Detection of apoB-100 R3500Q mutation by competitive allele-specific polymerase chain reaction

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...

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Bibliographic Details
Published in:	Journal of clinical laboratory analysis Vol. 15; no. 5; pp. 256 - 259
Main Authors:	Horvath, Anelia D., Kirov, Steven A., Karaulanov, Emil E., Ganev, Varban S.
Format:	Journal Article
Language:	English
Published:	New York John Wiley & Sons, Inc 2001
Subjects:	Alleles Amino Acid Substitution apoB-100 R3500Q Apolipoprotein B-100 Apolipoproteins B - genetics Arginine - genetics competitive allele-specific PCR DNA Mutational Analysis - methods Glutamine - genetics Humans Metabolic Diseases - genetics Original Polymerase Chain Reaction - methods primer competition
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Summary:	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.
Bibliography:	ark:/67375/WNG-71X47V18-R ArticleID:JCLA1037 istex:E1B76A038906C6BEAA56F69AB7E77DE1DBAEF056 Medical Research Council at the Medical University of Sofia - No. 0024/2000 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0887-8013 1098-2825
DOI:	10.1002/jcla.1037