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Method and kit for detecting one or more target sequence of multiple samples based on high-throughput sequencing

A target sequence, high-throughput technology, applied in the field of capturing and labeling one or more target sequences of multiple samples, methods or kits, can solve the problems of low throughput and high sequencing cost

Active Publication Date: 2016-10-05
大连晶泰医学检验实验室有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to use high-throughput sequencing technology to simultaneously sequence and analyze target sequence-related sequences in a large number of samples, solve the problems of low throughput and high sequencing costs in traditional methods, and expand the application of second-generation sequencing technology in the field of PCR sequencing

Method used

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  • Method and kit for detecting one or more target sequence of multiple samples based on high-throughput sequencing
  • Method and kit for detecting one or more target sequence of multiple samples based on high-throughput sequencing
  • Method and kit for detecting one or more target sequence of multiple samples based on high-throughput sequencing

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Embodiment 1

[0070] 21 exons (EXON1, EXON2, EXON3, EXON4, EXON5, EXON6, EXON7, EXON8, EXON9, EXON10, EXON11, EXON12, EXON13, EXON14, EXON15, EXON10, EXON11, EXON12, EXON13, EXON14, EXON15, EXON16, EXON17, EXON18, EXON19, EXON20 and EXON21) sequencing, a total of 10 clinical samples:

[0071] 1) Primer and linker design:

[0072] The corresponding capture primers were designed for the 21 exons of the ATP7B gene. Relevant parameters: Tm value 58.0℃-65.0℃, GC value 40.0%-60.0%, primer size 23±3bp, target fragment size 150-300bp, so The designed primers are as follows:

[0073]

[0074]

[0075] Design 2 adapters based on 10 samples, the adapters are single-stranded DNA, and the sequence contains a palindrome sequence of two regions (each region is 15bp in length), a U base and a 3'T end, which can form a stable hairpin structure , the designed linker sequence is as follows:

[0076] ADP1: 5'P-AGGACAGAAGCTCGA-U-TCGAGCTTCTGTCCT-s-T-3'

[0077] ADP2: 5'P-ACCTTGAGCGATCGA-U-TCGATCGCTCAAG...

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Abstract

The invention provides a method or a kit for capturing and labeling one or more target sequences of multiple samples based on high-throughput sequencing. A novel labeling technical scheme is provided by integrating a multiplex amplification technology, a ligation technology and a PCR-index (polymerase chain reaction-index) technology; the target sequences are amplified by virtue of specific capturing primers; by virtue of a ligation reaction, an adaptor, which is provided with a U base and has a known sequence, is introduced to each of two ends of a PCR product; the U bases are cut open by virtue of a USER enzyme; PCR primers are designed in accordance with the known sequences of the adaptors; tag primers are separately introduced to two ends of the PCR product by virtue of a PCR reaction; sample information of the PCR product can be specifically obtained by virtue of the sequences of the tag primers which are introduced to two ends of the PCR product; and then, in accordance with the sequence of the capturing primer of each of the target sequences, a corresponding sequencing result is correspondingly applied to each of the target sequences of the samples.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a method and kit for capturing and labeling target sequences of multiple samples, in particular to a method for capturing and labeling one or more target sequences of multiple samples based on high-throughput sequencing or Reagent test kit. Background technique [0002] The emergence of a new generation of high-throughput sequencers has greatly reduced the cost of nucleic acid sequencing, which has the characteristics of high-throughput, low-cost, and low sequencing error rate. Using the second-generation high-throughput sequencing technology, the mixed nucleic acid molecules can be sequenced, and each independent sequence can be resolved and detected at the same time, making it possible to develop this sequencing technology in high-throughput markers. [0003] With the widespread development of high-throughput sequencing technology in human disease research, microbial genom...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 刘琦许立志胡小许郑新杨文辉其他发明人请求不公开姓名
Owner 大连晶泰医学检验实验室有限公司
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