WAY OF CONDUCTING REAL-TIME PCR FOR GENOTYPING BOS TAURUS BY THE CSN3 GENER.R. VAFIN, S.V. TYULKIN, I.V. RZHANOVA, F.F. ZINNATOVA,N.YU. SAFINA, F.F. ZINNATOV, SH.K. SHAKIROV AND M. SH. TAGIROV
The kappa casein gene is uniquely associated with the signs of protein-lactic and technological properties of milk. Therefore, studies on development of molecular genetic approaches to assess the allelic polymorphism of the CSN3 gene are relevant and are of scientific and practical interest. The main objective of the study was to develop and test an effective method for genotyping cattle using the CSN3 gene based on the real-time PCR with hybridization-fluorescent detection. Approbation of the developed method of Real-time PCR for genotyping cattle on the A and B alleles of the CSN3 gene was carried out on DNA samples from 70 stud bulls, confirming the reliability of results obtained by the well-known PCR-RFLP method. The proposed method for real-time conduction of PCR for genotyping cattle for A and B alleles of the CSN3 gene in the hybridization-fluorescent detection format involves the use of two 5/ -fluorescencelabeled direct allele-specific primers, one reverse common primer, and one anti-primer labeled with a fluorescence quencher from the 3/-end of the oligonucleotide. As a result of practical studies aimed at approbation of the developed method, we obtained the technical result provided by the proposed method, which is expressed in effective identification of desired genotypes due to correct interpretation of fluorescence intensity increase data curve, reliability of results of which was also confirmed by the wellknown method of PCR-RFLP analysis for the genotyping of Bos taurus on similar alleles of the kappa-casein gene. The offered invention is an effective method for genotyping cattle based on CSN3 gene based on Realtime PCR in a hybridization-fluorescent detection format.
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