WO2018040961A1 - 一种建库方法及snp分型方法 - Google Patents

一种建库方法及snp分型方法 Download PDF

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WO2018040961A1
WO2018040961A1 PCT/CN2017/098213 CN2017098213W WO2018040961A1 WO 2018040961 A1 WO2018040961 A1 WO 2018040961A1 CN 2017098213 W CN2017098213 W CN 2017098213W WO 2018040961 A1 WO2018040961 A1 WO 2018040961A1
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sequencing
library
nucleic acid
snp
site
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PCT/CN2017/098213
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French (fr)
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盛司潼
黄思强
卫明
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广州康昕瑞基因健康科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the present invention relates to the field of molecular biology, and more particularly to a method of building a database and a method for typing a SNP.
  • SNP Single Nucleotide Polymorphism
  • the second-generation high-throughput sequencing technology has expanded its application range due to its accurate and sensitive characteristics. It has involved various aspects of life science research and medical research, using second-generation high-throughput sequencing technology to perform SNP sites. The detection is also one of the current research hotspots.
  • the SNP typing method of the second-generation high-throughput sequencing technology when the distance between the SNP site to be tested and the end of the sequencing primer is long, the detection time is greatly prolonged, and is limited by the read length of the sequencing method.
  • the accuracy is greatly reduced; in addition, when multiple SNP sites of multiple susceptibility genes are simultaneously detected, it is usually necessary to design a plurality of different sequencing primers for sequences near different SNP sites to be tested, but different sequencing primers It is easy to cause mutual interference, and it is difficult for the sequencing primer to be accurately anchored at a specific position, thereby increasing the design difficulty of the sequencing primer and possibly reducing the accuracy of the SNP typing detection.
  • the object of the present invention is to provide a database construction method and a SNP classification method, which aims to solve the problem that the SNP classification accuracy in the prior art is affected by the sequencing read length and the detection of multiple SNP sites simultaneously in the same system.
  • the present invention provides a method of building a database, comprising the following steps:
  • the product comprises a IIS type restriction endonuclease cleavage site, and the distance between the IIS type restriction endonuclease cleavage site and the SNP site to be tested is 0 to 5 bases;
  • the first nucleic acid fragment is ligated to the sequencing linker at the first end by a ligase to obtain a library molecule.
  • the specific amplification primer containing the IIS type restriction endonuclease recognition sequence further comprises U, and the positions of the U and the SNP site to be tested on the amplification product are respectively located in the IIS type.
  • the restriction endonuclease cleaves both sides of the site; after the end of the step B, the step of adding the USER enzyme to the reaction system is also included.
  • the 5' end of the specific amplification primer containing the IIS type restriction endonuclease recognition sequence is modified with phosphoric acid, and the phosphate-modified 5' end and the SNP site to be tested are in the amplification product.
  • the upper positions are respectively located on both sides of the IIS type restriction endonuclease cleavage site; after the step B is completed, the step of adding a lambda exonuclease to the reaction system is further included.
  • the sequencing linker has a sequencing primer binding site and a tag sequence.
  • the first end is a sticky end
  • the sequencing linker is a double-stranded nucleic acid molecule comprising a second end, the second end being completely complementary to the first end.
  • the library molecule contains a biotin label on the end which is pre-immobilized on streptavidin- or avidin-labeled magnetic beads.
  • the present invention also provides a SNP typing method comprising the step of sequencing a library molecule prepared according to any of the above methods.
  • the method further comprises the step of immobilizing the library molecule on the solid support.
  • the library is separately constructed according to the different samples to be sequenced, and a plurality of library molecules are obtained, and then the plurality of library molecules are mixed together for sequencing.
  • the plurality of library molecules each contain a different tag sequence.
  • the method for building a library of the present invention by designing a specific amplification primer containing a restriction endonuclease recognition sequence of type IIS, introducing a type IIS restriction endonuclease recognition sequence by complementary pairing of a primer and a template strand during PCR
  • the distance between the sequencing linker on the obtained library molecule and the SNP site to be tested is 0 to 5 bases.
  • Subsequent library molecules containing different SNP sites to be tested can be mixed and then sequenced.
  • the sequencing primers are completely complementary to the sequences on the sequencing linker in the library molecule, so that multiple SNP sites to be tested are sequenced.
  • the sequencing primers can be the same, which reduces the design difficulty of the sequencing primers, ensures the consistency of the sequencing primer anchoring efficiency of each SNP site, avoids the mutual interference caused by the different primers in the sequencing process, and improves the sequencing. Accuracy; in addition, only a small number of sequencing steps can be completed to complete the detection, greatly shortening the sequencing time, and the detection of the SNP site to be tested is not limited by the read length of the sequencing instrument, and can also improve the accuracy.
  • FIG. 1 is a diagram showing the detection of polyacrylamide gel electrophoresis of a Chinese library molecule according to a second embodiment of the present invention.
  • FIG. 2 is a polyacrylamide gel electrophoresis detection diagram of a Chinese library molecule according to a fifth embodiment of the present invention.
  • the present invention provides a first embodiment, a method of building a database, comprising the following steps:
  • the first nucleic acid fragment is ligated to the sequencing linker at the first end by a ligase to obtain a library molecule.
  • the present invention designs a specific amplification primer comprising a restriction endonuclease recognition sequence of type IIS, and introduces a restriction endonuclease recognition sequence of IIS into the amplification product by complementary pairing of the primer and the template strand during the PCR process.
  • the distance between the IIS type endonuclease cleavage site and the SNP site to be tested is 0 to 5 bases, so that the distance between the sequencing linker in the obtained library molecule and the SNP site to be tested is 0 to 5 bases.
  • Subsequent library molecules containing different SNP sites to be tested can be mixed and then sequenced, and the sequencing primers are completely complementary to the sequences on the sequencing linker in the library molecule, so the sequencing primers for various SNP sites to be tested can be The same, the design difficulty of the sequencing primer is reduced, the consistency of the sequencing primer anchoring efficiency of each SNP site is ensured, the mutual interference caused by the different primers in the sequencing process is avoided, and the accuracy of the sequencing is improved.
  • the SNP site to be detected can be detected by only one connection sequencing, which shortens the sequencing time and improves the detection accuracy.
  • the Type IIS restriction endonuclease is used to recognize the Type IIS restriction endonuclease recognition sequence and to cleave at the Type IIS restriction endonuclease cleavage site.
  • the type IIS restriction endonuclease is a restriction endonuclease having a cleavage site outside the recognition sequence, including but not limited to: Acu I, Alw I, Bbs I, BbV I, Bcc I, BceA I, BciV I , BfuA I, Bmr I, Bpm I, BpuE I, Bsa I, BseM II, BseR I, Bsg I, BsmA I, BsmB I, BsmF I, BspCN I, BspM I, BspQ I, BtgZ I, Ear I, Eci I, EcoP15 I, Fau I, Fok I, Hga I, Hph I, HpyA V
  • the sample to be sequenced is a nucleic acid molecule containing a SNP site to be tested, including but not limited to a DNA molecule, a cDNA molecule or an RNA molecule.
  • the PCR amplification described in step A can be either single molecule amplification or non-single molecule amplification.
  • the single molecule amplification is emulsion PCR, bridge PCR or emulsion bridge PCR.
  • the non-single molecule amplification is common PCR amplification, real-time fluorescent quantitative PCR, asymmetric PCR, solid phase PCR, in situ PCR, reverse transcription PCR, nested PCR, degenerate primer PCR, immunoPCR, and reverse PCR or decrement PCR.
  • the method further comprises the steps of: maintaining the reaction system at 80 ° C for 5 minutes and then naturally cooling to room temperature; the solution is cleavage by IIS restriction endonuclease
  • the obtained second nucleic acid fragment different from the first nucleic acid fragment becomes a 3' overhang, so that it is possible to effectively avoid reconnection between the cleavaged first nucleic acid fragment and the second nucleic acid fragment in the subsequent ligation step.
  • the specific amplification primer containing the IIS type restriction endonuclease recognition sequence further comprises U, and the positions of the U and the SNP site to be tested on the amplification product are respectively located in the IIS type restriction endonuclease Both sides of the enzyme cleavage site; after the end of the step B reaction, the USER enzyme is added to the reaction system.
  • U is located on the second nucleic acid fragment different from the first nucleic acid fragment obtained after cleavage, and the single strand containing U on the second nucleic acid fragment is cleaved in the presence of USER enzyme. Fragment, thereby avoiding reconnection between the cleaved first nucleic acid fragment and the second nucleic acid fragment in a subsequent ligation step.
