title: Detection of apoB-100 R3500Q mutation by competitive allele-specific polymerase chain reaction.
abstract: The apolipoprotein B-100 mutation R3500Q is one of the most common inherited defects causing abnormality of the lipid metabolism. We describe a one-step, single-tube PCR technique for detection of the mutation based on competition between allele-specific primers. Three oligonucleotides are used: two allele-specific primers differing in their 3' nucleotide (for the wild-type and the mutant allele) together with a common primer, resulting in simultaneous amplification of both alleles. This provided internal control of successful amplification and is expected to result in increased specificity. The allele-specific primers differ also in length, allowing us to distinguish both alleles by their size in a single electrophoretic run. For optimization of the protocol, DNAs genotyped before by oligonucleotide ligation assay were used. The individual genotypes obtained by CAS-PCR coincided fully with the ones from a referent OLA test: seven heterozygous individuals were found, 4 of them among 150 unrelated hypercholesterolemic individuals studied and other three in the pedigrees of heterozygous carriers. On the overall 160 genotypes were determined, neither false-positive (0 out of 153 non-carriers) nor false-negative (0 out of 7 carriers) results were obtained. No homozygous mutant genotypes were identified in this sample.
authors.affiliation: Department of Chemistry and Biochemistry, Medical University, Sofia, Bulgaria.
authors.forename: A D
authors.initials: AD
authors.lastname: Horvath
authors.forename: S A
authors.initials: SA
authors.lastname: Kirov
authors.forename: E E
authors.initials: EE
authors.lastname: Karaulanov
authors.forename: V S
authors.initials: VS
authors.lastname: Ganev
mesh_terms: D000483:Alleles; D019943:Amino Acid Substitution; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000235:genetics*; D001120:Arginine / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D005973:Glutamine / Q000235:genetics; D006801:Humans; D008659:Metabolic Diseases / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D005973:Glutamine; D001120:Arginine