METHOD OF allele-specific PCR for genotyping CATTLE IN A AND B alleles kappa-casein gene

摘要

A method of allele-specific PCR for genotyping cattle alleles A and kappa-casein gene, comprising preparing a nucleic acid sample, introducing said sample into a reaction mixture for PCR consisting of dNTPs, buffer system, Taq DNA polymerase, 4 primers; PCR; detecting the amplified DNA fragments by gel electrophoresis, characterized in that use different primer sequences, all of which are allele-specific, with no additional non-complementary to any of the alleles of the kappa-casein bovine "mismatch-nucleotides" in position - 2 from the 3'-terminal nucleotide primers, but having the 5 'non-complementary portions (n) of length 5-3 nucleotides: B-allele-specific primers "BF-hs": 5'-cccccGTGAGCCTACAAGTACACCTACCAT-3' and "BR-hs" : 5'-cCcccGATGTCTCCTTAGAGTATTTAGACC-3 ', which initiate t amplification B-allele-specific DNA fragment of the gene of kappa-casein bovine size 156 bp; A-allele-specific primers AF-hs: 5'-gggggCTGTTCACACACAAAAACAGTAAAG-3 'and AR-hs: 5'-gggGGGTGCCTAACCTTATACAGCCTTTCG-3', which initiate amplification of A-allele-specific DNA fragment of the gene of kappa-casein bovine size 242 bp ; 2 is carried-stage PCR, the annealing temperature registration and synthesis at 72 ° C.