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Deaf-related gene mutation and its detecting method

A gene and mutation site technology, applied in the field of kits for detecting POU3F4 mutant genes

Inactive Publication Date: 2007-10-03
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] There is no report on the discovery of the POU3F4925T→C mutation in Chinese deaf patients

Method used

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  • Deaf-related gene mutation and its detecting method
  • Deaf-related gene mutation and its detecting method
  • Deaf-related gene mutation and its detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] 1. Preparation of blood sample DNA of the subject to be tested

[0066] 1. Research object

[0067] A Chinese family with X-linked genetic congenital profound hearing loss 021 showed congenital profound hearing loss. Temporal bone CT examination showed that the internal auditory canal was enlarged, showing an X-linked recessive inheritance pattern. A total of 31 members of the 021 family were investigated, including 17 males and 14 females. Eight patients, all male, had the same audiological phenotype. The POU3F4 gene mutation was detected in the 31 family members according to the following method, and all 8 male patients were found to be hemizygous for the mutation, and 8 heterozygous carriers were detected among the females. No mutation was found in 15 family members. In addition, 110 normal controls with normal hearing and no genetic background of hearing loss were selected for POU3F4 gene mutation detection, and no mutation was found.

[0068] All participants w...

Embodiment 2

[0099] 1. Purification of PCR products——96-well plate method

[0100] 1. Add 50 μl sterile water to the 96-well plate containing the PCR product and mix well.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 5. Store in a -20°C refrigerator.

[0105] 2. Quantification by electrophoresis

[0106] 1. Sample preparation

[0107] Take a 96-well spotting plate, add 6 μl of sample buffer to each well, remove the PCR product (2 μl) from each well of the cavity plate containing the PCR product, transfer t...

Embodiment 3

[0120] 1. Purity and dosage requirements of PCR product DNA template

[0121] DNA purity: OD 260 / OD 280 = 1.6 to 2.0.

[0122] DNA concentration: PCR product 10ng / μl.

[0123] DNA consumption:

[0124] PCR product

[0125] 100-200bp 1-3ng

[0126] 200-500bp 3-10ng

[0127] 500-1000bp 5-20ng

[0128] 1000-2000bp 10-40ng

[0129] >2000bp 40-100ng

[0130] 2. Sequencing reaction

[0131] 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.

[0132] 2. In order to ensure the freshness of sequencing samples and reaction reagents, it should be operated on ice when adding samples.

[0133] 3. The current reaction system is 5 μl, and the amount of various reagents added is shown in Tabl...

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Abstract

The invention relates to POU3F4 mutant gene having 925T-G mutation, wherein the mutation gene is related to human genetic deafness, the invention also provides a method for diagnosing the cause and type of the deaf occurrence through determining the existence of the mutation gene in the body of the patients, the mutation gene and the detection method can be used for POU3F4 mutation screening for deaf patients clinically.

Description

technical field [0001] The invention relates to a detection method for diagnosing human hereditary deafness by applying POU3F4 mutation gene. [0002] The invention also relates to a kit for detecting POU3F4 mutant gene. [0003] The invention also relates to a new POU3F4 mutant gene and its application in diagnosing and / or treating human hereditary deafness. Background technique [0004] Hereditary deafness is divided into nonsyndromic hearing impairment (NSHI) and syndromic hearing impairment (SHI). All NSHI and most SHI are Mendelian monogenic diseases, and very few SHI are chromosomal diseases. Deafness is the most common cause of communication impairment. It is estimated that about 700 million people in the world have a hearing loss of at least 55dB. Hearing loss of 25dB and above accounts for about 1% of young people, about 10% of people aged 60, and 50% at the age of 75. The incidence of pre-language deafness is 1 / 1000, about half of which are caused by genetic f...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王秋菊李庆忠韩东一
Owner GENERAL HOSPITAL OF PLA
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