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Primer pair and method for detecting cow GART (genotypic antiretroviral resistance testing) mutant genetype

A mutated gene, GART-F technology, applied in the field of molecular biology, can solve the problem of high cost, achieve the effects of low cost, avoid false positives, and simple operation

Inactive Publication Date: 2015-10-07
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is not only expensive due to the dependence on microarray detection, but also needs to establish a genotype database of the reference population in advance

Method used

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  • Primer pair and method for detecting cow GART (genotypic antiretroviral resistance testing) mutant genetype
  • Primer pair and method for detecting cow GART (genotypic antiretroviral resistance testing) mutant genetype
  • Primer pair and method for detecting cow GART (genotypic antiretroviral resistance testing) mutant genetype

Examples

Experimental program
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Effect test

Embodiment 1

[0035] In this example, the frozen semen samples of 6 Holstein cattle were tested.

[0036] (1) DNA extraction

[0037] The high-salt method was used to extract genomic DNA from the bovine frozen semen of each sample, and the specific steps were as follows:

[0038] 1) Prepare frozen semen lysate (80mL): first add 60mL of double-distilled water, add 0.5mol / L EDTA (PH=8.0) 2mL, 1mol / L Tris.CL (PH=8.0) 1mL, 10% SDS 10mL, DTT 0.77g, NaCl 0.29g, dilute to 80mL;

[0039] 2) Transfer the thin tube of frozen semen to a 2mL centrifuge tube, add 500 μL of normal saline, vortex centrifuge, discard the waste liquid and keep the precipitate, repeat the above operation once;

[0040] 3) Add 400 μL of frozen semen lysate and 100 μL of 20% SDS to the precipitate, vortex, and finally add 10 μL of proteinase K (20 mg / mL), mix well, and digest at 56 ° C for 20 to 22 hours;

[0041] 4) After digestion, add 300 μL of saturated saline, invert for 2-3 minutes, centrifuge at 12,000 rpm for 10 min...

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Abstract

The invention relates to molecular biology, and particularly provides a primer pair for detecting cow GART (genotypic antiretroviral resistance testing) mutant genetype. The primer pair is shown in SEQ ID No.1-2. The invention also provides a method for detecting the cow GART mutant genetype. The method comprises the following steps of PCR (polymerase chain reaction) amplification, enzyme digestion of amplification products, electrophoresis detection of enzyme digestion products and judgment of the genetype of a sample to be detected. By the primer pair, objective mutation sites of cow GART genes can be amplified, an amplified fragment comprises two other identical enzyme digestion sites, and a false positive result caused by incompletion of enzyme digestion or failing of an enzyme digestion reaction can be avoided, so that accuracy of detection is ensured.

Description

technical field [0001] The invention relates to molecular biology, in particular to a pair of primers and a detection method for detecting bovine GART mutation genotype. Background technique [0002] In recent decades, the improvement of dairy cow populations has achieved remarkable results. Through intensive selection of milk production performance, economic traits such as milk production, milk fat percentage, and milk protein percentage of dairy cows have been greatly improved. However, during the breeding process, the selection of breeding bulls is always limited to some excellent families, resulting in the gradual shrinking of the gene pool of dairy cows. At the same time, the widespread use of artificial insemination technology not only increases the degree of inbreeding in dairy cow populations, but also increases the risk of rapid spread of genetic defect genes within the population. Since the 1990s, a variety of genetic defects have been reported, including vertebra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 张毅劳兰兰刘林石万海孙东晓俞英王雅春张胜利
Owner CHINA AGRI UNIV
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