Technical note: Use of PCR-single-strand conformation polymorphism analysis for detection of bovine beta-casein variants A(1), A(2), A(3), and B

We have optimized the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique to screen the most frequent variants (A(1 ), A(2), A(3), and B) of the bovine beta-casein gene. Five partly overlappi ng PCR products (233, 234, 265, 466, and 498 bp) of Exon VII of the p-casei n gene that encompass the target point mutations were heat-denatured, separ ated on nondenaturing polyacrylamide gels, and silver-stained. Simultaneous detection of all variants in reference samples of known genotypes (A(1)A(2 ), A(2)A(2), A(1)A(3), A(1)B, and A(2)B) was best achieved on 17% polyacryl amide (100:1 acrylamide:bis-acrylamide ratio) gels with the PCR product of 234 bp. These results were confirmed by sequencing the allele-specific SSCP bands directly excised from polyacrylamide gels. A population of 65 anonym ous samples belonging to various breeds was then analyzed twice, without di screpancies in a blind trial. Routine beta-casein genotyping using PCR-SSCP is proposed as a cost-effective, fast, and sensitive technique.