CN106702498B - 一种构建测序用dna文库的方法 - Google Patents
一种构建测序用dna文库的方法 Download PDFInfo
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- CN106702498B CN106702498B CN201510791719.6A CN201510791719A CN106702498B CN 106702498 B CN106702498 B CN 106702498B CN 201510791719 A CN201510791719 A CN 201510791719A CN 106702498 B CN106702498 B CN 106702498B
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- 238000012163 sequencing technique Methods 0.000 title claims abstract description 125
- 238000000034 method Methods 0.000 title abstract description 40
- 239000012634 fragment Substances 0.000 claims abstract description 97
- 108020004414 DNA Proteins 0.000 claims description 144
- 102000053602 DNA Human genes 0.000 claims description 17
- 230000003321 amplification Effects 0.000 claims description 17
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 17
- 238000012408 PCR amplification Methods 0.000 claims description 9
- 238000010276 construction Methods 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims 1
- 239000000047 product Substances 0.000 description 16
- 239000011324 bead Substances 0.000 description 11
- 102000012410 DNA Ligases Human genes 0.000 description 8
- 108010061982 DNA Ligases Proteins 0.000 description 8
- 239000012149 elution buffer Substances 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 102000003960 Ligases Human genes 0.000 description 3
- 108090000364 Ligases Proteins 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 102000004594 DNA Polymerase I Human genes 0.000 description 2
- 108010017826 DNA Polymerase I Proteins 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- 108010010677 Phosphodiesterase I Proteins 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000004904 shortening Methods 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 1
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 1
- 108091060592 XDNA Proteins 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
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- 239000012154 double-distilled water Substances 0.000 description 1
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- 238000007481 next generation sequencing Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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Abstract
Description
步骤(1)得到的DNA片段 | 42μL |
dNTPs的混合物(10mM) | 1μL |
T4 DNA聚合酶 | 1μL |
T4 PNK(T4多核苷酸激酶) | 1μL |
PNK缓冲液 | 5μL |
共有 | 50μL |
步骤(2)获得的平末端DNA片段 | 19.5μL |
Klenow缓冲液(10×) | 2.5μL |
dATP(1mM) | 2.5μL |
Klenow片段(缺失3′到5′外切酶活性) | 0.05μL |
ddH<sub>2</sub>O | 0.45μL |
共有 | 25μL |
步骤(3)中获得的DNA片段 | 25μL |
DNA连接酶缓冲液(2×) | 25μL |
T4DNA连接酶(1单位/μL) | 1μL |
Adapter(0.04pmol/μL) | 1μL |
共有 | 52μL |
两端加接头的DNA片段 | 17μL |
2×DNA聚合酶扩增混合物 | 25μL |
PCR引物1(10pmol/μL) | 4μL |
PCR引物2(10pmol/μL) | 4μL |
共有 | 50μL |
Claims (4)
Priority Applications (1)
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CN201510791719.6A CN106702498B (zh) | 2015-11-17 | 2015-11-17 | 一种构建测序用dna文库的方法 |
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CN201510791719.6A CN106702498B (zh) | 2015-11-17 | 2015-11-17 | 一种构建测序用dna文库的方法 |
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CN106702498A CN106702498A (zh) | 2017-05-24 |
CN106702498B true CN106702498B (zh) | 2020-03-24 |
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103917654A (zh) * | 2011-04-01 | 2014-07-09 | 桑特里莱恩科技控股公司 | 用于对长核酸进行测序的方法和系统 |
CN104372079A (zh) * | 2014-10-24 | 2015-02-25 | 中国科学院遗传与发育生物学研究所 | 一种甲基化dna的测序方法及其应用 |
CN104947197A (zh) * | 2015-07-15 | 2015-09-30 | 北京中科紫鑫科技有限责任公司 | 一种构建高通量测序文库的方法 |
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2015
- 2015-11-17 CN CN201510791719.6A patent/CN106702498B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103917654A (zh) * | 2011-04-01 | 2014-07-09 | 桑特里莱恩科技控股公司 | 用于对长核酸进行测序的方法和系统 |
CN104372079A (zh) * | 2014-10-24 | 2015-02-25 | 中国科学院遗传与发育生物学研究所 | 一种甲基化dna的测序方法及其应用 |
CN104947197A (zh) * | 2015-07-15 | 2015-09-30 | 北京中科紫鑫科技有限责任公司 | 一种构建高通量测序文库的方法 |
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