  • the USER enzyme may be added to the reaction system before the IIS type restriction enzyme is added, or may be added to the reaction system simultaneously with the IIS type restriction enzyme, or may be added to the reaction system after the end of the step B reaction; Preferably, the USER enzyme is added to the reaction system simultaneously with a Type IIS restriction enzyme. This protocol simplifies the procedure compared to the technical solution of stepwise addition of the USER enzyme and the IIS type restriction enzyme.
  • the 5' end of the specific amplification primer containing the type IIS restriction endonuclease recognition sequence is phosphate-modified, and the phosphate-modified 5' end and the test SNP site are respectively located in the The IIS type restriction endonuclease cleaves both sides of the site; after the end of the step B reaction, a lambda exonuclease is added to the reaction system.
  • the 5' end of the phosphoric acid modification is located on the second nucleic acid fragment different from the first nucleic acid fragment obtained after cleavage, and the second nucleic acid fragment is present in the presence of the ⁇ exonuclease.
  • the single chain containing the 5'-end phosphate-modified one is degraded into small fragments, thereby avoiding reconnection between the cleaved first nucleic acid fragment and the second nucleic acid fragment in the subsequent ligation step.
  • the 5' end of the specific amplification primer containing the IIS type restriction endonuclease recognition sequence contains a biotin label
  • the 5' end containing the biotin label and the SNP position to be tested are respectively located Two of the IIS type restriction endonuclease cleavage sites
  • the 5' end of the specific amplification primer is preliminarily immobilized on the magnetic beads containing streptavidin or avidin by specific binding of biotin labeling to streptavidin or avidin, or After the end of the reaction in Step B, magnetic beads containing streptavidin or avidin are added to the reaction system.
  • the biotin-labeled 5' end is located on the second nucleic acid fragment different from the first nucleic acid fragment obtained after the cleavage, and the second nucleic acid adsorbed on the magnetic beads after the end of the step B reaction
  • the fragment is separated from the reaction system to avoid reconnection between the cleaved first nucleic acid fragment and the second nucleic acid fragment in a subsequent ligation step.
  • the ligase is not particularly limited, and DNA fragment ligation can be achieved, for example, E. coli DNA ligase, T4 DNA ligase, thermostable DNA ligase, Tth DNA ligase. More preferably, it is a T4 DNA ligase, which has wide applicability and can be attached to both the sticky end and the blunt end.
  • the sequencing linker is a double-stranded nucleic acid molecule containing a sequencing primer binding site; the present invention mixes a library molecule containing different SNP sites to be tested by ligation of a sequencing linker on the first nucleic acid fragment, and then performs sequencing
  • the sequencing primers of the SNPs to be tested are unified, the design difficulty of the sequencing primers is reduced, the consistency of the sequencing primers of each SNP site is ensured, and the mutual generation due to the different primers in the sequencing process is avoided. Interference increases the accuracy of sequencing.
  • the sequencing primer binding site is located at the second end of the sequencing adaptor, the second end is for linking with the first end; and the sequencing primer binding site is located at other positions of the sequencing link.
  • the program shortens the distance between the SNP site to be tested and the sequencing primer, and reduces the sequencing step, thereby shortening the detection time and improving the detection accuracy.
  • the first end may be a blunt end or a sticky end.
  • the first end is an viscous end; at this time, the second end is an viscous end that is completely complementary to the first end, and the binding efficiency of the library molecule formed by the present scheme is compared with the blunt end of the first end. higher.
  • the end of the library molecule contains a biotin label, which may be located at the opposite end of the second end of the sequencing linker, or at the opposite end of the link end of the library molecule and the sequencing linker;
  • a biotin label may be located at the opposite end of the second end of the sequencing linker, or at the opposite end of the link end of the library molecule and the sequencing linker;
  • the library molecules can be conveniently purified after the end of step C.
  • the library molecule is preliminarily immobilized on a solid phase carrier containing streptavidin or avidin by biotin labeling thereon, or may be immobilized on a solid phase carrier by biotin labeling after the ligation reaction of step C is completed. on.
  • the present invention proposes a second embodiment, which constructs a library of MTHFR gene fragments containing the rs1801133 locus using the human whole blood genome as a template, and the steps of building the database are as follows:
  • the centrifuge tube was placed in a PCR machine, and the reaction procedure was set: at 94 ° C for 4 minutes; at 94 ° C for 20 seconds, at 56 ° C for 20 seconds, and at 72 ° C for 1 minute for a total of 30 cycles; The reaction was continued for 3 minutes at 72 ° C; after the completion of the PCR reaction, the first round of PCR product was obtained;
  • 1.0 ⁇ L of the first round of PCR amplification product at a concentration of 0.1 ng/ ⁇ L was added to a 200 ⁇ L centrifuge tube; 2 ⁇ long Taq Mix 10 ⁇ L; upstream primer at a concentration of 10 ⁇ M (SEQ ID NO: 3) 0.4 ⁇ L; 0.4 ⁇ L of a 10 ⁇ M downstream primer (SEQ ID NO: 4); 20 ⁇ L of deionized water; mixed and centrifuged.
  • the centrifuge tube was placed in a PCR machine, and the reaction procedure was set: 4 minutes at 94 ° C; 20 seconds at 94 ° C, 20 seconds at 55 ° C, and 20 seconds at 72 ° C for 30 cycles; After 72°C for 3 minutes; after the PCR reaction is completed, a second round of PCR product is obtained; wherein the upstream primer (SEQ ID NO: 3) contains the BceA I enzyme recognition sequence ACGGC, and the BceA I enzyme cleavage site is located in BceAI enzyme recognition.
  • the length between the sequence and the rs1801133 site, and the SNP site to be tested is 4 bp;
  • step A The second round of PCR product in step A was cleaved by BceA I enzyme, and the reaction system was prepared as follows:
  • step B After the end of the reaction in step B, the following components were added to the system: 0.4 ⁇ L of sequencing linker at a concentration of 2.5 pmol/ ⁇ L; 1 ⁇ L of T4 DNA ligase at a concentration of 2 units/ ⁇ L; 0.3 ⁇ L of ATP at a concentration of 100 mM; mass concentration 10 ⁇ L of 30% polyethylene glycol 6000; deionized water was added to 40 ⁇ L; and reacted at room temperature for 1 hour.
  • the sequencing linker consists of SEQ ID NO: 5 and SEQ ID NO: 6 and is fully complementary to the first terminus, wherein the AGTCGCTGAAGTAGTCGGT sequence on SEQ ID NO: 5 is the sequencing primer binding sequence, the 5' end of SEQ ID NO: Containing biotin labeling, and pre-fixed on streptavidin-containing magnetic beads; after the reaction is completed, the centrifuge tube is placed on a magnetic rack, and the supernatant is separated and removed to obtain a sequencing linker and adsorbed on the magnetic beads.
  • a library molecule of the rs1801133 locus is the sequencing primer binding sequence, the 5' end of SEQ ID NO: Containing biotin labeling, and pre-fixed on streptavidin-containing magnetic beads
  • the library molecules were verified and the following reaction system was prepared to amplify the library molecules:
  • Dilute 100-fold magnetic bead suspension 1 ⁇ L; 2 ⁇ long Taq Mix 10 ⁇ L; concentration 10 ⁇ M upstream primer (SEQ ID NO: 7) 0.4 ⁇ L; concentration 10 ⁇ M downstream primer (SEQ ID NO: 4) 0.4 ⁇ L; 20 ⁇ L deionized Water; mix and centrifuge. Place the centrifuge tube in the PCR machine and set the reaction procedure: 2 minutes at 94 °C; 20 seconds at 94 °C, 20 seconds at 54 °C, and 10 seconds at 72 °C for a total of 25 cycles The temperature was maintained at 72 ° C for 3 minutes; after the completion of the PCR reaction, the verified product was obtained.
  • Fig. 1 The results of the polyacrylamide gel electrophoresis of the verification product are shown in Fig. 1.
  • 0 is a molecular size marker
  • lane 1 is a verification product.
  • the verified product shows a target band near the 110 bp position, and the theoretical expected library.
  • the molecular size is completely consistent, indicating that the method of the present invention can achieve the construction of the sample to be sequenced.
