Simultaneous analysis of bovine kappa-casein and BLAD alleles by multiplex PCR followed by parallel digestion with two restriction enzymes

• Published in: Animal genetics Vol. 27; no. 3; p. 207
• Main Authors: Zsolnai, A, Fésüs, L
• Format: Journal Article
• Language: English
• Published: England 01.06.1996
• Subjects: Alleles; Animals; Base Sequence; Caseins - genetics; Cattle - genetics; CD18 Antigens - genetics; Molecular Sequence Data; Polymerase Chain Reaction - methods; Polymorphism, Restriction Fragment Length
• ISSN: 0268-9146
• DOI: 10.1111/j.1365-2052.1996.tb00954.x

An improved and simplified method allowing simultaneous genetic typing of kappa-casein and CD 18 (bovine leucocyte adhesion deficiency; BLAD) loci has been developed. The method is based on the simultaneous amplification of fragments of the two groups of alleles by multiplex PCR, and on a concurrent, parallel digestion of the products by two restriction enzymes (PstI and HaeIII) in the same incubation buffer. Digestion with PstI distinguishes kappa-casein A and B alleles and does not cut within any of the BLAD alleles, while digestion with HaeIII allows the differentiation between normal and mutant allele variants of the CD18 locus. All combinations of the known mutants of the two alleles, characterized to the regions amplified and resulting in phenotypic effect, could be detected by electrophoretic separation performed on the same agarose gel owing to the vast differences in the length of the restriction fragments.