, semiconductor sequencing, and CG sequencing are now the mainstream second-generation sequencing technologies. Third-generation sequencing technologies are broadly divided into two categories: single-molecule fluorescence sequencing, represented by TSMS and SMRT, and nanopore single-molecule sequencing. Compared with the previous two generations, the most significant feature of third-generation sequencing is single-molecule sequencing. Although third-generation sequencing has made some progress, the mainstream sequencing technology at this stage is still second-generation sequencing technology.

The whole-genome amplification technology currently available cannot directly be used for second-generation sequencing technology. Therefore, whether the whole-genome sequence is applied to the sequencing-by-synthesis technology, semiconductor sequencing technology, or CG sequencing technology in the second-generation sequencing technology, a library preparation process is required before sequencing on the machine. Each sequencing technology has its own corresponding library preparation method. The library preparation of the sequencing-by-synthesis platform is mainly divided into two categories, one is the technology of adding Y-shaped adapters or neck-loop adapters after end-repair of fragmented DNA, and the other is transpson technology. The library preparation of the semiconductor sequencing platform is also divided into two categories, one is the technology of adding adapters after end-repair of fragmented DNA, and the other is transpson technology. The library preparation process of the CG platform is relatively complicated. After end-repair of the fragmented DNA, enzyme digestion and two circularization processes are required. The operation is cumbersome and time-consuming.

When products amplified by current mainstream amplification methods are used in the aforementioned sequencing technologies, either additional library construction is required or the sequencing results are poor. Therefore, there is an urgent need for an improved amplification method that can overcome one, multiple, or all of the drawbacks of mainstream amplification methods.

Summary of the Invention

The invention provides a method for amplifying cell genomic DNA and a kit for amplifying genomic DNA.

In one aspect of the present application, a method for amplifying genomic DNA is provided, the method comprising: (a) providing a first reaction mixture, wherein the first reaction mixture comprises a sample comprising the genomic DNA, a first primer, a nucleotide monomer mixture, and a nucleic acid polymerase, wherein the first primer comprises a universal sequence and a first variable sequence from the 5' end to the 3' end, the first variable sequence comprises a first random sequence, wherein the first random sequence is X _a1 X _a2 ... X _an from the 5' end to the 3' end, and X _ai (i=1-n) of the first random sequence all belong to the same set, the set being selected from B, or D, or H, or V, wherein B={T, G, C}, D={A, T, G}, H={T, A, C}, V={A, C, G}, wherein X _ai represents the i-th nucleotide at the 5' end of the first random sequence, n is a positive integer selected from 3-20, optionally, the first reaction mixture further comprises a third primer, wherein the third primer comprises the universal sequence and a third variable sequence from the 5' end to the 3' end, the third variable sequence comprises a third random sequence, wherein the third random sequence is X _b1 X _b2 ... X _bn from the 5' end to the 3' end, X _bi (i=1-n) of the third random sequence all belong to the same set, the set is selected from B, or D, or H, or V, wherein B={T, G, C}, D={A, T, G}, H={T, A, C}, V={A, C, G}, and X _bi (i=1-n) and X _ai (i=1-n) belong to different sets, wherein X _bi represents the i-th nucleotide at the 5' end of the third random sequence, and n is a positive integer selected from 3-20; (b) placing the first reaction mixture in a first temperature cycling program for pre-amplification to obtain a pre-amplification product; (c) providing a second reaction mixture, the second reaction mixture comprising the pre-amplification product obtained in step (b), a second primer, a nucleotide monomer mixture, and a nucleic acid polymerase, wherein the second primer comprises or consists of a specific sequence and the universal sequence from the 5' end to the 3'end; (d) placing the second reaction mixture in a second temperature cycling program for amplification to obtain an amplified product.

In some implementations, X _ai (i=1-n) of the first random sequence all belong to set B, and X _bi (i=1-n) of the third random sequence all belong to set D.

In some embodiments, the first variable sequence and the third variable sequence further include a fixed sequence at their 3' end, wherein the fixed sequence can improve the base combination of genome coverage. In some embodiments, the fixed sequence is selected from CCC, AAA, TGGG, GTTT, GGG, TTT, TNTNG or GTGG.

In some embodiments, the first variable sequence is selected from _{Xal Xa2} ... _Xan TGGG or _{Xal Xa2} ... _Xan GTTT, and the third variable sequence is selected from _{Xb1 Xb2} ... _Xbn TGGG or _{Xb1 Xb2} ... Xbn _GTTT .

In some embodiments, the universal sequence is selected so that it will not be combined with genomic DNA to produce amplification, and the universal sequence length is 6-60bp. In some embodiments, the universal sequence is selected so that the amplified product can be directly sequenced. In some embodiments, the universal sequence is selected from SEQ ID NO:1[TTGGTAGTGAGTG], SEQ ID NO:2[GAGGTGTGATGGA], SEQ ID NO:3[GTGATGGTTGAGGTA], SEQ ID NO:4[AGATGTGTATAAGAGACAG], SEQ ID NO:5[GTGAGTGATGGTTGAGGTAGTGTGGAG] or SEQ ID NO:6[GCTCTTCCGATCT].

In some embodiments, the universal sequence and the first variable sequence are directly connected, or the universal sequence and the first variable sequence are connected through a first spacer sequence, and the first spacer sequence is _Ya1 ... _Yam , where _Yaj (j=1-m)∈{A, T, G, C}, where _Yaj represents the jth nucleotide at the 5' end of the spacer sequence, and m is a positive integer selected from 1-3.

In some embodiments, the universal sequence and the third variable sequence are directly connected, or the universal sequence and the third variable sequence are connected through a third spacer sequence, and the third spacer sequence is Y _b1 ...Y _bm , where Y _bj (j=1-m)∈{A, T, G, C}, where Y _bj represents the j-th nucleotide at the 5' end of the spacer sequence, and m is a positive integer selected from 1-3.

In some embodiments, m=1.

In some embodiments, the first primer includes GCTCTTCCGATCTY _a1 X _a1 X _a2 X _a3 X _a4 X _a5 TGGG, GCTCTTCCGATCTY _a1 X a1 X _a2 X _a3 X _{a4 X a5 GTTT} , or a mixture thereof, and the third primer includes GCTCTTCCGATCTY _b1 X _b1 X _b2 X _b3 X _b4 X _b5 TGGG, GCTCTTCCGATCTY _b1 X _b1 X _b2 X _b3 X _b4 X _b5 GTTT, or a mixture thereof, wherein _{Y a1} ∈ {A, T, G, C}, Y _b1 ∈ {A, T, G, C}, X _ai (i=1-5) ∈ {T, G, C}, and X _bi (i=1-5) ∈ {A, T, G}.

In some embodiments, the method further comprises the step of sequencing the amplified product obtained in step (d), wherein the second primer comprises a sequence that is complementary to or identical to part or all of the sequencing primer.

In some embodiments, the universal sequence includes a sequence that is complementary to or identical to part or all of a sequencing primer.

In some embodiments, the specific sequence of the second primer includes a sequence that is complementary to or identical to part or all of the sequencing primer.

In some embodiments, the specific sequence of the second primer further includes a sequence that is partially or completely complementary or identical to a capture sequence of a sequencing platform.

In some embodiments, the specific sequence of the second primer comprises a sequence that is complementary or identical to part or all of the sequencing primer and comprises or consists of SEQ ID NO: 31 [ACACTCTTTCCCTACACGAC] or SEQ ID NO: 32 [GTGACTGGAGTTCAGACGTGT].

In some embodiments, the specific sequence of the second primer comprises a sequence that is partially or completely complementary or identical to a capture sequence of a sequencing platform and comprises or consists of SEQ ID NO: 33 [AATGATACGGCGACCACCGAGATCT] or SEQ ID NO: 34 [CAAGCAGAAGACGGCATACGAGAT].

In some embodiments, the specific sequence of the second primer further includes an identification sequence, and the identification sequence is located between the sequence that is partially or fully complementary or identical to the capture sequence of the sequencing platform and the sequence that is partially or fully complementary or identical to the sequencing primer.

In some embodiments, the second primer comprises a primer mixture having the same universal sequence and different specific sequences, wherein the different specific sequences are respectively complementary to or identical to part or all of the different primers in the sequencing primer pair used in the same sequencing.

In some embodiments, the second primer comprises a mixture of the sequences shown in SEQ ID NO: 35 [AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT] and SEQ ID NO: 36 [CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT].

In some embodiments, the nucleic acid polymerase has thermostability and/or strand displacement activity. In some embodiments, the nucleic acid polymerase is selected from the group consisting of: Phi29 DNA polymerase, Bst DNA polymerase, Pyrophage 3137, Vent polymerase, TOPOTaq DNA polymerase, 9.Nm polymerase, Klenow Fragment DNA polymerase I, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, T7 phase DNA polymerase variant, super-fidelity DNA polymerase, Taq polymerase, Bst DNA polymerase, E. coli DNA polymerase, LongAmp Taq DNA polymerase, OneTaq DNA polymerase, DeepVent DNA polymerase, Vent (exo-) DNA polymerase, Deep Vent (exo-) DNA polymerase, and any combination thereof.

In some embodiments, step (b) enables the variable sequence of the first type of primer to pair with the genomic DNA and amplify the genomic DNA to obtain a genomic pre-amplification product, wherein the 5' end of the genomic pre-amplification product contains the universal sequence and the 3' end contains the complementary sequence of the universal sequence.

In some embodiments, the first temperature cycling program includes: (b1) a temperature program capable of opening the double-stranded DNA to obtain a single-stranded DNA template; (b2) a temperature program capable of causing the first primer and an optional third primer to bind to the single-stranded DNA template; (b3) a temperature program capable of extending the length of the first type of primer bound to the single-stranded DNA template under the action of the nucleic acid polymerase to produce a pre-amplification product; and (b4) repeating steps (b1) to (b3) to a specified number of first cycles, wherein the specified number of first cycles is greater than 1.

In some embodiments, during the first cycle, the double-stranded DNA in step (b1) is a double-stranded genomic DNA, and the temperature program includes a denaturation reaction at a temperature between 90° C. and 95° C. for 1-20 minutes. In some embodiments, after the first cycle, the temperature program in step (b1) includes a melting reaction at a temperature between 90° C. and 95° C. for 3-50 seconds.

In some embodiments, after the second cycle, the pre-amplification product comprises a genomic pre-amplification product comprising the universal sequence at the 5' end and a complementary sequence of the universal sequence at the 3' end.

In some embodiments, after step (b1) and before step (b2), an additional step (b2') of placing the first reaction mixture in an appropriate temperature program is not included so that the 3' end and the 5' end of the genomic pre-amplification product hybridize and bind to form a hairpin structure. In some embodiments, the step (b2) includes placing the reaction mixture in more than one temperature program to encourage the first type of primer to fully and effectively bind to the DNA template. In some embodiments, the more than one temperature program includes: a first temperature between 10-20°C, a second temperature between 20-30°C, and a third temperature between 30-50°C. In some embodiments, the step (b2) includes annealing for 3-60 seconds at the first temperature, annealing for 3-50 seconds at the second temperature, and annealing for 3-50 seconds at the third temperature. In some embodiments, the temperature program in step (b3) includes an extension reaction between 60-80°C for 10 seconds to 15 minutes. In some embodiments, the number of the first cycles in step (b4) is 2-40.

In some embodiments, step (d) enables the universal sequence of the second primer to pair with the 3' end of the genomic pre-amplification product and amplify the genomic pre-amplification product to obtain an amplified genomic amplification product.

In some embodiments, the step (d) includes: (d1) a temperature program capable of opening the double-stranded DNA; (d2) a temperature program capable of further opening the double-stranded DNA; (d3) a temperature program capable of causing the second primer to bind to the single-stranded genomic pre-amplification product obtained in step (b); (d4) a temperature program capable of causing the second primer bound to the single-stranded genomic pre-amplification product to extend its length under the action of the nucleic acid polymerase; (d5) repeating steps (d2) to (d4) to a specified number of second cycles, wherein the specified number of second cycles is greater than 1.

In some embodiments, the double-stranded DNA in step (d1) is the genomic pre-amplification product, and the double-stranded DNA includes a double-stranded DNA contained in a DNA hairpin structure, and the temperature program includes a denaturation reaction at a temperature between 90-95°C for 5 seconds to 20 minutes.

In some embodiments, the temperature program in step (d2) comprises a melting reaction at a temperature between 90°C and 95°C for 3-50 seconds. In some embodiments, the temperature program in step (d3) comprises an annealing reaction at a temperature between 45°C and 65°C for 3-50 seconds. In some embodiments, the temperature program in step (d4) comprises an extension reaction at a temperature between 60°C and 80°C for 10 seconds to 15 minutes.

In some embodiments, the method further includes analyzing the amplified product to identify sequence features associated with a disease or phenotype. In some embodiments, the sequence features associated with the disease or phenotype include chromosome level abnormalities, chromosome ectopy, aneuploidy, deletion or duplication of part or all of the chromosomes, fetal HLA haplotypes and paternal mutations, or the disease or phenotype is selected from the group consisting of β-thalassemia, Down syndrome, cystic fibrosis, sickle cell disease, Tay-Sachs disease, fragile X syndrome, spinal muscular atrophy, hemoglobinopathy, α-thalassemia, X-linked diseases (diseases dominated by genes on the X chromosome), spina bifida, anencephaly, congenital heart disease, obesity, diabetes, cancer, fetal sex, fetal RHD. In some embodiments, the genomic DNA is derived from blastomeres, blastocyst trophoblasts, cultured cells, extracted gDNA, or blastocyst culture fluid.

One aspect of the present application provides a method for amplifying genomic DNA, the method comprising: (a) providing a first reaction mixture, wherein the first reaction mixture comprises a sample comprising the genomic DNA, a first primer, a nucleotide monomer mixture, and a nucleic acid polymerase, wherein the first primer comprises a universal sequence and a variable sequence from the 5' end to the 3' end, wherein the first primer comprises a universal sequence and a first variable sequence from the 5' end to the 3' end, and the first variable sequence comprises a first random sequence, wherein the first random sequence is X _a1 X _a2 ... X _an from the 5' end to the 3' end, and X _ai (i=1-n) of the first random sequence all belong to the same set, wherein the set is selected from B, or D, or H, or V, wherein B={T, G, C}, D={A, T, G}, H={T, A, C}, V={A, C, G}, wherein X _ai represents the i-th nucleotide at the 5' end of the first random sequence, n is a positive integer selected from 3-20, wherein the universal sequence and the first variable sequence are directly connected, or the universal sequence and the first variable sequence are connected via a first spacer sequence, the first spacer sequence is _Ya1 ... _Yam , wherein _Yaj (j=1-m)∈{A, T, G, C}, wherein _Yaj represents the j-th nucleotide at the 5' end of the spacer sequence, optionally, wherein the first reaction mixture further comprises a third primer, wherein the third primer comprises the universal sequence and a third variable sequence from the 5' end to the 3' end, the third variable sequence comprises a third random sequence, wherein the third random sequence is _{Xb1 Xb2} ... _Xbn from the 5' end to the 3' end, _Xbi (i=1-n) of the third random sequence all belong to the same set, the set being selected from B, or D, or H, or V, wherein B＝{T, G, C}, D＝{A, T, G}, H＝{T, A, C}, V＝{A, C, G}, and _Xbi (i=1-n) and X _ai (i=1-n) belong to different sets, wherein X _bi represents the i-th nucleotide at the 5' end of the first random sequence, and n is a positive integer selected from 3-20, wherein the universal sequence and the third variable sequence are directly connected, or the universal sequence and the third variable sequence are connected through a third spacer sequence, and the third spacer sequence is Y _b1 ...Y _bm , wherein Y _bj (j=1-m)∈{A, T, G, C}, wherein Y _bj represents the jth nucleotide at the 5' end of the spacer sequence, and m is a positive integer selected from 1-3; (b) placing the first reaction mixture in a first temperature cycling program so that the first variable sequence of the first primer and the optional third variable sequence of the third primer can pair with the genomic DNA and amplify the genomic DNA to obtain a genomic pre-amplification product, wherein the 5' end of the genomic pre-amplification product comprises the universal sequence and the 3' end comprises the complementary sequence of the universal sequence; wherein the first temperature cycling program comprises: (b1) a first cycle of a first denaturation temperature reaction at a temperature between 90-95°C for 1-20 minutes, followed by a second melting temperature reaction at a temperature between 90-95°C for 3-50 seconds; (b2) a first annealing temperature reaction at a temperature between 10-20°C for 3-60 seconds, a second annealing temperature reaction at a temperature between 20-30°C for 3-50 seconds, and a third annealing temperature reaction at a temperature between 30-50°C for 3-50 seconds; (b3) a first extension temperature reaction at a temperature between 60-80°C for 10 seconds to 15 minutes; (b4 ) repeating steps (b1) to (b3) for 2-40 cycles; (c) providing a second reaction mixture, wherein the second reaction mixture comprises the genomic pre-amplification product obtained in step (b), a second primer, a nucleotide monomer mixture, and a nucleic acid polymerase, wherein the second primer comprises or consists of a specific sequence and the universal sequence from the 5' end to the 3'end; (d) placing the second reaction mixture in a second temperature cycle program so that the universal sequence of the second primer can pair with the 3' end of the genomic pre-amplification product and amplify the The genomic pre-amplification product is used to obtain an amplified genomic amplification product, wherein the second temperature cycling program includes: (d1) reacting at a second denaturation temperature between 90-95°C for 5 seconds to 20 minutes; (d2) reacting at a second melting temperature between 90-95°C for 3-50 seconds; (d3) reacting at a fourth annealing temperature between 45-65°C for 3-50 seconds; (d4) reacting at a second extension temperature between 60-80°C for 10 seconds to 15 minutes; and (d5) repeating steps (d2) to (d4) for 2-40 cycles.

In some embodiments, the universal sequence comprises or consists of SEQ ID NO: 6; X _ai (i=1-n) of the first random sequence all belong to D, and X _bi (i=1-n) of the third random sequence all belong to B.

In some embodiments, the amplified product obtained in step (d) has completed library construction.

