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A method for building a library and a method for SNP typing

A typing method and library technology, applied in the field of molecular biology, can solve problems such as the influence of SNP typing accuracy and the mutual interference of different sequencing primers.

Active Publication Date: 2021-11-09
WUHAN CONSIDERIN GENE & HEALTH TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for building a library and a method for SNP typing, aiming to solve the problem that the accuracy of SNP typing in the prior art is affected by the sequencing read length, and the difference in the detection of multiple SNP sites in the same system at the same time. The problem of mutual interference between sequencing primers

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  • A method for building a library and a method for SNP typing
  • A method for building a library and a method for SNP typing

Examples

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no. 1 example

[0023] The present invention proposes a first embodiment, a method for building a database, including the following steps:

[0024] A. Use the specific amplification primer set to perform PCR amplification on the sample to be sequenced containing the SNP site to be detected to obtain the amplified product; at least one of the amplification primers in the specific amplification primer set contains IIS type A restriction endonuclease recognition sequence, the amplified product contains an IIS type restriction endonuclease cleavage site, the distance between the IIS type restriction endonuclease cleavage site and the SNP site to be detected is 0 to 5 bases;

[0025] B. Digest the amplified product with an IIS type restriction endonuclease to obtain a first nucleic acid fragment containing the SNP site to be detected, and the first nucleic acid fragment is digested to form a first end;

[0026] C. Under the action of ligase, the first nucleic acid fragment is ligated with a seque...

no. 6 example

[0075] The present invention also proposes a sixth embodiment, a SNP typing method, including the step of sequencing the library molecules prepared by the library construction method in any of the above embodiments.

[0076] Because the library molecule of the present invention contains sequencing adapters, the sequencing primers of multiple different SNP sites to be tested in the same system are unified, and mutual interference between sequencing primers is avoided.

[0077] Preferably, the method further includes the step of addressably immobilizing library molecules on a solid support.

[0078] The addressable fix refers to a fix that can determine location information. That is, the library molecules immobilized at each specific position on the solid phase support can be clearly distinguished from the library molecules immobilized at other positions.

[0079] Furthermore, the library molecules containing the sequencing adapters can be addressably immobilized on the solid p...

no. 7 example

[0089] The present invention also proposes a seventh embodiment, a method for detecting the rs1801133 site of the MTHFR gene. This embodiment further includes the following steps on the basis of the second embodiment:

[0090] Add 20 μL of 0.1M NaOH solution to the library molecules adsorbed on the magnetic beads obtained in step C to denature the template into a single strand, separate and remove the supernatant, and use 20 μL of 1×TE (containing 0.01% triton) was washed twice, 20 μL 1×TE was washed once, and finally resuspended in 10 μL 1×TE as a sequencing template; the high-throughput gene sequencer Pstar IIA of Shenzhen Huayinkang Gene Technology Co., Ltd. was used for ligation sequencing For sequencing, the 5' end of the sequencing primer (SEQ IDNO:12) is modified with phosphoric acid, and fixed on the sequencing primer binding sequence of the sequencing adapter, and the degenerate nonamer NNNNXNNNN with a fluorescent group complementary to the detection site is used as t...

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Abstract

The invention provides a method for building a library, comprising: A, using a specific amplification primer set to perform PCR amplification on a sample to be sequenced containing a SNP site to be tested to obtain an amplification product; at least one of the primer sets Contain IIS type restriction endonuclease recognition sequence on the kind of primer, contain IIS type restriction endonuclease cleavage site on the described amplified product, described IIS type restriction endonuclease cleavage site and described SNP to be tested The distance between the sites is 0 to 5 bases; B. The amplified product is digested with an IIS type restriction endonuclease to obtain the first nucleic acid fragment containing the SNP site to be detected, and the first The first end is formed by enzymatic cleavage on the nucleic acid fragment; C. Under the action of ligase, the first nucleic acid fragment is connected to a sequencing adapter at the first end to obtain library molecules. The invention also provides a SNP typing method. The invention shortens the site detection time, improves the detection accuracy, and unifies the sequencing primers for multiple site detection in the same system.

Description

technical field [0001] The invention relates to the field of molecular biology, and more specifically, relates to a method for building a library and a method for SNP typing. Background technique [0002] Single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) refers to the existence of changes such as conversion, transversion, insertion, and deletion at a single nucleotide position on the genome, with a large number and rich polymorphisms. SNP is considered as a genetic marker, and many phenotypic differences in the human body, susceptibility to drugs or diseases, etc. may be related to SNP, so the typing of SNP has positive significance for the treatment and medication of many diseases. [0003] For gene detection, the second-generation high-throughput sequencing technology has been continuously expanded due to its accuracy and sensitivity, and has been involved in various aspects of life science research and medical research. Using the second-generation high-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806C12Q1/6869C40B50/06
CPCC12Q1/6806C12Q1/6869C40B50/06C12Q2531/113C12Q2525/191C12Q2521/313C12Q2535/122C12Q1/68
Inventor 盛司潼黄思强
Owner WUHAN CONSIDERIN GENE & HEALTH TECH CO LTD
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