Method for detecting mutation of deafness susceptibility genes through combination of overlapping extension PCR and Sanger sequencing

A technology of sequencing and primer combination, applied in the field of gene detection, can solve the problems of gene chip method, such as many detection sites, high cost, cumbersome detection operation, etc., to reduce the number of PCR reactions and sequencing reactions, reduce the detection cost, and simplify the operation. effect of steps

Active Publication Date: 2019-05-21
GUANGZHOU HYBRIBIO MEDICINE TECH LTD +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fluorescent PCR method can detect quickly, low cost, no special equipment is needed, and can avoid pollution, but there are few sites, so follow-up detection is required; gene chip method has many detection sites, but requires special equipment, and the cost is high; ARMS-PCR method is low in cost, However, the detection operation is cumbersome, electrophoresis is required, and there are few sites covered, so follow-up detection is required; high-throughput sequencing method can detect other mutations, but requires

Method used

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  • Method for detecting mutation of deafness susceptibility genes through combination of overlapping extension PCR and Sanger sequencing
  • Method for detecting mutation of deafness susceptibility genes through combination of overlapping extension PCR and Sanger sequencing
  • Method for detecting mutation of deafness susceptibility genes through combination of overlapping extension PCR and Sanger sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 A kit for detecting deafness-related genes based on Sanger sequencing

[0074] 1. Composition

[0075] A combination of primers with nucleotide sequences as shown in SEQ ID No.1-26, sequencing primers with nucleotide sequences as shown in SEQ ID Nos.27-28 and reagents for PCR reactions, wherein the reagents for PCR reactions include: GeneAmp TM 10×PCRBufferI (ABI TM 4379876), 25mM Mg 2+ , 25mM dNTPs, Primers, Roche Taq Enzyme (Product No.: 03501221190), H 2 O; The reagent for Sanger sequencing is BigDye Terminator v3.1 Cycle Sequencing Kit (including: BigDye, 5×seq Buffer).

[0076] 2. How to use

[0077] S1. Collect EDTA anticoagulated whole blood samples;

[0078] S2. extracting DNA by column method;

[0079] S3. PCR reaction, there are five independent PCR systems, using primer mix 1-5 respectively:

[0080] Primer mixture 1 includes nucleotides such as primers shown in SEQ ID No.1-6;

[0081] The primer mixture 2 includes nucleotides such as prim...

Embodiment 2

[0098] Example 2 Detection of deafness-related gene mutation sites

[0099] 1. Experimental method

[0100] A kit for detecting deafness-related mutation sites based on the Sanger sequencing method in Example 1 was used to detect the blood of a known positive sample to obtain DNA with a sample concentration of 32 ng / μl.

[0101] The corresponding relationship between the mutation status of known positive samples and primers and primer mixes is shown in Table 2 to Table 6:

[0102] Table 2:

[0103]

[0104] table 3:

[0105]

[0106]

[0107] Table 4:

[0108]

[0109] table 5:

[0110]

[0111] Table 6:

[0112]

[0113] 2. Experimental results

[0114] The result is as Figures 1 to 11 As shown, all the 43 sites detected can be successfully covered and detected. Separate fragment sequencing was performed on each single site of the same sample, and all sites were the same as the overlapping results.

Embodiment 3

[0116] 1. Experimental method

[0117] Utilize a Sanger sequencing method based on overlap extension PCR in the embodiment 1 to detect deaf susceptibility related four gene common loci (total 5 reactions), by the detection of 20 examples of known result samples, and with the common PCR The single locus Sanger sequencing method was used to compare the experimental results of four common loci (13 reactions in total) related to deafness susceptibility to determine the consistency.

[0118] 2. Experimental results

[0119]

[0120]

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Abstract

The invention discloses a method for detecting mutation of deafness susceptibility genes through combination of overlapping extension PCR and Sanger sequencing. The method is based on a primer combination which is used for amplifying deafness-relevant mutation sites of GJB2 genes, SLC26A4 genes, mitochondrial DNA and GJB3 genes, wherein the nucleotide sequences of the primer combination are shownin SEQ ID No.1-26. By means of the built detection method, the deafness susceptibility genes are detected through the combination of overlapping extension PCR and Sanger sequencing, only 5 PCR reactions and sequencing reactions are needed, the PCR reaction number and sequencing reaction number are greatly reduced, main 43 mutation sites of the GJB2 genes, SLC26A4 genes, mitochondrial DNA and GJB3genes can be detected, and new mutation sites in amplification fragments can be found. The detection technology has important clinical significance when applied to detecting the mutation of the deafness susceptibility genes, the operation step is simplified, the detection cost is reduced, the detection efficiency is improved, and the method is worthy of wide popularization.

Description

technical field [0001] The invention relates to the technical field of gene detection, more specifically, to a method for detecting deafness susceptibility gene mutations by combining overlap extension PCR with Sanger sequencing. Background technique [0002] Deafness is the most common severe sensory system defect. In 2005, at least 278 million people worldwide had disabling hearing loss. Hereditary deafness is divided into non-syndromic deafness (NSHI) and syndromic deafness (SHI). Most of NSHI and SHI are caused by single gene abnormalities. At present, one out of every 500 newborns is diagnosed as binaural congenital sensorineural deafness, of which about 50% to 70% are caused by genetic factors. Common deafness-causing genes include GJB2, SLC26A4, mitochondrial DNA12SrRNA, and GJB3 genes, and the inheritance methods of different genes are different. [0003] Deafness gene detection is of great significance: ①Clarify the cause of congenital hereditary deafness and impr...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
Inventor 郑焱卢炫廷邱美兰谢龙旭邱雪莲吴丹叶徐爱娟
Owner GUANGZHOU HYBRIBIO MEDICINE TECH LTD
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