In another aspect of the present application, a kit for amplifying genomic DNA is provided, the kit comprising a first primer, wherein the first primer comprises a universal sequence and a first variable sequence from the 5' end to the 3' end, the first variable sequence comprising a first random sequence, wherein the first random sequence is X _a1 X _a2 ... X _an from the 5' end to the 3' end, and X _ai (i=1-n) of the first random sequence all belong to the same set, the set being selected from B, or D, or H, or V, wherein B={T, G, C}, D={A, T, G}, H={T, A, C}, V={A, C, G}, wherein X _ai represents the i-th nucleotide at the 5' end of the first random sequence, and n is a positive integer selected from 3-20, wherein the universal sequence and the first variable sequence are directly connected, or the universal sequence and the first variable sequence are connected via a first spacer sequence, the first spacer sequence being Y _a1 ... Y _am , wherein Y _aj (j=1-m)∈{A, T, G, C}, wherein Y _aj represents the jth nucleotide at the 5' end of the spacer sequence, m is a positive integer selected from 1-3, optionally, wherein the first reaction mixture further comprises a third primer, wherein the third primer comprises the universal sequence and a third variable sequence from the 5' end to the 3' end, the third variable sequence comprises a third random sequence, wherein the third random sequence is X _b1 X _b2 ... X _bn from the 5' end to the 3' end, X _bi (i=1-n) of the third random sequence all belong to the same set, the set is selected from B, or D, or H, or V, wherein B={T, G, C}, D={A, T, G}, H={T, A, C}, V={A, C, G}, and X _bi (i=1-n) and X _ai (i=1-n) belong to different sets, wherein X _bi represents the i-th nucleotide at the 5' end of the first random sequence, n is a positive integer selected from 3-20, wherein the universal sequence and the third variable sequence are directly connected, or the universal sequence and the third variable sequence are connected through a third spacer sequence, and the third spacer sequence is Y _b1 ...Y _bm , wherein Y _bj (j=1-m)∈{A, T, G, C}, wherein Y _bj represents the j-th nucleotide at the 5' end of the spacer sequence, and m is a positive integer selected from 1-3.

In some embodiments, the universal sequence comprises or consists of SEQ ID NO: 6; the X _ai (i=1-n) of the first random sequence all belong to D, and the X _bi (i=1-n) of the third random sequence all belong to B. In some embodiments, the universal sequence comprises or consists of SEQ ID NO: 1; the X _ai (i=1-n) of the first random sequence all belong to D, and the X _bi (i=1-n) of the third random sequence all belong to B. In some embodiments, the universal sequence comprises or consists of SEQ ID NO: 2; the X _ai (i=1-n) of the first random sequence all belong to D, and the X _bi (i=1-n) of the third random sequence all belong to B.

In some embodiments, the kit is used to construct a whole-genomic DNA library.

In some embodiments, the kit further comprises a nucleic acid polymerase, wherein the nucleic acid polymerase is selected from the group consisting of: Phi29 DNA polymerase, Bst DNA polymerase, Pyrophage 3137, Vent polymerase, TOPO Taq DNA polymerase, 9. Nm polymerase, Klenow Fragment DNA polymerase I, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, T7 phase DNA polymerase variant, super-fidelity DNA polymerase, Taq polymerase, Bst DNA polymerase, E. coli DNA polymerase, LongAmp Taq DNA polymerase, OneTaq DNA polymerase, Deep Vent DNA polymerase, Vent (exo-) DNA polymerase, Deep Vent (exo-) DNA polymerase, and any combination thereof.

In some embodiments, the kit further comprises one or more reagents comprising one or more components selected from the group consisting of a nucleotide monomer mixture, Mg ²⁺ , dTT, bovine serum albumin, pH adjuster, DNase inhibitor, RNase, SO ₄ ²⁻ , Cl ⁻ , K ⁺ , Ca ^{2 +} , Na ⁺ , and (NH ₄ ) ⁺ .

In some embodiments, the mixture further comprises a cell lysis agent, and the cell lysis agent is selected from one or more of proteinase K, pepsin, papain, NP-40, Tween, SDS, TritonX-100, EDTA and guanidine thiocyanate.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other features of the present application will be more fully described by the following description and the appended claims, taken in conjunction with the accompanying drawings. It should be understood that these drawings only depict several embodiments of the present application and, therefore, should not be construed as limiting the scope of the present application. The present application will be more clearly and fully illustrated by the accompanying drawings.

FIG1 shows the basic principle of the amplification method of the present application.

FIG2 shows a schematic structural diagram of the first type of primers (linear amplification primers) used in the amplification method of the present application.

Figure 3 shows the results of amplifying 50 pg of human genomic DNA using different first-class primer mixtures and subjecting the resulting amplified products to gel electrophoresis, wherein lane 1 from left to right is a molecular weight marker (M), lanes 2-13 are amplified samples obtained by amplifying gDNA using primer mixtures of experimental groups 1-12 (see Table 1 for details), and lane 14 is a molecular weight marker.

FIG4 shows the distribution of A, T, C, and G at each read position in the amplification products obtained in experimental groups 1-12 during SBS sequencing.

Figure 5 shows the amplification results of normal human epidermal fibroblasts (AFP cells) as the starting sample using the primer mixtures of experimental groups 1-12 shown in Table 1. From left to right, lane 1 is a molecular weight marker, lanes 2-11 are single cell amplification samples, and lane 12 is a molecular weight marker.

Figure 6 shows the results of gel electrophoresis of amplified products obtained by amplifying normal human dermal fibroblasts (AFP cells) using the primer mixtures of experimental groups 9/10 and 11/12 shown in Table 1. From left to right, lane 1 is a molecular weight marker, lanes 2-11 are amplified samples obtained by amplifying single cells using the primer mixture of experimental group 11/12, lane 12 is a molecular weight marker, lanes 13-22 are amplified samples obtained by amplifying single cells using the primer mixture of experimental group 9/10, and lane 23 is a molecular weight marker.

FIG. 7 shows the data volume of each sample 1_1, 1_2 ... 1_10 and 2_1, 2_2 ... 2_10 in FIG. 6 in SBS sequencing (sequencing was performed using equal volumes of amplification products).

FIG8 shows the copy number variation coefficient of each sample 1_1, 1_2 ... 1_10 and 2_1, 2_2 ... 2_10 in FIG6 during SBS sequencing.

9A-9D show the copy number of each chromosome in SBS sequencing for each sample 1_1, 1_2 ... 1_10 and 2_1, 2_2 ... 2_10 in FIG6 .

Figure 10 shows the results of PCR amplification of samples 1_1, 1_2, and 2_1, 2_2 in Figure 6 targeting the 35 pathogenicity loci listed in Table 8, followed by gel electrophoresis of the amplified products. From left to right, the lanes represent a molecular weight marker, amplification results targeting pathogenicity loci 1-23 listed in Table 8, a molecular weight marker, amplification results targeting pathogenicity loci 24-35 listed in Table 8, and a molecular weight marker.

Figure 11 shows the results of gel electrophoresis of amplified products obtained from normal human dermal fibroblasts (AFP cells) using the primer mixture of experimental group 9/10 shown in Table 1 as the starting sample. From left to right, each lane represents a molecular weight marker, an amplified sample (four parallel experimental wells) obtained by amplifying a single cell using the primer mixture of experimental group 9/10, and a molecular weight marker.

Figure 12 shows the results of PCR amplification of samples 1 and 2 in Figure 11 targeting the 35 pathogenicity loci listed in Table 8, followed by gel electrophoresis of the amplified products. From left to right, the lanes represent a molecular weight marker, amplification results targeting pathogenicity loci 1-23 listed in Table 8, a molecular weight marker, amplification results targeting pathogenicity loci 24-35 listed in Table 6, and a molecular weight marker.

FIG. 13 shows the copy number of each chromosome in semiconductor sequencing of the amplified sample in FIG. 11 .

FIG14 shows the chromosome copy numbers obtained by amplifying DNA from blastocyst culture medium using the primer mixture of experimental group 9/10 shown in Table 1 and performing SBS sequencing on the amplified sample.

DETAILED DESCRIPTION

The present invention provides a method for amplifying genomic DNA, in particular a method for amplifying the whole genome DNA of a single cell.

Before the present invention, it is usually to build a library after gene amplification is completed, and then sequence it after building the library is completed. This method has a complicated process and consumes a long time. The inventors of the present application design a primer of a special structure and optimize the process of amplification so that after single cell amplification, the library can be directly built into a library, thereby significantly reducing the time required for the construction of a single cell whole genome DNA library. Although some designs of primers have been reported in some documents, these designs all have defects of one kind or another. For example, when carrying out the single cell whole genome pre-amplification step in WO2012/166425, the random sequence of primers is selected from four bases (i.e., A, T, C and G), but when using this method to directly amplify and build a library, it will inevitably form a loop or dimer from or each other, thereby significantly reducing the efficiency of amplification. For another example, US Pat. No. 8,206,913 reports that the random sequence in the primers is selected from two bases (i.e., G and T, G and A, A and C, C and T) to avoid self- or mutual looping. However, because the base randomness before the target sequence in the sequences amplified using such primers is very poor, a positive control must be added to correct for base randomness when performing SBS sequencing on a whole plate; otherwise, detection cannot be performed. Therefore, this method inevitably wastes a certain amount of data. Unlike the above-mentioned prior art, the primers involved in the present invention, while having high base randomness, basically do not form loops or dimers within the primers themselves or between primers, or form very few loops or dimers compared to four-base random primers. In addition, the libraries constructed in the present invention have high base randomness before the target sequence. Therefore, the amplified products obtained by amplification according to the method of the present invention have few dimers, can be directly libraryed, can be used for whole plate sequencing, and produce good sequencing results.

In one aspect, the present application provides a method for amplifying genomic DNA, the method comprising: (a) providing a first reaction mixture, wherein the first reaction mixture comprises a sample comprising the genomic DNA, a first primer, a nucleotide monomer mixture, and a nucleic acid polymerase, wherein the first primer comprises a universal sequence and a first variable sequence from the 5' end to the 3' end, the first variable sequence comprises a first random sequence, wherein the first random sequence is X _a1 X _a2 ... X _an from the 5' end to the 3' end, and X _ai (i=1-n) of the first random sequence all belong to the same set, the set being selected from B, or D, or H, or V, wherein B={T, G, C}, D={A, T, G}, H={T, A, C}, V={A, C, G}, wherein X _ai represents the i-th nucleotide at the 5' end of the first random sequence, n is a positive integer selected from 3-20, optionally, the first reaction mixture further comprises a third primer, wherein the third primer comprises the universal sequence and a third variable sequence from the 5' end to the 3' end, the third variable sequence comprises a third random sequence, wherein the third random sequence is X _b1 X _b2 ... X _bn from the 5' end to the 3' end, X _bi (i=1-n) of the third random sequence all belong to the same set, the set is selected from B, or D, or H, or V, wherein B={T, G, C}, D={A, T, G}, H={T, A, C}, V={A, C, G}, and X _bi (i=1-n) and X _ai (i=1-n) belong to different sets, wherein X _bi represents the i-th nucleotide at the 5' end of the third random sequence, and n is a positive integer selected from 3-20; (b) subjecting the first reaction mixture to a first temperature cycle program for pre-amplification to obtain a pre-amplification product; (c) providing a second reaction mixture, the second reaction mixture comprising the pre-amplification product obtained in step (b), a second primer, a nucleotide monomer mixture, and a nucleic acid polymerase, wherein the second primer comprises or consists of a specific sequence and the universal sequence from the 5' end to the 3'end; (d) subjecting the second reaction mixture to a second temperature cycle program for amplification to obtain an amplified product. A diagram of one embodiment of the method provided herein is shown in Figure 1.

Step (a): Providing a first reaction mixture

The method of the present application is widely applicable to the amplification of genomic DNA, especially the amplification of trace amounts of genomic DNA.

i. Genomic DNA

The method of the present application is preferably applicable to genomic DNA.In certain embodiments, the starting amount of the genomic DNA included 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 10 pg.

Genomic DNA can be obtained from a biological sample, such as a biological tissue or a body fluid containing cells or free DNA. Samples containing genomic DNA can be obtained by known methods, such as oral mucosal samples, nasal samples, hair, mouthwash, umbilical cord blood, plasma, amniotic fluid, fetal tissue, endothelial cells, nail samples, hoof samples, etc. The biological sample can be provided in any suitable form, such as paraffin-embedded form, freshly isolated form, etc. The genomic DNA can be from any species or biological type, such as, but not limited to, humans, mammals, cattle, pigs, sheep, horses, rodents, birds, fish, zebrafish, shrimp, plants, yeast, viruses, or bacteria.

In certain embodiments, the genomic DNA is the genomic DNA from a single cell, or the genomic DNA from two or more cells of the same type. The single cell or cells of the same type can be derived from, for example, a pre-implantation embryo, an embryonic cell in the peripheral blood of a pregnant woman, a single sperm, an egg cell, a fertilized egg, a cancer cell, a bacterial cell, a circulating tumor cell, a tumor tissue cell, or a single or multiple cells of the same type obtained from any tissue. The method of the present application can be used to amplify the DNA in some valuable samples or low-starting samples, such as human egg cells, germ cells, circulating tumor cells, tumor tissue cells, etc.

In some embodiments, the genomic DNA is derived from blastomeres, blastocyst trophoblasts, cultured cells, extracted gDNA, or blastocyst culture fluid.

Methods for obtaining single cells are also well known in the art, and can be performed, for example, by flow cytometry (Herzenberg et al., Proc Natl Acad Sci USA 76:1453-55, 1979; Iverson et al., Prenatal Diagnosis 1:61-73, 1981; Bianchi et al., Prenatal Diagnosis 11:523-28, 1991), fluorescence-activated cell sorting, magnetic bead separation (MACS, Ganshirt-Ahlert et al., Am J Obstet Gynecol 166:1350, 1992), using a semi-automated cell picker (e.g., Quixell ^™ Cell Transfer System manufactured by Stoelting), or a combination of these methods. In some embodiments, gradient centrifugation and flow cytometry can be used to improve separation and sorting efficiency. In some embodiments, specific cell types can be selected based on the different properties of individual cells, such as cells expressing a specific biomarker.

The method for obtaining genomic DNA is also well known in the art. In certain embodiments, the genomic DNA can be obtained by lysing cells from a biological sample or in a single cell and releasing the obtained genomic DNA. Any suitable method well known in the art can be used to crack, for example, by thermal cracking, alkaline cracking, enzyme cracking, mechanical cracking, or any combination thereof (specifically referring to, for example, U.S. 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 lysis includes methods that use mechanical force to disrupt cells, including ultrasound, high-speed stirring, homogenization, pressurization (e.g., French press), decompression, and grinding. The most commonly used mechanical lysis method is liquid homogenization, which forces a cell suspension through a very narrow space, thereby applying shear forces to the cell membrane (e.g., as described in WO2013153176A1).

In certain embodiments, a mild lysis method can be used. For example, cells can be heated at 72°C for 2 minutes in a solution containing Tween-20, at 65°C for 10 minutes in water (Esumi et al., Neurosci Res 60(4):439-51 (2008), at 70°C for 90 seconds in PCR buffer II (Applied Biosystems) containing 0.5% NP-40 (Kurimoto et al., Nucleic Acids Res 34(5):e42 (2006), or lysed using a protease (e.g., proteinase K) or a free salt solution (e.g., guanidine isothiocyanate) (e.g., as described in U.S. Patent Application No. US 20070281313).

Thermal cracking includes heating and repeated freezing and thawing. In some embodiments, the thermal cracking includes a temperature between 20-100 ° C and a cracking time of 10-100 minutes. In some embodiments, the temperature of the thermal cracking can be any temperature between 20-90, 30-90, 40-90, 50-90, 60-90, 70-90, 80-90, 30-80, 40-80, 50-80, 60-80 or 70-80 ° C. In some embodiments, the temperature of the thermal cracking is not less than 20, 30, 40 or 50 ° C. In some embodiments, the temperature of the thermal cracking is not higher than 100, 90 or 80 ° C. In some embodiments, the thermal cracking time can be any time between 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 or 30-40 minutes. In some embodiments, the thermal cracking time is not less than 20, 30, 40, 50, 60, 70, 80 or 90 minutes. In some embodiments, the thermal cracking time is not more than 90, 80, 70, 60, 50, 40, 30 or 20 minutes. In some embodiments, the thermal cracking temperature varies over time. In some embodiments, the thermal cracking temperature is maintained at 30-60°C for 10-30 minutes, and then maintained at 70-90°C for 5-20 minutes.

In some embodiments, the thermal lysis is carried out in the presence of a lytic agent. When a lytic agent is present, the time required for lysis can be reduced or the temperature required for lysis can be reduced. The lytic agent can disrupt protein-protein, lipid-lipid and/or protein-lipid interactions, thereby promoting the release of genomic DNA from cells.

In some embodiments, the lysis reagent includes a surfactant and/or a lyase. Surfactants can be divided into ionic, amphoteric and non-ionic surfactants. Generally, the lysis efficiency of amphoteric and non-ionic surfactants is weaker than that of ionic surfactants. Exemplary surfactants include, but are not limited to, one or more of NP-40, Tween, SDS, GHAPS, TritonX-100, TritonX-114, EDTA, sodium deoxycholate, sodium cholate, and guanidine isothiocyanate. Those 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 0.01%-5%, 0.1%-3%, 0.3%-2% or 0.5-1%.

Exemplary lytic enzymes can be proteinase K, pepsin, papain, etc., or any combination thereof. In some embodiments, the working concentration of the lytic enzyme is 0.01%-1%, 0.02%-0.5%, 0.03%-0.2% or 0.4-0.1%.

In the methods provided herein, the lysate containing genomic DNA can be used directly in the first reaction mixture. For example, the biological sample can be pre-lysed to obtain a lysate, which is then mixed with the other components of the first reaction mixture. If necessary, the lysate can be further treated to isolate the genomic DNA therein, which is then mixed with the other components of the first reaction mixture to obtain the first reaction mixture.

In some embodiments, the cleaved nucleic acid sample can be amplified without purification. In some embodiments, the cleaved nucleic acid sample is purified before amplification. In some embodiments, the DNA has already undergone varying degrees of fragmentation during the cleavage process and can be amplified without a special shearing step. In some embodiments, the cleaved nucleic acid sample is sheared before amplification.

The application also provides a more simple method, that is, directly the cell comprising genomic DNA is mixed with other components required for amplification to obtain the first reaction mixture, that is to say, the genomic DNA in the first reaction mixture is present in the cell interior. In such a case, the first reaction mixture can also further contain a surfactant (such as but not limited to one or more of NP-40, Tween, SDS, TritonX-100, EDTA, guanidine isothiocyanate) and/or a lyase (such as one or more of proteinase K, pepsin, papain) that can crack the cell. Like this, the cracking of cell and the pre-amplification of genomic DNA are all carried out in the same reaction mixture, which can improve reaction efficiency and shorten reaction time.

