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Kit for detecting A75S mutation of SCN4B gene

A kit and detection method technology, applied in the field of molecular biology, can solve the problems that hinder the research and judgment of atrial fibrillation, and achieve the effects of improving diagnostic efficiency, high accuracy, and easy operation

Inactive Publication Date: 2018-05-01
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the control of environmental factors can delay the onset and prognosis of atrial fibrillation to a certain extent, the lack of understanding of inherent genetic factors has greatly hindered the research and judgment of atrial fibrillation

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] (1) Extract the blood DNA of the subject (who may carry the SCN4B A75S gene mutation) and dilute it to 100ng / μL;

[0040] (2) Prepare 20 μL ARMS-PCR reaction system, including: 10×PCR Buffer 2 μL, FI 0.5 μL, RI 0.5 μL, FO 2.5 μL, RO 2.5 μL, dNTP mixture 1.6 μL, Taq DNA polymerase 0.4 μL, Mg 2+ 1.6 μL, 1 μL of DNA template, add ddH 2 0 to 20 μL;

[0041] (3) Put the mixed reaction solution into the PCR instrument, and set the program as follows: pre-denaturation at 95°C for 10 minutes, followed by 1 cycle, denaturation at 95°C for 15 s, annealing at 45°C for 45 s, extension at 72°C for 45 s, a total of 32 cycles , after that, continue to extend at 72°C for 8min, 1 cycle. PCR reaction products were analyzed by gel electrophoresis using 3% agarose gel.

Embodiment 2

[0043] (1) Extract the blood DNA of the subject (who may carry the SCN4B A75S gene mutation) and dilute it to 100ng / μL;

[0044] (2) Prepare 20 μL ARMS-PCR reaction system, including: 10×PCR Buffer 2 μL, FI 0.5 μL, RI 0.5 μL, FO 0.5 μL, RO 0.5 μL, dNTP mixture 1.6 μL, Taq DNA polymerase 0.4 μL, Mg 2+1.6 μL, 1 μL of DNA template, add ddH 2 0 to 20 μL;

[0045] (3) Put the mixed reaction solution into the PCR instrument, and set the program as follows: pre-denaturation at 95°C for 10 minutes, followed by 1 cycle, denaturation at 95°C for 15 s, annealing at 45°C for 45 s, extension at 72°C for 45 s, a total of 32 cycles , after that, continue to extend at 72°C for 8min, 1 cycle. PCR reaction products were analyzed by gel electrophoresis using 3% agarose gel.

[0046] The ratio of endogenous primer and exogenous primer in embodiment 2 is 1:1.

Embodiment 3

[0048] (1) Extract the blood DNA of the subject (who may carry the SCN4B A75S gene mutation) and dilute it to 100ng / μL;

[0049] (2) Prepare 20 μL ARMS-PCR reaction system, including: 10×PCR Buffer 2 μL, FI 0.5 μL, RI 0.5 μL, FO 5 μL, RO 5 μL, dNTP mixture 1.6 μL, Taq DNA polymerase 0.4 μL, Mg 2+ 1.6 μL, 1 μL of DNA template, add ddH 2 0 to 20 μL;

[0050] (3) Put the mixed reaction solution into the PCR instrument, and set the program as follows: pre-denaturation at 95°C for 10 minutes, followed by 1 cycle, denaturation at 95°C for 15 s, annealing at 45°C for 45 s, extension at 72°C for 45 s, a total of 32 cycles , after that, continue to extend at 72°C for 8min,

[0051] 1 cycle. PCR reaction products were analyzed by gel electrophoresis using 3% agarose gel.

[0052] The ratios of endogenous primers and exogenous primers in Examples 2 and 3 are 1:1 and 1:10 respectively, and the brightness of the 300bp wild-type or 150bp positive mutant products produced is only 1 in th...

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Abstract

The invention discloses a kit for detecting A75S mutation of an SCN4B gene and belongs to the field of molecular biologics. The kit comprises Taq DNA polymerase, 10*PCR buffer liquid, Mg<2+>(MgCl2), dNTP mixture, H2O, an A75S endogenesis upstream primer F1, an A75S endogenesis downstream primer R1, an exogenous upstream primer FO and exogenous downstream primer RO, wherein the sequences of the primers are as shown in SEQ ID NO.1-4. The kit is applicable to nearly all PCR instruments and is low in equipment requirement, the operation feasibility of the kit is greatly improved, and the cost of the kit is also greatly reduced when being compared with a detection method which needs precise equipment. The kit can be applied to primary detection on A75S mutation of the SCN4B gene, diagnosis bases are provided for SCN4B A75S gene mutation associated auricular fibrillation diseases, and the diagnosis efficiency is improved.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a kit for detecting the A75S mutation of the SCN4B gene. Background technique [0002] Atrial fibrillation (AF) is one of the most common arrhythmia diseases clinically, and is an independent risk factor for various cardiovascular diseases. Serious consequences such as tachycardia, ventricular arrhythmia and even death are extremely harmful to public health. [0003] With the development of molecular medicine, atrial fibrillation is considered to be the result of the joint action of genetic factors and environmental factors. Although the control of environmental factors can delay the onset and prognosis of atrial fibrillation to a certain extent, the lack of understanding of inherent genetic factors has greatly hindered the research and judgment of atrial fibrillation. [0004] Studies in recent years have shown that multiple molecular markers, such as KCNQ1, SCN5A, KCNJ2, SCN3...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/156C12Q2535/137
Inventor 黄渊唐景峰陈兴珍周策凡
Owner HUBEI UNIV OF TECH
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