  • the present invention proposes a third embodiment, which constructs a library of MTHFR gene fragments containing the rs1801133 locus using the human whole blood genome as a template, which differs from the second embodiment in that the second round of PCR amplification amplification primers in step A is : upstream primer (SEQ ID NO: 8), downstream primer (SEQ ID NO: 4); wherein the upstream primer (SEQ ID NO: 8) contains the Acu I recognition sequence CTGAAC, and the Acu I cleavage site is located at Acu I Between the enzyme recognition sequence and the rs1801133 site, and the length between the SNP site to be tested is 4 bp;
  • step A The amplification product in step A is cleaved by using Acu I enzyme, and the reaction system is prepared as follows:
  • the sequencing linker in step C consists of SEQ ID NO: 9 and SEQ ID NO: 10 and is fully complementary to the first terminus, wherein the AGTCGCTGAAGTAG TCGGT sequence on SEQ ID NO: 9 is a sequencing primer binding sequence.
  • the amplification reaction system is prepared by the upstream primer (SEQ ID NO: 11) and the downstream primer (SEQ ID NO: 12) and amplified to obtain a verification product.
  • the product was verified by polyacrylamide gel electrophoresis, and it was verified that the target band appeared near 110 bp, which was completely consistent with the theoretical expected molecular size of the library, indicating that the method of the present invention can realize the construction of the sample to be sequenced.
  • the present invention proposes a fourth embodiment, which constructs a library of MTHFR gene fragments containing the rs1801133 locus using the human whole blood genome as a template to prepare two different reaction systems, which differs from the second embodiment in the reactions in the step A.
  • the primers amplified by the second round of PCR were: upstream primer (SEQ ID NO: 13) and downstream primer (SEQ ID NO: 4); upstream primer (SEQ ID NO: 14) and downstream primer (SEQ ID NO: 4).
  • the upstream primer (SEQ ID NO: 13) is a DNA sequence comprising the BsmFI enzyme recognition sequence GGGAC and two U; the upstream primer (SEQ ID NO: 14) is a DNA sequence containing the BtgZI enzyme recognition sequence GCGATG And two U.
  • the sequencing linkers of the two reaction systems in step C are composed of SEQ ID NO: 5 and SEQ ID NO: 15;
  • the verification product of the library molecule of the present example was subjected to polyacrylamide gel electrophoresis, and it was verified that the target band appeared near the 110 bp position, which completely coincided with the theoretical expected library molecular size, indicating that the method of the present invention can realize the construction of the sample to be sequenced.
  • the present invention proposes a fifth embodiment, which uses the human whole blood genome as a template to establish five different reaction systems, and respectively constructs a CYP2C9 gene fragment containing the rs1799853 locus, a CYP2C9 gene fragment containing the rs1057910 locus, and a rs9923231 locus.
  • the VKORC1 gene fragment, the CYP2C19 gene fragment containing the rs4244285 locus, and the gene library containing the CYP2C19 gene fragment of the rs4986893 locus are constructed as follows:
  • 1.0 ⁇ L of human whole blood DNA molecule at a concentration of 50 ng/ ⁇ L was added to a 200 ⁇ L centrifuge tube; 10 ⁇ L of 2 ⁇ long Taq Mix; 0.4 ⁇ L of upstream primer at a concentration of 10 ⁇ M; and a downstream primer at a concentration of 10 ⁇ M 0.4 ⁇ L; 20 ⁇ L of deionized water; mix and centrifuge.
  • the centrifuge tube was placed in a PCR machine, and the reaction procedure was set: at 94 ° C for 4 minutes; at 94 ° C for 20 seconds, at 49 ° C for 20 seconds, and at 72 ° C for 1 minute for a total of 30 cycles; The reaction was continued for 3 minutes at 72 ° C; after the completion of the PCR reaction, the first round of PCR product was obtained.
  • the first round of PCR amplification amplification primers in each reaction system are: upstream primer (SEQ ID NO: 16) and downstream primer (SEQ ID NO: 17); upstream primer (SEQ ID NO: 18) and downstream Primer (SEQ ID NO: 19); upstream primer (SEQ ID NO: 20) and downstream primer (SEQ ID NO: 21); upstream primer (SEQ ID NO: 22) and downstream primer (SEQ ID NO: 23); upstream Primer (SEQ ID NO: 24) and downstream primer (SEQ ID NO: 25);
  • 1.0 ⁇ L of the first round of PCR amplification product at a concentration of 0.1 ng/ ⁇ L was added to a 200 ⁇ L centrifuge tube; 2 ⁇ L of Long Taq Mix; 0.4 ⁇ L of the upstream primer at a concentration of 10 ⁇ M; and a concentration of 10 ⁇ M.
  • the centrifuge tube was placed in a PCR machine, and the reaction procedure was set: at 94 ° C for 4 minutes; at 94 ° C for 20 seconds, at T ° C for 20 seconds, and at 72 ° C for 20 seconds for a total of 30 cycles; After 72 ° C for 3 minutes; after the completion of the PCR reaction, a second round of PCR product was obtained; wherein the annealing temperatures T in each reaction system were 50 ° C, 50 ° C, 53 ° C, 45 ° C and 45 ° C, respectively.
  • the second round of PCR amplification amplification primers were: upstream primer (SEQ ID NO: 26) and downstream primer (SEQ ID NO: 27); upstream primer (SEQ ID NO: 28) and downstream primer (SEQ ID NO: 29); upstream primer (SEQ ID NO: 30) and downstream primer (SEQ ID NO: 31); upstream primer (SEQ ID NO: 32) and downstream primer (SEQ ID NO: 33); upstream primer (SEQ ID NO: 34) and downstream primer (SEQ ID NO: 35); wherein the upstream primers are SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 contains the BceA I enzyme recognition sequence ACGGC, the BceA I enzyme cleavage site is located between the BceA I enzyme recognition sequence and the SNP site to be tested, and the length between the SNP site and the SNP site to be tested is 4 bp;
  • step A The second round of PCR product in step A was cleaved by BceA I enzyme, and the reaction system was prepared as follows:
  • Purified second round PCR product 200 ng; concentration of 2 units/ ⁇ L BceAI enzyme 1 ⁇ L; 10 ⁇ NEBuffer 3.1 2.5 ⁇ L, deionized water 1.5 ⁇ L; reaction at 37 ° C for 1 hour, 65 ° C inactivation for 20 minutes, a first nucleic acid fragment comprising a SNP site to be tested, and the first nucleic acid fragment is cleaved to form a first end having 2 protruding bases at the 5' end;
  • step B After the completion of the reaction in step B, the following components were added to each reaction system: 0.4 ⁇ L of a sequencing linker at a concentration of 2.5 pmol/ ⁇ L, 1 ⁇ L of T4 DNA ligase at a concentration of 2 units/ ⁇ L, and 0.3 ⁇ L of ATP at a concentration of 100 mM.
  • SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42 respectively contain the tag sequences ACGT, TGCA, GTAC, CATG, AGTC, and both contain sequencing
  • the primer binding site AGTCGCTGAAGTAGTCGGT has a biotin label at the 5' end and is pre-fixed on the magnetic beads containing streptavidin; after the reaction is completed, the centrifuge tube is placed on a magnetic rack, and the supernatant is separated and removed. Library molecules adsorbed on the magnetic beads containing the SNP site to be tested and the sequencing linker are respectively obtained;
  • the primers for amplification reaction in each system are: upstream primer (SEQ ID NO: 7) and downstream primer (SEQ ID NO: 44); upstream primer (SEQ ID NO: 7) and downstream primer (SEQ ID NO: 29); upstream primer (SEQ ID NO: 7) and downstream primer (SEQ ID NO: 31); upstream primer (SEQ ID NO: 7) and downstream primer (SEQ ID NO: 45); upstream primer (SEQ ID NO: 7) and downstream primers (SEQ ID NO: 35);
  • Fig. 2 The results of the agarose gel electrophoresis of the verification product are shown in Fig. 2.
  • 0 is a molecular size marker
  • lanes 1-5 are the verification products of the above five reaction systems, and targets appear near the positions of 82 bp, 67 bp, 121 bp, 127 bp, and 112 bp.
  • the band which is fully consistent with the theoretical predicted library molecule, demonstrates that the method of the present invention enables the construction of a sample to be sequenced.
  • the present invention also proposes a sixth embodiment, a SNP typing method comprising the step of sequencing a library molecule prepared according to the database construction method of any of the above embodiments.
  • the library molecule of the invention contains a sequencing linker, and the measurement of a plurality of different SNP sites in the same system is unified.