In certain embodiments, the methods provided herein may further comprise subjecting the reaction mixture to a lysis temperature cycle program after step (a) and before step (b) to lyse the cells and release the genomic DNA. A person skilled in the art may select an appropriate lysis temperature cycle program based on the lysis components contained in the reaction mixture, the type of cells, and the like. An exemplary cleavage temperature cycle program includes placing the reaction mixture at 50° C. for 3 minutes to 8 hours (e.g., any time between 3 minutes to 7 hours, 3 minutes to 6 hours, 3 minutes to 5 hours, 3 minutes to 4 hours, 3 minutes to 3 hours, 3 minutes to 2 hours, 3 minutes to 1 hour, 3 minutes to 40 minutes, 3 minutes to 20 minutes, such as 10 minutes, 20 minutes, 30 minutes, etc.), and then placing it at 80° C. for 2 minutes to 8 hours (e.g., any time between 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, 2 minutes to 40 minutes, 2 minutes to 20 minutes, such as 10 minutes, 20 minutes, 30 minutes, etc.). The cleavage temperature program can be performed in one cycle or, if desired, in two or more cycles, depending on the specific cleavage conditions.

ii. First class primers

The method described in this application involves two major categories of primers, wherein the first category of primers comprises a universal sequence and a variable sequence from the 5' end to the 3' end, and the second category of primers comprises a specific sequence and a universal sequence, but does not comprise any variable sequence. The "first primer" and "third primer" described herein both belong to the above-mentioned first category of primers. The first primer included in the first reaction mixture comprises a universal sequence and a first variable sequence from the 5' end to the 3' end; and the third primer optionally included in the first reaction mixture comprises a universal sequence and a third variable sequence from the 5' end to the 3' end. In some embodiments, the first category of primers consists of a universal sequence and a variable sequence. In other embodiments, the first category of primers consists of a universal sequence, a variable sequence, and a spacer sequence.

universal sequence

Universal sequence refers to the nucleotide sequence that first class primer and second class primer all have at its 5 ' end in the present application.The length of universal sequence can be for example, 6-60,8-50,9-40,10-30,10-15 or 25-30 base.In the present application, select suitable universal sequence, make to be combined with genomic dna and produce amplification basically, and avoid polymerization between first class primer and the first class primer (for example, between first primer and the first primer, between the 3rd primer and the 3rd primer or between first primer and the 3rd primer) and the looping of first class primer self (for example, the partial sequence at 5 ' end of first primer and the partial sequence at 3 ' end are complementary and the first primer self forms hairpin structure or the partial sequence at 5 ' end of the 3rd primer and the partial sequence at 3 ' end are complementary and the 3rd primer self forms hairpin structure), and polymerization between first class primer and second class primer or the situation of looping.

In some embodiments, the universal sequence comprises all 4 types of bases A, T, C, and G. In some embodiments, the universal sequence only comprises three or two types of bases with weaker self-complementary pairing abilities, and does not contain another type or two types of bases. In some embodiments, the universal sequence is composed of three types of bases: G, A, and T, i.e., the universal sequence does not contain a C base. In some embodiments, the universal sequence is composed of three types of bases: C, A, and T, i.e., the universal sequence does not contain a G base. In some embodiments, the universal sequence is composed of two types of bases: A and T, A and C, A and G, T and C, or T and G, i.e., the universal sequence does not contain G and C bases at the same time. Without wishing to be bound by theory, it is believed that if the universal sequence contains C or G bases, it may cause mutual polymerization between primers, produce polymers, thereby weakening the amplification ability of genomic DNA. Preferably, the universal sequence does not have a sequence that can self-pair, a sequence that can cause pairing between primers, or a plurality of continuous homologous bases.

In certain embodiments, the base sequence of an appropriate universal sequence and the ratio of each base therein can be selected to ensure that the universal sequence itself does not base pair with the genomic DNA template sequence or cause amplification.

In some embodiments, the universal sequence can be selected so that amplified production can directly be sequenced.Do not wish to be bound by theory, the universal sequence can be designed to comprise partial or complete complementarity or identical sequence (for example, identical with the part of sequencing primer, all identical, partially complementary or entirely complementary sequence) of sequencing primer. In some embodiments, universal sequence is selected specifically according to different sequencing platforms. In some embodiments, universal sequence is selected specifically according to second generation or third generation sequencing platform. In some embodiments, universal sequence is selected specifically according to the NGS sequencing platform of Illumina. In some embodiments, universal sequence is selected specifically according to the Ion torrent sequencing platform.

In certain embodiments, the common sequence is selected from the group consisting of SEQ ID NO: 1 [TTGGTAGTGAGTG], SEQ ID NO: 2 [GAGGTGTGATGGA], SEQ ID NO: 3 [GTGATGGTTGAGGTA], SEQ ID NO: 4 [AGATGTGTATAAGAGACAG], SEQ ID NO: 5 [GTGAGTGATGGTTGAGGTAGTGTGGAG], and SEQ ID NO: 6 [GCTCTTCCGATCT].

Mutable Sequence

The first class primer comprises universal sequence and variable sequence (for example the first primer/the 3rd primer comprises the first/the 3rd variable sequence respectively) from 5 ' end to 3 ' end, and wherein the universal sequence in the first class primer is all identical, but variable sequence may be different.For example, in some embodiments, the first/the 3rd primer is respectively a primer mixture comprising identical universal sequence and different variable sequences.Variable sequence refers to a section of base sequence that sequence is not fixed in the application, and it can comprise random sequence (for example the first/the 3rd variable sequence comprises the first/the 3rd random sequence respectively).In some embodiments, variable sequence is made up of random sequence.In other embodiments, variable sequence is made up of random sequence and fixed sequence.

a) Random sequence

A random sequence means that the bases at each base position in the sequence are independently and randomly selected from a specific set, so the above random sequence represents a set of base sequences composed of different base combinations.

Specifically, for example, the first variable sequence may include a first random sequence, wherein the number of bases in the first random sequence is n, where n is a positive integer selected from 3-20, and the sequence of the first random sequence from the 5' end to the 3' end can be expressed as X _a1 X _a2 ... X _an , and the base at any base position i (i.e., the i-th nucleotide at the 5' end of the first random sequence, i=1-n) can be represented by X _ai , wherein each X _ai is randomly selected from a specific set, for example, a set consisting of specific two or three nucleotides among A, T, G, and C. The selectable sets at any of the above-mentioned base positions can usually be represented by a degenerate identification method. For example, the set containing only two nucleotides, A and G, can be represented as R (i.e., R = {A, G}). Other sets that can be represented by degenerate identification include: Y = {C, T}, M = {A, C}, K = {G, T}, S = {C, G}, W = {A, T}, H = {A, C, T}, B = {C, G, T}, V = {A, C, G}, D = {A, G, T}, N = {A, C, G, T}.

The random sequence (i.e., any base position in the random sequence) can be selected in a completely random manner, or certain restrictions can be added to the randomness to exclude some undesirable situations or increase the degree of matching with the target genomic DNA. In some embodiments, to avoid complementary pairing between the variable sequence and the universal sequence, when the universal sequence contains a large number of Gs, any base position in the random sequence is selected from set D (i.e., not C); or when the universal sequence contains a large number of Cs, any base position in the random sequence is selected from set H (i.e., not G); when the universal sequence contains a large number of Ts, any base position in the random sequence is selected from set B (i.e., not A); or when the universal sequence contains a large number of As, any base position in the random sequence is selected from set V (i.e., not T).

The random sequence can have an appropriate length, for example 2-20 bases, 2-19 bases, 2-18 bases, 2-17 bases, 2-16 bases, 2-15 bases, 2-14 bases, 2-13 bases, 2-12 bases, 2-11 bases, 2-12 bases, 2-11 bases, 2-10 bases, 2-9 bases, 2-8 bases, 3-18 bases, 3-16 bases, 3-14 bases, 3-12 bases, 3-10 bases, 4-16 bases, 4-12 bases, 4-9 bases, or 5-8 bases. In certain embodiments, the length of the random sequence is 5 bases. In certain embodiments, the length of the random sequence is 8 bases. Theoretically, if each base position in the random sequence is randomly selected from the three bases A, T, and G, then a variable sequence of 4 bases can combine to form 3 ⁴ = 81 possible random sequences, a random sequence of 5 bases can combine to form 3 ⁵ = 243 possible random sequences, and so on. These random sequences can complement each other with corresponding sequences at different locations on the genomic DNA, thereby initiating replication at different locations on the genomic DNA.

In one embodiment, the base X _ai (i=1-n) at each arbitrary base position i in the first random sequence belongs to the same set, and the set is selected from one of B, D, H, or V. As a non-limiting example, a first primer has a universal sequence and a first random sequence, where n=5, and each arbitrary X _ai (i=1-5) of the random sequence belongs to the same set B, that is, the random sequence can be represented as BBBBB or (B) ₅ , and the random sequence can be selected from {TTTTT, TGTTT, TCTTT, TTGTT, TTCTT...}, for a total of 3 ⁵ = 243 sequence combinations. In a specific first reaction mixture including such first primers, these first primers all have the same universal sequence and the above-mentioned first random sequence, that is, the first primers in this specific first reaction are a group of primers, these primers all have the same universal sequence and have the same or different random sequences composed of bases selected from set B.

Unless otherwise explicitly stated, all descriptions herein of the first primer and its various parts are applicable to the third primer and its corresponding parts. Similarly, when the first reaction mixture further comprises a third primer, the third variable sequence in the third primer may include a third random sequence, wherein the third random sequence is X _b1 X _b2 ... X _bn from the 5' end to the 3' end, preferably, X _bi (i=1-n) of the third random sequence all belong to the same set, the set being selected from B, or D, or H, or V, wherein B={T, G, C}, D={A, T, G}, H={T, A, C}, V={A, C, G}, and X _bi (i=1-n) and X _ai (i=1-n) belong to different sets, wherein X _bi represents the i-th nucleotide at the 5' end of the third random sequence, and n is a positive integer selected from 3-20. In a specific first reaction mixture, a certain amount of first primers is included, each of which has the same universal sequence and a first random sequence of length n, wherein each base X _ai of the first random sequence belongs to the same set, and wherein the set is selected from B, D, H, or V; and the first reaction mixture further includes a certain amount of third primers, each of which has the same universal sequence and a first random sequence of length n, wherein each base X _bi of the first random sequence belongs to the same set, and wherein the set is selected from B, D, H, or V, and X _bi and X _ai belong to different sets. In some embodiments, the first random sequence and the third random sequence are the same length. In other embodiments, the first random sequence and the third random sequence are different lengths.

b) Fixed sequence

Variable sequence can further include fixed sequence at its 3 ' end, and described fixed sequence can be selected from any base combination that can improve genome coverage.Fixed sequence described in the present application includes but is not limited to the sequence selected from CCC, AAA, TGGG, GTTT, GGG, TTT, TNTNG or GTGG.The N used when describing fixed sequence in the present application represents any mononucleotide selected from A, T, C, G, rather than the random sequence selected from N. In the same primer set, for example, in the first primer, from 5 ' end to 3 ' end, can include successively identical universal sequence, random sequence containing different sequence combinations and identical fixed sequence (for example, all first primers include any one of TGGG or GTTT at its 3 ' end). Or, in the same primer set, for example, in the first primer, from 5 ' end to 3 ' end, can include successively identical universal sequence, random sequence containing different sequence combinations and different fixed sequences (for example, in the first primer, include the primer mixture that all includes TGGG at its 3 ' end and or include the primer mixture that all includes GTTT at its 3 ' end). In some embodiments, _the first reaction mixture includes a first primer and a third primer, wherein the first variable sequence in the first primer is selected from _Xa1Xa2 ... _XanGGG , _Xa1Xa2 ... _XanTTT , _Xa1Xa2 ... _XanTGGG , or _Xa1Xa2 ... _XanGTTT , _and the _third variable sequence in _the third primer is selected _{from Xb1Xb2} ... _XbnGGG , _Xb1Xb2 ... _XbnTTT , _{Xb1Xb2 ... XbnTGGG , or Xb1Xb2} ... _XbnGTTT .

In certain embodiments, statistical calculations can be used to select variable sequences that are more evenly distributed on the genome and have higher coverage, thereby increasing the chances of recognition between the variable sequences and genomic DNA.

In certain embodiments, the variable sequence is selected from the group consisting of (B) _n CCC, (B) _n AAA, (B) _n TGGG, (B) _n GTTT, (B) _n GGG, (B) _n TTT, (B) _n TNTNG, (B) _n GTGGGGG, (D) _n CCC, (D) _n AAA, (D) _n TGGG, (D) _n GTTT, (D) _n GGG, (D) _n TTT, (D) _n TNTNG, (D) _n GTGGGGG, (H) n CCC, (H) _n AAA, (H) _n TGGG, (H) _n GTTT, (H) _n GGG, (H) _n TTT, (H) _n TNTNG, (H) _n GTGGGGG, (V) _n CCC, (V) _n AAA, (V) _n TGGG, (V) _n GTTT, (V) _n In some embodiments, the first variable sequence in the first primer may have one or more of the following sequences: (B) _n CCC, (B) _n AAA, (B) _n TGGG, (B) _n GTTT, (B) _n GGG _, (B) _n TTT, ( _B ) _n TNTNG, (B) _n GTGGGGG. In some embodiments, the third variable sequence in the third primer may have one or more of the following sequences: (D) _n CCC, (D) _n AAA, (D) _n TGGG, (D) _n GTTT, (D) _n GGG _, (D) _n TTT, (D) _n TNTNG, (D) _n GTGGGGG.

spacer sequence

The universal sequence and variable sequence of the first class primer can be directly adjacent, or also can have the spacer sequence of one or more bases.In some embodiments, universal sequence and variable sequence link to each other through the spacer sequence of m, and wherein m is the positive integer that is selected from 1-3.When the random sequence in the variable sequence is restricted to a certain degree in order to get rid of some undesirable situations (for example primer dimer etc.) or in order to increase with the matching degree of target gene group dna, can in universal sequence and variable sequence, introduce m base (length is the spacer sequence of m) that is selected from A, T, G, C completely at random, to further increase the coverage ratio of the first class primer on target gene group dna when not increasing primer dimer and producing degree.

In some embodiments, the universal sequence and the first variable sequence in the first primer are connected by a first spacer sequence, wherein the first spacer sequence is Y _a1 …Y _am , where _Yaj (j=1-m)∈{A, T, G, C}, where _Yaj represents the jth nucleotide at the 5' end of the first spacer sequence, and m is a positive integer selected from 1-3. In some embodiments, the universal sequence and the third variable sequence in the third primer are connected by a third spacer sequence, wherein the first spacer sequence is Y _b1 …Y _bm , where Y _bj (j=1-m)∈{A, T, G, C}, where Y _bj represents the jth nucleotide at the 5' end of the third spacer sequence, and m is a positive integer selected from 1-3. In some embodiments, m is 1, i.e., the universal sequence and the first variable sequence in the first primer are connected by a base selected from set N, and the universal sequence and the third variable sequence in the third primer are connected by a base selected from set N.

In certain embodiments, the first primer (and optional third primer) is designed so that its amplification product can be directly used in the Illumina NGS sequencing platform, wherein the first primer comprises a sequence represented by GCTCTTCCGATCTY _a1 X _a1 X _a2 X _a3 X _a4 X _a5 TGGG, GCTCTTCCGATCTY _a1 X _a1 X _a2 X _a3 X _a4 X _a5 GTTT, or a mixture thereof, and the third primer comprises GCTCTTCCGATCTY _b1 X _b1 X _b2 X _b3 X _b4 X _b5 TGGG, GCTCTTCCGATCTY _b1 X _b1 X _b2 X _b3 X _b4 X _b5 GTTT, or a mixture thereof, wherein each base X _ai (i=1-n) at any base position i belongs to the same set, wherein the set is selected from one of B, D, H, or V, and each base X _bi (i=1-n) at any base position i belongs to the same set, wherein the set is selected from one of B, D, H, or V, and X _bi (i=1-n) and X _ai (i=1-n) belong to different sets; wherein _Ya1 ∈ {A, T, G, C}, Y _b1 ∈ {A, T, G, C}. In certain specific embodiments, the above X _ai (i=1-5) ∈ {T, G, C}, X _bi (i=1-5) ∈ {A, T, G}, that is, the first primer includes the sequence shown in SEQ ID NO:7, SEQ ID NO:11, or a mixture thereof, and the third primer includes the sequence shown in SEQ ID NO:8, SEQ ID NO:12, or a mixture thereof.

In certain embodiments, the first class of primers comprises or consists of a sequence selected from SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14, wherein the common sequence of each first class of primers comprises or consists of SEQ ID NO:6. In certain embodiments, the first class of primers comprises a primer consisting of the sequence set forth in SEQ ID NO:7 and/or a primer consisting of the sequence set forth in SEQ ID NO:11. In certain embodiments, the first class of primers comprises a primer consisting of the sequence set forth in SEQ ID NO:8 and a primer consisting of the sequence set forth in SEQ ID NO:12. In certain embodiments, the first class of primers comprises a primer consisting of the sequence set forth in SEQ ID NO:7 or a primer consisting of the sequence set forth in SEQ ID NO:11; and a primer consisting of the sequence set forth in SEQ ID NO:8 or a primer consisting of the sequence set forth in SEQ ID NO:12. In certain embodiments, the first type of primer comprises a primer consisting of the sequence shown in SEQ ID NO:7, a primer consisting of the sequence shown in SEQ ID NO:11, a primer consisting of the sequence shown in SEQ ID NO:8, and a primer consisting of the sequence shown in SEQ ID NO:12.

