SNP (single-nucleotide polymorphism) detection method based on high-flux sequencing

A technology for sequencing and detecting probes, which is applied in biochemical equipment and methods, microbiological measurement/inspection, etc. It can solve the problems of nucleic acid sample sequencing with unfavorable concentration, library construction quality, hybridization efficiency, waste of sequencing space, etc.

Active Publication Date: 2015-02-25
CAPITALBIO CORP +1
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

This method needs to design different probes according to the measured gene sequence. The probe design is more complicated, and the quality of library construction will be greatly affected by hybridization efficiency.
When using this method to detect low-frequency mutations, because most of the sequencing results are wild-type sequences, it is necessary to increase the sequencing depth to achieve the required sensitivity, which invisibly wastes the sequencing space
At the same time, when using this method for large-scale population mutation gene screening, wild-type sequences will occupy most of the sequencing space, resulting in higher sequencing costs
In addition, this library construction method requires up to hundreds of ng of nucleic acid per sample, which is not conducive to the sequencing of some low-concentration, rare or difficult-to-obtain nucleic acid samples

Method used

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  • SNP (single-nucleotide polymorphism) detection method based on high-flux sequencing
  • SNP (single-nucleotide polymorphism) detection method based on high-flux sequencing
  • SNP (single-nucleotide polymorphism) detection method based on high-flux sequencing

Examples

Experimental program
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Effect test

Embodiment 1

[0106] Example 1. SNP detection based on high-throughput sequencing technology mixed plasmid samples of 5 deafness-related mutation sites

[0107] This example detects all deafness sites. All the plasmids related to deafness sites were constructed by Boao Biological Group Co., Ltd. All plasmids have been sequenced to verify that their sequences are correct. The relevant plasmid sequence information is shown in Table 1. The mutation types include SNP mutation or Deletion / Insertion Mutations:

[0108] Table 1 shows the sequence information of plasmids related to deafness sites

[0109]

[0110] Note: WT means this site is wild type, del means deletion mutation, > means SNP mutation.

[0111] Plasmid pGEMT-299WT was obtained by using primers XPMS0299F / XPMS0299R to amplify the fragment 299WT containing 235, 176, and 299 sites using human genomic DNA as a template, and then cloned into pGEMT-easy vector.

[0112] The 235, 176, and 299 positions in the fragment 299WT sequence w...

Embodiment 2

[0183] Embodiment 2, SNP detection human genome DNA sample based on high-throughput sequencing technology

[0184] 1. Design of probes and preamplification primers

[0185] 1. Preparation of samples to be tested

[0186] Blood genomic DNA of deaf patients (235 purified mutant genomic DNA has been verified) and normal human blood genomic DNA (wild-type genomic DNA), each sample is set to repeat 3 times, and the concentration of nucleic acid used is 10n g / uL.

[0187] 2. Design of probes and preamplification primers

[0188] 1) Probe design

[0189] The design principle is the same as in Example 1, and the probes are as follows in Table 5:

[0190] Table 5 is the probe

[0191]

[0192] 2), the design of pre-amplification primers

[0193] The design principles are the same as in Example 1, and the primers are GJB234F1 / GJB234R1-B.

[0194] Two, preamplification: same as embodiment 1;

[0195] Three, hybridization: same as embodiment 1;

[0196] Four, connection: same...

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Abstract

The invention discloses an SNP (single-nucleotide polymorphism) detection method based on high-flux sequencing. The method comprises the following steps: designing probes, carrying out pre-amplification and biotin labeling, hybridizing, connecting, carrying out Barcode specific primer amplification, sequencing, and analyzing SNP site information of the sample according to the sequencing result. The experiment proves that the method enhances the sensitivity of the system, is simple in sample labeling, eliminates the influence of the non-specific amplification product in the pre-amplification so as to be beneficial to data analysis, is simple in the library establishment method, lowers the probability of template random mutation, greatly lowers the sequencing cost, is easy for data analysis, is flexible for site selection and is very suitable for large-scale sample screening on a medium quantity of mutant sites.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a SNP detection method based on high-throughput sequencing. Background technique [0002] SNP has extremely important application value in molecular diagnosis, clinical testing, pathogen detection, forensic science, genetic disease research, guidance of individualized medicine and new drug research and development (GayetAgeron et al.2009). SNP detection is the main research content of gene diagnosis at present. At the same time, the genetic diagnosis represented by SNP detection has gradually become one of the important means of genetic disease screening for newborns or specific populations. Therefore, an easy-to-operate, low-cost and high-throughput SNP detection technology is the key to current gene detection. [0003] Compared with other high-throughput genetic testing technologies, the second-generation high-throughput sequencing technology is more accurate, sensitive, and has ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2565/30C12Q1/6827C12Q1/6858C12Q1/6883C12Q2600/156C12Q2525/155C12Q2525/161C12Q2535/131C12Q2561/125C12Q2563/179C12Q1/6853C12Q1/6876C12Q2600/16
Inventor 王栋王辉张岩项光新孙义民邢婉丽程京
Owner CAPITALBIO CORP
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