Analysis of genotyping features of bovine cattle individuals at the CSN2 locus using ACRS-PCR methods

Abstract

In the context of solving the problem of obtaining high quality dairy products from livestock, the issue of determining the type of beta-casein (A1 and A2) in the protein fraction of milk becomes essential. Purpose – to analyse the use of ACRS-PCR methods for differentiation of A1 and A2 alleles of bovine beta-casein locus. Genotyping features were analysed using the artificially created restriction site polymerase chain reaction method utilising TaqI and DdeI restriction endonucleases. The electrophoretic distribution of DNA fragments in agarose gels of various concentrations was used to analyse restriction patterns. Based on the results of bioinformatic analysis of the nucleotide reference sequences of the experimental fragment of the beta-casein gene, it was found that the primer system for the ACRS-PCR DdeI method is characterised by higher parameters of flanking efficiency of the target DNA site compared to the ACRS-PCR TaqI system due to significantly greater effectiveness of hybridisation of oligonucleotides on the target DNA. Based on the results of laboratory tests of both methods, it is proposed to use an additional procedure for analysing the fluorescence intensity of individual elements of restriction patterns, which allows reducing the number of false genotyping that occurs in both cases (based on the results of using both methods) due to the appearance of non-specific amplification/restriction fragments within the size of target restrictions. The application of the ACRS-PCR DdeI method provides more differentiated patterns of the corresponding genotypes in agarose gel compared to the ACRS-PCR TaqI method, but leads to higher material costs for conducting research. These disadvantages of using primer systems for ACRS-PCR of the beta-casein locus determine the relevance of developing alternative methods for typing A1 and A2 alleles which include allele-specific PCR. The use of results is promising for solving the problems of genotyping cattle individuals of different breeds by A1 and A2 alleles of the beta-casein locus