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Primer in use for in vitro diagnosing GJB2 mutation of deaf gene of autosomal recessive inheritance in non-syndrome

An autosomal recessive and syndromic technology, applied in the field of genetic engineering, can solve the problems of high cost, complicated operation, expensive equipment, etc., and achieve the effect of reducing the chance of birth, simple operation steps and improving work efficiency.

Inactive Publication Date: 2006-12-06
山东三月三基因技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above method is not suitable for screening and promotion due to its high cost, expensive equipment, complicated operation, high technical requirements, and time-consuming and labor-intensive use.
[0006] Since we found that the mutation hotspot of the Chinese GJB2 gene is at the 233-235 site, and deaf patients with this site mutation can confirm that their deafness is caused by the GJB2 gene mutation; after the deletion of base C in the 233-235 site, It leads to a change in the sequence of the 230-235 position, so compared with the wild-type sample, the sample with the delC mutation at the 230-235 position of the GJB2 gene cannot be digested by the endonuclease ApaI

Method used

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  • Primer in use for in vitro diagnosing GJB2 mutation of deaf gene of autosomal recessive inheritance in non-syndrome
  • Primer in use for in vitro diagnosing GJB2 mutation of deaf gene of autosomal recessive inheritance in non-syndrome
  • Primer in use for in vitro diagnosing GJB2 mutation of deaf gene of autosomal recessive inheritance in non-syndrome

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Identification of non-syndromic autosomal recessive deafness gene GJB2 gene mutation by ApaI digestion method

[0043] 1. Test samples

[0044] A total of 998 children with non-syndromic deafness were collected from deaf schools across China. The coding region of the GJB2 gene was amplified by PCR, and the known 233-235 sites were preliminarily analyzed by enzyme digestion. At the same time, 204 normal hearing children were taken as the control group. Forward and reverse sequencing was performed on children with 233-235delC heterozygous mutations detected by enzyme digestion, and 40-50 cases were selected from various places for mutation detection in the entire coding region of the GJB2 gene. At the same time, 100 cases of normal hearing children were selected for control detection.

[0045] 2. Primer Design

[0046] Use the GenetoolLite program to assist in the design of improved primers. According to the published mitochondrial gene Cambridge sequence (Cam...

Embodiment 2

[0070] Example 2. The reliability of the direct sequencing method to test the ApaI enzyme digestion method

[0071] Steps 1-4 are the same as Steps 1-4 in Example 1, except that the root blood DNA is extracted from the sample. The following is the procedure for direct sequencing, in which the aforementioned kits are used.

[0072] 5. PCR product purification 1

[0073] The PCR amplification product of the tested sample was purified in the first step according to the following conditions:

[0074] Add 0.5 times the volume of sodium acetate (PH=5.2), 2.5 times the volume of 100% alcohol, leave it at room temperature for 20 minutes, centrifuge at 5700 rpm for 20 minutes, and remove the supernatant;

[0075] Add 200ul of 75% alcohol, centrifuge at 5700 rpm for 15 minutes, remove the supernatant, and dry at 95°C for 1 minute;

[0076] Add 20ul double distilled water to dissolve; after the DNA content is determined by gel electrophoresis, it is diluted to 5ng / ul.

[0077] 6. Seq...

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Abstract

This invention provides primers for in vitro diagnosis of mutation of autosomal recessive nonsyndromic hearing loss gene GJB2. The primers can be used for completely amplifying the coding region and upstream or downstream important splicing sequences of GJB2 gene. This invention also provides a test kit for containing the primers and ApaI restriction endonuclease and their application for in vitro detection of 233-235delC mutation of the complete GJB2 coding region. The test kit can also be used for detecting the mutation sites of the GJB2 coding region through sequencing by the forward primer, and detecting deletion or insertion heterozygous mutation through sequencing by the reverse primer. The test kit is suitable for large-scale screening of autosomal recessive nonsyndromic hearing loss gene GJB2 mutation and genetic counseling.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a primer for in vitro diagnosis of mutations in the entire coding region of the non-syndromic autosomal recessive deafness gene GJB2 gene and 233-235delC hotspot mutations. The present invention also relates to the application of the primer in the preparation of a kit or similar product for the in vitro diagnosis of mutations in the entire coding region of the non-syndromic autosomal recessive deafness gene GJB2, particularly a kit or similar product containing the primer , which can be used for in vitro diagnosis of mutations in the entire coding region of the non-syndromic autosomal recessive deafness gene GJB2 gene and 233-235delC hotspot mutations. Background technique [0002] Currently, 1 / 1000-3 / 1000 children are born with profound deafness. More than half of childhood deafness is hereditary deafness, and most of them are autosomal recessive and non-syndromic d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q2600/156C12Q1/6883
Inventor 戴朴于飞韩东一金政策
Owner 山东三月三基因技术有限公司
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