  • the primers avoid mutual interference between the sequencing primers.
  • the method further comprises the step of immobilizing the library molecule on the solid support.
  • the addressable fixed means that the position information can be fixed. That is, the library molecules immobilized at each specific position on the solid phase carrier can be clearly distinguished from the library molecules immobilized at other positions.
  • the library molecule containing the sequencing linker can be immobilized on the solid phase carrier by direct or indirect means.
  • the present invention provides an embodiment in which a library of molecules containing a sequencing linker hybridizes to a flow cell to achieve addressable immobilization of library molecules containing sequencing linkers
  • the present invention also proposes another embodiment in which a library molecule containing a sequencing linker is immobilized on a microsphere, and the microsphere is preliminarily immobilized on a solid phase carrier, thereby realizing addressable fixation of the library molecule containing the sequencing linker.
  • the present invention provides an embodiment in which a library molecule containing a sequencing linker is first immobilized on a microsphere, and then the microsphere is immobilized on a solid phase carrier. Thereby an addressable fixation of the library molecule containing the sequencing linker is achieved.
  • the library is separately constructed according to the different samples to be sequenced, and a plurality of library molecules are obtained, and then the plurality of library molecules are mixed and sequenced in the same system.
  • the plurality of library molecules respectively contain different tag sequences, and the program can distinguish the sequencing results of different library molecules by using the tag sequences on the library molecules.
  • the tag sequence is located on a sequencing linker on the library molecule.
  • the method of sequencing is a second generation high throughput gene sequencing technology, including but not limited to ligation sequencing or synthetic sequencing.
  • the ligation sequencing method is based on the fidelity of a ligase in a ligation reaction between nucleic acid fragments, using a nucleic acid fragment to be sequenced as a template, a sequencing primer and an oligonucleotide probe (the probe).
  • the ligation reaction is carried out with a fluorescent label at a specific position, and the information of the sequence corresponding to the specific position of the fluorescent probe with the fluorescent label is determined by detecting the fluorescent label on the ligation product.
  • connection sequencing methods commonly used in the market, including but not limited to: Pstar connection sequencing method of Shenzhen Huayinkang Gene Technology Co., Ltd., ABI's connection sequencing method, and Complete Genomics' connection sequencing method.
  • the synthetic sequencing method is based on the fidelity of the polymerase in the process of extending the nucleic acid strand, and the nucleic acid fragment to be sequenced is used as a template, and the anchor primer (also referred to as a sequencing primer, which is complementary to the strand of the nucleic acid fragment to be sequenced) is complementary. Binding to the nucleic acid fragment to be sequenced, sequence information of the corresponding position on the nucleic acid fragment to be sequenced is determined by detecting a signal generated during the extension.
  • synthetic sequencing methods commonly available on the market, including but not limited to: Illumina's Solexa synthetic sequencing method, Roche's 454 synthetic sequencing method, Life Technologies' Iontorrent, and Ion Proton synthetic sequencing method.
  • any single strand of the sequencing adaptor can be immobilized on a solid support and retained as a sequencing template.
  • the present invention also provides a seventh embodiment, a method for detecting the rs1801133 site of the MTHFR gene.
  • the present embodiment further includes the following steps on the basis of the second embodiment:
  • the 5' end of the sequencing primer (SEQ ID NO: 12) was modified with phosphoric acid and immobilized on the sequencing primer binding sequence of the sequencing linker, using a degenerate nine-band NNNNXNNNN with a fluorescent group complementary to the detection site.
