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Method for constructing high-throughput sequencing library and kit

A sequencing library and kit technology, applied in the biological field, can solve the problems of high cost, long time-consuming, and many reagents

Active Publication Date: 2017-04-05
DALIAN GENTALKER BIO-TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional steps take a long time, require more reagents, and cost more

Method used

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  • Method for constructing high-throughput sequencing library and kit
  • Method for constructing high-throughput sequencing library and kit
  • Method for constructing high-throughput sequencing library and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Sequencing of 50 exons of BRCA-1 and BRCA-2 genes (including 23 exons of BRCA1 gene and 27 exons of BRCA2 gene) related to hereditary breast cancer, a total of 10 clinical cases sample:

[0122] 1) Primer design:

[0123] Corresponding amplification primers were designed for the 50 exons of the two genes, and the related parameters were: Tm value 58.0°C-62.0°C, GC value 40.0%-60.0%, primer size 22±3bp. At the 5' end of the forward amplification primer and the reverse amplification primer, respectively add a sequence identical to the general sequencing primer sequence of the 3' end high-throughput sequencing of the D5 adapter primer and the D7 adapter primer. The designed primers are as follows , where the underline is the general sequence primer sequence for high-throughput sequencing introduced:

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Abstract

The invention relates to a method for constructing a high-throughput sequencing library capable of capturing a plurality of amplified fragments at the same time and a kit. The method comprises the following steps: first round amplification; primer digestion; second round amplification; purification and recovery; sequencing; and analysis. By virtue of reasonable primer design and PCR policy, a D5 adapter primer sequence and a D7 adapter primer sequence are directly added to the 5' tail end of a PCR product. By introducing a differentiable label sequence for each of samples, the sequencing result of each of the samples during detection by a second generation high-throughput sequencing technology can be found through a unique label sequence thereof. The method can be applied to detecting a plurality of different genetic loci of a large number of samples at the same time, so that the sequencing cost is greatly lowered.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and a kit for constructing a sequencing library capable of simultaneously capturing multiple amplified fragments. Background technique [0002] The emergence of second-generation sequencing technology has greatly reduced the cost of sequencing. It has the characteristics of high throughput, low cost, and low sequencing error rate, and has developed rapidly in recent years. With the application of the second-generation high-throughput sequencing technology, it is possible to sequence mixed nucleic acid molecules, distinguish and measure each independent sequence at the same time, so that large-scale target sequence sequencing can be performed simultaneously. [0003] At present, the traditional next-generation sequencing library uses steps such as performing end repair on nucleic acid fragments, adding A treatment, connecting universal adapter primers (adaptor), and enriching...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/68
CPCC12Q1/6869C40B50/06C12Q2535/122C12Q2537/143C12Q2531/113
Inventor 刘琦赵金银邢晓星许立志于闯李杰明鸿博
Owner DALIAN GENTALKER BIO-TECH CO LTD
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