Marker based on high-throughput sequencing and method and kit for capturing one or multiple specific genes of multiple samples

A gene-specific, high-throughput technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of high sequencing cost and low throughput

Active Publication Date: 2016-04-27
DALIAN GENTALKER BIO-TECH CO LTD
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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 specific gene-related sequences in a large number of sample

Method used

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  • Marker based on high-throughput sequencing and method and kit for capturing one or multiple specific genes of multiple samples
  • Marker based on high-throughput sequencing and method and kit for capturing one or multiple specific genes of multiple samples
  • Marker based on high-throughput sequencing and method and kit for capturing one or multiple specific genes of multiple samples

Examples

Experimental program
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Effect test

Embodiment 1

[0065] 27 exons of pheochromocytoma-related SDHB, SDHC, SDHD, RET, and VHL genes were sequenced, with a total of 25 clinical samples:

[0066] 1) Primer design:

[0067] Corresponding amplification primers were designed for 27 exons of the 5 genes, and the related parameters were: Tm value 55.0°C-60.0°C, GC value 40.0%-60.0%, primer size 20±3bp. A general sequence is added to the 5' ends of the forward amplification primer and the reverse amplification primer respectively. The designed primers are as follows, and the underlined is the general sequence introduced:

[0068]

[0069]

[0070] Design the tag sequence according to the following rules: the number of forward tag primers × the number of reverse tag primers ≥ the number of samples, in this example, design 5 pairs of tag sequences based on 25 samples, and the 3' end plus the universal sequence to form the second The index primers used in the round of amplification are amplified through the combination of 5 pairs ...

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Abstract

The invention relates to a marker based on high-throughput sequencing and a method and a kit for capturing one or multiple specific genes of multiple samples. The method includes steps of primary amplification, primer digestion, secondary amplification, mixing, recycling, sequencing and analyzing. In the method, a mixture of a universal sequence and specific primers of all genes is adopted for primary amplification, specific sequences of all genes act in multiple amplification at this moment, and the universal sequence is added in front of each gene; a tag sequence and the universal sequence are adopted for secondary amplification, the universal sequence acts in amplification at this moment, and the tag sequence which is recognizable is added in front of a final product. By introducing a unique primer tag sequence into each sample, when the samples are detected through second-generation high-throughput sequencing technology, a sequencing result of each sample can be retrieved through the corresponding unique primer tag sequence, and the method can be applied to detecting multiple different genetic loci of a lot of samples at the same time, so that sequencing cost is lowered greatly.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a method and a kit for labeling and capturing specific genes in multiple samples, in particular, to a method for labeling and capturing one or more specific genes in multiple samples based on high-throughput sequencing and kits. 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. With the application of the second-generation high-throughput sequencing technology, the mixed nucleic acid molecules can be sequenced, and each independent sequence can be resolved and measured at the same time, making it possible for this sequencing technology to be developed in high-throughput markers. [0003] With the widespread development of high-throughput sequencing technology in human disease research a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 刘琦郑新赵金银郭丽萍许立志其他发明人请求不公开姓名
Owner DALIAN GENTALKER BIO-TECH CO LTD
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