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Kit for detecting SLC26A4 gene mutation and application of kit

A kit and gene technology, applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of high cost, high technical requirements, and high equipment requirements, and achieve low cost and simple detection operation. , the effect of low equipment requirements

Active Publication Date: 2015-04-22
BIOSINO BIO TECH & SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are dozens of methods for single-nucleotide polymorphism (Single-nucleotide polymorphism, SNP) gene mutation detection, such as gene chip method, fluorescent quantitative PCR method, restriction fragment length polymorphism (RFLP) method and the direct sequencing method of gold standard PCR products, etc., but these methods generally have the characteristics of high technical requirements, high equipment requirements, and high cost, so their use at the grassroots level has certain limitations

Method used

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  • Kit for detecting SLC26A4 gene mutation and application of kit
  • Kit for detecting SLC26A4 gene mutation and application of kit
  • Kit for detecting SLC26A4 gene mutation and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The design of the primer of embodiment 1

[0033]Based on the analysis of the IVS7-2 site sequence of the SLC26A4 gene, the present invention uses software to design primers, and after multiple rounds of screening, it is found that the following primers can quickly and accurately detect the IVS7-2A>G mutation of the SLC26A4 gene.

[0034] Three kinds of primers were selected for PCR amplification experiments, and the selected primers were primer combinations designed for the sequence of the IVS7-2 site of the SLC26A4 gene. Its sequence is shown in Table 1.

[0035] Table 1 Sequences of three sets of primer combinations targeting the IVS7-2 site of SLC26A4 gene

[0036]

[0037] Table 2 reaction system

[0038] sample DNA

5μL

10×PCR buffer, containing Mg 2+

2μL

2.5mM dNTPs

0.3μL

20 μM S-1 / 5

1μL

20 μM S-2 / 6

1μL

20 μM S-3 / 7 / 8

1μL

20 μM S-4 / 9 / 10

1μL

5U / μL Taq DNA polymerase

0.4μ...

Embodiment 2

[0047] The establishment of embodiment 2 PCR detection method

[0048] 1. The best primer set (combination two) determined in Example 1 is used to detect the PCR reaction system screening test of SLC26A4 gene mutation

[0049] Table 4 Reaction System 1

[0050] sample DNA

5μL

10×PCR buffer, containing Mg 2+

2μL

2.5mM dNTPs

0.3μL

20 μM S1

1μL

20 μM S2

1μL

20 μM S3

1μL

20 μM S4

1μL

5U / μL Taq DNA polymerase

0.4μL

ddH2O

Make up to 20 μL.

[0051] Table 5 Reaction System II

[0052] sample DNA

5μL

10×PCR buffer, containing Mg 2+

2μL

2.5mM dNTPs

0.3μL

15 μM S1

1μL

20 μM S2

1μL

15 μM S3

1μL

20 μM S4

1μL

5U / μL Taq DNA polymerase

0.4μL

[0053] ddH2O

Make up to 20 μL.

[0054] Table 6 Reaction System III

[0055] sample DNA

5μL

10×PCR bu...

Embodiment 3

[0068] Embodiment 3 clinical application effect

[0069] Step 1, select the sample to be tested: the sample to be tested is a DNA sample detected by the "PCR amplification-sequencing" technical method. The steps are: take a certain amount of subject's blood sample DNA as a template, and synthesize a pair of universal SLC26A4 gene primer, the sequence is as follows:

[0070] Sequencing forward primer S5: 5'-AAGTTCAGCATTATTTGGTTGACA-3' (SEQ ID NO.5)

[0071] Sequencing reverse primer S2: 5'-TGGTTGTTTCTTCCAGATCACA-3' (SEQID NO.6)

[0072] PCR amplification was carried out, and after the reaction was completed, the PCR amplification products were sequenced, and the genotyping was carried out at the IVS7(-2) site of the SLC26A4 gene according to the sequencing results.

[0073] Select 4 people with normal IVS7(-2) site of SLC26A4 gene, 4 homozygous mutations of IVS7(-2) site of SLC26A4 gene and 4 heterozygous mutations of IVS7(-2) site of SLC26A4 gene as subjects .

[0074] Ste...

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PUM

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Abstract

The invention provides a kit for detecting SLC26A4 gene mutation and an application of the kit, and belongs to the technical field of gene detection. According to a tetras-primer amplification refractory mutation system-PCR technology, a group of specific primers are designed with respect to the condition that an SLC26A4 gene IVS7 (-2) site is greater than G mutation; a normal outer primer sequence is as shown in SEQ ID NO.1; a mutant outer primer sequence is shown in SEQ ID NO.2; a normal inter primer sequence is as shown in SEQ ID NO 3; and a mutant inter primer sequence is as shown in SEQ ID NO.4. PCR detection is carried out on a to-be-detected sample by virtue of four primers; and the genetype of the SLC26A4 gene in the to-be-detected sample is judged according to the agarose electrophoresis results. The primer disclosed by the invention is good in specificity; the detection method is fast, simple, high in accuracy, and good in sensitivity; and a detection method is provided for the condition that SLC26A4 gene IVS7-2A is greater than G mutation.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a kit for detecting deafness-related gene SLC26A4IVS7-2A>G mutation and its application. Background technique [0002] Solute transporter family 26, member 4 (solute carrier family 26, member 4; SLC26A4) gene is located at 7q31, encoding ion transport-related proteins, and is responsible for SO 4 2- , HCO 3- , I - , Cl - It transports a variety of monovalent or divalent ions, and plays an important role in the maintenance of the balance of ion components in the body. The inner ear has a complex and delicate fluid balance system, so any pathogenic gene mutation will lead to changes in the ionic composition and osmotic pressure of the inner ear, which will have an important impact on a person's hearing and body balance. Homozygous or heterozygous mutations in SLC26A4 are well-established causes of large vestibular aqueduct syndrome. [0003] The incidence of this ge...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2535/137C12Q2549/119
Inventor 黄茜及思莹杨君何犇易翔
Owner BIOSINO BIO TECH & SCI
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