  • the sequencing probe was sequenced and the rs1801133 locus of the MTHFR gene was determined to be T.
  • the present invention also provides an eighth embodiment, a method for detecting the rs1801133 site of the MTHFR gene.
  • the present embodiment further includes the following steps on the basis of the third embodiment:
  • the library molecule prepared in step C was hybridized to the flow cell; 20 ⁇ L of a 0.1 M NaOH solution was added to the library molecule to denature the template into a single strand, and the supernatant was separated and removed, using 20 ⁇ L of 1 ⁇ TE (including mass concentration). Washed twice with 0.01% triton), washed once with 20 ⁇ L of 1 ⁇ TE, finally resuspended in 10 ⁇ L of 1 ⁇ TE for use as a sequencing template; immobilized the sequencing primer (SEQ ID NO: 12) on the sequencing adaptor using an illumina sequencer On the sequencing primer binding sequence, after four sequencing experiments, the rs1801133 locus of the MTHFR gene was determined to be T.
  • the present invention also provides a ninth embodiment, which simultaneously detects the rs1799853 site, the rs1057910 site on the CYP2C9 gene fragment, the rs9923231 site on the VKORC1 gene fragment, the rs4244285 site on the CYP2C19 gene fragment, and the rs4986893 site.
  • the fourth embodiment differs in that it further includes the following steps:
  • the library molecules adsorbed on the magnetic beads obtained in each reaction system in the step C were mixed, and 20 ⁇ L of a 0.1 M NaOH solution was added thereto to denature the template into a single strand, and the supernatant was separated and removed, and 20 ⁇ L of 1 ⁇ TE was used. Washed twice with triton containing v/v concentration of 0.01%, washed once with 20 ⁇ L of 1 ⁇ TE, and finally resuspended in 10 ⁇ L of 1 ⁇ TE for sequencing template; using high-throughput gene of Shenzhen Huayinkang Gene Technology Co., Ltd.
  • the sequencer Pstar IIA was sequenced by ligation sequencing, and the sequencing primer (SEQ ID NO: 43) was immobilized on the sequencing primer binding site of the sequencing linker. After one ligation and ligation, the SNPs were determined to be C, A, T, G, G.
  • sequencing primers for detecting multiple SNP sites in the same system are unified, thereby avoiding mutual interference between different primers.

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Abstract

提供了一种建库方法，包括：A、利用特异性扩增引物组对含待测SNP位点的待测序样本进行PCR扩增，得到扩增产物；所述引物组中的至少一种引物上含有IIS型限制性内切酶识别序列，所述扩增产物上含有IIS型限制性内切酶切割位点，所述IIS型限制性内切酶切割位点与所述待测SNP位点之间的距离为0至5个碱基；B、采用IIS型限制性内切酶对扩增产物进行酶切，得到含有待测SNP位点的第一核酸片段，且所述第一核酸片段上经酶切形成第一末端；C、在连接酶作用下，所述第一核酸片段在第一末端处连接测序接头，得到文库分子。还提供了一种SNP分型方法。所述方法缩短了位点检测时间，提高了检测的准确性，且统一了同一体系中进行多个位点检测的测序引物。

Description

一种建库方法及SNP分型方法 技术领域
本发明涉及分子生物学领域，更具体地说，涉及一种建库方法及SNP分型方法。
背景技术
单核苷酸多态性(Single Nucleotide Polymorphism，SNP)是指基因组上单个核苷酸位置上存在转换、颠换、插入、缺失等变化，其数量很多，多态性丰富。SNP被认为是遗传标志，人体许多表型差异、对药物或疾病的易感性等等都可能与SNP有关，因此SNP的分型对诸多疾病的治疗和用药有着积极的意义。
针对基因检测，二代高通量测序技术因其准确、灵敏的特性，应用范围不断扩大，已涉及生命科学研究以及医学研究的各个不同方面，利用二代高通量测序技术来进行SNP位点的检测也是目前的研究热点之一。但是，基于二代高通量测序技术的SNP分型方法，当待测SNP位点与测序引物末端之间距离较长时，检测时间会大大延长，且受限于测序方法的读长，检测的准确性会大大降低；此外，当同时对多个易感基因的多个SNP位点检测时，通常需要针对不同待测SNP位点附近的序列设计多种不同的测序引物，但不同测序引物之间容易产生相互干扰，测序引物难以准确锚定在特定位置，从而增加了测序引物的设计难度，可能降低SNP分型检测的准确率。
因此，需要一种新的建库方法及SNP分型方法，使得对待测SNP位点检测的准确性不受测序读长的影响；且能够避免在同一体系中同时对多个待测SNP位点进行检测时，不同测序引物之间相互干扰的现象。
发明内容
本发明的目的在于提供一种建库方法及SNP分型方法，旨在解决现有技术中SNP分型准确性受测序读长影响，以及在同一体系中同时对多个SNP位点检测时不同测序引物之间相互干扰的问题。
为了实现发明目的，本发明提供了一种建库方法，包括以下步骤：
A、利用特异性扩增引物组对含待测SNP位点的待测序样本进行PCR扩增，得到扩增产物；所述特异性扩增引物组中的至少一种扩增引物上含有IIS型限制性内切酶识别序列，所述扩增 产物上含有IIS型限制性内切酶切割位点，所述IIS型限制性内切酶切割位点与所述待测SNP位点之间的距离为0至5个碱基；
B、采用IIS型限制性内切酶对扩增产物进行酶切，得到含有待测SNP位点的第一核酸片段，且所述第一核酸片段上经酶切形成第一末端；
C、在连接酶作用下，所述第一核酸片段在第一末端处连接测序接头，得到文库分子。
优选的，所述含有IIS型限制性内切酶识别序列的特异性扩增引物上还含有U，所述U和所述待测SNP位点在扩增产物上的位置分别位于所述IIS型限制性内切酶切割位点的两侧；所述步骤B结束后，还包括向反应体系中加入USER酶的步骤。
优选的，所述含有IIS型限制性内切酶识别序列的特异性扩增引物的5’端经过磷酸修饰，所述经磷酸修饰的5’端和所述待测SNP位点在扩增产物上的位置分别位于所述IIS型限制性内切酶切割位点的两侧；所述步骤B结束后，还包括向反应体系中加入λ核酸外切酶的步骤。
优选的，所述测序接头上有测序引物结合位点和标签序列。
优选的，所述第一末端为粘性末端；所述测序接头为含有第二末端的双链核酸分子，所述第二末端与第一末端完全互补配对。
优选的，所述文库分子的末端上含有生物素标记，其被预先固定在含链霉亲和素或亲和素标记的磁珠上。
本发明还提供了一种SNP分型方法，包括对按上述任一种建库方法制得的文库分子进行测序的步骤。
优选的，所述方法还包括将文库分子可寻址的固定在固相载体上的步骤。
