Application of PCR site-directed mutagenesis for a rapid and accurate detection of mutation 3500 (Arg-->Gln) of human apolipoprotein B-100.

A rapid detection of the Arg3500-->Gln mutation of human apolipoprotein B-100 is of particular interest because of its prevalence in familial forms of hypercholesterolemia. A simple procedure, based on amplification by polymerase chain reaction (PCR) and NlaIII endonuclease restriction cleavage, allows this diagnosis without ambiguity. By using two oligonucleotide primers carrying one mismatch each, two permanent restriction sites were generated in the normal allele, while one of them disappeared in the mutant allele. Thus, the two alleles can be differentiated by their specific N/aIII restriction profile.