Kit and PCR amplification method for detection of one pathogenic mutation site of male infertility Dmc1 gene

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

Inactive Publication Date: 2014-07-09
GENEHEAL BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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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 and PCR amplification method for detection of one pathogenic mutation site of male infertility Dmc1 gene
  • Kit and PCR amplification method for detection of one pathogenic mutation site of male infertility Dmc1 gene
  • Kit and PCR amplification method for detection of one pathogenic mutation site of male infertility Dmc1 gene

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Experimental program
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Effect test

Embodiment 1

[0036] Using the DNA sequence of exon 11 of the Dmc1 gene, which is the pathogenic site G817C of the human male infertility Dmc1 gene, was used as a template, primer premier 5.0 was used to design primers.

[0037] The base sequence of the primer to detect the mutation site of G817C:

[0038] Normal template matching primer G817C N F: 5'-TCAAATGACTGCCGA-3'; mutant template matching primer G817C M F: 5'-TCAAATGACTGCCCA-3'; common downstream primer G817C R: 5'-AGGTTCAAGTGATTC-3'. Wherein, the normal template paired primer G817C NF and the mutant template paired primer G817C MF have a phosphodiester bond between the -3 and -2 bases at the 3'end, or the -2 base is modified by phosphorothioate Locked nucleic acid (LNA) modification.

Embodiment 2

[0040] Take the blood sample of the tested patient and extract the DNA before using the primer kit. The PCR products were identified by a gel electrophoresis system, and the genotype of the G817C mutation site of Dmc1 gene was judged according to the presence or absence of electrophoretic bands. 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 performs PCR amplification on an unknown DNA template, if the target electrophoresis 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 performs PCR amplification on an unknown DNA template, if the target electrophoresis band is observed, it indicates that the unknown DNA template contains a normal gene at the corresponding detection site.

[0043] 3. When the above two pai...

Embodiment 3

[0046] After taking a blood sample from the tested patient and extracting the DNA, the kit uses the process operation. The PCR products were identified by a gel electrophoresis system, and the genotype of the G817C mutation site of Dmc1 gene was judged according to the presence or absence of electrophoretic bands. The specific judgment method is the same as in the first embodiment. The PCR amplification results are attached Figure II Shown. According to the electrophoresis band status of the PCR product, the G817C site in the Dmc1 gene of sample 1 is normal homozygous; the genotype of sample 2 is mutation homozygous; the genotype of sample 3 is heterozygous. The fragment was sent for sequencing, and the sequencing result confirmed that the PCR detection result was correct. Judging from this, the probability of the carrier of sample 1 suffering from genetic male infertility is very low; the probability of carrier of sample 2 suffering from genetic male infertility is extreme...

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Abstract

The invention discloses a kit and a PCR amplification method for detection of one pathogenic mutation site of a male infertility Dmc1 gene. The method is characterized in that a modified mutation-detection primer resistant to 3'-5' exonuclease enzyme digestion is used in a PCR reaction in which a high-fidelity DNA polymerase participates, so that the specificity of PCR is improved; and the existing condition of a gel electrophoresis product band can be directly observed to judge the genotype of the Dmc1 gene corresponding mutation site. The detection kit has low requirements on instruments and equipment, is simple in operation, is economic, has no need of sequencing, and can be operated in common 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 the pathogenic mutation site of the human male infertility gene. [0002] The invention provides a PCR kit for detecting mutations in human male infertility genes. The content of the present invention relates to a kit for detecting a pathogenic mutation site in the Dmc1 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 has not conceived his healthy spouse within one year when he has regular sex without contraception. Male Infertility seriously affects the health of modern men and family happiness. According to WTO statistics, about 15% of couples of childbearing age are infertile, of which 50% are male factors. The causes of male infertility are complex ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q1/686C12Q2600/156
Inventor 朱威贺雄雷潘武广郭蕾雷楗勇潘岷溟
Owner GENEHEAL BIOTECH
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