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Kit for detecting SLC26A4 gene c.665G>T mutation

A kit and gene technology, applied in the field of kits for detecting SLC26A4 gene mutations, can solve problems such as changes in membrane labyrinth structure

Inactive Publication Date: 2013-01-02
王秋菊 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And due to toxic mechanisms of altered osmotic pressure and altered composition lead to altered membrane labyrinth structure

Method used

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  • Kit for detecting SLC26A4 gene c.665G>T mutation
  • Kit for detecting SLC26A4 gene c.665G>T mutation
  • Kit for detecting SLC26A4 gene c.665G>T mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] 【Example 1】 Extraction of blood samples to be tested and PCR amplification of SLC26A4 gene coding region

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

[0069] 1. Research object

[0070] 272 patients with sporadic vestibular aqueduct enlargement and 100 normal hearing controls without family history were screened for SLC26A4 gene according to the following method. Among the 272 patients, 2 patients with enlarged vestibular aqueduct were found to be compound heterozygotes of the c.665G>T mutation and another known pathogenic mutation (c.235delC) through SLC26A4 gene detection. No c.665G>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 signing the informed consent form, 5-10ml blood samples we...

Embodiment 2

[0101] [Example 2] Purification and Quantification of PCR Amplified Products of Coding Region of SLC26A4 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...

Embodiment 3

[0121] [Example 3] Direct Sequencing of PCR Amplified Product of Purified SLC26A4 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]

[0129] 2. Sequencing reaction

[0130] 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.

[0131] 2. In order to ensure the freshness of sequencing samples and reaction reagents, it should be operated on ice when adding samples.

[0132] 3. The current reaction system is 5 μl, and the amount of various reagents added is shown in Table 2.

[0133] Table 2 Sequencing reaction system of PCR ampli...

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Abstract

The invention provides a kit used for detecting SLC26A4 gene c.665G>T mutation. Through the detections upon whether the mutation gene exists in a patient, occurrence causes and types of enlarged vestibular aqueduct can be diagnosed. The mutation gene and the detection method assist in promoting clinical implementations of SLC26A4 mutation screening of enlarged vestibular aqueduct patients, and in providing a basis for diagnosis and treatments for enlarged vestibular aqueduct patients.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a kit for detecting SLC26A4 gene mutation, and also relates to a new SLC26A4 mutation gene and its application in diagnosing and / or treating vestibular aqueduct enlargement diseases. Background technique [0002] The SLC26A4 gene is located at 7q31, which was originally located by Everett et al., and found that the gene mutation can lead to an autosomal recessive genetic disease: Pendred syndrome, clinical manifestations of goiter, and combined vestibular aqueduct enlargement or Mondini malformation ( Enlarged vestibular aqueduct combined with cochlear hypoplasia) sensorineural deafness. Later, Usami et al. screened the SLC26A4 gene in 6 families with simple vestibular aqueduct enlargement and showed that the mutation of this gene can also lead to simple vestibular aqueduct enlargement, and its inheritance mode is also recessive. Thus, mutations in the SLC26A4 gene can lead to enla...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王秋菊
Owner 王秋菊
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