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Method for detecting cattle SMC2 gene recessive lethal mutation

A lethal mutation and gene technology, applied in the field of molecular biology, can solve the problem of high cost, achieve high accuracy, improve seed conservation efficiency, and simple operation.

Active Publication Date: 2016-10-12
CHINA AGRI UNIV +1
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  • Application Information

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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; in addition, because it is essentially an indirect inference of the genotype of the disease-causing mutation site using the surrounding SNP markers, the result is not accurate. 100% accurate

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  • Method for detecting cattle SMC2 gene recessive lethal mutation
  • Method for detecting cattle SMC2 gene recessive lethal mutation
  • Method for detecting cattle SMC2 gene recessive lethal mutation

Examples

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Embodiment 1

[0032] Example 1 Establishment of Pyrosequencing Method for Detecting Recessive Lethal Mutation of Bovine SMC2 Gene

[0033] 1. Genomic DNA extraction The high-salt method was used to extract genomic DNA from frozen bovine semen.

[0034] 2. Primer screening and PCR amplification

[0035] The target DNA sequence (UMD3.1:8:95410307:95410707:1) was obtained from the ensembl database (http: / / asia.ensembl.org / ), and PCR amplification primers and sequencing primers were designed. The position of the following primers and sequencing sequences on the bovine SMC2 gene sequence is shown in figure 1 .

[0036] Table 1 Primer combination for detection of recessive lethal mutation of bovine SMC2 gene

[0037]

[0038] Among them, the upstream primer (SMC2-F) and the downstream primer (SMC2-R) will amplify the SMC2 gene fragment containing the recessive lethal mutation site; the sequence of the universal primer (SMC2-U) and the sequence of the downstream primer (SMC2-R) The first 20...

Embodiment 3

[0056] Embodiment 3 The practical application of the inventive method

[0057] Using the method described in Example 1 of the present invention, 152 Holstein bulls in the Beijing Dairy Center were clinically tested, and 15 lethal mutation carriers were found, accounting for 9.9% of the total. Based on the test results, a selection and mating plan for these bulls was formulated, thereby gradually eliminating carriers of harmful genes and significantly improving the efficiency of selection.

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Abstract

The invention provides a method for detecting cattle SMC2 gene recessive lethal mutation, and relates to the field of molecular biology. The method provided by the invention is based on a pyrosequencing technology, a pair of primers, a universal primer and a sequencing primer having the amino acid sequences as shown in SEQ ID No.1, 2, 3 and 4 respectively are adopted for analyzing single nucleotide polymorphism of a 95410507 bp locus of a cattle No.8 chromosome, and results find out that a wild-type homozygote has a T / T genotype at a mutation locus, a pyrosequencing result pattern indicates that the peak height ratio of the T base to the C base is 3:1; a lethal mutation carrier peak pattern has a T / C genotype at the mutation locus, and the pyrosequencing result pattern indicates that the peak height ratio of the T base to the C base is 5:3. The method provided by the invention is simple to operate, high in sensitivity, strong in accuracy, high in throughput and low in detection cost, and has importable application value in cattle breed-conservation and breeding processes.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a SNP molecular marker for detecting the bovine SMC2 genotype and a method for detecting the recessive lethal mutation of the bovine SMC2 gene. Background technique [0002] Single nucleotide polymorphism markers (SNPs) mainly refer to DNA sequence polymorphisms caused by single nucleotide mutations on the genome. Detection of genotype by detection of single nucleotide polymorphism markers is a method that has emerged in recent years. Molecular markers have been used in animal breeding for some time. Compared with traditional breeding methods, molecular breeding greatly speeds up the efficiency of breeding, saves breeding time, and enables breeders to continuously explore and select better animals at the molecular level. Livestock breeds. [0003] A variety of methods have been used for SNP detection. Currently, gene chip methods, DNA sequencing methods, mass spectrom...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 张毅劳兰兰肖炜俞英唐韶青刘林王雅春孙东晓张胜利
Owner CHINA AGRI UNIV
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