优选的，当检测的待测序样本有多个时，根据待测序样本的不同分别进行建库，获得多种文库分子，再将多种文库分子混合在一起进行测序。
优选的，所述多种文库分子上分别含有不同的标签序列。
本发明的建库方法，通过设计含有IIS型限制性内切酶识别序列的特异性扩增引物，通过在PCR过程中引物与模板链的互补配对将IIS型限制性内切酶识别序列引入至扩增产物上，使获得的文库分子上的测序接头与待测SNP位点之间的距离为0至5个碱基。后续可将含有不同待测SNP位点的文库分子混合，然后进行测序，测序时，测序引物与文库分子中的测序接头上的序列完全互补配对，这样，对多种待测SNP位点进行测序的测序引物可以是相同的，降低了测序引物的设计难度，保证了各SNP位点的测序引物锚定效率的一致性，避免了测序过程中由于引物不同而产生的相互干扰，提高了测序的准确性；另外，只需进行较少次数的测序步骤即可完成检测，大大缩短了测序时间，且使对待测SNP位点的检测不受测序仪器读长的限制，也能提高准确性。
附图说明
图1是本发明第二实施例中文库分子的聚丙烯酰胺凝胶电泳检测图。
图2是本发明第五实施例中文库分子的聚丙烯酰胺凝胶电泳检测图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。
本发明提出第一实施例，一种建库方法，包括以下步骤：
A、利用特异性扩增引物组对含待测SNP位点的待测序样本进行PCR扩增，得到扩增产物；所述特异性扩增引物组中的至少一种扩增引物上含有IIS型限制性内切酶识别序列，所述扩增产物上含有IIS型限制性内切酶切割位点，所述IIS型限制性内切酶切割位点与所述待测SNP位点之间的距离为0至5个碱基；
B、采用IIS型限制性内切酶对扩增产物进行酶切，得到含有待测SNP位点的第一核酸片段，且所述第一核酸片段上经酶切形成第一末端；
C、在连接酶作用下，所述第一核酸片段在第一末端处连接测序接头，得到文库分子。
本发明设计含有IIS型限制性内切酶识别序列的特异性扩增引物，通过在PCR过程中引物与模板链的互补配对，将IIS型限制性内切酶识别序列引入至扩增产物上，使IIS型内切酶切割位点与待测SNP位点之间的距离为0至5个碱基，从而使获得的文库分子中的测序接头与待测SNP位点之间的距离为0至5个碱基。后续可将含有不同待测SNP位点的文库分子混合，然后进行测序，测序引物与文库分子中的测序接头上的序列完全互补配对，因此对多种待测SNP位点进行的测序引物可以是相同的，降低了测序引物的设计难度，保证了各SNP位点的测序引物锚定效率的一致性，避免了测序过程中由于引物不同而产生的相互干扰，提高了测序的准确性。此外，若采用合成测序法，只需要进行较少次数的测序步骤即可完成对待测SNP位点的检测，缩短了检测时间，提高了检测的准确性；采用连接测序法，可以根据待测SNP位点离测序引物的距离设计测序探针，只需进行一次连接测序即可完成对待测SNP位点的检测，缩短了测序时间，提高了检测的准确性。
所述IIS型限制性内切酶用于识别IIS型限制性内切酶识别序列并在IIS型限制性内切酶切割位点处进行切割。所述IIS型限制性内切酶为切割位点在识别序列之外的限制性内切酶，包括但不限于：Acu Ⅰ、Alw Ⅰ、Bbs Ⅰ、BbV Ⅰ、Bcc Ⅰ、BceA Ⅰ、BciV Ⅰ、BfuA Ⅰ、Bmr Ⅰ、Bpm Ⅰ、BpuE Ⅰ、Bsa Ⅰ、BseM Ⅱ、BseR Ⅰ、Bsg Ⅰ、BsmA Ⅰ、BsmB Ⅰ、BsmF Ⅰ、BspCN Ⅰ、 BspM Ⅰ、BspQ Ⅰ、BtgZ Ⅰ、Ear Ⅰ、Eci Ⅰ、EcoP15 Ⅰ、Fau Ⅰ、Fok Ⅰ、Hga Ⅰ、Hph Ⅰ、HpyA V、Mbo Ⅱ、Mly Ⅰ、Mme Ⅰ、Mnl Ⅰ、NmeAⅢ、Ple Ⅰ、Sap Ⅰ、SfaN Ⅰ和TspDT Ⅰ。
所述待测序样本为含待测SNP位点的核酸分子，包括但不限于DNA分子、cDNA分子或RNA分子。
步骤A中所述的PCR扩增可为单分子扩增，也可为非单分子扩增。
优选的，所述单分子扩增为乳液PCR、桥式PCR或乳液桥式PCR。
优选的，所述非单分子扩增为普通PCR扩增、实时荧光定量PCR、不对称PCR、固相PCR、原位PCR、反转录PCR、巢式PCR、兼并引物PCR、免疫PCR、反向PCR或递减PCR。
优选的，步骤B中经IIS型限制性内切酶后，还包括将反应体系在80℃下维持5分钟，然后自然冷却至室温的步骤；本方案使得经IIS型限制性内切酶酶切获得的不同于第一核酸片段的第二核酸片段变为3’突出端，从而可以有效避免在后续的连接步骤中，切割后的第一核酸片段与第二核酸片段之间重新连接。
优选的，所述含有IIS型限制性内切酶识别序列的特异性扩增引物上还含有U，所述U和待测SNP位点在扩增产物上的位置分别位于IIS型限制性内切酶切割位点的两侧；步骤B反应结束后，向反应体系中加入USER酶。本方案使得步骤B反应结束后，U位于切割后得到的不同于第一核酸片段的第二核酸片段上，在USER酶存在的条件下，第二核酸片段上含U的单链被切割为小片段，从而能够避免在后续的连接步骤中，切割后的第一核酸片段和第二核酸片段之间重新连接。
进一步的，当U靠近扩增引物的5’端，IIS型限制性内切酶识别序列靠近扩增引物的3’端，且所述U与所述IIS型限制性内切酶识别序列之间的距离至少为3bp，USER酶可以在IIS型限制性内切酶加入之前加入反应体系、还可以与IIS型限制性内切酶同时加入反应体系、还可以在步骤B反应结束后加入反应体系；优选的，所述USER酶与IIS型限制性内切酶同时加入反应体系。与分步加入USER酶和IIS型限制性内切酶的技术方案相比，本方案简化了操作步骤。
优选的，所述含有IIS型限制性内切酶识别序列的特异性扩增引物的5’端经过磷酸修饰，所述经磷酸修饰的5’端和所述待测SNP位点分别位于所述IIS型限制性内切酶切割位点的两侧；步骤B反应结束后，向反应体系中加入λ核酸外切酶。本方案使得步骤B反应结束后，经磷酸修饰的5’端位于切割后得到的不同于第一核酸片段的第二核酸片段上，在λ核酸外切酶存在的条件下，第二核酸片段上含有5’端经过磷酸修饰的单链被降解为小片段，从而能够避免在后续的连接步骤中，切割后的第一核酸片段和第二核酸片段之间重新连接。
优选的，所述含有IIS型限制性内切酶识别序列的特异性扩增引物的5’端含有生物素标记，所述含生物素标记的5’端和所述待测SNP位点分别位于所述IIS型限制性内切酶切割位点的两 侧；所述特异性扩增引物的5’端通过生物素标记与链霉亲和素或亲和素的特异性结合预先固定在含链霉亲和素或亲和素的磁珠上，或在步骤B反应结束后，向反应体系中加入含链霉亲和素或亲和素的磁珠。本方案使得步骤B结束后，含生物素标记的5’端位于切割后得到的不同于第一核酸片段的第二核酸片段上，步骤B反应结束后，可以吸附在磁珠上的第二核酸片段从反应体系中分离出来，从而能够避免在后续的连接步骤中，切割后的第一核酸片段和第二核酸片段之间重新连接。
优选的，所述连接酶无特殊限制，能够实现DNA片段连接即可，例如：E.coli DNA连接酶、T4 DNA连接酶、热稳定DNA连接酶、Tth DNA连接酶。更优选为T4 DNA连接酶，其适用性广，且既可连接粘性末端，又能连接平末端。
所述测序接头为含有测序引物结合位点的双链核酸分子；本发明通过在第一核酸片段上连接测序接头，后续将含有不同待测SNP位点的文库分子混合，然后进行测序的过程中，统一了多种待测SNP位点的测序引物，降低了测序引物的设计难度，保证了各SNP位点的测序引物锚定效率的一致性，避免了测序过程中由于引物不同而产生的相互干扰，提高了测序的准确性。
优选的，所述测序引物结合位点位于所述测序接头的第二末端，所述第二末端用于与所述第一末端连接；与测序引物结合位点位于测序接头其他位置的技术方案相比，本方案缩短了待测SNP位点与测序引物之间的距离，减少了测序步骤，从而缩短了检测时间，提高了检测的准确性。
所述第一末端可以为平末端，也可以为粘性末端。
优选的，所述第一末端为粘性末端；此时，所述第二末端为与第一末端完全互补配对的粘性末端，与第一末端为平末端相比，本方案形成文库分子的连接效率更高。
优选的，所述文库分子的末端上含有生物素标记，所述生物素标记可以位于所述测序接头的第二末端的相对末端上，也可以位于文库分子与测序接头连接端的相对末端上；本方案中，利用该生物素标记，可以在步骤C结束后，很方便的将文库分子纯化出来。
所述文库分子通过其上的生物素标记被预先固定在含链霉亲和素或亲和素的固相载体上，也可以在步骤C的连接反应结束后通过生物素标记固定在固相载体上。
本发明提出第二实施例，以人类全血基因组为模板，构建含rs1801133位点的MTHFR基因片段文库，建库步骤如下：
A、制备扩增产物，配制巢式扩增反应体系如下：
第一轮PCR扩增，在200μL离心管中加入浓度为50ng/μL的人类全血DNA分子1.0μL；2×long Taq Mix(深圳华因康基因科技有限公司生产)10μL；浓度为10μM的上游引物(SEQ ID  NO:1)0.4μL；浓度为10μM的下游引物(SEQ ID NO:2)0.4μL；20μL去离子水；混匀并离心。将离心管置于PCR仪中，设置反应程序：94℃条件下持续4分钟；94℃条件下持续20秒，56℃条件下持续20秒，72℃条件下持续1分钟，一共30个循环；72℃条件下持续3分钟；PCR反应完成后，得到第一轮PCR产物；
第二轮PCR扩增，在200μL离心管中加入浓度为0.1ng/μL的第一轮PCR扩增产物1.0μL；2×long Taq Mix 10μL；浓度为10μM的上游引物(SEQ ID NO:3)0.4μL；0.4μL浓度为10μM的下游引物(SEQ ID NO:4)；20μL去离子水；混匀并离心。将离心管置于PCR仪中，设置反应程序：94℃条件下持续4分钟；94℃条件下持续20秒，55℃条件下持续20秒，72℃条件下持续20秒，一共30个循环；72℃条件下持续3分钟；PCR反应完成后，得到第二轮PCR产物；其中，上游引物(SEQ ID NO:3)上含有BceA Ⅰ酶识别序列ACGGC，BceA Ⅰ酶切割位点位于BceAⅠ酶识别序列和rs1801133位点之间，且与待测SNP位点之间的长度为4bp；