In certain embodiments, the first class of primers comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 15-22, wherein the common sequence of each first class of primers comprises or consists of SEQ ID NO: 1. In certain embodiments, the first class of primers comprises a primer consisting of a sequence set forth in SEQ ID NO: 15 and/or a primer consisting of a sequence set forth in SEQ ID NO: 19. In certain embodiments, the first class of primers comprises a primer consisting of a sequence set forth in SEQ ID NO: 16 and/or a primer consisting of a sequence set forth in SEQ ID NO: 20. In certain embodiments, the first class of primers comprises a primer consisting of a sequence set forth in SEQ ID NO: 15 or a primer consisting of a sequence set forth in SEQ ID NO: 19; and a primer consisting of a sequence set forth in SEQ ID NO: 16 or a primer consisting of a sequence set forth in SEQ ID NO: 20. In certain embodiments, the first class of primers comprises a primer consisting of a sequence set forth in SEQ ID NO: 15, a primer consisting of a sequence set forth in SEQ ID NO: 19, a primer consisting of a sequence set forth in SEQ ID NO: 16, and a primer consisting of a sequence set forth in SEQ ID NO: 20.

In certain embodiments, the first class of primers comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 23-30, wherein the common sequence of each first class of primers comprises or consists of SEQ ID NO: 2. In certain embodiments, the first class of primers comprises one or both of a primer consisting of a sequence set forth in SEQ ID NO: 23 and/or a primer consisting of a sequence set forth in SEQ ID NO: 27. In certain embodiments, the first class of primers comprises one or both of a primer consisting of a sequence set forth in SEQ ID NO: 24 and/or a primer consisting of a sequence set forth in SEQ ID NO: 28. In certain embodiments, the first class of primers comprises a primer consisting of a sequence set forth in SEQ ID NO: 23 or a primer consisting of a sequence set forth in SEQ ID NO: 27; and a primer consisting of a sequence set forth in SEQ ID NO: 24 or a primer consisting of a sequence set forth in SEQ ID NO: 28. In certain embodiments, the first type of primer comprises a primer consisting of the sequence shown in SEQ ID NO:23, a primer consisting of the sequence shown in SEQ ID NO:27, a primer consisting of the sequence shown in SEQ ID NO:24, and a primer consisting of the sequence shown in SEQ ID NO:28.

In some embodiments, the total concentration of the first and third primers in the first reaction mixture is 10-150 ng/μL. In some embodiments, the total concentration of the first and third primers in the first reaction mixture is 10-120 ng/μL, 10-100 ng/μL, 10-90 ng/μL, 10-80 ng/μL, 10-70 ng/μL, 10-60 ng/μL, 10-50 ng/μL, 10-40 ng/μL, 20-120 ng/μL, 20-100 ng/μL, 20-80 ng/μL, 20-70 ng/μL, 20-60 ng/μL, 20-50 ng/μL, 30-140 ng/μL, 30-120 ng/μL, 30-100 ng/μL, 30-80 ng/μL, 30-60 ng/μL, or 30-40 ng/μL. In some embodiments, the concentration of the first and third primers in the first reaction mixture is respectively 10-140ng/μL, 10-120ng/μL, 10-100ng/μL, 10-80ng/μL, 10-60ng/μL, 10-30ng/μL, 10-20ng/μL, 20-120ng/μL, 20-100ng/μL, 20-80ng/μL, 20-60ng/μL, 20-40ng/μL or 20-30ng/μL. In some embodiments, the concentration of the first and third primers in the first reaction mixture is respectively 15ng/μL, 30ng/μL or 60ng/μL. In some embodiments, the concentration of the first primer and the third primer in the first reaction mixture is the same. In some embodiments, the first and third primers in the first reaction mixture are respectively 100-800pmol. In some embodiments, the first and third primers are present in a total amount of 400-600 pmol in the first reaction mixture.

iii. Other ingredients

The first reaction mixture may further comprise other components required for DNA amplification, such as nucleic acid polymerase, nucleotide monomer mixture, and appropriate metal ions and buffer components required for enzyme activity, etc. At least one or more of these components may use reagents known in the art.

As used herein, a nucleic acid polymerase refers to an enzyme capable of synthesizing new nucleic acid chains. Any nucleic acid polymerase suitable for the methods of the present application may be used. DNA polymerase is preferably used. In certain embodiments, the methods of the present application utilize thermostable nucleic acid polymerases, such as those whose polymerase activity does not decrease, or decreases by less than 1%, 3%, 5%, 7%, 10%, 20%, 30%, 40%, or 50%, at the temperature of PCR amplification (e.g., 95 degrees Celsius). In certain embodiments, the nucleic acid polymerase used in the methods of the present application possesses strand displacement activity. "Strand displacement activity," as used herein, refers to an activity of a nucleic acid polymerase that can separate a nucleic acid template from its paired complementary strand, with this separation occurring in a 5' to 3' direction and accompanied by the generation of a new nucleic acid strand complementary to the template. Nucleic acid polymerases with strand displacement ability and their use are known in the art, for example, see U.S. Patent No. 5,824,517, the entire contents of which are incorporated herein by reference. Suitable nucleic acid polymerases include, but are not limited to, one or more of Phi29 DNA polymerase, Bst DNA polymerase, Bst 2.0 DNA polymerase, Pyrophage 3137, Vent polymerase (e.g., Vent polymerase, Deep Vent polymerase, Vent (-exo) polymerase, Deep Vent (-exo) polymerase from Thermococcus litoralis), TOPO Taq DNA polymerase, ^9.Nm polymerase, Klenow Fragment DNA polymerase I, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, T7 phase DNA polymerase variant (lacking 3'-5' exonuclease activity), super-fidelity DNA polymerase, Taq polymerase, Psp GBD (exo-) DNA polymerase, Bst DNA polymerase (full length), E. coli DNA polymerase, Long Amp Taq DNA polymerase, and One Taq DNA polymerase.

The nucleotide monomer mixture in this application refers to a mixture of dATP, dTTP, dGTP, and dCTP.

In certain embodiments, the first reaction mixture contains one or more of Vent polymerase, Deep Vent polymerase, Vent(-exo) polymerase, or Deep Vent(-exo) polymerase from Thermococcus litoralis. In certain embodiments, the reaction mixture contains Vent polymerase from Thermococcus litoralis. Vent polymerase from Thermococcus litoralis refers to a natural polymerase isolated from Thermococcus litoralis. In certain embodiments, the reaction mixture contains Deep Vent polymerase. Deep Vent polymerase refers to a natural polymerase isolated from Pyrococcus species GB-D. In certain embodiments, the reaction mixture contains Vent(-exo) polymerase. Vent(-exo) polymerase refers to an enzyme in which Vent polymerase from Thermococcus litoralis has been genetically modified with D141A/E143A. In certain embodiments, the reaction mixture contains Deep Vent(-exo) polymerase. Deep Vent (-exo) polymerase refers to an enzyme that has been genetically modified with D141A/E143A to Deep Vent polymerase. The various Vent polymerases described in this application can be obtained from commercial sources, such as New England Biolabs.

The first reaction mixture may further include appropriate metal ions required for the nucleic acid polymerase to exert enzymatic activity (e.g., an appropriate concentration of Mg ²⁺ ions (e.g., a final concentration of about 1.5 mM to about 8 mM), a nucleotide monomer mixture (e.g., dATP, dGTP, dTTP, and dCTP), bovine serum albumin (BSA), dTT (e.g., a final concentration of about 2 mM to about 7 mM), pure water, etc.

In certain embodiments, the first reaction mixture may further include a pH regulator to maintain the pH of the mixture between 7.0 and 9.0. Suitable pH regulators may include, for example, Tris HCl and Tris SO ₄ . In certain embodiments, the first reaction mixture may further include one or more other components, such as a DNase inhibitor, RNase, SO ₄ ^2- , Cl ^- , K ⁺ , Ca ²⁺ , Na ⁺ , and/or (NH ₄ ) ⁺ .

Step (b): Place in the first temperature cycle program

The method provided in the present application includes step (b): placing the first reaction mixture in a first temperature cycle program so that the variable sequence of the first type of primer (the first primer or the first primer and the third primer) can bind to the genomic DNA through base pairing, and replicate the genomic DNA under the action of nucleic acid polymerase.

" Amplification " refers to in the present application, under the action of nucleic acid polymerase, adding nucleotides complementary to the nucleic acid template at the 3 ' end of the primer, so as to synthesize a new nucleic acid chain complementary to the 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 all known in the art, and can be found in, for example, U.S. Patent Nos. 4,683,195 and 4,683,202, and 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 full contents of these documents and patents are incorporated herein by reference.

During amplification, the reaction mixture is placed in an appropriate temperature cycling program to allow the double-stranded DNA template to unwind into single strands, allowing the first/third primers to hybridize to the single-stranded template, and then be extended at the 3' end of the primers by the action of a DNA polymerase. Therefore, the temperature cycling program typically includes: a denaturation or melting temperature, at which the double-stranded DNA template unwinds into single strands; an annealing temperature, at which the primers specifically hybridize to the single-stranded DNA template; and an extension temperature, at which the DNA polymerase adds nucleotides complementary to the bases of the DNA template to the 3' end of the primer, allowing the primers to be extended, resulting in a new DNA strand complementary to the DNA template.

In the first cycle of the first temperature cycling program, the first reaction mixture is first placed in a temperature program capable of opening the double-stranded genomic DNA (step (b1)). In the first round of cycles, in order to ensure that the double-stranded genomic DNA is completely unwound into a single strand (i.e., denaturation/melting), a higher reaction temperature (e.g., 90°C-95°C) can be used, and a longer reaction time (e.g., 1-20 minutes at a temperature between 90-95°C) can be maintained. In subsequent cycles, the double strands that need to be unwound are the double strands generated during the amplification process. In this case, as long as the double strands of the half-amplicons or full-amplicons to be amplified can be denatured into single strands, the required melting time does not need to be very long (e.g., 3-50 seconds for melting reaction at a temperature between 90-95°C).

Then, the first reaction mixture is placed in a temperature program that enables the first type of primer (the first primer or the first primer and the third primer) to bind to the DNA single-stranded template (step (b2)). In this temperature program, the variable sequence in the first type of primer binds to the complementary sequence at different positions in the genomic DNA through base complementarity (i.e., annealing), and thus initiates replication at different positions of the genomic DNA. Since the variable sequences in the first type of primers are different, there are differences in the base ratio and sequence therein, and therefore the optimal temperature for each variable sequence to bind to the genomic DNA is also quite different. In this way, at a certain specific annealing temperature, only a part of the primers may be able to bind well to the genomic DNA, while the binding of another part of the primers to the genomic DNA may not be ideal. In some embodiments, the step (b2) includes placing the reaction mixture in a program of more than one temperature to encourage the first type of primer to fully and effectively bind to the DNA template. For example, the reaction mixture for DNA denaturation can be rapidly cooled to a low temperature, such as about 10°C-20°C, and then the temperature can be increased in a gradient manner so that the reaction mixture reacts at different annealing temperatures for an appropriate time, thereby ensuring that as many primers as possible are paired and bound to the genomic DNA. In certain embodiments, step (b2) includes reacting at a first annealing temperature between 10-20°C (e.g., 15°C) for an appropriate time (e.g., 3-60 seconds), reacting at a second annealing temperature between 20-30°C (e.g., 25°C) for an appropriate time (e.g., 3-50 seconds), and reacting at a third annealing temperature between 30-50°C (e.g., 35°C) for an appropriate time (e.g., 3-50 seconds).

It is well known in the art that the annealing temperature of a primer is usually not more than 5°C lower than the primer Tm value, and an annealing temperature that is too low will cause nonspecific binding between primers, resulting in the formation of primer polymers and nonspecific amplification products. Therefore, low temperatures such as 10°C-20°C are usually not used in the primer annealing temperature. However, the inventors of the present application unexpectedly found that even if the temperature is gradually increased from a low temperature (e.g., 10°C-20°C), the pairing between the primer and the genomic DNA can still maintain good specificity, and the amplification results still maintain very low variability, indicating that the amplification results are accurate and reliable. At the same time, since the primer annealing temperature covers the low temperature situation, it is possible to ensure that a wider range of primer sequences are combined with the genomic DNA, thereby providing better genome coverage and amplification depth. After the annealing temperature program, the reaction mixture is placed in a temperature program that allows the first type of primer bound to the DNA single-stranded template to extend its length under the action of the nucleic acid polymerase to produce an amplified product (step (b3)).

The extension temperature is usually related to the optimum temperature of the DNA polymerase, and those skilled in the art can make a specific selection based on the specific reaction mixture. In some embodiments, the DNA polymerase in the reaction mixture can have a strand displacement activity, so that if the primer encounters a primer or amplicon bound to a downstream template during the extension process, the strand displacement activity of the DNA polymerase can separate these downstream bound primers from the template chain, thereby ensuring that the primer in the extension can continue to extend to obtain a longer amplified sequence. DNA polymerases with strand displacement activity include, but are not limited to, for example, phi29 DNA polymerase, T5 DNA polymerase, SEQUENASE 1.0 and SEQUENASE 2.0. In some embodiments, the DNA polymerase in the reaction mixture is a thermostable DNA polymerase. Thermostable DNA polymerases include, but are not limited to, for example, Taq DNA polymerase, OmniBase ^™ sequenase, Pfu DNA polymerase, TaqBead ^™ hot start polymerase, Vent DNA polymerase (for example Vent DNA polymerase, Deep Vent polymerase, Vent (-exo) polymerase, Deep Vent (-exo) polymerase of Thermococcus litoralis), Tub DNA polymerase, TaqPlus DNA polymerase, Tfl DNA polymerase, Tli DNA polymerase and Tth DNA polymerase. In some embodiments, the DNA polymerase in the reaction mixture can be a DNA polymerase that is thermostable and has strand displacement activity. In some embodiments, the DNA polymerase in the reaction mixture is selected from: Phi29 DNA polymerase, Bst DNA polymerase, Pyrophage 3137, Vent DNA polymerase (for example Vent DNA polymerase, Deep Vent polymerase, Vent (-exo) polymerase, Deep Vent (-exo) polymerase of Thermococcus litoralis), TOPO Taq DNA polymerase, 9. One or more of Nm polymerase, Klenow Fragment DNA polymerase I, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, T7phase DNA polymerase variant (lacking 3'-5' exonuclease activity), high-fidelity DNA polymerase, Taq polymerase, Bst DNA polymerase (full length), E. coli DNA polymerase, LongAmp Taq DNA polymerase, and OneTaq DNA polymerase.

In certain embodiments, step (b3) comprises reacting at an extension temperature between 60-90°C (e.g., at 65-90°C, 70-90°C, 75-90°C, 80-90°C, 60-85°C, 60-80°C, 60-75°C, 70-80°C, or at 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75°C) for 10 seconds to 15 minutes (e.g., 1-14, 1-13, 1-12, 1-14). In some embodiments, step (b3) comprises reacting at one or more temperatures between 60-80° C. for 30 seconds to 2 minutes. In some embodiments, step (b3) comprises reacting at 65° C. for 40 seconds. In some embodiments, step (b3) comprises reacting at 75° C. for 40 seconds. In certain embodiments, step (b3) comprises reacting at 65°C for 40 seconds and then reacting at 75°C for 40 seconds.

After the primer extension procedure, steps (b1) to (b3) are repeated for the designated first number of cycles. As described above, in subsequent cycles, the melting temperature in step (b1) is similar to that in the first cycle, but the reaction time may be slightly shorter. In certain embodiments, in cycles subsequent to the first cycle, step (b1) comprises a reaction at a temperature between 90°C and 95°C for 10-50 seconds.

The first cycle number of times described in the present application is at least 2. During the first circulation, the sequence at the 3 ' end of the variable sequence of the first class primer is extended, and the amplified product obtained is a universal sequence at the 5 ' end, and the 3 ' end is a sequence complementary to the genomic template single-stranded sequence, and such an amplified product is also referred to as a half-amplicon. During the second circulation, the previous half-amplicon itself can also be combined with the variable sequence in the first class primer as a DNA template, and the primer extends to the 5 ' end of the amplified product under the action of nucleic acid polymerase, until the universal sequence at the 5 ' end of the amplified product is copied, thus obtaining a universal sequence at the 5 ' end, and a genomic amplified product with a complementary sequence to the universal sequence at the 3 ' end, and such an amplified product is also referred to as a full amplicon. The pre-amplified product described in the present application mainly refers to a full amplicon with a universal sequence at the 5 ' end and a complementary sequence to the universal sequence at the 3 ' end.

In the subsequent amplification after the first cycle, 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 obtained by amplification. The original genomic DNA template and the half-amplicon produced in the initial amplification can be used as new DNA templates again to bind to the primers and start a new round of DNA synthesis. However, since the full amplicon contains complementary sequences at both ends (the universal sequence contained at the 5' end and the complementary sequence of the universal sequence contained at the 3' end), it will form a hairpin structure by itself, and thus cannot be used as a new DNA template again in the next reaction cycle for a new round of DNA synthesis.

In certain embodiments, the number of first cycles is controlled within an appropriate range to ensure that there are sufficient pre-amplification products for subsequent reactions, while not affecting the reaction time of the entire process due to excessive number of cycles. In certain embodiments, the number of first cycles is 2-40 cycles (e.g., 2-40, 4-40, 6-40, 8-40, 10-40, 12-40, 14-40, 16-40, 18-40, 20-40, 15-40, 20-40, 25-40, 30-40, 5-35, 10-35, 15-35, 20-35, 25-35, 30-35, 10-30, 15-30, 20-30, 25-30, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6 , 2-4, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 6-20, 6-18, 6-16, 6-14, 6-12, 6-10, 6-8, 8-20, 8-18, 8-16, 8-14, 8-12, 8-10, 10-20, 10-18, 10-16, 10-14, 10-12, 12-20, 12-18, 12-16, 12-14, 14-20, 1-18, 14-16, 16-20, 16-18, and 18-20 cycles). For example, the number of first cycles is at least 3, at least 4, at least 5, or at least 6, at least 7, at least 8, at least 9, or at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16, at least 17, at least 18, at least 19 or at least 20, or preferably no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, or no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, or no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, no more than 30, no more than 31, no more than 32, no more than 33, no more than 34, no more than 35, no more than 36, no more than 37, no more than 38, no more than 39 or no more than 40. If the number of first cycles is too low, the amount of pre-amplification product obtained will be small. To obtain sufficient amplification product, the number of cycles in amplification step (d) will need to be increased, which will reduce the accuracy of the amplification results. If the number of first cycles is too high, the entire process will take too long due to the long reaction time.

In certain embodiments, step (b3) is followed by a further step (b3'), wherein the reaction mixture is subjected to a suitable temperature program to allow the 3' and 5' ends of the full amplicon in the genomic pre-amplification product to hybridize and bind to form a circular structure. It was previously believed that step (b3') protects the ends of the full amplicon, thereby preventing end-to-end polymerization between two or more full amplicons and thus preventing the joining of two sequences that were originally non-adjacent on the genome. This helps improve the accuracy of the amplification results.

