Method for detecting base mutation polymorphism of goat gonadotropin releasing hormone and growth differentiation factor 9

Abstract

The invention discloses a method for detecting the polymorphism of three base mutations of the gene exon 4 of goat gonadotropin releasing hormone and the gene exon 2 of the growth differentiation factor 9. The method comprises the following steps: using the DNA sequence of goat genome as template, using 2 to amplify the primer P1 and P2 under PCR condition in the presence of Taq DNA polymerase, buffer environment, Mg2+ and dNTPs, using agarose gel electrophoresis to judge the size of the target fragments; using polyacrylamide gel electrophoresis to detect the PCR amplified product of the primer P1 and P2 to find that the two amplification sites have three base mutations, then performing genotyping and gene frequency analysis to the SNPs of the two genes, and performing association analysis with the fecundity traits of Boer goat and Xinong Saanen dairy goat. The result shows that the SNPs sites detected by the primer P1 and P2, of the GnRH gene exon 4 (containing partial intron 3 and 3' non-translational region) and the GDF9 gene exon 2, thus the method can be used for molecular marking of fecundity trait selection of goat.

Description

technical field [0001] The invention belongs to the field of molecular genetics, and in particular relates to a three-way test for detecting goat gonadotropin-releasing hormone gene exon 4 (including part of intron 3 and 3' untranslated region) and growth differentiation factor 9 gene exon 2 A base mutation polymorphism method, which uses polyacrylamide gel electrophoresis (PAGE), has the characteristics of rapidity, accuracy and simplicity. Background technique [0002] Single Nucleotide Polymorphisms (Single Nucleotide Polymorphisms, SNP) refers to the polymorphism caused by the replacement of a single nucleotide (A / T / C / G) in the genomic DNA sequence. SNPs can be polymorphisms of two or more alleles. Therefore, commonly referred to as SNPs include base insertions, deletions, insertions / deletions, and changes in the copy number of repeated sequences. A SNP represents a nucleotide change at a certain position in the genome, mainly consisting of single base conversions (one ...

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Problems solved by technology

Among these SNP detection techniques, DNA sequence determination is the most accurate SNP detection method, but its detection cost is extremely expensive, and large-scale instruments such as DNA sequencers are required. At the same time, very skilled technicians and Experience, so DNA sequence determination is not an ideal SNP detection method for actual production; of course, the combination of PCR-SSCP and DNA sequencing can be used to detect SNPs can appropriately reduce the detection cost, but the experimental process of PCR-SSCP is relatively long , the operation is cumbersome, and there are false negative problems in the experimental process, so it is not an ideal SNP detection method; as a new SNP detection method, the AS-PCR method has very broad prospects in the future application field, but , this method needs to design special primers, and it can only target specific gene loci. At the same time, there is a probability of false detection in the detection process. Therefore, it is not generally applicable at present; Ligation reaction technology to detect SNP sites requires detection platforms such as plate reader, gene chip, microsphere array technology and mass spectrometer, which is not very practical for general molecular laboratories

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