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Kit for detecting male infertility Fkbp6 gene mutation site, and PCR amplification method thereof

A technology for male infertility and mutation sites, applied in the field of biomedicine, can solve the problems such as the inability of the PCR reaction to proceed normally, the immature termination of the polymerization reaction, etc., and achieve the effect of high specificity and simple operation.

Inactive Publication Date: 2016-02-10
GENEHEAL BIOTECH
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  • Abstract
  • Description
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Problems solved by technology

[0016] When the high-fidelity DNA polymerase is performing DNA amplification, when the primers are completely matched with the template, the polymerization reaction will be carried out directly in the polymerization center of the polymerase; when the primers are not completely matched with the template, it will be sent to the enzyme center for correction. The corrected primers are sent back to the polymerization center for the polymerization reaction; if the primers cannot be corrected immediately, the PCR reaction cannot proceed normally, thereby triggering the immature termination of the polymerization reaction

Method used

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  • Kit for detecting male infertility Fkbp6 gene mutation site, and PCR amplification method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] human male infertility Fkbp6 Where is the gene pathogenic locus T141G located? Fkbp6 The DNA sequence of the second exon of the gene was used as a template, and the primers were designed using the software primerpremier5.0.

[0037] The base sequence of the primer for detecting the T141G mutation site:

[0038] Normal template matching primer T141GNF: 5’-TCCGAGAAGGAGCTG-3’; mutant template matching primer T141GMF: 5’-TCCGAGAAGGAGCGG-3’; common downstream primer T141GR: 5’-AGGCTGAGGCAGGAG-3’. Wherein, the phosphodiester bond between the -3 and -2 bases at the 3' end of the normal template matching primer T141GNF and the mutant template matching primer T141GMF is modified with phosphorothioate, or the -2 base is locked by nucleic acid (LNA) modification.

Embodiment 2

[0040] Take the blood sample of the patient under test and extract the DNA before using the primer kit to operate the process. PCR products are identified by gel electrophoresis system, and judged according to the presence or absence of electrophoresis bands Fkbp6 The genotype of the gene T141G mutation site. The specific judgment method is as follows:

[0041] 1. When a pair of primers consisting of a mutant template paired primer and a downstream primer is used for PCR amplification of an unknown DNA template, if the target electrophoretic band is observed, it indicates that the unknown DNA template contains a mutant gene at the corresponding detection site.

[0042] 2. When a pair of primers consisting of a normal template paired primer and a downstream primer is used for PCR amplification of an unknown DNA template, if the target electrophoretic band is observed, it indicates that the unknown DNA template contains a normal gene at the corresponding detection site.

[00...

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Abstract

The present invention discloses a kit for detecting one pathogenic mutation site in male infertility Fkbp6 gene, and a PCR amplification method thereof. The method is characterized in that mutation detection primers resisting 3'-5'exonuclease digestion modification are introduced into a high fidelity DNA involved PCR reaction so as to improve the PCR specificity, and the product band of the gel electrophoresis can be directly observed so as to determine the genotype of the corresponding mutation site of the Fkbp6 gene. According to the present invention, the detection kit has characteristics of low instrument requirements, simple operation, economy, no requirement of sequencing, and capability of being operated in general molecular biology laboratories.

Description

technical field [0001] The invention belongs to the clinical detection technology in the field of biomedicine, and relates to a detection method for a pathogenic mutation site of a human male infertility gene. [0002] The invention provides a PCR kit for detecting mutations of human male infertility pathogenic genes. The content of the present invention relates to a kit for detecting a disease-causing mutation site in the Fkbp6 gene of male infertility and a PCR amplification method thereof, and the detection result can be used for clinical auxiliary detection of male infertility. Background technique [0003] Male infertility refers to the fact that a man fails to impregnate his healthy spouse within one year when he has regular sex life and does not use contraception. Male infertility (Male Infertility) seriously affects modern men's health and family happiness. According to WTO statistics, about 15% of couples of childbearing age are infertile, of which male factors acc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 朱威贺雄雷郭蕾潘武广
Owner GENEHEAL BIOTECH
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