In certain embodiments, the method proceeds directly to the subsequent step (b1) or (c) without further steps (e.g., step (b3')) after step (b3). In this way, the full amplicon does not undergo a specific step to avoid end-to-end aggregation. Therefore, in theory, such an amplification result should have certain defects in accuracy. However, unexpectedly, in the method of the present application, even if the full amplicon is not circularized after step (b3) without a specific step, the final amplification result still has a fairly high accuracy, which is similar to the method using step (b3'). This simplifies the reaction steps while still maintaining the specificity of the reaction.

Step (c): Providing a second reaction mixture

In step (c), the second reaction mixture comprises the pre-amplification product obtained in step (b), the second primer, a mixture of nucleotide monomers and a nucleic acid polymerase, and the second primer comprises a specific sequence and the universal sequence from the 5' end to the 3' end. Since the universal sequence is substantially not complementary to the genomic sequence, if the other parts of the second class primer are designed to be substantially not complementary to the genomic sequence, the second class primer will not directly pair with the genomic DNA and start the replication of the genomic DNA. Therefore, in certain specific embodiments, the second reaction mixture can be obtained by directly adding the second primer to the reaction mixture obtained after the end of step (b). In other embodiments, the reaction mixture obtained after the end of step (b) is purified before step (c) to obtain a purified pre-amplification product, which is then mixed with the second primer, the mixture of nucleotide monomers and a nucleic acid polymerase, and optionally with any other reagents known in the art that can be used for amplification reactions to obtain a second reaction mixture.

i. Second type of primers

The "second primer" described herein belongs to the second class of primers described above. The second class of primers contains the universal sequence of the first class of primers, so that the second class of primers can bind to the complementary sequence of the universal sequence at the 3' end of the full amplicon, thereby further replicating the full amplicon and greatly increasing its number.

In certain embodiments, the second class primers include or consist of a specific sequence and a universal sequence from 5' to 3'. The second class primers can be selected specifically according to different sequencing platforms. In certain embodiments, the second class primers are selected specifically according to a second generation sequencing platform. In certain embodiments, the second class primers are selected specifically according to an NGS sequencing platform of Illumina (such as but not limited to Hiseq, Miseq, etc.) or an Ion torrent of Life technologies. In certain embodiments, the second class primers include sequences that are complementary or identical to part or all of the sequencing primers. In certain embodiments, the sequences that are complementary or identical to part or all of the sequencing primers in the above-mentioned second class primers include or consist of the universal sequence.

The second primer described in this application can be a pair of primers having the structural characteristics of the second class primer or a single primer having the same structure and sequence. In some embodiments, the specific sequence of the second primer includes a sequence at its 3' end that is complementary or identical to part or all of the sequencing primer. In some embodiments, the sequence included in the specific sequence of the second primer that is complementary or identical to part or all of the sequencing primer includes or consists of SEQ ID NO:31 [ACACTCTTTCCCTACACGAC] or SEQ ID NO:32 [GTGACTGGAGTTCAGACGTGT]. In some embodiments, the specific sequence in the second primer further includes a sequence at its 5' end that is complementary or identical to part or all of the capture sequence of the sequencing platform. The capture sequence refers to the sequence included on the sequencing plate in the sequencing platform for capturing the fragment to be sequenced. In some embodiments, the specific sequence of the second primer comprises a sequence that is partially or completely complementary or identical to the capture sequence of the sequencing platform and comprises or consists of SEQ ID NO: 33 [AATGATACGGCGACCACCGAGATCT] or SEQ ID NO: 34 [CAAGCAGAAGACGGCATACGAGAT]. In some embodiments, the specific sequence of the second primer further comprises an identifier sequence (barcode sequence) between the sequence that is partially or completely complementary or identical to the capture sequence of the sequencing platform and the sequence that is partially or completely complementary or identical to the sequencing primer. The identifier sequence is a sequence used to identify a specific set of fragments to be sequenced. When the sequencing platform simultaneously sequences multiple sets of sequencing fragments, the sequencing data can be distinguished by screening the sequencing results for the identifier sequence carried by each set.

In some embodiments, the second primer comprises a primer pair having the same universal sequence and different specific sequences, wherein the different specific sequences each comprise sequences complementary to or identical to part or all of a capture sequence pair used in the same sequencing platform, and/or the different specific sequences each comprise specific sequences complementary to or identical to part or all of different primers in a sequencing primer pair used in the same sequencing run. In some embodiments, the second primer comprises a mixture of the sequences set forth in SEQ ID NO: 35 [AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT] and [CAAGCAGAAGACGGCATACGAGAT X…X GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT], wherein X…X represents an identifier sequence. Those skilled in the art can select the length and specific sequence of the identifier sequence based on actual needs. In some embodiments, the second primer comprises a mixture of the sequences set forth in SEQ ID NO: 35 [AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT] and SEQ ID NO: 36 [CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT]. In some embodiments, the second primer comprises a mixture of the sequences set forth in SEQ ID NO: 37 [CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATGCTCTTCCGATCT] and SEQ ID NO: 38 [CCATCTCATCCCTGCGTGTCTCCGACTCAGCTAAGGTAACGATGCTCTTCCGATCT]. In some embodiments, the second primer includes a mixture of the sequences set forth in SEQ ID NO: 39 [CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATTTGGTAGTGAGTG] and SEQ ID NO: 40 [CCATCTCATCCCTGCGTGTCTCCGACTCAGCTAAGGTAACGATTTGGTAGTGAGTG].

In some embodiments, the concentration of the second primer in the second reaction mixture is 1-15 ng/μL. In some embodiments, the concentration of the second primer in the second reaction mixture is 1-12 ng/μL, 1-10 ng/μL, 1-8 ng/μL, 1-7 ng/μL, 1-6 ng/μL, 1-5 ng/μL, 1-4 ng/μL, 2-3 ng/μL, 2-12 ng/μL, 2-10 ng/μL, 2-8 ng/μL, 2-6 ng/μL, 2-5 ng/μL, 2-4 ng/μL, 2-3 ng/μL, 3-12 ng/μL, 3-10 ng/μL, 3-8 ng/μL, 3-6 ng/μL, or 3-4 ng/μL. In some embodiments, the concentration of the second primer in the second reaction mixture is 2-3 ng/μL. In some embodiments, the second primer in the second reaction mixture is 5-50 pmol. In some embodiments, the second primer is present in the second reaction mixture at 10 pmol, 15 pmol, or 20 pmol.

ii. Other ingredients

In certain embodiments, the nucleic acid polymerase contained in the second reaction mixture is one or more selected from Vent polymerase, Deep Vent polymerase, Vent (-exo) polymerase or Deep Vent (-exo) polymerase of Thermococcus litoralis. In certain embodiments, the second reaction mixture contains Vent polymerase of Thermococcus litoralis. In certain embodiments, the second reaction mixture contains Deep Vent polymerase. In certain embodiments, the second reaction mixture contains Vent (-exo) polymerase. In certain embodiments, the second reaction mixture contains Deep Vent (-exo) polymerase. Various polymerases described in this application can all be obtained from commercial sources, for example, from New England Biolabs.

In certain embodiments, the second reaction mixture may further include appropriate metal ions required for the enzymatic activity of the nucleic acid polymerase (e.g., an appropriate concentration of Mg2 ⁺ ions (e.g., a final concentration of about 1.5 mM to about 8 mM); a nucleotide monomer mixture (e.g., dATP, dGTP, dTTP, and dCTP), bovine serum albumin (BSA), dTT (e.g., a final concentration of about 2 mM to about 7 mM), pure water, appropriate buffer components (e.g., pH adjusters, such ^as Tris HCl and Tris _SO4 ), or one or more other components commonly used in the art (e.g., DNase inhibitors, RNase, _SO42- , ^Cl- , K ⁺ , Ca2 ⁺ , Na ⁺ , and/or (NH4) ⁺ , etc.), etc.).

Step (d): Place in the second temperature cycle program

The method provided in the present application also includes step (d): placing the second reaction mixture obtained in step (c) under a second temperature cycle program, so that the universal sequence of the second type of primer can pair with the 3' end of the genomic pre-amplification product and amplify the genomic pre-amplification product to obtain an amplified genomic amplification product.

Since the genomic pre-amplification product obtained in step (b), i.e., the full amplicon, has a complementary sequence to the universal sequence at the 3' end, it can be complementary to the universal sequence of the second type of primer. Under the action of nucleic acid polymerase, the second type of primer is extended to replicate the full length of the full amplicon.

In the second temperature cycle program, the reaction mixture is first placed in a temperature program capable of opening the DNA double strand (step (d1)). The DNA double strand here mainly refers to the double strand (including the single-stranded hairpin structure molecule of the full amplicon) of the genomic pre-amplification product (i.e., the full amplicon) obtained in step (b). Although the original genomic DNA may still be present in the second reaction mixture at this time, since the second type of primer does not basically bind to the genomic DNA pairing, the original genomic DNA is not the DNA template to be amplified in step (d). A higher reaction temperature (e.g., 90°C-95°C) can be used to react for an appropriate time so that the full amplicon double strand/hairpin structure to be amplified can be denatured into a linear single strand. In certain embodiments, in the temperature program in step (d1), the reaction mixture is placed in a temperature reaction capable of opening the DNA double strand for a sufficient time to ensure that the template DNA double strand or hairpin structure is completely denatured into a single strand, and the temperature program includes a denaturation temperature reaction between 90-95°C (e.g., 95°C) for 5 seconds to 20 minutes (e.g., 30 seconds or 3 minutes). After step (d1), the reaction mixture is placed in a temperature program (step (d2)) capable of melting the double-stranded amplification product generated in the x-th round (x is an integer ≥ 1) of amplification contained therein into a single-stranded template, i.e., reacting at a melting temperature between 90° C. and 95° C. (e.g., 95° C.) for 3-50 seconds (e.g., 20 seconds). It should be understood that step (d2) is not required in the first cycle, but because the temperatures used in the denaturation and melting procedures are similar and the melting time is very short relative to the denaturation time, it can be considered as a delay of step (d1) in the first cycle.

After step (d2), the reaction mixture is placed in a temperature program that allows the second class primer to bind to the DNA single strand obtained in step (d1) or (d2) (step (d3)). Based on the base composition in the second class primer, the Tm value of the second class primer can be calculated, and based on the Tm value, a suitable annealing temperature for the second class primer is found. In certain embodiments, the temperature program in step (d3) includes reacting at an annealing temperature between 45-65°C (e.g., 63°C) for 3-50 seconds (e.g., 40 seconds). In certain embodiments, the second class primer is a mixture of SEQ ID NO:35 and SEQ ID NO:36, and the temperature program in step (d3) includes reacting at 63°C for 3-50 seconds. In certain embodiments, the annealing temperature in step (d3) is higher than the annealing temperature in step (b2). During step (d3), the reaction mixture may still contain unreacted first-class primers from step (b). The variable sequences in these first-class primers may bind to the single-stranded DNA template obtained in step (d3), thereby producing incomplete amplified sequences. When the annealing temperature in step (d3) is higher than the suitable annealing temperature for the first-class primers, binding of the first-class primers to the single-stranded DNA template can be reduced or avoided, thereby selectively allowing the second-class primers to amplify.

After primer annealing is complete, the reaction mixture is subjected to a temperature program that allows the second type of primer bound to the single-stranded amplification product to be extended in length by the action of the nucleic acid polymerase. In certain embodiments, the temperature program in step (d4) includes a reaction at an extension temperature between 60°C and 80°C (e.g., 72°C) for 10 seconds to 15 minutes (e.g., 40 seconds or 3 minutes).

Steps (d2) to (d4) can be repeated for a second number of cycles to obtain the desired amplified genomic amplification product. In this process, the genomic amplification product obtained in step (b) is further replicated and amplified, and the quantity is greatly increased to provide sufficient genomic DNA sequences for subsequent research or operation. In some embodiments, the second number of cycles in step (d5) is greater than the first number of cycles in step (b4). In some embodiments, the number of the second cycle is controlled within an appropriate range so that it can provide a sufficient amount of DNA without affecting the accuracy of amplification due to an excessive number of cycles. In certain embodiments, the second cycle number is 2-40 cycles (e.g., 2-40, 4-40, 6-40, 8-40, 10-40, 12-40, 14-40, 16-40, 18-40, 20-40, 15-40, 20-40, 25-40, 30-40, 5-35, 10-35, 15-35, 20-35, 25-35, 30-35, 1 30, 20-30, 24-30, 26-30, 28-30, 32-40, 32-38, 32-36, or 32-34 cycles).

In certain embodiments, step (d) further comprises, after the second temperature cycling program, subjecting the reaction mixture to the same temperature program as step (d4) (e.g., 72° C.) for an appropriate reaction time (e.g., 40 seconds). The reaction mixture is then placed at 4° C. to terminate the reaction. In certain embodiments, after the reaction in step (d) is completed, the reaction mixture is directly placed at 4° C. to terminate the reaction.

In certain specific embodiments, the present application also provides a method for amplifying a cell genome, the method comprising:

(a) providing a first reaction mixture, wherein the first reaction mixture comprises the genomic DNA, a first primer, a nucleotide monomer mixture, and a nucleic acid polymerase, wherein the first primer comprises a universal sequence and a variable sequence from the 5' end to the 3' end, wherein the first primer comprises a universal sequence and a first variable sequence from the 5' end to the 3' end, and the first variable sequence comprises a first random sequence, wherein the first random sequence is X _a1 X _a2 ... X _an in order from the 5' end to the 3' end, and X _ai (i=1-n) of the first random sequence all belong to the same set, which is selected from B, or D, or H, or V, wherein B={T, G, C}, D={A, T, G}, H={T, A, C}, and V={A, C, G}, wherein X _ai represents the i-th nucleotide at the 5' end of the first random sequence, and n is a positive integer selected from 3-20, wherein the universal sequence and the first variable sequence are directly connected, or the universal sequence and the first variable sequence are connected via a first spacer sequence, and the first spacer sequence is _Ya1 ... _Yam , wherein _Yaj (j=1-m)∈{A, T, G, C}, where _Yaj represents the jth nucleotide at the 5' end of the spacer sequence,

Optionally, the first reaction mixture further comprises a third primer, wherein the third primer comprises the universal sequence and a third variable sequence from the 5' end to the 3' end, the third variable sequence comprises a third random sequence, wherein the third random sequence is X _b1 X _b2 ... X _bn from the 5' end to the 3' end, X _bi (i=1-n) of the third random sequence all belong to the same set, the set is selected from B, or D, or H, or V, wherein B={T, G, C}, D={A, T, G}, H={T, A, C}, V={A, C, G}, and X _bi (i=1-n) and X _ai (i=1-n) belong to different sets, wherein X _bi represents the i-th nucleotide at the 5' end of the first random sequence, and n is a positive integer selected from 3-20, wherein the universal sequence and the third variable sequence are directly connected, or the universal sequence and the third variable sequence are connected via a third spacer sequence, the third spacer sequence is Y _b1 ... Y _bm , wherein Y _bj (j=1-m)∈{A, T, G, C}, where Y _bj represents the jth nucleotide at the 5' end of the spacer sequence, and m is a positive integer selected from 1-3;

(b) subjecting the first reaction mixture to a first temperature cycling program, so that the variable sequences of the first primer and the third primer can 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 comprises the universal sequence and the 3' end comprises a complementary sequence to the universal sequence; wherein the first temperature cycling program comprises:

(b1) reacting at a first denaturation temperature between 90° C. and 95° C. for 1 to 10 minutes (in the first cycle) or 10 to 50 seconds (in subsequent cycles);

(b2) a first annealing temperature between 5-15° C. for 3-50 seconds, a second annealing temperature between 15-25° C. for 3-50 seconds, and a third annealing temperature between 30-50° C. for 3-50 seconds;

(b3) reacting at a first extension temperature(s) between 60° C. and 80° C. for 10 seconds to 15 minutes;

(b4) repeating steps (b1) to (b3) for 2-40 cycles;

(c) providing a second reaction mixture, the second reaction mixture comprising the genomic pre-amplification product obtained in step (b), a second primer, a nucleotide monomer mixture, and a nucleic acid polymerase, wherein the second primer comprises a specific sequence and the universal sequence from the 5' end to the 3' end;

(d) subjecting the second reaction mixture to a second temperature cycling program, so that the universal sequence of the second primer can pair with the 3' end of the genomic pre-amplification product and amplify the genomic pre-amplification product to obtain an amplified genomic amplification product, wherein the second temperature cycling program comprises:

(d1) reacting at a second denaturation temperature between 90° C. and 95° C. for 5 seconds to 20 minutes;

(d2) reacting at a second melting temperature between 90°C and 95°C for 3-50 seconds;

(d3) reacting at a fourth annealing temperature between 45° C. and 65° C. for 3-50 seconds;

(d4) reacting at a second extension temperature between 60° C. and 80° C. for 10 seconds to 15 minutes;

(d5) Repeat steps (d2) to (d4) for 2-40 cycles to obtain a genome amplification product.

In certain embodiments, the genomic DNA in the reaction mixture of step (a) exists inside the cells, that is, the reaction mixture contains cells, and the genomic DNA to be amplified is contained in the cells. In certain embodiments, the reaction mixture of step (a) contains cells and further contains components capable of lysing cells, such as surfactants and/or lysing enzymes. Suitable surfactants can be used, such as one or more of NP-40, Tween, SDS, TritonX-100, EDTA, and guanidine isothiocyanate. Suitable lysing enzymes can also be selected, such as one or more of proteinase K, pepsin, and papain. In such an embodiment, the above-mentioned method for amplifying the cell genome further includes placing the reaction mixture in a lysis temperature cycle program (for example, placing the reaction mixture at 50°C for 20 minutes and then at 80°C for 10 minutes) after step (a) and before step (b), so that the cells are lysed and the genomic DNA is released.

application

In certain embodiments, the product obtained by amplification of the present application method can be further used for sequencing, such as whole-genome sequencing. Since various sequencing analysis platforms such as next-generation sequencing (NGS), gene chips (Microarray), fluorescent quantitative PCR, etc. have high requirements for the starting amount of samples to be analyzed (more than 100ng), if it is necessary to obtain sufficient nucleic acid material for analysis from a single human cell (about 6pg) or a small amount of starting sample, whole-genome amplification is required. The genomic DNA in a biological sample (such as a single cell) can be amplified by the method of the present application, and the amplified product can be sequenced by an appropriate sequencing method in the art. Exemplary sequencing methods include sequencing by hybridization (SBH), sequencing by ligase (SBL), quantitative incremental fluorescent nucleic acid addition sequencing (QIFNAS), stepwise ligation and cleavage, molecular beacon, pyrosequencing, fluorescence in situ sequencing (FISSEQ), fluorescence resonance energy transfer (FRET), multiplex sequencing (U.S. patent application 12/027039; Porreca et al. (2007) NAT. Methods 4:931), polymeric population (POLONY) sequencing (U.S. 6,432,360, U.S. 6,485,944 and PCT/US05/06425), wobble sequencing (PCT US05/27695), TaqMan reporter probe digestion, microparticle rolling cycle sequencing (ROLONY) (U.S. patent application 12/120541), FISSEQ beads (U.S. 7,425,431) and allele-specific oligonucleotide ligation analysis, etc.

