Fast characterisation of selected β-casein and β-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 described by means of detection of distinct mutations in the nucleotide sequence and verified using isoelectric focusing of the corresponding proteins. With the inherent positive effect of certain genetic variants on cheese-making 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 protein sequence information available from databases, deduced gene fragments containing the variant-specific mutations were generated using PCR. A partial nucleotide sequence of the beta -LG-gene fragment D, containing allele-specific point mutations, could be determined. For P-CN those mutations occur at amino acid residues 67 and 122 (beta -CN gene locus 8101+8267) whereas for the beta -LG variants specific mutations occur at amino acid residues 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 specific PCR fragments generated from milk cell DNA, genotyping of alleles of beta -CN and beta -LG or admixtures becomes efficient and simultaneous. Hence, a real-time PCR approach was established specifically distinguishing three important beta -CN milk protein variants with remarkable benefits when compared to other DNA-based mutation detection systems.