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Genetic deafness associated gene detection kit and specific primer set

A hereditary deafness and gene detection technology, applied in the direction of combinatorial chemistry, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of specificity, accuracy, low uniformity, and difficulty in implementing multiple PCR capture technology, etc. Achieve the effects of good uniformity and stability, high utilization rate and detection accuracy, and low amount of template DNA

Active Publication Date: 2019-01-18
CAPITALBIO GENOMICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, generally speaking, the more target regions to be captured by multiplex PCR, the lower the specificity, accuracy, and uniformity of amplification, and the more difficult it is to realize multiplex PCR capture technology; in addition, based on different high-throughput sequencing platforms , the implementation of multiplex PCR capture technology is also different

Method used

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  • Genetic deafness associated gene detection kit and specific primer set
  • Genetic deafness associated gene detection kit and specific primer set
  • Genetic deafness associated gene detection kit and specific primer set

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. Specific primer set for detection of hereditary deafness-related genes

[0027] The inventor screened 33 mutation sites of 5 genes related to hereditary deafness from the database, and designed specific primers according to the sequence information of the mutation sites and the 300 bp flanking each side, and after a large number of tests, screening, optimization, and verification, finally 21 pairs of primers with high amplification efficiency and good specificity were preferably selected (as shown in Table 1).

[0028] The 33 mutation sites in 5 related genes of hereditary deafness are: c.235delC, c.299_300delAT, c.35delG, c.176_191del16, c.512insAACG, c.427C>T, c.257C>G, c. .35insG, c.109G>A site, c.538C>T site in GJB3 gene, c.917insG, IVS7-2A>G, c.2168A>G, c.1229C>T, c.1174A in SLC26A4 gene >T, c.1975G>C, c.2027T>A, c.589G>A, c.1226G>A, c.281C>T, IVS15+5G>A, c.2086C>T, c.754T>C , c.1079C>T, c.1343C>T, c.1336C>T, c.387delC, IVS14+1G>A, IVS13+9C>T, c.1693in...

Embodiment 2

[0032] Example 2, hereditary deafness-related gene detection kit

[0033] According to the semiconductor sequencing platform, this embodiment exemplarily provides a genetic deafness-related gene detection kit based on the semiconductor sequencing method. The genetic deafness-related gene detection kit based on sequencing should also be within the scope of protection of this application, and will not be exemplified here.

[0034] Genetic detection kits related to hereditary deafness, including:

[0035](1) Specific primer set: add a common primer to the 5' end of each specific primer shown in SEQ ID NO:1~SEQ ID NO:42, and the common primer sequence is: 5'-AAATGGGCGGTAGGCTTG-3 '(SEQ ID NO: 43);

[0036] (2) Sequencing adapter: 5'-CCTCTCTATGGGCAGTCGGTGAT-common primer-3' (SEQ ID NO:44);

[0037] (3) Index adapter: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAGNNNNNNNNGAT-common primer-3' (SEQ ID NO: 45); N in the sequence represents any base of AGCT, and NNNNNNNNN is used to identify libra...

Embodiment 3

[0040] Example 3, library construction and sequencing of genes related to hereditary deafness

[0041] In this example, the kit in Example 2 was used to build a library and sequence genes related to hereditary deafness using 35 cases of detection limit reference products (L01-L35) and 3 cases of negative reference products (N01-N03) as templates. The steps are as follows:

[0042] 1. One-step multiplex PCR

[0043] Prepare a 25 μL multiplex PCR reaction system, including: template DNA 5-20 ng, specific primer set 0.2 μL, sequencing adapter 0.5 μL, index adapter 0.5 μL, amplification buffer 12.5 μL, nuclease-free water balance; different templates use different label connector.

[0044] Amplification was performed according to the following procedure: ① 95°C for 3 min; ② 35 cycles of amplification, each cycle: 95°C for 15 s, 60°C for 90 s, 72°C for 30 s; ③ 72°C for 1 min, and ④ 16°C to hold.

[0045] 2. Amplified product mixing and purification

[0046] Take 10 μL of the amp...

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Abstract

The invention discloses a genetic deafness associated gene detection kit and a specific primer set. The invention designs a specific primer set for 33 mutation sites of genetic deafness-related genes, and a detection kit for genes associated with genetic deafness was prepared, the kit and the specific primer set of the present invention can be used quickly, Accurate, one-time detection of 33 genetic mutations associated with genetic deafness, and the use of specific primers for the construction of a library of multiple PCR homogeneity and stability, the amount of required initial template DNA, the library obtained from the utilization of sequencing data and detection accuracy is high, applicable to a variety of sample types.

Description

technical field [0001] The invention belongs to the field of gene detection, and more specifically relates to a genetic detection kit and a specific primer set related to hereditary deafness. Background technique [0002] Deafness is the most common communication disorder, which has a significant impact on personal life and social development. The number of hearing disabled people in my country is close to 30 million, accounting for 33% of the total disabled population in the country, ranking first in the country's disabled population. The number of deaf carriers in the normal hearing population is as high as 80 million; among newborns, the proportion of deafness is about 1 to 3 ‰, it is estimated that there are more than 30,000 newborn deaf children every year, about 60% of which are caused by genetic factors, and the rest are caused by environmental factors or the combined effects of environmental factors and genetic factors. Deafness is divided into congenital deafness, d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6806C40B50/06C12N15/10C12N15/11
Inventor 黄铨飞朱鹏远吴春求黄晓燕王杨
Owner CAPITALBIO GENOMICS
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