B、采用BceA Ⅰ酶对步骤A中第二轮PCR产物进行切割，配制反应体系如下：
纯化的第二轮PCR产物200ng，浓度为2units/μL的BceA Ⅰ酶1μL，10×NEBuffer 3.1(NEB公司生产)2.5μL，去离子水1.5μL，在37℃条件下反应1小时，65℃下灭活20分钟，得到含待测SNP位点的第一核酸片段，且第一核酸片段上经切割形成5’端有2个突出碱基的第一末端；
C、步骤B反应结束后，向体系中加入以下组分：浓度为2.5pmol/μL的测序接头0.4μL；浓度为2units/μL的T4 DNA连接酶1μL；浓度为100mM的ATP 0.3μL；质量浓度为30％的聚乙二醇6000 10μL；去离子水加至40μL；室温下反应1小时。测序接头由SEQ ID NO:5和SEQ ID NO:6组成，与第一末端完全互补配对，其中，SEQ ID NO:5上的AGTCGCTGAAGTAGTCGGT序列为测序引物结合序列，SEQ ID NO:5的5’端含有生物素标记，且被预先固定在含链霉亲和素的磁珠上；反应完成后，将离心管置于磁架上，分离去除上清，得到吸附在磁珠上的含有测序接头和rs1801133位点的文库分子；
对文库分子进行验证，配制以下反应体系，扩增文库分子：
稀释100倍的磁珠悬液1μL；2×long Taq Mix 10μL；浓度为10μM上游引物(SEQ ID NO:7)0.4μL；浓度为10μM下游引物(SEQ ID NO:4)0.4μL；20μL去离子水；混匀并离心。将离心管置于PCR仪中，设置反应程序：94℃条件下持续2分钟；94℃条件下持续20秒，54℃条件下持续20秒，72℃条件下持续10秒分钟，一共25个循环；72℃条件下持续3分钟；PCR反应完成后，得到验证产物。
验证产物聚丙烯酰胺凝胶电泳结果如图1所示，0为分子大小标记物，泳道1为验证产物，从图中可以看出，验证产物在110bp位置附近出现目标条带，与理论预期文库分子大小完全相符，说明本发明的方法可以实现对待测序样本的建库。
本发明提出第三实施例，以人类全血基因组为模板，构建含rs1801133位点的MTHFR基因片段文库，与第二实施例的区别在于，步骤A中第二轮PCR扩增的扩增引物为：上游引物(SEQ ID NO:8)、下游引物(SEQ ID NO:4)；其中，上游引物(SEQ ID NO:8)上含有Acu Ⅰ酶识别序列CTGAAC，Acu Ⅰ酶切割位点位于Acu Ⅰ酶识别序列和rs1801133位点之间，且与待测SNP位点之间的长度为4bp；
B、采用Acu Ⅰ酶对步骤A中的扩增产物进行切割，配制反应体系如下：
纯化的第二轮PCR产物200ng；浓度为2units/μL的Acu Ⅰ酶0.5μL；10×
Figure PCTCN2017098213-appb-000001
Buffer2.5μL；3.2mM S-腺苷甲硫氨酸0.5μl,去离子水1.5μL；在37℃条件下反应1小时，65℃下灭活20分钟，得到含待测SNP位点的第一核酸片段，且第一核酸片段上经切割形成3’端有2个突出碱基的第一末端；
步骤C中的测序接头由SEQ ID NO:9和SEQ ID NO:10组成，与第一末端完全互补配对，其中，SEQ ID NO:9上的AGTCGCTGAAGTAGTCGGT序列为测序引物结合序列。
对文库分子进行验证的步骤中，以上游引物(SEQ ID NO:11)、下游引物(SEQ ID NO:12)配制扩增反应体系并进行扩增得到验证产物。
验证产物经聚丙烯酰胺凝胶电泳，验证产物在110bp附近出现目标条带，与理论预期文库分子大小完全相符，说明本发明的方法可以实现对待测序样本的建库。
本发明提出了第四实施例，以人类全血基因组为模板，构建含rs1801133位点的MTHFR基因片段文库，配制两个不同的反应体系，与第二实施例的区别在于，步骤A中各反应体系第二轮PCR扩增的引物分别为：上游引物(SEQ ID NO:13)和下游引物(SEQ ID NO:4)；上游引物(SEQ ID NO:14)和下游引物(SEQ ID NO:4)；其中上游引物(SEQ ID NO:13)为DNA序列，其上含有BsmFⅠ酶识别序列GGGAC及两个U；上游引物(SEQ ID NO:14)为DNA序列，其上含有BtgZⅠ酶识别序列GCGATG及两个U。
B、分别配制两个如下的反应体系并反应：纯化的第二轮PCR产物200ng；浓度为2units/μL的BsmFⅠ酶1μL；10×
Figure PCTCN2017098213-appb-000002
Buffer 1μL；去离子水1.5μL；在65℃条件下反应1小时，65℃下灭活20分钟，得到含待测SNP位点的第一核酸片段及含两个U的第二核酸片段，且第一核酸片段上经切割形成5’端有4个突出碱基的第一末端；切割反应完成后，向反应体系中加入USER酶，室温下作用30分钟，第二核酸片段上含U的单链发生降解，本方案可以有效防止第一核酸片段和第二核酸片段之间重新连接，有利于文库分子的制备；
纯化的第二轮PCR产物200ng；浓度为2units/μL的BtgZⅠ酶1μL；10×
Figure PCTCN2017098213-appb-000003
Buffer0.5μL；去离子水1.5μL；在60℃条件下反应1小时，80℃下灭活20分钟，得到含待测SNP位点的第一核酸片段及含两个U的第二核酸片段，且第一核酸片段上经切割形成5’端有4个突出碱 基的第一末端；切割反应完成后，向反应体系中加入USER酶，室温下作用30分钟，第二核酸片段上含U的单链发生降解，本方案可以有效防止第一核酸片段和第二核酸片段之间重新连接，有利于文库分子的制备；
步骤C中两个反应体系的测序接头均由SEQ ID NO:5和SEQ ID NO:15组成；
本实施例文库分子的验证产物经聚丙烯酰胺凝胶电泳，验证产物在110bp位置附近出现目标条带，与理论预期文库分子大小完全相符，说明本发明的方法可以实现对待测序样本的建库。
本发明提出了第五实施例，以人类全血基因组为模板，建立五个不同的反应体系，分别构建含rs1799853位点的CYP2C9基因片段，含rs1057910位点的CYP2C9基因片段，含rs9923231位点的VKORC1基因片段，含rs4244285位点的CYP2C19基因片段，含rs4986893位点的CYP2C19基因片段的基因文库，建库步骤如下：
A、制备扩增产物，配制巢式扩增反应体系如下：
第一轮PCR扩增，在200μL离心管中加入浓度为50ng/μL的人类全血DNA分子1.0μL；2×long Taq Mix 10μL；浓度为10μM的上游引物0.4μL；浓度为10μM的下游引物0.4μL；20μL去离子水；混匀并离心。将离心管置于PCR仪中，设置反应程序：94℃条件下持续4分钟；94℃条件下持续20秒，49℃条件下持续20秒，72℃条件下持续1分钟，一共30个循环；72℃条件下持续3分钟；PCR反应完成后，得到第一轮PCR产物。其中，各反应体系中第一轮PCR扩增的扩增引物分别为：上游引物(SEQ ID NO:16)和下游引物(SEQ ID NO:17)；上游引物(SEQ ID NO:18)和下游引物(SEQ ID NO:19)；上游引物(SEQ ID NO:20)和下游引物(SEQ ID NO:21)；上游引物(SEQ ID NO:22)和下游引物(SEQ ID NO:23)；上游引物(SEQ ID NO:24)和下游引物(SEQ ID NO:25)；
第二轮PCR扩增，在200μL离心管中加入浓度为0.1ng/μL的第一轮PCR扩增产物1.0μL；2×long Taq Mix 10μL；浓度为10μM的上游引物0.4μL；浓度为10μM的下游引物0.4μL；20μL去离子水；混匀并离心。将离心管置于PCR仪中，设置反应程序：94℃条件下持续4分钟；94℃条件下持续20秒，T℃条件下持续20秒，72℃条件下持续20秒，一共30个循环；72℃条件下持续3分钟；PCR反应完成后，得到第二轮PCR产物；其中，各反应体系中的退火温度T分别为50℃，50℃，53℃，45℃和45℃。各反应体系中，第二轮PCR扩增的扩增引物分别为：上游引物(SEQ ID NO:26)和下游引物(SEQ ID NO:27)；上游引物(SEQ ID NO:28)和下游引物(SEQ ID NO:29)；上游引物(SEQ ID NO:30)和下游引物(SEQ ID NO:31)；上游引物(SEQ ID NO:32)和下游引物(SEQ ID NO:33)；上游引物(SEQ ID NO:34)和下游引物(SEQ ID NO:35)；其中上游引物SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、 SEQ ID NO:34上含有BceA Ⅰ酶识别序列ACGGC，BceA Ⅰ酶切割位点位于BceA Ⅰ酶识别序列和待测SNP位点之间，且与待测SNP位点之间的长度为4bp；
B、分别采用BceA Ⅰ酶对步骤A中第二轮PCR产物进行切割，配制反应体系如下：
纯化的第二轮PCR产物200ng；浓度为2units/μL的BceAI酶1μL；10×NEBuffer 3.1 2.5μL，去离子水1.5μL；在37℃条件下反应1小时，65℃下灭活20分钟，得到含待测SNP位点的第一核酸片段，且第一核酸片段上经切割形成5’端有2个突出碱基的第一末端；
C、步骤B反应结束后，向各反应体系中加入以下组分：浓度为2.5pmol/μL的测序接头0.4μL，浓度为2units/μL的T4 DNA连接酶1μL，浓度为100mM的ATP 0.3μL，质量浓度为30％的聚乙二醇6000 10μL，去离子水加至40μL，室温下反应1小时；各反应体系的测序接头分别由SEQ ID NO:36和SEQ ID NO:37、SEQ ID NO:38和SEQ ID NO:37、SEQ ID NO:39和SEQ ID NO:40、SEQ ID NO:41和SEQ ID NO:40、SEQ ID NO:42和SEQ ID NO:43组成。其中，SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:42上分别含有标签序列ACGT、TGCA、GTAC、CATG、AGTC，且均含有测序引物结合位点AGTCGCTGAAGTAGTCGGT，其5’端为含有生物素标记，且被预先固定在含链霉亲和素的磁珠上；反应完成后，将离心管置于磁架上，分离去除上清，分别得到吸附在磁珠上含有待测SNP位点和测序接头的文库分子；