In certain embodiments, the sequencing of the amplified product of the present application method can be achieved with a high-throughput method. The high-throughput method is usually fragmented (e.g., by means of enzymatic hydrolysis or mechanical shearing) by the nucleic acid molecules to be sequenced to form a large amount of short fragments of tens of bp to hundreds of bp in length. By sequencing tens of thousands, hundreds of thousands, millions, tens of millions, or even hundreds of millions of such short fragments in parallel in a sequencing reaction, the throughput of sequencing can be greatly improved and the time required for sequencing can be shortened. The sequence of the short fragments measured is processed by software and can be spliced into a complete sequence. Various 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 technologies are also known in the art, such as those described in Landegren et al. (1998) Genome Res. 8:769-76, Kwok (2000) Pharmacogenomics 1:95-100, and Shi (2001) Clin. Chem. 47:164-172.

In certain embodiments, the products amplified by the methods of the present application can also be used to analyze the genotype or genetic polymorphism in genomic DNA, such as single nucleotide polymorphism (SNP) analysis, short tandem repeat (STR) analysis, restriction fragment length polymorphism (RFLP) analysis, variable number tandem repeat (VNTRs) analysis, complex repeat (CTR) analysis or microsatellite analysis, etc. For example, reference can be made to Krebs, J.E., Goldstein, E.S. and Kilpatrick, S.T. (2009). Lewin’s Genes X (Jones & Bartlett Publishers), the disclosure of which is incorporated herein by reference in its entirety.

In certain embodiments, the amplified product obtained by the method of the present application can also be used for medical analysis and/or diagnostic analysis. For example, the biological sample of an individual can be amplified using the method of the present application, and the abnormalities such as mutation, deletion, insertion or fusion between chromosomes in the gene or DNA sequence of interest in the amplified product are analyzed to assess the risk of the individual suffering from a certain disease, the progression stage of the disease, the genotyping of the disease, the severity of the disease or the possibility of the individual's reaction to a certain therapy. Appropriate methods known in the art can be used to analyze the gene or DNA sequence of interest, such as, but not limited to, by nucleic acid probe hybridization, primer-specific amplification, sequencing of the sequence of interest, single-stranded conformation polymorphism (SSCP) etc.

In certain embodiments, 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 are differences between the genomes of different single cells from the same individual, such as between tumor cells and normal cells, the methods of the present application can be used to amplify the genomic DNA of each of the different single cells, and the amplified products can be further analyzed, for example, by sequencing analysis and comparison, or by comparative genomic hybridization (CGH) analysis. Reference may be made to Fan, H.C., Wang, J., Potanina, A. and Quake, S.R. (2011). Whole-genome molecular haplotyping of single cells. Nature Biotechnology 29, 51–57. and 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, the disclosures of which are incorporated herein by reference in their entirety.

In certain embodiments, the method of the present application can be used to identify the haploid structure or haploid genotype in homologous chromosomes.Haploid genotype refers to the combination of alleles on multiple loci inherited together on the chromosome of the same haploid.Biological samples (such as single cells from individual diploids) can be divided into enough parts so that the DNA sequences on the two haploids of homology are statistically separated into different parts.Each part is configured into a reaction mixture, and each reaction mixture is amplified by the method of the present application, and then the amplified product is subjected to sequence analysis and compared with the genome sequence of reference (such as the standard genome sequence of disclosed people, see: International Human Genome Sequencing Consortium, Nature 431,931-945 (2004)), to identify single nucleotide mutations therein.If there is no ready-made reference genome sequence, a region of appropriate length can also be assembled from multiple fragment sequences of genome by the method of de-novo genome assembly for comparison.

In certain embodiments, the product amplified by the method of the present application can be further used for analysis such as gene cloning and fluorescent quantitative PCR.

In certain embodiments, the method of the present application can further include analyzing the amplified product to identify the sequence signature relevant to the disease or phenotype. In some embodiments, analyzing the amplified product includes genotyping the DNA amplified product. In other embodiments, analyzing the amplified product includes identifying the polymorphism of the DNA amplified product, such as single nucleotide polymorphism analysis (SNP). SNP can be detected by some well-known methods, such as oligonucleotide ligation assay (OLA), single base extension method, allele-specific primer extension method, mismatch hybridization method, etc. Disease can be diagnosed by comparing the relationship between SNP and known disease phenotype.

In some embodiments, the sequence features associated with the disease or phenotype include chromosome-level abnormalities, chromosome translocation, aneuploidy, deletion or duplication of part or all of a chromosome, fetal HLA haplotype, and paternal mutation.

In some embodiments, the disease or phenotype can be beta-thalassemia, Down syndrome, cystic fibrosis, sickle cell disease, Tay-Sachs disease, fragile X syndrome, spinal muscular atrophy, hemoglobinopathy, alpha-thalassemia, X-linked diseases (diseases dominated by genes on the X chromosome), spina bifida, anencephaly, congenital heart disease, obesity, diabetes, cancer, fetal sex, or fetal RHD.

Reagent test kit

In another aspect of the present application, a test kit for genomic DNA amplification is provided, comprising the first primer. In some embodiments, the test kit comprises the first primer and the third primer simultaneously. In some embodiments, the test kit further comprises a nucleic acid polymerase, wherein the nucleic acid polymerase is selected from: Phi29 DNA polymerase, Bst DNA polymerase, Pyrophage 3137, Vent polymerase, TOPO Taq DNA polymerase, 9.Nm polymerase, Klenow Fragment DNA polymerase I, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, T7 phase DNA polymerase variant, super-fidelity DNA polymerase, Taq polymerase, Bst DNA polymerase, E.coli DNA polymerase, LongAmp Taq DNA polymerase, OneTaq DNA polymerase, Deep Vent DNA polymerase, Vent (exo-) DNA polymerase, Deep Vent (exo-) DNA polymerase, and any combination thereof. In certain embodiments, the kit further comprises one or more components selected from the group consisting of a nucleotide monomer mixture (e.g., dATP, dGTP, dTTP, and dCTP, e.g., at a total concentration of 1 mmol-8 mmol/μL), dTT (e.g., at a concentration of 1 mmol-7 mmol/μL), a Mg ²⁺ solution (e.g., at a concentration of 2 mmol-8 mmol/μL), bovine serum albumin (BSA), a pH adjuster (e.g., Tris HCl), a DNase inhibitor, an RNase, SO ₄ ^2- , Cl ^- , K ⁺ , Ca ²⁺ , Na ⁺ , and/or (NH ₄ ) ⁺ . In certain embodiments, the kit further comprises a component capable of lysing cells, e.g., one or more surfactants (e.g., NP-40, Tween, SDS, Triton X-100, EDTA, guanidine isothiocyanate), and/or one or more lytic enzymes (e.g., proteinase K, pepsin, papain). In some embodiments, the kit further comprises a second type of primer (i.e., a second primer). It should be understood that the first primer, the second primer and the third primer in the kit all have the structural and sequence characteristics as specifically described above.

In some embodiments, all components of the test kit are stored in separate containers. In some embodiments, all components of the test kit are stored together in the same container. In some embodiments, each primer in the test kit is stored in a separate container, while all other components except the primers are stored in the same container. When the test kit includes a nucleic acid polymerase, the nucleic acid polymerase can be stored in a separate container in a substantially pure form, or alternatively can be mixed with other components.

In some embodiments, the kit may include a mixture containing all reactants other than genomic DNA required for a linear amplification reaction. When such a kit is used for the linear amplification reaction described herein, the sample containing genomic DNA can be directly mixed with the mixture in the kit, and optionally, an appropriate amount of pure water can be added to obtain the required reaction volume to obtain the first reaction mixture in step (a) of the method of the present application. In some embodiments, the kit may include a mixture containing all reactants other than the amplification template required for an exponential amplification reaction. When such a kit is used for the exponential amplification reaction described herein, the DNA template sample containing the amplified product in step (b) can be directly mixed with the mixture in the kit, and optionally, an appropriate amount of pure water can be added to obtain the required reaction volume to obtain the second reaction mixture in step (c) of the method of the present application. In other embodiments, the kit may include both a mixture containing all reactants other than the amplification template required for an exponential amplification reaction and a mixture containing all reactants other than the amplification template required for an exponential amplification reaction. The above mixtures may be two separate ones or a mixed one.

In another aspect of the present application, a test kit that can be used for genomic DNA amplification is also provided, wherein the test kit comprises a first class primer (for example, the first primer and/or the third primer) and a second class primer (for example, the second primer), and further comprises instructions for use, wherein the instructions for use have been recorded in the steps of mixing primers and other components to obtain the first/third reaction mixture before starting to carry out the amplification. In other embodiments, the instructions have also been recorded in the steps of how to carry out amplification described in the present application. The first class primer and the second class primer in the test kit can be placed in different containers respectively, but the instructions can include in the step of mixing the two in the same container before starting to amplify.

Specific embodiments

Example 1: Preliminary verification of amplification effects using different linear amplification primer mixtures

a) Verification using standard genomic DNA samples

The standard genomic DNA was previously extracted from human cells. Dilute the standard genomic DNA with nuclease-free water to a 50 pg/μL DNA solution. Add 1 μL of this solution (as the genomic DNA source) to a PCR tube. For each experimental group, add the primer mix and other relevant reagents listed in Table 1 to obtain the first reaction mixture (containing Na ⁺ , Mg ²⁺ , Cl ⁻ , Tris-Cl, Triton X-100, dNTPs, Vent polymerase, and primer mix).

Linear amplification

Each primer combination/mixture was tested in parallel using two experimental groups to ensure its accuracy, and the reaction mixture of each experimental group was placed in the first temperature control program as follows for reaction:

The primer mix used in each experimental group is shown in Table 1 below.

Table 1: Primers used in each experimental group

Experimental group Primers (or primer mixtures) used 1-2 Universal sequence +NNNNNTTT and universal sequence +NNNNNGGG 3-4 Universal sequence +NNNNNGTTT and universal sequence +NNNNNTGGG 5-6 SEQ ID NO: 7 and SEQ ID NO: 11 7-8 SEQ ID NO:8 and SEQ ID NO:12 9-10 SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11 and SEQ ID NO:12 11-12 Universal sequence + KKKKK

The total amount of the first type primer used in each experimental group was 600 pmoles (if multiple primers were included, the total amount was 600 pmoles, with the same amount of each primer). Different universal sequences can be designed based on different sequencing platforms. In experiments 1-12 of this invention, SEQ ID NO: 6 was selected as the universal sequence based on the Illumina platform.

After the linear amplification process, 1 μl of 10 pmol/μl of the second primer was added to each reaction system to obtain 12 sets of second reaction mixtures. The reaction mixtures of each experimental group were placed in the following second temperature control program for reaction:

Different second primer sequences can be designed based on different sequencing platforms. In experiments 1-12, the following second primer mixture was used based on the Illumina platform:

Second primer-1 (SEQ ID NO: 35): GCT CTT CCG ATCT

Second primer-2 (SEQ ID NO: 36): GCT CTT CCG ATCT

Among them, the bases marked with double underlines in the second primer include the part corresponding to the capture sequence of the sequencing platform, the bases marked in italics are the part corresponding to the sequencing sequence of the sequencing platform, the part marked with a dot is the identification sequence part, which can be replaced with other identification sequences as needed, and the part marked with a single underline is the universal sequence part.

After completing the above amplification temperature control program, each experimental group obtained the amplification product.

Gel electrophoresis (qualitative)

5 μl of unpurified amplified product from each experimental group in Example 1 was taken and added with 1 μl of 6xDNA loading buffer (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., Catalog No. CW0610A) for sample loading. A 1% agarose gel was used, and a DM2000 marker (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., Catalog No. CW0632C) was used. For the electropherogram, see Figure 3 . From left to right, lane 1 is a molecular weight marker, lanes 2-13 are genomic DNA amplified samples, and lane 14 is a molecular weight marker.

As shown in the electrophoresis diagram in Figure 3, the products of experimental groups 1-4 have a clear band near 100 bp, but the product content is relatively low between 100-500 bp. The products of other experimental groups 5-12 are concentrated around 500 bp, and experimental group 5-10 has an unclear band near 100 bp and its concentration is very low compared to experimental groups 1-4. Figure 3 shows that experimental groups 1-4 have more primer aggregates and relatively low reaction efficiency. However, the reaction efficiency of the first type of primers containing the fixed sequence TGGG or GTTT in the variable sequence (experimental groups 3-4) is higher than that of the primers with the fixed sequence GGG or TTT (experimental groups 1-2). The amplification products of experimental groups 5-12 are not much different from each other, but are significantly higher than those of experimental groups 1-4, indicating that the degree of primer aggregate generation is lower than that of groups 1-4.

Purified product (quantitative)

50 μl of unpurified amplified product was purified using a magnetic bead DNA purification and recovery kit (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., Cat. No. CW2508). Purification steps were performed according to the kit instructions. Elution was performed using 20 μl of EB. After purification, 2 μl of the purified product was collected and the concentration was determined using a Nanodrop (AOSHENG, NANO-100). The concentration results are shown in Table 2.

Table 2: Concentration of recovered samples in Figure 3

Experimental group Concentration (ng/μl) Volume (μl) Experimental Group 1 16.647 20 Experimental Group 2 20.158 20 Experimental Group 3 82.085 20 Experimental Group 4 79.712 20 Experimental Group 5 130.52 20 Experimental Group 6 130.024 20 Experimental Group 7 144.456 20 Experimental Group 8 128.041 20 Experimental Group 9 118.145 20 Experimental group 10 101.04 20 Experimental Group 11 126.059 20 Experimental group 12 130.079 20

The amplification efficiency of each experimental group was estimated based on the concentration of the purified amplified product. The concentration test results shown in Table 2 indicate that experimental group 1-2 had the lowest amplification efficiency. Experimental groups 3-4 had relatively lower amplification efficiencies than experimental groups 5-12, but higher than experimental group 1-2. With the exception of experimental groups 1-4, the total amount of amplified product in the other experimental groups was comparable, with no significant differences.

Sequencing (qualitative)

The purified amplified products of the 12 experimental groups were sequenced using Illumina's NGS sequencing platform hiSeq2500 sequencer in a shallow sequencing manner, and the sequenced sequences were aligned to the human reference genome.

Table 3 provides various indicator parameters of high-throughput sequencing results.

Among the various metrics in Table 3, the unique_mapped_of_raw ratio (i.e., the proportion of data that can be mapped to a unique location in the human genome) is the most important metric. As shown in Table 3, the unique_mapped_of_raw ratio for experimental groups 5-12 ranged from 83% to 86%, with little difference between the groups. However, the unique_mapped_of_raw ratio for experimental groups 1-4 was relatively low, ranging from 67% to 79%.

Another important indicator parameter is the mapping ratio in the raw data (mapped_of_raw, that is, the proportion of data in the raw data that can be mapped to a certain position in the human genome). Similarly, as shown in the data in Table 3, the mapped_of_raw ratio in experimental groups 5-12 ranged from 89% to 93%, with little difference between the groups. However, the mapped_of_raw ratio in experimental groups 1-4 was relatively low, ranging from 73% to 86%.

In addition, the data in Table 3 also show that the reading data quality of experimental groups 1-4 is also lower than that of other experimental groups. For example, the proportion of high-quality data in the original data of experimental groups 3 and 4 is only 77.08% and 76.99%, while the proportion of high-quality data in experimental groups 5-12 is between 94% and 96%.

Figure 4 shows the nucleotide readings at the start position of the sequence reads in the sequencing library. As can be seen from Figure 4, the start read regions in experimental groups 1-4 and 9-10 include four bases: A, T, C, and G. The start read regions in experimental groups 5-7 lack A or C, and the start read regions in experimental groups 11-12 lack A and C. Those skilled in the art will know that when sequencing, especially when using SBS sequencing, the first few bases of the sequencing are required to have a high degree of randomness. When the randomness of the first few bases of the sequencing sample as a whole is low, it is necessary to add a certain amount of positive quality control to each loading well when loading the whole plate to increase the base randomness, but this is bound to waste a certain amount of data. For example, when using the library prepared in experimental group 11-12 for whole plate sequencing, due to the lack of A and C in the start read region, a certain amount of positive quality control needs to be added. According to experimental experience, it is generally necessary to add at least 20% of the sample amount of positive quality control to ensure the smooth progress of SBS sequencing.

Table 3: Main quality indicators of high-throughput sequencing results of experimental groups 1-12

b) Validation using AFP single cell samples

The sample to be tested is AFP single cell. Human epidermal fibroblasts (AFP) in good culture state are digested with trypsin, and the digested cells are collected into a 1.5ml EP tube. The collected cells are centrifuged and rinsed with a 1x PBS solution. After rinsing, 1x PBS is added to suspend the cells. Use a pipette to extract a portion of the suspension containing cells, and use a mouth pipette to pick up single cells under a 10x microscope. The volume of the PBS solution absorbed does not exceed 1 microliter, and the picked single cells are transferred into a PCR tube containing 5 microliters of lysis buffer (containing Tris-Cl, KCl, EDTA, Triton X-100 and Qiagen Protease). After a brief centrifugation, the PCR tube is placed on a PCR instrument to perform the lysis program. The specific program is shown in Table 4.

Table 4: Lysis procedure

The lysed reaction solution was used as the genomic DNA source, replacing the standard genomic DNA in Example 1a). Other components and the temperature control procedures for linear and exponential amplification were the same as in Example 1a). The resulting amplified products were detected by gel electrophoresis using the conditions described above. See Figure 5 for the gel electrophoresis diagram, where, from left to right, lane 1 is a molecular weight marker, lanes 2-13 are genomic DNA amplified samples, and lane 14 is a molecular weight marker.

