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ARMS fluorescent quantitative PCR-based gene mutation kit and method thereof

A fluorescence quantitative and kit technology, applied in the field of molecular biology, can solve the problems of unfavorable result judgment and high background, and achieve the effects of accurate and reliable result judgment, reduced pollution, and fast and convenient detection

Active Publication Date: 2014-09-10
JIANGSU MACRO&MICRO TEST MED TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the concentration of human genomic DNA molecular template is generally high, the background of ARMS primers after selective amplification is still high, and for specific sequences, ARMS primers are not enough to form obvious amplification differences, which is not conducive to the judgment of results

Method used

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  • ARMS fluorescent quantitative PCR-based gene mutation kit and method thereof
  • ARMS fluorescent quantitative PCR-based gene mutation kit and method thereof
  • ARMS fluorescent quantitative PCR-based gene mutation kit and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Detection of GJB235delG deletion mutations associated with deafness using conventional ARMS primers and enhanced ARMS primers of the present invention

[0051] In the present invention, on the basis of conventional ARMS primers, a mismatched base is set in the primer at the 6th to 8th upstream base of the deletion site in the direction of extension, thereby reducing the number of wild-type bases in real-time fluorescent quantitative PCR. The amplification efficiency of enhanced ARMS primers for mutant samples and the amplification efficiency of mutant enhanced ARMS primers for wild-type samples make the results easier to judge.

[0052] 1) Design of ARMS primers and enhanced ARMS primers

[0053] Three pairs of wild-type ARMS primers, three pairs of mutant ARMS primers, one pair of wild-type enhanced ARMS primers and one pair of mutant enhanced ARMS primers were designed in the experiment. 60.0°C, GC value 40.0%-60.0%, primer size 20±3bp. According to the sp...

Embodiment 2

[0072] Example 2 Detection of deafness-related gene 12SrRNA1494C>T point mutation using conventional ARMS primers and enhanced ARMS primers of the present invention

[0073] 1) Design of ARMS primers and enhanced ARMS primers

[0074]One pair of wild-type ARMS primers, one pair of mutant ARMS primers, one pair of wild-type enhanced ARMS primers and one pair of mutant enhanced ARMS primers were designed in the experiment. The common upstream primer was 12SrRNAF. 60.0°C, GC value 40.0%-60.0%, primer size 20±3bp. The 3' terminal bases of ARMS primers and enhanced ARMS primers are located at the sites to be tested and are matched with wild-type and mutant genes respectively. In order to improve specificity, introduce One mismatched base, and a mismatched base was introduced at the 4th base and the 13th base at the 3' end of the enhanced ARMS primer respectively. The designed ARMS primer and enhanced ARMS primer sequences are as follows:

[0075] Downstream ARMS primers:

[0076...

Embodiment 3

[0090] Example 3 Using conventional ARMS primers and enhanced ARMS primers of the present invention to detect warfarin medication-related sites VKORC13730G>A

[0091] 1) Design of ARMS primers and enhanced ARMS primers

[0092] One pair of wild-type ARMS primers, one pair of mutant-type ARMS primers, one pair of wild-type enhanced ARMS primers and one pair of mutant-type enhanced ARMS primers were designed in the experiment, and the common downstream primer was V3R. 60.0°C, GC value 40.0%-60.0%, primer size 20±3bp. The 3' terminal bases of ARMS primers and enhanced ARMS primers are located at the site to be tested and are matched with wild-type and mutant genes respectively. In order to improve specificity, a Mismatched bases, and a mismatched base was introduced at the 2nd base and the 7th base at the 3′ end of the enhanced ARMS primer. The designed ARMS primer and enhanced ARMS primer sequences are as follows:

[0093] Upstream ARMS primers:

[0094] V3WF1CCCTCCTCCTGCCATA...

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Abstract

The invention relates to an ARMS fluorescent quantitative PCR-based gene mutation kit and a method thereof. Compared with present gene polymorphism detection kits and methods thereof, the enhanced ARMS fluorescent quantitative PCR-based gene polymorphism mutation kit and the method thereof have the advantages of strong specificity, high sensitivity, low cost, highly reliable result, low background, easy determination of the result, ascendant operation and use, fast and convenient fluorescent quantitative PCR, closed tube detection in the whole course, pollution reduction, and reduction of the unnecessary cost and workload. The fluorescent quantitative PCR can realize the characterization of the amplification efficiency by using a CT value, so the method can realize the detection of the nuclear gene polymorphism heterozygote genotype conditions, and makes the result accurately and reliably determined.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a kit and method for detecting gene mutations based on ARMS fluorescence quantitative PCR. Background technique [0002] Human gene polymorphisms play an important role in elucidating the susceptibility and tolerance of the human body to diseases and poisons, the diversity of clinical manifestations of diseases, and the responsiveness to drug treatment. Through the study of gene polymorphism, the nature of differences in the functions and effects of biologically active substances among different human individuals can be revealed from the gene level. By studying the link between genetic polymorphisms and disease susceptibility, for example, deafness caused by mutations in the mitochondrial genes mtDNA A1555G and C1494T is mainly associated with aminoglycoside use, the vitamin K epoxide reductase complex subunit 1 gene (VKORC1) gene polymorphism and cytochrome P4502C9 ge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
CPCC12Q1/6858C12Q2535/137C12Q2531/113C12Q2561/101
Inventor 刘琦谢飞飞徐磊张森林其他发明人请求不公开姓名
Owner JIANGSU MACRO&MICRO TEST MED TECH CO LTD
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