SNP (single nucleotide polymorphism) marks of Yuxi fat-tailed sheep as well as screening method and application of SNP marks

A screening method and labeling technology, which can be used in biochemical equipment and methods, recombinant DNA technology, DNA/RNA fragments, etc. , the effect of no interference between sites

Inactive Publication Date: 2017-08-25
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Microsatellite markers were first used for individual identification, but the disadvantage is that it is difficult to achieve standardization and scale due to the different conditions in different laboratories

Method used

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  • SNP (single nucleotide polymorphism) marks of Yuxi fat-tailed sheep as well as screening method and application of SNP marks
  • SNP (single nucleotide polymorphism) marks of Yuxi fat-tailed sheep as well as screening method and application of SNP marks
  • SNP (single nucleotide polymorphism) marks of Yuxi fat-tailed sheep as well as screening method and application of SNP marks

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Determination of Yuxi fat-tailed sheep breeds by pyrophosphoric acid method

[0036] (1) Reagents: GoTaqMaster Mix solution produced by Promega Company; specific amplification primers and sequencing primers synthesized by Shanghai Sangon; Sepharose Bead produced by Biotage Company.

[0037] (2) Amplification reaction system and amplification program: the total volume of the amplification reaction is 50 μL, and its various components are: 2×GoTaqMaster Mix 25 μL, 10 μmol / L primers 1 μL each, DNA template 2 μL for the test variety and the control variety (Cucumber seed DNA), make up to 50 μL with sterilized deionized water; reaction program: pre-denaturation at 95°C for 10 minutes, denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, extension at 72°C for 45 seconds, 50 cycles, and finally extension at 72°C for 7 minutes , stored at 4°C.

[0038] (3) Sequencing reaction system: Add 47 μL Binding buffer and 3 μL Sepharosebeads to 50 μL PCR prod...

Embodiment 2

[0041] Example 2PCR amplification method for determination of Yuxi fat-tailed sheep breeds

[0042] Randomly select 24 Altay sheep, 24 Bayinbulak sheep and 29 Yuxi fat-tailed sheep as sample individuals, collect the blood of these sheep, anticoagulate with heparin, and extract genomic DNA with Tiangen blood genome kit, 0.8% Agarose gel electrophoresis detection, nucleic acid protein quantification instrument to measure its concentration and purity, diluted to 100ng / ul, and stored for future use. Use the primers listed in Table 2 to perform PCR amplification using the genomic DNA of the sample to be tested as a template. The PCR amplification reaction system is: add 0.4 μL of Taq DNA polymerase (5U / μL), 5 μL of 10×PCR Buffer, and 4 μL of MgCl2 (25mmol / μL), 1μL of Forward primer (10pmol / μL), 1μL of Reverse primer (10pmol / μL), 4μL of dNTP (2.5mmol / μL), 2μL of template DNA and 32.6μL of ddH2O, a total of 50μL.

[0043] PCR products were recovered and purified using an agarose gel...

Embodiment 3

[0044] Example 3 Determination of Yuxi fat-tailed sheep breeds by gene chip method

[0045] Step 1, taking experimental animals, taking blood from ear veins, and extracting genomic DNA from the blood samples.

[0046] Step 2. Take the genomic DNA obtained in step 1 and hybridize it with the nucleic acid chip with inherent 149 probes (30 probes are the single-stranded DNA molecules shown in sequence 1 to sequence 30 in Sequence Table 2).

[0047] Step 3. After step 2 is completed, the ends of each point in the nucleic acid chip are extended, so as to obtain the genotypes of 149 SNP sites in the genomic DNA.

[0048] Step 4, taking the genomic DNA obtained in step 1, performing whole genome sequencing, and obtaining the genotypes of 149 SNP sites in the genomic DNA. The result shows that the result of step 3 is exactly the same as the result of step 4. Among the 149 SNP sites of 48 experimental animals of Yuxi fat-tailed sheep, at least 99-136 sites could be matched. The matc...

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Abstract

The invention discloses SNP (single nucleotide polymorphism) marks of Yuxi fat-tailed sheep as well as a screening method and an application of the SNP marks. SNP genotyping analysis is performed according to specific SNP marks of Dorper sheep with a pyrosequencing method, a sanger sequencing method, a genechip method or time-of-flight mass spectrometry, and whether an experimental variety is the Dorper sheep or not is identified. Compared with a conventional determination method, the variety authenticity identifying method is simple and convenient to operate, free of interference among loci and suitable for high-flux operation. Based on the pyrosequencing method, the method has very high accuracy, variety identification can be finished in 5 h, result judgment is simple and an identification result can be directly read according to signal peaks of visible light.

Description

technical field [0001] The invention belongs to the technical field of sheep breed identification, and in particular relates to a SNP marker of Yuxi fat-tailed sheep, a screening method and application thereof. Background technique [0002] Yuxi fat-tailed sheep (Yuxi Fat-tailed sheep) is a fine local breed in Henan Province, which was compiled into the book "Records of Livestock and Poultry Breeds in Henan Province" in 1985. Over the years, due to the small impact of the environment, the quality of Yuxi fat-tailed sheep has not changed significantly, and its excellent traits of resistance to rough feeding, strong disease resistance, heat resistance and fast fattening have been preserved. It has strong adaptability, rapid growth and development, early sexual maturity, high lamb survival rate, good meat quality, and a slaughter rate of up to 57%. Its meat is delicious, tender, rich in nutrition, unique in flavor, good in color and taste, warm but not hot, oily but not greasy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/156
Inventor 王文姜雨周东珂侯雨楠郝志强袁圣钧
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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