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Sheep back fat trait-related SNP and application thereof

A technology of sheep and chain reaction, applied in the field of bioengineering, can solve problems such as unclear knowledge, marker-assisted selection and breeding obstacles of sheep, and less research on CAST genes

Inactive Publication Date: 2011-01-12
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] It is currently known that there are four transcripts of the CAST gene in cattle, but there are few studies on the CAST gene in sheep, and the sequence on GenBank cannot clearly know which transcript it belongs to.
The research on the detection and functional verification of sheep CAST gene SNP is still blank, which brings great obstacles to the marker-assisted selection and breeding of sheep

Method used

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  • Sheep back fat trait-related SNP and application thereof
  • Sheep back fat trait-related SNP and application thereof
  • Sheep back fat trait-related SNP and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] (1) Fully cut up about 0.3 g of muscle tissue with ophthalmic scissors, put it in a 1.5 ml centrifuge tube, and dry it for 20 minutes.

[0019] (2) Add 500 μl of tissue DNA extraction solution to the above centrifuge tube.

[0020] (3) Add RNase solution to the above centrifuge tube to a final concentration of 20 μg / ml, mix thoroughly, and digest at 37°C for 1-2 hours.

[0021] (4) Add proteinase K to the above centrifuge tube to a final concentration of 150 μg / ml, mix thoroughly, and digest at 55°C for 12-24 hours.

[0022] (5) Add an equal volume of Tris saturated phenol to the above centrifuge tube, slowly invert the centrifuge tube for 10 minutes to fully mix the two phases of the solution at 4°C, centrifuge at 12000rpm for 10 minutes, then transfer the supernatant to a clean centrifuge tube . repeat.

[0023] (6) Add an equal volume of Tris-saturated phenol:chloroform:isoamyl alcohol (25:24:1), mix well, and centrifuge at 12,000 rpm for 10 minutes at 4°C.

[00...

Embodiment 2

[0030] The PCR primers for PCR-SSCP are shown in the table below.

[0031] 1. PCR amplification

[0032] Table 1 5 pairs of specific primer sequences, annealing temperature and target fragment length

[0033]

[0034] Add to PCR tube:

[0035] Template cDNA 1.0μl

[0036] Primer 1 (20pmol / μl) 0.3μl

[0037] Primer 2 (20pmol / μl) 0.3μl

[0038] 10mM dNTP Mix 2.0μl

[0039] 10× PCR buffer 2.5 μl

[0040] Taq DNA polymerase 0.30μl

[0041] wxya 2 O make up to 25 μl

[0042] PCR amplification temperature setting: pre-denaturation at 95°C for 3min

[0043] 35 cycles: denaturation at 95°C for 25 sec; annealing temperature see the above table, annealing for 25 sec; extension at 72°C for 20 sec;

[0044] Extend at 72°C for 10min

[0045] Cool down to 4°C for storage

[0046] 2% agarose gel was used to detect the results by electrophoresis. The results showed that the PCR products of the designed 3 pairs of primers were specific amplification, the fragment length was con...

Embodiment 3

[0065] According to the method described in Example 2, PCR amplification was performed on the total DNA of 450 offspring of Dorper sheep×Hu sheep, 145 offspring of Dorset sheep×Hu sheep, and 45 offspring of Suffolk sheep×Xinjiang fine-wool sheep. For the CAST-S3 amplification product, the HinfI enzyme can recognize the mutation at position 162, and the PCR product is digested with HinfI enzyme and typed ( image 3 ).

[0066] The digestion system is 20 μl, which contains 10 μl of PCR product, 2 μl of 10× restriction endonuclease-specific buffer, 4 U of restriction endonuclease, and high-pressure sterilized double-distilled water to make up to 20 μl. After mixing, digest at 37°C for about 4 hours according to the instructions of the enzyme. The digested products were electrophoresed on 2.0% agarose gel at 5V / cm, observed under ultraviolet light, and photographed.

[0067] The linear model software in SASv8.02 (Statistical Analysis System) was used to analyze the correlation b...

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PUM

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Abstract

The invention relates to the field of biological engineering, in particular to a method for predicating sheep back fat trait by using single nucleotide polymorphism. The method comprises the following steps of: performing PCR amplification on total DNA of the sheep by using primers in SEQ ID No: 2 and SEQ ID No: 3; and detecting the single nucleotide polymorphism by using PCR amplified products, and determining whether the 162nd position basic group at the 5' end of SEQ ID No: 1 in a sequence list is G or T, wherein the back fat thickness of the GT gene type is obviously thicker than that of the GG gene type. The detected polymorphic loci provide new materials for molecular breeding and provide scientific evidence for marker-assisted selection of the sheep back fat thickness.

Description

technical field [0001] The invention relates to a method for predicting sheep backfat traits by using single nucleotide polymorphism in the field of bioengineering. Background technique [0002] Designing degenerate primers based on known gene sequences of one species is a way to obtain unknown genes of another species. After the gene is obtained, the function of the new gene can be predicted by means of bioinformatics, and the tissue expression of the gene can be analyzed by semi-quantitative RT-PCR and fluorescent quantitative PCR, and verified by polymorphism analysis. Calpastatin (Calpastatin, CAST) gene has different transcripts in species such as mouse, human, rabbit and cattle, and the expression of different transcripts in different tissues is different (Emori et al., 1987, Proc Natl Acad Sci USA 84(11):3590-3594; Killefer and Koohmaraie, 1994, J Anim Sci 72(3):606-614; Takano et al., 2000, J Biochem 128(1):83-92). Zhu et al. found that there is a new subtype of CA...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 杜立新魏彩虹张莉张菊路国彬
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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