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

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...

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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 special equipment, high cost, and long follow-up data analysis time, which is more suitable for one-time analysis For the detection of the whole genome; Sanger sequencing is the gold standard, with low cost and low technical requirements, and is suitable for the detection of large quantities of clinical samples. However, due to the limited read length of Sanger sequencing, a sequencing reaction can only detect 800bp, or so, usually only A site can be detected, and a PCR reaction is required for each site detected

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