SIMULTANEOUS ANALYSIS OF BOVINE KAPPA-CASEIN AND BLAD ALLELES BY MULTIPLEX PCR FOLLOWED BY PARALLEL DIGESTION WITH 2 RESTRICTION ENZYMES

An improved and simplified method allowing simultaneous genetic typing of kappa-casein and CD18 (bovine leucocyte adhesion deficiency; BLAD) loci has been developed. The method is based on the simultaneous ampl ification of fragments of the two groups of alleles by multiplex PCR, and on a concurrent, parallel digestion of the product's by two restri ction enzymes (PstI and HaeIII) in the same incubation buffer. Digesti on with PstI distinguishes kappa-casein A and B alleles and does not c ut within any of the BLAD alleles, while digestion with Haem allows th e differentiation between normal and mutant allele variants of the CD1 8 locus. All combinations of the known mutants of the two alleles, cha racterized to the regions amplified and resulting in phenotypic effect , could be detected by electrophoretic separation performed on the sam e agarose gel owing to the vast differences in the length of the restr iction fragments.