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Specific primersand detection method of Tibetan mastiff molecular marker

A technology of molecular markers and primer sets, which is applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve problems such as genetic molecular markers of Tibetan mastiffs that have not yet been seen, and achieve the effect of enriching polymorphism

Active Publication Date: 2019-07-09
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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Problems solved by technology

At present, there is no relevant report on the development of genetic molecular markers for Tibetan mastiffs

Method used

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  • Specific primersand detection method of Tibetan mastiff molecular marker
  • Specific primersand detection method of Tibetan mastiff molecular marker
  • Specific primersand detection method of Tibetan mastiff molecular marker

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Experimental program
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Effect test

Embodiment

[0068] 1. Extraction of Tibetan Mastiff Genomic DNA

[0069] 1. Reagent preparation:

[0070] Digestion buffer: Weigh Tris1.2114g, EDTA37.224g, SDS5g, dilute to 1000mL with sterilized ultrapure water (final concentration is Tris10mmol / L, EDTA0.1mol / L, SDS0.5%), adjust to pH To 8.0, 4 degrees to save for later use.

[0071] 2. DNA extraction:

[0072] Extract 200 μL of blood samples from 100 domestic dogs, add 400 μL of digestion buffer, 40 μL of proteinase K (10 mg / mL), mix well and digest at 55°C for 5 hours; after the digestion is clear, add 600 μL of water-saturated phenol, and gently invert Homogenize for 30 minutes, centrifuge at 6000rpm / min for 10 minutes, extract the supernatant; then add 300μL water-saturated phenol, 300μL chloroform / isoamyl alcohol (24:1), gently invert and mix for 30 minutes, and centrifuge at 6000rpm / min for 10 minutes. Extract the supernatant; then add 600 μL of chloroform / isoamyl alcohol (24:1), gently invert and mix for 30 minutes, centrifuge ...

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Abstract

The invention relates to the technical field of specific primers and a detection method of single nucleotide variation and microsatellite markers of a dog variety of Tibetan mastiff. The method comprises the following steps: 1) extracting Tibetan mastiff genome DNA and other dog genome DNA; 2) taking the genome DNA extracted in the step (1) as a template, and respectively carrying out PCR amplification by using SNP molecular marker primers or primer groups; 3) sequencing a PCR amplification product by using a sequencer; 4) detecting the variation condition of single nucleotide polymorphic sites. The specific primers for the Tibetan mastiff molecular marker has a detailed detection method, and has an important application value for analyzing genetic characteristics, pedigree geographic patterns, population structures of Tibetan mastiff dogs and scientific protection and utilization of germplasm resources.

Description

technical field [0001] The invention belongs to the technical field of molecular markers, in particular to the technical field of specific primers and detection methods for single nucleotide variation and microsatellite markers of domestic dog breed Tibetan Mastiff. Background technique [0002] Tibetan Mastiff (Tibetan Mastiff) belongs to the domestic dog subspecies (Canis lupus familiaris) of the canidae canis gray wolf species of the order Carnivora. It is a unique domestic dog breed on the Qinghai-Tibet Plateau. Dan. Historically, Tibetan Mastiffs were mainly bred by local herders to protect livestock from predators in the wild. In recent years, due to its unique phenotype and ability to adapt to high altitudes, it has been highly sought after by the market. Market behaviors such as excessive trade, wrong introduction, random breeding, and artificial hype have made Tibetan Mastiff germplasm resources face a huge threat. In order to scientifically protect Tibetan mastif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156
Inventor 李艳
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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