对各反应体系中制备的文库分子进行验证，首先配制以下反应体系，扩增文库分子：稀释100倍的磁珠悬液1μL，2×long Taq Mix 10μL，浓度为10μM上游引物0.4μL，浓度为10μM下游引物(SEQ ID NO:4)0.4μL，20μL去离子水，混匀并离心；将离心管置于PCR仪中，设置反应程序：94℃条件下持续2分钟；94℃条件下持续20秒，54℃条件下持续20秒，72℃条件下持续10秒分钟，一共25个循环；72℃条件下持续3分钟；PCR反应完成后，得到验证产物。其中，各体系中扩增反应的引物分别为：上游引物(SEQ ID NO:7)和下游引物(SEQ ID NO:44)；上游引物(SEQ ID NO:7)和下游引物(SEQ ID NO:29)；上游引物(SEQ ID NO:7)和下游引物(SEQ ID NO:31)；上游引物(SEQ ID NO:7)和下游引物(SEQ ID NO:45)；上游引物(SEQ ID NO:7)和下游引物(SEQ ID NO:35)；
验证产物经琼脂糖凝胶电泳结果如图2所示，0为分子大小标记物，泳道1-5分别为上述五个反应体系验证产物，在82bp、67bp、121bp、127bp、112bp位置附近出现目标条带，与理论预期文库分子完全相符，说明本发明的方法可以实现对待测序样本的建库。
本发明还提出了第六实施例，一种SNP分型方法，包括对按上述任一实施例中的建库方法制得的文库分子进行测序的步骤。
本发明的文库分子上由于含有测序接头，统一了同一体系中多个不同待测SNP位点的测 序引物，避免了测序引物之间的相互干扰。
优选的，所述方法还包括将文库分子可寻址的固定在固相载体上的步骤。
所述可寻址的固定，是指能够确定位置信息的固定。即固相载体上每一具体位置上所固定的文库分子与其它位置上所固定的文库分子之间是能够明确区分的。
进一步的，含测序接头的文库分子可通过直接或间接的方式可寻址的固定在固相载体上。
针对通过直接的方式实现含测序接头的文库分子的可寻址固定，本发明提出一实施例：含测序接头的文库分子杂交至流动小室上，从而实现含测序接头的文库分子的可寻址固定；本发明还提出另一实施例，含测序接头的文库分子固定在微球上，微球预先固定在固相载体上，从而实现含测序接头的文库分子的可寻址固定。
针对通过间接的方式实现含测序接头的文库分子的可寻址固定，本发明提出一实施例：含测序接头的文库分子先固定在微球上，然后再将微球固定在固相载体上，从而实现含测序接头的文库分子的可寻址固定。
当检测的待测序样本有多个时，根据待测序样本的不同分别进行建库，获得多种文库分子，再将多种文库分子混合，在同一体系中进行测序。
优选的，所述多种文库分子分别含有不同的标签序列，本方案通过文库分子上的标签序列，可以区分出不同文库分子的测序结果。
进一步的，所述标签序列位于所述文库分子上的测序接头上。
优选的，所述测序的方法为第二代高通量基因测序技术，包括但不限于连接测序法或合成测序法。
所述连接测序法是基于连接酶在核酸片段之间进行连接反应的过程中的保真性来实现的，以待测序核酸片段为模板，测序引物和寡聚核苷酸探针(该探针的特定位置上带有荧光标记)进行连接反应，通过检测连接产物上的荧光标记从而确定寡核苷酸探针上带有荧光标记的特定位置对应的序列的信息。目前，市场上常见的连接测序法有多种，包括但不限于：深圳华因康基因科技有限公司的Pstar连接测序法、ABI公司的连接测序法、Complete Genomics公司的连接测序法。
所述合成测序法是基于聚合酶在延伸核酸链过程中的保真性来实现的，以待测序核酸片段为模板，锚定引物(又称测序引物，其与待测序核酸片段所在链互补)互补结合至待测序核酸片段上，通过检测在延伸过程中产生的信号来确定待测序核酸片段上相应位置的序列信息。目前，市场上常见的合成测序法有多种，包括但不限于：Illumina公司的Solexa合成测序法、Roche公司的454合成测序法、Life Technologies公司的Iontorrent、Ion Proton合成测序法。
需要说明的是，采用合成测序法进行SNP分型检测时，所述测序接头的3’端固定在固相载体上，保留固定在固相载体上的单链核酸分子，对待测SNP位点进行检测。采用连接测序法进行SNP分型检测时，所述测序接头的任一条单链均可固定在固相载体上，并保留作为测序模板。
本发明还提出第七实施例，一种对MTHFR基因rs1801133位点检测的方法，本实施例在第二实施例的基础上，还包括以下步骤：
向步骤C中得到的吸附在磁珠上的文库分子中加入浓度为0.1M的NaOH溶液20μL，使模板变性为单链，分离去除上清，用20μL 1×TE(含质量浓度为0.01％的triton)洗涤两遍，20μL1×TE洗涤一遍，最后重悬于10μL 1×TE中用作测序模板；采用深圳华因康基因科技有限公司的高通量基因测序仪Pstar IIA，以连接测序法进行测序，测序引物(SEQ ID NO:12)5’端经磷酸修饰，且固定在测序接头的测序引物结合序列上，采用与检测位点互补的带有荧光基团的简并九聚物NNNNXNNNN作为测序探针，经过一次连接测序，确定MTHFR基因的rs1801133位点为T。
本发明还提出第八实施例，一种对MTHFR基因rs1801133位点检测的方法，本实施例在第三实施例的基础上，还包括以下步骤：
将步骤C制得的文库分子杂交至流动小室上；向文库分子中加入浓度为0.1M的NaOH溶液20μL，使模板变性为单链，分离去除上清，用20μL 1×TE(含质量浓度为0.01％的triton)洗涤两遍，20μL 1×TE洗涤一遍，最后重悬于10μL 1×TE中用作测序模板；采用illumina测序仪，将测序引物(SEQ ID NO:12)固定在测序接头的测序引物结合序列上，经过四次合成测序，确定MTHFR基因的rs1801133位点为T。
本发明还提出第九实施例，同时检测CYP2C9基因片段上的rs1799853位点、rs1057910位点，VKORC1基因片段上的rs9923231位点，CYP2C19基因片段上的rs4244285位点、rs4986893位点，本实施例在第四实施例的区别在于，还包括以下步骤：
将步骤C中各反应体系得到的吸附在磁珠上的文库分子混匀，向其中加入浓度为0.1M的NaOH溶液20μL，使模板变性为单链，分离去除上清，用20μL 1×TE(含v/v浓度为0.01％的triton)洗涤两遍，20μL 1×TE洗涤一遍，最后重悬于10μL 1×TE中用作测序模板；采用深圳华因康基因科技有限公司的高通量基因测序仪Pstar IIA，以连接测序法进行测序，将测序引物(SEQ ID NO:43)固定在测序接头的测序引物结合位点上，经过一次连接测序，确定上述待测SNP位点分别为C、A、T、G、G。
本实施例通过在文库分子上连接相同的测序接头，统一了同一体系中多个待测SNP位点检测的测序引物，避免了不同引物之间的相互干扰。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种建库方法，其特征在于，包括以下步骤：
    A、利用特异性扩增引物组对含待测SNP位点的待测序样本进行PCR扩增，得到扩增产物；所述特异性扩增引物组中的至少一种扩增引物上含有IIS型限制性内切酶识别序列，所述扩增产物上含有IIS型限制性内切酶切割位点，所述IIS型限制性内切酶切割位点与所述待测SNP位点之间的距离为0至5个碱基；
    B、采用IIS型限制性内切酶对扩增产物进行酶切，得到含有待测SNP位点的第一核酸片段，且所述第一核酸片段上经酶切形成第一末端；
    C、在连接酶作用下，所述第一核酸片段在第一末端处连接测序接头，得到文库分子。
  2. 根据权利要求1所述的建库方法，其特征在于，所述含有IIS型限制性内切酶识别序列的特异性扩增引物上还含有U，所述U和所述待测SNP位点在扩增产物上的位置分别位于所述IIS型限制性内切酶切割位点的两侧；所述步骤B结束后，还包括向反应体系中加入USER酶的步骤。
  3. 根据权利要求1所述的建库方法，其特征在于，所述含有IIS型限制性内切酶识别序列的特异性扩增引物的5’端经过磷酸修饰，所述经磷酸修饰的5’端和所述待测SNP位点在扩增产物上的位置分别位于所述IIS型限制性内切酶切割位点的两侧；所述步骤B结束后，还包括向反应体系中加入λ核酸外切酶的步骤。
  4. 根据权利要求1所述的建库方法，其特征在于，所述测序接头上含有测序引物结合位点，所述测序引物结合位点位于所述测序接头的第二末端处，所述第二末端用于与所述第一末端连接。
  5. 根据权利要求4所述的建库方法，其特征在于，所述第一末端为粘性末端；所述第二末端为与第一末端完全互补配对的粘性末端。
  6. 根据权利要求4所述的建库方法，其特征在于，所述文库分子的末端上含有生物素标记，其被预先固定在含链霉亲和素或亲和素标记的磁珠上。
  7. 一种SNP分型方法，其特征在于，包括对按权利要求1至6中任一项所述的建库方法制得的文库分子进行测序的步骤。
  8. 根据权利要求7所述的SNP分型方法，其特征在于，所述方法还包括将文库分子可寻址的固定在固相载体上的步骤。
  9. 根据权利要求7所述的SNP分型方法，其特征在于，当检测的待测序样本有多个时，根据待测序样本的不同分别进行建库，获得多种文库分子，再将多种文库分子混合后进行测序。
  10. 根据权利要求9所述的SNP分型方法，其特征在于，所述多种文库分子上分别含有不同的标签序列。
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