As shown in the electrophoresis diagram in Figure 5, the experimental results are similar to those shown in Figure 3. The products of experimental groups 1-4 showed a distinct band around 100 bp, but the product content was relatively low between 100 and 500 bp. The products of the other experimental groups 5-12 were concentrated around 500 bp, and experimental group 5-12 showed no distinct band around 100 bp. Figure 5 shows that experimental groups 1-4 had a high concentration of primer aggregates, indicating a relatively low reaction efficiency. However, the reaction efficiency of the first-class primers containing the fixed sequence TGGG or GTTT within the variable sequence (experimental groups 3-4) was higher than that of the primers containing the fixed sequence GGG or TTT (experimental groups 1-2). The amplification product yields of experimental groups 5-10 were not significantly different among the groups, but were higher than those of experimental groups 1-4, indicating that the degree of primer aggregates was lower than that of groups 1-4, but slightly lower than that of experimental groups 11-12.

The amplified products were purified and sequenced according to the procedures described above. The sequencing results are shown in Table 5 below. The unique_mapped_of_raw percentage for experimental groups 5-12 ranged from 78% to 85%, with little difference between the groups. However, the unique_mapped_of_raw percentage for experimental groups 1-4 was relatively low, ranging from 63% to 70%. As shown in Table 5, the mapped_of_raw percentage for experimental groups 5-12 ranged from 84% to 92%, with little difference between the groups. However, the mapped_of_raw percentage for experimental groups 1-4 was relatively low, ranging from 73% to 86%. Furthermore, the data in Table 3 also indicate that the read quality of experimental groups 1-4 was lower than that of the other experimental groups. For example, the proportion of high-quality raw data in experimental groups 3 and 4 was only 68.97% and 72.29%, respectively, while the proportion of high-quality data in experimental groups 5-12 ranged from 94% to 96%.

Table 5: Main quality indicators of high-throughput sequencing results of experimental groups 1-12

The above results indicate that the linear amplification primer mixture used in Experimental Groups 1-4 produced a high amount of primer aggregates, significantly reducing amplification efficiency and correspondingly producing lower data volumes under equivalent amplification conditions. Although the A, T, C, and G sequences in the initial sequencing regions of Experimental Groups 1-4 were evenly distributed, the alignment ratios, particularly the unique alignment ratio, were low, making subsequent processing of the sequencing data more difficult.

Example 2: Further verification of the amplification effect using different linear amplification primer mixtures

Human epidermal fibroblasts were isolated and lysed according to the method described in Example 1b) to obtain single-cell genomic DNA, and amplification was performed using the primer mixture used in experimental groups 11/12 and the primer mixture used in experimental groups 9/10 in Table 1, respectively. Ten parallel experiments were performed for each primer mixture (represented by 1_1, 1_2...1_10 and 2_1, 2_2...2_10, respectively). Amplification was performed according to the procedure described in Example 1a) to obtain amplified products, and the amplified products were detected by gel electrophoresis. The electrophoresis detection results are shown in Figure 6. The concentrations of the amplified products in experimental groups 2_1, 2_2...2_10 were slightly lower than those in experimental groups 1_1, 1_2...1_10.

The amplified products were purified according to the procedure described in Example 1a) and the same volume of the purified amplified products was sequenced. The relevant index parameters of the sequencing results are shown in Tables 6-7 below.

Table 6: Main quality indicators of high-throughput sequencing results of experimental groups 1_1, 1_2…1_10

Table 7: Main quality indicators of high-throughput sequencing results of experimental groups 2_1, 2_2…2-10

The data in Tables 6 and 7 show that for experimental groups 2_1, 2_2, …, and 2_10, unique_mapped_of_raw values ranged from approximately 83% to 84%, and mapped_of_raw values ranged from approximately 90% to 91%. For experimental groups 1_1, 1_2, …, and 1_10, unique_mapped_of_raw values ranged from approximately 84% to 85%, and mapped_of_raw values ranged from approximately 91% to 92%, with little difference between the two groups. The statistical results of the amount of data loaded on the machine for each sample in each group are shown in Figure 7. With the exception of experimental group 1_1 (possibly due to errors in the data collection process resulting in extremely low data volumes), the data volumes for experimental groups 1_1, 1_2, …, and 1_10 ranged from 1.5 to 2 MB. The average amount of data loaded on the machine for experimental groups 1_2, 1_3, …, and 1_10 was approximately 1.7 MB. The data volumes for experimental groups 2_1, 2_2, …, and 2_10 ranged from 1.5 to 2.5 megabytes. The average data volume for these groups was approximately 1.8 megabytes, with slightly higher volumes for these groups. Furthermore, the copy number variation coefficients (CVs) for the experimental group data are summarized in Figure 8 : Excluding the significantly abnormal experimental group 1_1, the average CVs for experimental groups 1_2, 1_3, …, and 1_10 were approximately 0.046, while the average CVs for experimental groups 2_1, 2_2, …, and 2_10 were approximately 0.049. There was no significant difference in the CVs between the two experimental groups. Figures 9A-9D show the copy number variation plots for each experimental group. The ordinate represents the chromosome copy number (normal is 2), while the abscissa represents chromosomes 1-22 and the sex chromosomes. As shown in the figure, chromosomes 1-22 in each experimental group were present in approximately two copies except for a few individual data points, while sex chromosomes X and Y were present in approximately one copy each.

Example 3: Detection of pathogenic sites and quality control primers

Pathogenic site detection

35 pathogenicity sites were randomly selected (see Table 8 below for the selected sites) and primers were designed. The selected pathogenicity sites and their corresponding primers are shown in Tables 8 and 9, respectively.

Table 8: 35 randomly selected pathogenic sites

Pathogenic site name Chromosome location 1 SMN1-1 chr5 2 SMN1-2 chr5 3 SMN1-3 chr5 4 SMN1-4 chr5 5 SMN1-1R chr5 6 SMN1-2R chr5 7 SMN1-3R chr5 8 SMN1-4R chr5 9 PDS-IV15 chr7 10 PDS-EXON5 chr7 11 PDS-EXON7+8 chr7 12 PDS-EXON10 chr7 13 PDS-EXON17 chr7 14 PDS-EXON19 chr7 15 HBB3 chr11 16 HBB chr11 17 MMACHC chr1 18 HBA2 chr16 19 GJB2 chr13 20 GJB2-C796 chr13 twenty one ATP7B-8 chr13 twenty two PKHD1-3681 chr6 twenty three PKHD1-1713 chr6 twenty four WASP-C21 chrX 25 WASP-C12 chrX 26 DMD-13exe chrX 27 GJB2 chr13 28 GJB2-c79 chr13 29 PDS-7+8 chr7 30 PDS-10 chr7 31 CFTR-IVS13 chr17 32 IL2RG chrX 33 IL2RGIVS4 chrX 34 FLG-c3319 chr1 35 IDS chrX

Table 9: Primers corresponding to the pathogenicity sites in Table 8

Amplification products from experimental groups 1_1, 1_2, 2_1, and 2_2 in Example 2 were randomly selected as template DNA. PCR was performed on the template DNA using 2x Goldstar Master Mix (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., catalog number CW0960). The amplification system composition is shown in Table 10, and the amplification procedure is shown in Table 11.

Table 10: PCR reaction system for pathogenicity site detection

Components Volume (μl) 2xGoldstarMasterMix 25 Forword primer (10uM) 1 Reverse primer (10uM) 1 Template DNA (10 ng/ul) 1 Warter-Rnase free twenty two total 50

Table 11: PCR amplification procedures for pathogenicity site detection

The amplification results are shown in the gel electrophoresis diagram in Figure 10. The amplification results show that in samples 1_1 and 1_2, pathogenicity sites 4 and 13 were not amplified in either sample, while pathogenicity site 21 was not amplified in sample 1_1, and pathogenicity sites 20, 29, and 31 were not amplified in sample 1_2. In samples 2_1 and 2_2, pathogenicity site 31 was not amplified in either sample, while pathogenicity sites 18, 21, 32, and 35 were not amplified in sample 2_1, and pathogenicity sites 8 and 22 were not amplified in sample 1_2. The results show that there was no significant difference in amplification accuracy or the amount of amplified product between the two primer sets (1_1, 1_2 and 2_1, 2_2).

Quality control primer q-PCR detection

The amplified products, positive controls (same concentration of gDNA), and negative controls (no template) in the experimental groups 1_1, 1_2, 2_1, and 2_2 in Example 2 above were used as template DNA, respectively. 6 sets of quality control primers as shown in Table 12 were used to perform q-PCR detection on the template DNA for DNA sequences on different chromosomes, respectively. 2xFast SYBR Mixture (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., article number CW0955) was used in fluorescent quantitative PCR. The amplification system composition is shown in Table 13, and the amplification program is shown in Table 14.

Table 12: Information of 6 pairs of random primers used in quality control

Table 13: Reaction system for qPCR detection

Components Volume (μl) 2xFastSYBR Mixture 25 Forword primer (10uM) 1 Reverse primer (10uM) 1 Template DNA (10 ng/ul) 1 Warter-Rnase free twenty two total 50

Table 14: Amplification procedures for qPCR detection

The amplification results are shown in Table 15, which lists the q-PCR data for primer pairs CH1, CH2, CH4, CH5, CH6, and CH7 for each set of template DNA. A higher Ct value indicates a lower number of templates corresponding to that primer pair, corresponding to poorer amplification efficiency in gDNA amplification. The amplification results show that CH1, CH2, CH4, CH5, CH6, and CH7 in sample 2_1 all had high amplification efficiencies. Sample 2_2 also had high amplification efficiencies for CH1, CH2, CH4, CH5, CH6, and CH7. There was no substantial difference in amplification between the results for samples 1-1 and 1-2.

Table 15: Amplification efficiency of the four samples in Figure 6 using qPCR detection using the six primer pairs in Table 12

Example 4: The amplification method of the present application is used for the Ion torrent sequencing platform

Fresh blood was drawn and lymphocytes were isolated using lymphocyte separation medium, wherein a portion of the cell suspension was withdrawn with a pipette, and approximately 3 white blood cells were picked using a mouth pipette under a 10x microscope, with the volume of PBS solution not exceeding 1 μl. The picked 3 or so white blood cells were transferred into a PCR tube containing 4 μl of lysis buffer containing Tris-Cl, KCl, EDTA, Triton X-100, and Qiagen Protease, and lysed according to the steps described in Example 1b). The genomic DNA was linearly amplified using the primer mixture used in experimental group 9/10 in Table 1, and the following second primer mixture was used: Second Primer-1 (SEQ ID NO: 37): CCA CTA CGC CTC CGC TTT CCTCTC TAT GGG CAG TCG GTG AT G CTC TTC CGA TCT;

The second primer-2 (SEQ ID NO: 38): T CAGGA T GC TCT TCC GAT CT ; (wherein, the double-underlined bases in the second primer correspond to the capture sequence of the sequencing platform; the portion marked with a dot is the identifier sequence and can be replaced with another identifier sequence as needed; the single-underlined portion is the universal sequence.) Exponential amplification was performed to obtain amplified products. Four sets of experiments were performed in parallel. All other reaction conditions were consistent with those described in Example 1b). The amplification results are shown in the gel electrophoresis in Figure 11.

Homogeneity testing

Two samples amplified according to Example 4 (shown as Sample 1 and Sample 2 in Figure 11) were randomly selected as template DNA. PCR was performed on the template DNA using 2x Goldstar Master Mix (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., Catalog No. CW0960). The primers listed in Table 9 were used to amplify the 35 pathogenic loci listed in Table 8. The amplification system composition is shown in Table 10, and the amplification procedure is shown in Table 11.

The amplification results are shown in Figure 12. The amplification results showed that the 35 pathogenic sites were well amplified in both amplification product samples, and there was no significant difference in amplification accuracy and amplification product quantity between the two samples.

Gene sequencing

After purification, equal volumes of the four samples shown in Figure 11 were taken and sequenced using the Life Technologies Iontorrent sequencing platform PGM ^™ sequencer. The resulting sequences were aligned to the human reference genome. The sequencing results are shown in Table 16 and Figure 13.

Table 16: Sequencing results of semiconductor sequencing (PGM platform) of samples in Figure 11

The data in Table 16 show that for samples 1, 2, 3, and 4, unique_mapped_of_raw values were approximately 68%, mapped_of_raw values were approximately 72%-73%, the data size ranged from 0.38 to 0.53M, and the copy number variation coefficient (CV) for the on-machine data was approximately 0.06. Furthermore, the copy number variation plot for sequencing reads is shown in Figure 13. Except for a few individual data points, chromosomes 1-22 in each experimental group had approximately two copies, while sex chromosomes X and Y each had approximately one copy.

It is worth noting that in the Ion Torrent sequencing platform, there is no need to design a specific universal sequence for the sequencing platform. In principle, any sequence within the length range of 6-60bp that basically does not bind to genomic DNA to produce amplification can be selected as a universal sequence. We also used two other primer combinations for detection in the Ion Torrent sequencing platform: 1) using a first-class primer mixture similar to the first-class primer mixture used in experimental group 9/10 in Table 1: SEQ ID NO: 15, 16, 19 and 20 (but the universal sequence is SEQ ID NO: 1), the corresponding exponential amplification primer mixture is SEQ ID NO: 39 CCA CTA CGC CTC CGC TTT CCT CTC TAT GGG CAG TCG GTG AT T TGG TAG TGA GTG and SEQ ID NO: 40 T CAGGA T TT GGT AGT GAG TG ; 2) using a first-class primer mixture similar to the first-class primer mixture used in experimental group 9/10 in Table 1: SEQ ID NO: 23, 24, 27 and 28 (but the universal sequence is SEQ ID NO: 2), the corresponding exponential amplification primer mixture is SEQ ID NO: 41 CCA CTA CGC CTC CGC TTT CCT CTC TAT GGG CAGTCG GTG AT G AGG TGT GAT GGA and SEQ ID NO: 42 T CAGGA T GA GGT GTG ATG GA . The amplification and sequencing results were not significantly different from those shown in Figure 11 and Table 16 (specific data not provided herein).

Example 5: The amplification method of the present application is used for preimplantation chromosome detection in blastocyst culture medium

The fertilized egg is cultured in vitro, and multiple cells (about 3 cells) of the ectotrophoblast are collected at the blastocyst stage (day 3 of in vitro culture) to detect chromosome copy number abnormalities. The method for collecting ectotrophoblast cells from the blastocyst can be any method known to those skilled in the art, such as but not limited to the method described in Wang L, Cram DS et al. Validation of copy number variation sequencing for detecting chromosome imbalances in human preimplantation embryos. Biol Reprod, 2014, 91(2):37. The isolated trophoblasts were washed three times with PBS and resuspended in 25 μl of PBS solution. A portion of the cell suspension was pipetted and approximately three leukocytes were picked under a 10x microscope using a mouth pipette. The volume of PBS solution used was no more than 1 μl. The three or so leukocytes were transferred into a PCR tube containing 4 μl of lysis buffer containing Tris-Cl, KCl, EDTA, Triton X-100, and Qiagen Protease. The cells were lysed according to the steps described in Example 1b) and genomic DNA was amplified using the primer mixture used in experimental groups 9/10 in Table 1 (four experiments were performed in parallel). The amplified products were purified and sequenced as described in Example 1. The sequencing results are shown in Table 17 below.

Table 17: Sequencing results of blastocyst culture samples

The data in Table 17 show that unique_mapped_of_raw in samples 1, 2, and 3 ranges from 66% to 73%, mapped_of_raw ranges from 71% to 78%, and the data size ranges from 1.4 to 2.6M. Furthermore, the copy number variation plot of the sequencing reads is shown in Figure 14. Except for a few individual data points, chromosomes 1-22 in each experimental group have approximately two copies, while sex chromosomes X and Y each have approximately one copy.

Although the present invention has disclosed various aspects and embodiments, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustration only and are not intended to limit the present invention. The actual scope of protection of the present invention shall be determined by the claims.

