Library building kit for detecting hereditary deafness genes and application

A kind of hereditary deafness, kit technology, applied in the direction of chemical library, biochemical equipment and method, microbial determination/inspection, etc., can solve the problem of erroneous results, probe binding error, probe position mutation, etc., to reduce detection Cost, accurate and reliable test results

Inactive Publication Date: 2017-10-24
MGI TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, time-of-flight mass spectrometry relies on the specificity of extended primers. Probe binding errors or mutations in probe positions are prone to false negative results, and are also susceptible to false results due to interference substances.

Method used

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  • Library building kit for detecting hereditary deafness genes and application
  • Library building kit for detecting hereditary deafness genes and application
  • Library building kit for detecting hereditary deafness genes and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Primer Design

[0052] 1. Primer design specifically includes the following steps:

[0053] 21 mutation sites of GJB2, GJB3, SLC26A4 genes and 12s rRNA gene related to hereditary deafness were selected as detection sites, namely: 35delG, 167delT, 176-191del16, 299_300delAT and 235delC of GJB2 gene, 538C> of GJB3 gene T and 547G>A, 281C>T, 589G>A, IVS7-2A>G, 1174A>T, 1226G>A, 1229C>T, 1975G>C, 2027T>A, 2162C>T, 2168A>G of SLC26A4 gene With IVS15+5G>A, 1494C>T, 1555A>G, 1095T>C of mitochondrial genes. Use primer design software to design PCR amplification primers for the above gene loci, and the length of the amplification primers is about 20 bases. Use the software to design and evaluate the sample tag sequence. The tag sequence contains 7 bases. When designing the tag sequence, avoid sequences with high similarity with the sequencing primers and PCR amplification primers with tags to form hairpin structures or dimers. level structure. See Table 1 for the ...

Embodiment 2

[0059] Example 2 Construction of library and sequencing

[0060] In this example, 24 clinical DNA samples whose information on the above-mentioned sites to be detected (Sanger sequencing detection) is known (Sanger sequencing detection) were detected, numbered 1-24, of which samples 1-21 were mutation-positive samples, and samples 22-24 were mutation-positive samples. Mutation-negative samples. The principle of the detection is as figure 1 As shown, the detection process is as follows figure 2 shown.

[0061] Specifically include the following steps:

[0062] 2. PCR amplification reaction

[0063] 2.1 Prepare the PCR amplification reaction mixture in a suitable centrifuge tube according to the ratio in Table 3, mix well, and distribute 18 μL per reaction well on ice into a 96-well PCR reaction plate;

[0064] Table 3 PCR amplification reaction mixture formula

[0065]

[0066]

[0067] 2.2 Use an eight-channel pipette to pipette 2 μL of the PCR reaction primers st...

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Abstract

The invention relates to a kit for building a library for detecting hereditary deafness genes and its application. The kit includes an amplification primer set for amplifying hereditary deafness genes; Primers designed for the polymorphism of the gene and the mutation site of the 12s rRNA gene. The present invention adopts multiplex PCR technology, which can detect more than 20 mutation sites of deafness genes in a single reaction; in conjunction with the "double label" system, the DNA amplification product of each sample has two sets of independent label sequences, so The amplification products of all samples can be mixed and sequenced simultaneously to achieve high-throughput detection, thereby greatly reducing the detection cost of a single sample.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a library building kit and its preparation method and application, in particular to a library building kit for detecting hereditary deafness genes and its application. Background technique [0002] Deafness is a very common sensory disorder that seriously affects human life. Its etiology is complex. Single or multiple gene mutations are likely to cause severe deafness, which is affected by environmental factors, such as drugs, trauma, extreme environmental exposure, etc. , can also cause sudden deafness. According to the data of WHO in 2013, the number of hearing-impaired people in the world has reached 360 million. According to the results of the main data communiqué of the second national sample survey of disabled persons published in December 2006, the number of people with speech and hearing-impaired people in my country has reached 27.8 million. There were 20,00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10C40B50/06
CPCC12Q1/6883C12N15/1093C12Q1/6869C12Q2600/156C40B50/06
Inventor 邹婧李全侯强张春杨谭宏东
Owner MGI TECH CO LTD
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