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A SNP detection method based on high-throughput sequencing

A technique for sequencing and detecting probes, which is applied in biochemical equipment and methods, microbiological determination/inspection, etc. It can solve the problems of library construction quality, hybridization efficiency, complex probe design, and waste of sequencing space, etc.

Active Publication Date: 2016-09-21
CAPITALBIO CORP +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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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  • A SNP detection method based on high-throughput sequencing
  • A SNP detection method based on high-throughput sequencing
  • A SNP detection method based on high-throughput sequencing

Examples

Experimental program
Comparison scheme
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 a SNP detection method based on high-throughput sequencing. The method of the present invention comprises the following steps: probe design, pre-amplification and biotin labeling, hybridization, connection, Barcode-specific primer amplification, sequencing, and analyzing the SNP site information of the sample according to the sequencing result. Experiments of the present invention prove that the method of the present invention has the following advantages: (1) the sensitivity of the system is improved, (2) the sample labeling is simple, (3) the influence of non-specific amplification products in the pre-amplification is eliminated, and data analysis is beneficial , (4) The library construction method is simple, (5) The probability of template random mutation is reduced, (6) The cost of sequencing is greatly reduced, (7) Data analysis is easy, (8) Site selection is flexible, very suitable for a medium number of Large-scale sample screening for mutation 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 Patents(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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