Detection kit for c.403>T mutation of CDH23 gene

A kit and gene technology, applied in the field of kits for detection of CDH23 gene mutations, can solve problems such as inability to express, significant impact on protein function, etc.

Inactive Publication Date: 2012-10-17
王秋菊 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The applicant's research group carried out the screening of this gene on 108 Chinese patients with hereditary deafness and / or Usher 1D syndrome, and found a new mutation of c.403C>T(p.Q135X) in the CDH23 gene. Located in the extracellular functional domain of the gene product cadherin protein 23, between the first and second repeat regions of its 27 repeat sequences, it will cause the expression of the subsequent 26 extracellular repeat sequences, which has a major impact on protein function

Method used

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  • Detection kit for c.403>T mutation of CDH23 gene
  • Detection kit for c.403>T mutation of CDH23 gene
  • Detection kit for c.403>T mutation of CDH23 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] 【Example 1】 Extraction of blood samples to be tested and PCR amplification of CDH23 gene coding region

[0068] 1. Preparation of blood sample DNA of the subject to be tested

[0069] 1. Research object

[0070] 108 patients with sporadic hereditary deafness and / or Usher 1D syndrome and 100 normal hearing controls without family history were screened for CDH23 gene according to the following method. A CDH23 gene test in a deaf patient was found to be a c.403C>T homozygous mutation. This patient was also accompanied by retinitis pigmentosa and had been diagnosed with Usher 1D syndrome. In his family, the deafness phenotype co-segregated with this genotype. No c.403C>T mutation was found in the screening of 100 normal hearing persons.

[0071] All participants were investigated in detail about their medical history and family history, and a physical examination was performed on them. The otological examination included otoscopy and audiological evaluation. After sig...

Embodiment 2

[0101] [Example 2] Purification and Quantification of PCR Amplified Product of Coding Region of CDH23 Gene

[0102] 1. Purification of PCR products——96-well plate method

[0103] 1. Add 50 μl sterile water to the 96-well plate containing the PCR product and mix well.

[0104] 2. Transfer it to the Millipore purification plate, put it on the vacuum pump for about 3 minutes, and see that there is no water in the purification plate.

[0105] 3. Add 50 μl of deionized water to the purification plate again, and continue to filter until there is no water in the purification plate.

[0106] 4. Remove the purification plate from the vacuum pump, add 20 μl of deionized water to the plate, let it rest for 15 minutes, shake it for another 15 minutes, and then suck it into a new 96-well plate.

[0107] 5. Store in a -20°C refrigerator.

[0108] 2. Quantification by electrophoresis

[0109] 1. Sample preparation

[0110] Take a 96-well spotting plate, add 6 μl of sample buffer to ea...

Embodiment 3

[0121] [Example 3] Direct Sequencing of PCR Amplified Product of Purified CDH23 Gene Coding Region

[0122] 1. Purity and dosage requirements of PCR product DNA template

[0123] DNA purity: OD 260 / OD 280 =1.6~2.0.

[0124] DNA concentration: PCR product 10ng / μl.

[0125] DNA consumption:

[0126] PCR product

[0127]

[0128] 2. Sequencing reaction

[0129] 1. The reagents required for the sequencing reaction should be freshly prepared, and the reagents that need to be sterilized by autoclaving must be sterilized before use. The equipment required for the sequencing reaction (such as 384-well plates, tips, etc.) should also be clean and sterile.

[0130] 2. In order to ensure the freshness of sequencing samples and reaction reagents, it should be operated on ice when adding samples.

[0131] 3. The current reaction system is 5 μl, and the amount of various reagents added is shown in Table 2.

[0132] Table 2 The sequencing reaction system of the PCR amplificati...

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Abstract

The invention provides a detection kit for c.403>T mutation of the CDH23 gene. Causes and types of hereditary hearing loss and/or Usher ID syndrome are diagnosed through detecting whether patients have the mutation gene or not. The mutation gene and a detection method mentioned above are beneficial for clinical screening of CDH23 mutation for patients with hereditary hearing loss, providing bases for diagnosis and treatment of patients with hereditary hearing loss and Usher ID syndrome.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a kit for detecting CDH23 gene mutation, and also relates to the CDH23 mutation gene and its application in diagnosing and / or treating hereditary deafness and / or Usher 1D syndrome. Background technique [0002] Deafness is a common and highly disabling disease in the field of population and health, and is a major public health issue of global concern. According to WHO's 2005 estimate, the global hearing disabled population reached 278 million, accounting for 4.6% of the world's total population. In my country, 27.8 million people were disabled due to deafness, accounting for 33.52% of the total number of disabled people in my country. Deafness not only affects individuals and families, but also brings a heavy burden to the whole society. Deafness is a common hearing disease caused by auditory conduction pathway disorders caused by genetic or / and environmental factors. Current resea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 王秋菊
Owner 王秋菊
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