Fast characterisation of selected beta-casein and beta-lactoglobulin variants using specific single nucleotide polymorphisms derived from milk cell DNA: a novel real-time PCR approach

The determination of genetic variants of beta -cascin (beta -CN A(1), A(2), B) and beta -lactoglobulin (beta -LG A, B, C, D) directly from milk is des cribed by means of detection of distinct mutations in the nucleotide sequen ce and verified using isoelectric focusing of the corresponding proteins. W ith the inherent positive effect of certain genetic variants on cheese-maki ng properties, the information derived by the new technique is of interest for the dairy technology as well as for the milk production. Based on the p rotein sequence information available from databases, deduced gene fragment s containing the variant-specific mutations were generated using PCR. A par tial nucleotide sequence of the beta -LG-gene fragment D, containing allele -specific point mutations, could be determined. For P-CN those mutations oc cur at amino acid residues 67 and 122 (beta -CN gene locus 8101+8267) where as for the beta -LG variants specific mutations occur at amino acid residue s 45, 59, 64+118 (beta -LG gene locus 3662, 3706, 4581+4583). Additionally, seven mutations were found in intron 2 of the beta -LG gene. Based on spec ific PCR fragments generated from milk cell DNA, genotyping of alleles of b eta -CN and beta -LG or admixtures becomes efficient and simultaneous. Henc e, a real-time PCR approach was established specifically distinguishing thr ee important beta -CN milk protein variants with remarkable benefits when c ompared to other DNA-based mutation detection systems.