Primer system for detecting gene SNPs (single nucleotide polymorphisms) related to hereditary hearing loss and application of primer system

A hereditary deafness and gene technology, applied in the field of kits, can solve the problems of inability to cover deafness susceptibility loci, high cost, and limited detection capabilities.

Inactive Publication Date: 2013-09-04
向华
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method only targets the 233-235delC deletion of the GJB2 gene, the coverage of mutation hotspots is too small, and involves the use of expensive reagents such as fluorescent SYBR green I labeling, which does not meet the existing needs
[0008] In summary, the current technical problems are: the lack of methods and products that can simultaneously detect multiple deafness gene-related polymorphic sites at one time, common detection technologies, such as sequencing, real-time fluorescent quantitative PCR, etc. The sites are detected one by one. When there are many sites, the operation is complicated and the cost is high
Due to the limitation of detection ability in these existing methods, it cannot cover the 10 common deafness susceptibility loci in the Chinese population

Method used

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  • Primer system for detecting gene SNPs (single nucleotide polymorphisms) related to hereditary hearing loss and application of primer system
  • Primer system for detecting gene SNPs (single nucleotide polymorphisms) related to hereditary hearing loss and application of primer system
  • Primer system for detecting gene SNPs (single nucleotide polymorphisms) related to hereditary hearing loss and application of primer system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Primer design and synthesis.

[0081] 针对GJB2基因rs80338939(35delG)、GJB2基因176del16、GJB2基因rs80338943(235delC)、GJB2基因rs111033204(299delAT)、GJB3基因rs74315319(538C>T)、GJB3基因rs74315318(547G>A)、SLC26A4基因rs121908362(2168A> G), SLC26A4 gene rs111033313 (IVS7-2A>G), mit12S rRNA1494C>T, mit12S rRNA1555A>G and other 10 gene polymorphic sites related to hereditary deafness were designed corresponding to specific PCR primer core sequences (SEQ ID No: 1 to SEQ ID No: 20) and specific extension primer core sequences (SEQ ID No: 21 to SEQ ID No: 30).

[0082] Among them, in order to prevent PCR primers from entering the detection window of the mass spectrometer and interfering with the detection effect, a certain number of bases can be added to the core sequence (SEQ ID No: 1 to SEQ ID No: 20) at the 5' end of each PCR primer , such as a 10bp tag (ACGTTGGATG), to increase the molecular weight of the PCR primers, thereby exceeding the detection window of the mass spectrometer. ...

Embodiment 2

[0083] Embodiment 2: sample DNA extraction.

[0084] A total of 10 deaf patients were collected. Among them, sample collection, DNA extraction, etc. were collected in accordance with the requirements of the instructions, and human venous blood was collected with EDTA anticoagulant tubes. According to the instructions, the collected blood should not be stored at 2-8°C for more than one week, and at -20°C for no more than one month, and can be transported in a curling box with ice or a foam box with ice. It is recommended to use fresh blood as much as possible. Genomic DNA extraction. Since this kit does not provide human genomic DNA extraction reagents, a commercial nucleic acid extraction kit (such as QIAGEN’s DNeasy Blood and Tissue kit) was used to extract human genomic DNA from 200ul whole blood of each patient, and the DNA was extracted using NanoDrop2000 ( Thermo company) and standardized to 30ng / ul (A1-A10 respectively). Among them, the kit is recommended to detect hu...

Embodiment 3

[0085] Embodiment three: Biological experiment.

[0086] Using ABI9700 PCR instrument, according to the instructions, 10 genetic polymorphic sites related to hereditary deafness were tested.

[0087] The components used in the kit for PCR, PCR product purification and single base extension are:

[0088] serial number

component name

main ingredient

Specification

1

PCR Primer Mix

PCR primers

24ul / tube x1 tube

2

PCR reaction solution

Taq enzyme, dNTP

72ul / tube x1 tube

3

Enzyme digestion reaction solution

SAP enzyme

48ul / tube x1 tube

4

Extension Primer Mix

extension primer

24ul / tube x1 tube

5

Extension reaction solution

Single base elongase, ddNTP

24ul / tube x1 tube

6

positive control

Human Genomic DNA (30ng / ul)

10ul / tube x1 tube

[0089] The concentration of each primer pair is 500nmol / L.

[0090] According to the manual, the specific oper...

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Abstract

The invention discloses a primer system which is used for simultaneously detecting 10 gene SNPs (single nucleotide polymorphisms) related to hereditary hearing loss. Based on a product prepared by using the primer system, the 10 gene SNPs related to the related to hereditary hearing loss can be simultaneously detected. The product can be used for detecting the gene types of 10 gene SNPs of a testee, and the detection results can be used as a reference to clinical diagnosis and also can be used in the fields, such as epidemiological investigation, prenatal screening and neonatal screening. According to the primer system, 10 gene SNPs on different genes can be simultaneously detected in one reaction system; and therefore compared with technologies, such as sequencing, real-time fluorogenic quantitative PCR (polymerase chain reaction), the primer system is lower in cost and simple and convenient to operate, and the accuracy and sensitivity are improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a detection method and product for determining genetic polymorphic sites (SNPs) related to hereditary deafness, specifically using multiplex PCR technology, single base extension technology and mass spectrometry technology, A method for detecting 10 gene polymorphic sites related to hereditary deafness and a corresponding kit. Background technique [0002] According to statistics from the Ministry of Health of my country, there are currently 120 million people with hearing impairment, and as many as 30 million people with hearing and speech disabilities, and the number of newborn deaf children is increasing at a rate of 30,000 every year, and more than 50% of the patients have hearing impairment. The etiology is related to genetic factors. According to the clinical statistics of foreign developed countries, patients with hereditary deafness account for 80% of the total. Therefore, the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 张学记马庆伟张海燕赵洪斌
Owner 向华
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