Claims (41)

1. A method for amplifying genomic DNA to obtain amplification products, the method comprising: (a) A first reaction mixture is provided, wherein the first reaction mixture comprises a sample containing the genomic DNA, a first primer, a mixture of nucleotide monomers, and a nucleic acid polymerase, wherein the first primer consists of a universal sequence and a first variable sequence from the 5' end to the 3' end, or consists of a universal sequence, a first variable sequence, and a first spacer sequence, wherein the universal sequence and the first variable sequence are directly linked, or the universal sequence and the first variable sequence are linked by the first spacer sequence, the first variable sequence consists of a first random sequence and a fixed sequence at the 3' end of the first variable sequence, wherein the universal sequence is selected such that it does not bind to the genomic DNA to generate amplification, the universal sequence being 6-60 bp in length, wherein the first random sequence is X _a1 X _a2 ... X _an from the 5' end to the 3' end, wherein X _ai (i=1-n) of the first random sequence all belong to the same set, the set being selected from B, or D, or H, or V, wherein B={T, G, C}, D={A, T, G}, H={T, A, C}, V={A, C, G}, wherein X _ai represents the i-th nucleotide at the 5' end of the first random sequence, n is a positive integer selected from 3 to 20, the fixed sequence is a combination of bases that can improve genome coverage, wherein the first spacer sequence is _Ya1 …… _Yam , where _Yaj (j=1-m)∈{A、T、G、C}, where _Yaj represents the j-th nucleotide at the 5' end of the spacer sequence, and m is a positive integer selected from 1 to 3; The first reaction mixture further includes a third primer, wherein the third primer, from its 5' end to its 3' end, is composed of the universal sequence and a third variable sequence, or of the universal sequence, the third variable sequence, and a third spacer sequence, wherein the universal sequence and the third variable sequence are directly linked, or the universal sequence and the third variable sequence are linked through the third spacer sequence, the third variable sequence being composed of a third random sequence and a fixed sequence at the 3' end of the third variable sequence, wherein the third random sequence, from its 5' end to its 3' end, is X _b1 X _b2 … X _bn , and the X _bi (i=1-n) of the third random sequence all belong to the same set, the set being selected from B, or D, or H, or V, where B={T, G, C}, D={A, T, G}, H={T, A, C}, V={A, C, G}, and X _bi (i=1-n) and X _ai (i=1-n) belong to different sets, wherein X _bi represents the i-th nucleotide at the 5' end of the third random sequence, n is a positive integer selected from 3 to 20, wherein the third spacer sequence is Y _b1 ……Y _bm , where Y _bj (j=1-m)∈{A、T、G、C}, where Y _bj represents the j-th nucleotide at the 5' end of the spacer sequence, and m is a positive integer selected from 1 to 3; (b) The first reaction mixture is subjected to a first temperature cycling program for pre-amplification to obtain a pre-amplified product, wherein step (b) enables the variable sequence of the first type of primer to pair with the genomic DNA and amplify the genomic DNA to obtain a genomic pre-amplified product, wherein the 5' end of the genomic pre-amplified product contains the universal sequence and the 3' end contains the complementary sequence of the universal sequence. (c) Providing a second reaction mixture comprising the pre-amplification product obtained in step (b), a second primer, a mixture of nucleotide monomers, and a nucleic acid polymerase, wherein the second primer consists of a specific sequence and the universal sequence from the 5' end to the 3' end, the specific sequence consisting of a sequence complementary to or identical to the sequencing primer, or consisting of a sequence complementary to or identical to the sequencing primer and a sequence complementary to or identical to the capture sequence of the sequencing platform, or consisting of a sequence complementary to or identical to the sequencing primer, a sequence complementary to or identical to the capture sequence of the sequencing platform, and a marker sequence; (d) The second reaction mixture is placed in a second temperature cycling program for amplification to obtain the amplification product. 2. The method according to claim 1, wherein the first random sequence X _{_ai} (i=1-n) all belong to set B, and the third random sequence X _{_bi} (i=1-n) all belong to set D. 3. The method according to claim 1, wherein the fixed sequence of the first primer is selected from CCC, AAA, TGGG, GTTT, GGG, TTT, TNTNG or GTGG, and the fixed sequence of the third primer is selected from CCC, AAA, TGGG, GTTT, GGG, TTT, TNTNG or GTGG. 4. The method according to claim 1, wherein the first variable sequence is selected from X _a1 X _a2 ……X _an TGGG or X _a1 X _a2 ……X _an GTTT, and the third variable sequence is selected from X _b1 X _b2 ……X _bn TGGG or X _b1 X _b2 ……X _bn GTTT. 5. The method of claim 1, wherein the universal sequence is selected such that the amplified product can be directly sequenced. 6. The method according to claim 1, wherein the universal sequence is selected from SEQ ID NO: 1 [TTGGTAGTGAGTG], SEQ ID NO: 2 [GAGGTGTGATGGA], SEQ ID NO: 3 [GTGATGGTTGAGGTA], SEQ ID NO: 4 [AGATGTGTATAAGAGACAG], SEQ ID NO: 5 [GTGAGTGATGGTTGAGGTAGTGTGGAG] or SEQ ID NO: 6 [GCTCTTCCGATCT]. 7. The method according to claim 1, wherein m=1. 8. The method according to claim 7, wherein the first primer is composed of GCTCTTCCGATCTY _{a1Xa1Xa2 Xa3} Xa4 _{Xa5 TGGG} , GCTCTTCCGATCTY _{a1Xa1Xa2 Xa3 Xa4 Xa5} GTTT or a mixture thereof, and the third primer is composed of GCTCTTCCGATCTY _b1Xb1Xb2 Xb3 _{Xb4 Xb5 TGGG} , GCTCTTCCGATCTY _{b1Xb1Xb2 Xb3 Xb4 Xb5} GTTT or a mixture thereof, wherein _Ya1 ∈ {A, T, G, C}, _Yb1 ∈ {A, T, G, C}, _Xai (i=1-5) ∈ {T, G, C}, and _Xbi (i=1-5) ∈ {A, T, G}. 9. The method according to claim 1, wherein the complementary or identical sequence to the sequencing primer in the specific sequence of the second primer consists of SEQ ID NO: 31 [ACACTCTTTCCCTACACGAC] or SEQ ID NO: 32 [GTGACTGGAGTTCAGACGTGT]. 10. The method according to claim 1, wherein the sequence in the specific sequence of the second primer that is complementary to or identical to the capture sequence of the sequencing platform consists of SEQ ID NO: 33 [AATGATACGGCGACCACCGAGATCT] or SEQ ID NO: 34 [CAAGCAGAAGACGGCATACGAGAT]. 11. The method of claim 1, wherein the identifier sequence is located between a sequence complementary to or identical to the capture sequence of the sequencing platform and a sequence complementary to or identical to the sequencing primer. 12. The method of claim 1, wherein the second primer comprises a mixture of primers having the same universal sequence and different specific sequences, the different specific sequences being complementary to or identical to different primers in the same sequencing primer pair used in sequencing. 13. The method of claim 1, wherein the second primer is composed of a mixture of the sequences shown in SEQ ID NO: 35 [AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC GCTCTTCCGATCT] and SEQ ID NO: 36 [CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT]. 14. The method according to claim 1, wherein the nucleic acid polymerase has thermal stability and/or strand displacement activity. 15. The method according to claim 1, wherein the nucleic acid polymerase is selected from: Phi29 DNA polymerase, Bst DNA polymerase, Pyrophage 3137, Vent polymerase, TOPOTaq DNA polymerase, ^9Nm polymerase, KlenowFragment DNA polymerase I, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, T7 phase DNA polymerase variant, ^Phusion® Ultra Fidelity DNA Polymerase, Taq polymerase, Bst DNA polymerase, E. coli DNA polymerase, LongAmp Taq DNA polymerase, OneTaq DNA polymerase, Deep Vent DNA polymerase, Vent (exo-) DNA polymerase, Deep Vent (exo-) DNA polymerase, and any combination thereof. 16. The method of claim 1, wherein the first temperature cycling procedure comprises: (b1) A temperature program capable of opening DNA double strands to obtain DNA single-stranded templates; (b2) A temperature program that enables the first primer and the third primer to bind to the DNA single-stranded template; (b3) A temperature program that enables the first type of primers bound to the single-stranded DNA template to be extended in length under the action of the nucleic acid polymerase to produce pre-amplified products; (b4) Repeat steps (b1) to (b3) for a specified first number of cycles, wherein the specified first number of cycles is greater than 1. 17. The method of claim 16, wherein during the first cycle, the DNA double strand in step (b1) is genomic DNA double strand, and the temperature program includes a denaturation reaction at a temperature between 90-95°C for 1-20 minutes. 18. The method of claim 17, wherein after the first cycle, the temperature program in step (b1) comprises a chain-breaking reaction at a temperature of 90-95°C for 3-50 seconds. 19. The method of claim 17, wherein after the second cycle, the pre-amplification product comprises a genomic pre-amplification product containing the universal sequence at the 5' end and a complementary sequence of the universal sequence at the 3' end. 20. The method of claim 16, wherein the additional step of subjecting the first reaction mixture to a suitable temperature program after step (b1) and before step (b2) to hybridize the 3' end to the 5' end of the pre-amplified genome product to form a hairpin structure is not included. 21. The method of claim 16, wherein step (b2) comprises subjecting the reaction mixture to more than one temperature program to induce sufficient and effective binding of the first type of primer to the DNA template. 22. The method of claim 21, wherein the more than one temperature program comprises: a first temperature between 10-20°C, a second temperature between 20-30°C, and a third temperature between 30-50°C. 23. The method according to claim 22, wherein the step (b2) comprises annealing at a first temperature for 3-60 seconds, annealing at a second temperature for 3-50 seconds, and annealing at a third temperature for 3-50 seconds. 24. The method of claim 16, wherein the temperature program in step (b3) comprises extending the reaction for 10 seconds to 15 minutes at a temperature between 60 and 80°C. 25. The method of claim 16, wherein the first cycle number in step (b4) is 2-40. 26. The method of claim 1, wherein step (d) enables the universal sequence of the second primer to pair with the 3' end of the pre-amplified genome product and amplify the pre-amplified genome product to obtain an amplified genome product. 27. The method of claim 1, wherein step (d) comprises: (d1) A temperature program capable of opening the DNA double strand; (d2) Further, the temperature program for opening the DNA double strand can be activated; (d3) A temperature program that enables the second primer to bind to the single strand of the pre-amplified genome product obtained in step (b); (d4) A temperature program that enables the second primer, which binds to the single strand of the pre-amplified genome product, to extend in length under the action of the nucleic acid polymerase; (d5) Repeat steps (d2) to (d4) for a specified second number of cycles, wherein the specified second number of cycles is greater than 1. 28. The method of claim 27, wherein the DNA double strand in step (d1) is the pre-amplified product of the genome, and the DNA double strand includes a double strand contained in a DNA hairpin structure, and the temperature program includes a denaturation reaction at a temperature between 90-95°C for 5 seconds to 20 minutes. 29. The method of claim 27, wherein the temperature program in step (d2) comprises a chain-breaking reaction at a temperature of 90-95°C for 3-50 seconds. 30. The method of claim 27, wherein the temperature program in step (d3) comprises an annealing reaction at a temperature between 45 and 65°C for 3 to 50 seconds. 31. The method of claim 27, wherein the temperature program in step (d4) comprises extending the reaction for 10 seconds to 15 minutes at a temperature between 60 and 80°C. 32. The method according to claim 1, wherein the genomic DNA is derived from blastomeres, trophoblasts, cultured cells, extracted gDNA, or blastoblast culture medium. 33. A method for amplifying genomic DNA to obtain an amplification product, the method comprising: (a) A first reaction mixture is provided, wherein the first reaction mixture comprises a sample containing the genomic DNA, a first primer, a mixture of nucleotide monomers, and a nucleic acid polymerase, wherein the first primer consists of a universal sequence and a first variable sequence from the 5' end to the 3' end, or consists of a universal sequence, a first variable sequence, and a first spacer sequence, wherein the universal sequence and the first variable sequence are directly linked, or the universal sequence and the first variable sequence are linked by the first spacer sequence, the first variable sequence consists of a first random sequence and a fixed sequence at the 3' end of the first variable sequence, wherein the universal sequence is selected such that it does not bind to the genomic DNA to generate amplification, the universal sequence being 6-60 bp in length, wherein the first random sequence is X _a1 X _a2 ... X _an from the 5' end to the 3' end, wherein X _ai (i=1-n) of the first random sequence all belong to the same set, the set being selected from B, or D, or H, or V, wherein B={T, G, C}, D={A, T, G}, H={T, A, C}, V={A, C, G}, wherein X _ai represents the i-th nucleotide at the 5' end of the first random sequence, n is a positive integer selected from 3 to 20, wherein the first spacer sequence is _Ya1 …… _Yam , where _Yaj (j=1-m)∈{A、T、G、C}, where _Yaj represents the j-th nucleotide at the 5' end of the spacer sequence, m is a positive integer selected from 1 to 3, and the fixed sequence is a base combination that can improve genome coverage; The first reaction mixture further includes a third primer, wherein the third primer, from its 5' end to its 3' end, is composed of the universal sequence and a third variable sequence, or the universal sequence, the third variable sequence, and a third spacer sequence, wherein the universal sequence and the third variable sequence are directly linked, or the universal sequence and the third variable sequence are linked through the third spacer sequence, the third variable sequence being composed of a third random sequence and a fixed sequence at the 3' end of the third variable sequence, wherein the third random sequence, from its 5' end to its 3' end, is sequentially _{Xb1 Xb2} … _Xbn , and the _Xbi (i=1-n) of the third random sequence all belong to the same set, the set being selected from B, or D, or H, or V, where B={T, G, C}, D={A, T, G}, H={T, A, C}, V={A, C, G}, and _Xbi (i=1-n) and _Xai (i=1-n) belong to different sets, wherein X _bi represents the i-th nucleotide at the 5' end of the first random sequence, n is a positive integer selected from 3 to 20, wherein the third spacer sequence is Y _b1 ……Y _bm , where Y _bj (j=1-m)∈{A、T、G、C}, where Y _bj represents the j-th nucleotide at the 5' end of the spacer sequence, and m is a positive integer selected from 1 to 3; (b) The first reaction mixture is placed in a first temperature cycling program such that the first variable sequence of the first primer and the third variable sequence of the third primer can pair with the genomic DNA and amplify the genomic DNA to obtain a genomic pre-amplification product, wherein the 5' end of the genomic pre-amplification product contains the universal sequence and the 3' end contains a complementary sequence to the universal sequence; wherein the first temperature cycling program includes: (b1) The first cycle is a reaction at the first denaturation temperature between 90-95°C for 1-20 minutes, followed by a reaction at the first denaturation temperature between 90-95°C for 3-50 seconds; (b2) React at a first annealing temperature between 10-20°C for 3-60 seconds, a second annealing temperature between 20-30°C for 3-50 seconds, and a third annealing temperature between 30-50°C for 3-50 seconds; (b3) React at a first extension temperature between 60-80°C for 10 seconds to 15 minutes; (b4) Repeat steps (b1) to (b3) for 2-40 cycles; (c) Providing a second reaction mixture comprising the genome preamplification product obtained in step (b), a second primer, a mixture of nucleotide monomers, and a nucleic acid polymerase, wherein the second primer consists of a specific sequence and the universal sequence from its 5' end to its 3' end, the specific sequence consisting of a sequence complementary to or identical to the sequencing primer, or consisting of a sequence complementary to or identical to the sequencing primer and a sequence complementary to or identical to the capture sequence of the sequencing platform, or consisting of a sequence complementary to or identical to the sequencing primer, a sequence complementary to or identical to the capture sequence of the sequencing platform, and a marker sequence; (d) The second reaction mixture is placed in a second temperature cycling program such that the universal sequence of the second primer pairs with the 3' end of the pre-amplified genome product and amplifies the pre-amplified genome product to obtain an amplified genome product, wherein the second temperature cycling program includes: (d1) React at a second denaturation temperature between 90-95°C for 5 seconds to 20 minutes; (d2) React at a second melting temperature between 90-95°C for 3-50 seconds; (d3) React at a fourth annealing temperature between 45-65°C for 3-50 seconds; (d4) React at a second extended temperature between 60-80°C for 10 seconds to 15 minutes; (d5) Repeat steps (d2) to (d4) for 2-40 cycles. 34. The method of claim 33, wherein the universal sequence comprises SEQ ID NO:6; X _ai (i=1-n) of the first random sequence all belong to D, and X _bi (i=1-n) of the third random sequence all belong to B. 35. The method of claim 1, wherein the amplification product obtained in step (d) has completed library construction. 36. A kit for amplifying genomic DNA, the kit comprising a first primer, wherein the first primer consists of a universal sequence and a first variable sequence from the 5' end to the 3' end, or consists of a universal sequence, a first variable sequence, and a first spacer sequence, wherein the universal sequence and the first variable sequence are directly linked, or the universal sequence and the first variable sequence are linked by the first spacer sequence, the first variable sequence consisting of a first random sequence and a fixed sequence at the 3' end of the first variable sequence, wherein the universal sequence is selected such that it does not bind to the genomic DNA to generate amplification, the universal sequence being 6-60 bp in length, wherein the first random sequence is X _a1 X _a2 ... X _an from the 5' end to the 3' end, wherein X _ai (i=1-n) of the first random sequence all belong to the same set, the set being selected from B, or D, or H, or V, wherein B={T, G, C}, D={A, T, G}, H={T, A, C}, V={A, C, G}, wherein X _ai represents the i-th nucleotide at the 5' end of the first random sequence, n is a positive integer selected from 3 to 20, wherein the first spacer sequence is _Ya1 …… _Yam , where _Yaj (j=1-m)∈{A、T、G、C}, where _Yaj represents the j-th nucleotide at the 5' end of the spacer sequence, m is a positive integer selected from 1 to 3, and the fixed sequence is a base combination that can improve genome coverage; The kit further includes a third primer, wherein the third primer consists of the universal sequence and a third variable sequence from the 5' end to the 3' end, or consists of the universal sequence, the third variable sequence, and a third spacer sequence, wherein the universal sequence and the third variable sequence are directly linked, or the universal sequence and the third variable sequence are linked through the third spacer sequence, wherein the third variable sequence consists of a third random sequence and a fixed sequence at the 3' end of the third variable sequence, wherein the third random sequence is X _b1 X _b2 ... X _bn from the 5' end to the 3' end, and X _bi (i=1-n) of the third random sequence all belong to the same set, the set being selected from B, or D, or H, or V, where B={T, G, C}, D={A, T, G}, H={T, A, C}, V={A, C, G}, and X _bi (i=1-n) and X _ai (i=1-n) belong to different sets, wherein X _bi represents the i-th nucleotide at the 5' end of the first random sequence, n is a positive integer selected from 3 to 20, wherein the third spacer sequence is Y _b1 ……Y _bm , where Y _bj (j=1-m)∈{A、T、G、C}, where Y _bj represents the j-th nucleotide at the 5' end of the spacer sequence, and m is a positive integer selected from 1 to 3. 37. The kit of claim 36, wherein the universal sequence comprises SEQ ID NO:6; X _ai (i=1-n) of the first random sequence all belong to D, and X _bi (i=1-n) of the third random sequence all belong to B. 38. The kit of claim 36, wherein the kit is used to construct a whole-genome DNA library. 39. The kit of claim 36, further comprising a nucleic acid polymerase selected from: Phi29 DNA polymerase, Bst DNA polymerase, Pyrophage 3137, Vent polymerase, TOPOTaq DNA polymerase, 9Nm polymerase, Klenow Fragment DNA polymerase ^I , MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, T7 phase DNA polymerase variant, ^Phusion® Ultra Fidelity DNA Polymerase, Taq polymerase, Bst DNA polymerase, E. coli DNA polymerase, LongAmp Taq DNA polymerase, OneTaq DNA polymerase, Deep Vent DNA polymerase, Vent (exo-) DNA polymerase, Deep Vent (exo-) DNA polymerase, and any combination thereof. 40. The kit according to any one of claims 36-39, wherein the kit further comprises one or more ingredients selected from the group consisting of: a mixture of nucleotide monomers, Mg2 ⁺ , dTT, bovine serum albumin, a pH adjuster, a DNase inhibitor, ^RNase , _SO42- , ^Cl- , K ⁺ , Ca2 ⁺ , Na ⁺ , and ( _NH4 ) ⁺ . 41. The kit of claim 36, wherein the kit further comprises a cell lysis agent selected from one or more of proteinase K, pepsin, papain, NP-40, Tween, SDS, Triton X-100, EDTA, and guanidine isothiocyanate.