Technical note: use of PCR-single-strand conformation polymorphism analysis for detection of bovine beta-casein variants A1, A2, A3, and B.
We have optimized the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique to screen the most frequent variants (A1, A2, A3, and B) of the bovine beta-casein gene. Five partly overlapping PCR products (233, 234, 265, 466, and 498 bp) of Exon VII of the beta-casein gene that encompass the target point mutations were heat-denatured, separated on nondenaturing polyacrylamide gels, and silver-stained. Simultaneous detection of all variants in reference samples of known genotypes (A1A2, A2A2, A1A3, A1B, and A2B) was best achieved on 17% polyacrylamide (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 anonymous samples belonging to various breeds was then analyzed twice, without discrepancies in a blind trial. Routine beta-casein genotyping using PCR-SSCP is proposed as a cost-effective, fast, and sensitive technique.
Barroso
A
A
Departamento de Producción Animal, Facultad de Veterinaria, Madrid, Spain.
Dunner
S
S
Cañón
J
J
D000818:Animals; D001483:Base Sequence; D002364:Caseins / Q000235:genetics*; D002417:Cattle / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods / Q000662:veterinary*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D018807:Polymorphism, Single-Stranded Conformational
D002364:Caseins