Open AccessDetection of apoB‐100 R3500Q mutation by competitive allele‐specific polymerase chain reaction
Author(s) -
Horvath Anelia D.,
Kirov Steven A.,
Karaulanov Emil E.,
Ganev Varban S.
Publication year - 2001
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.1037
Subject(s) - allele , genotype , genetics , biology , microbiology and biotechnology , polymerase chain reaction , oligonucleotide , primer (cosmetics) , mutation , mutant , point mutation , gene , chemistry , organic chemistry
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. J. Clin. Lab. Anal. 15:256–259, 2001. © 2001 Wiley‐Liss, Inc.