Calpastatin is an endogenous inhibitor of calpain which is responsible for the breakdown of myofibrillar proteins, The association of Single Nucleotide Polymorphism （SNP） in the calpastatin gene with meat tenderness...Calpastatin is an endogenous inhibitor of calpain which is responsible for the breakdown of myofibrillar proteins, The association of Single Nucleotide Polymorphism （SNP） in the calpastatin gene with meat tenderness is an important topic in meat production. Therefore efficient procedure to investigate the SNP is necessary. The objectives of this study were to detect the SNP of calpastatin gene at domain L marker （G/C transversion） of the Kamphaengsaen beef breed （KPS cattle; n = 26） by the Amplification Refractory Mutation System （ARMS） compared with the Restriction Fragment Length Polymorphism （RFLP） methods and to determine the genotypes of the KPS cattle at that marker. Genomic DNA of calpastatin gene extracted from blood of the KPS cattle was detected with ARMS and RFLP methods. The ARMS system has utilized two primer pairs to amplify the two different alleles of a polymorphism in single PCR reaction to detected single base mutation. In this method, the alleles-specific primers had a mismatch at 3＇ terminal base and a second deliberate mismatch at position -2 from 3＇ terminus. While the RFLP method detected a polymorphism using PCR-base technique follow by RsaI restriction enzyme. Amplification of the ARMS method revealed that the results were not different from the conventional method of RFLP. Analysis of genotypes revealed that the KPS cattle inherited the CC, CG and GG genotypes at domain L marker. These were reliable when verified by nucleotide sequence analysis of PCR products. The animals were genotyped and determined for tenderness phenotype with this marker that predicted variation of an intronic polymorphism at domain L of the calpastatin gene. Therefore, the ARMS method was simple, efficient technique, and suitable for detecting SNP at domain L marker of the calpastatin gene.展开更多
文摘Calpastatin is an endogenous inhibitor of calpain which is responsible for the breakdown of myofibrillar proteins, The association of Single Nucleotide Polymorphism （SNP） in the calpastatin gene with meat tenderness is an important topic in meat production. Therefore efficient procedure to investigate the SNP is necessary. The objectives of this study were to detect the SNP of calpastatin gene at domain L marker （G/C transversion） of the Kamphaengsaen beef breed （KPS cattle; n = 26） by the Amplification Refractory Mutation System （ARMS） compared with the Restriction Fragment Length Polymorphism （RFLP） methods and to determine the genotypes of the KPS cattle at that marker. Genomic DNA of calpastatin gene extracted from blood of the KPS cattle was detected with ARMS and RFLP methods. The ARMS system has utilized two primer pairs to amplify the two different alleles of a polymorphism in single PCR reaction to detected single base mutation. In this method, the alleles-specific primers had a mismatch at 3＇ terminal base and a second deliberate mismatch at position -2 from 3＇ terminus. While the RFLP method detected a polymorphism using PCR-base technique follow by RsaI restriction enzyme. Amplification of the ARMS method revealed that the results were not different from the conventional method of RFLP. Analysis of genotypes revealed that the KPS cattle inherited the CC, CG and GG genotypes at domain L marker. These were reliable when verified by nucleotide sequence analysis of PCR products. The animals were genotyped and determined for tenderness phenotype with this marker that predicted variation of an intronic polymorphism at domain L of the calpastatin gene. Therefore, the ARMS method was simple, efficient technique, and suitable for detecting SNP at domain L marker of the calpastatin gene.
