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Markus Nauck, Heinrich Wieland, Winfried März
Evaluation of the Roche Diagnostics LightCycler-Apo B 3500 Mutation Detection Kit
Familial defective apolipoprotein (apo) B-100 is an autosomal codominant disorder associated with hypercholesterolemia and an increased risk of coronary artery disease. Two independent mutations affecting the codon 3500 (Arg3500?Gln and Arg3500?Trp) have been shown to cause ligand-defective apo B-100. Identification of carriers of these mutations is an important step in the risk stratification of individuals and families with hypercholesterolemia.
We evaluated a homogeneous assay for detection of mutations at codon 3500 that combines rapid-cycle PCR with allele-specific fluorescent probe melting profiles for product genotyping. This single-tube analysis is performed on the LightCycler?, a microvolume fluorimeter integrated with a thermal cycler. Continuous acquisition of fluorescence data during a melting curve analysis at completion of PCR allows the detection of mutations, as loss of fluorescence occurs in an allele-specific manner. By plotting melting peaks, the three apo B-100 alleles were readily distinguishable. Using this method, genotyping of 32 samples is completed within 40 min without the need for any post-PCR sample manipulation, thereby eliminating the risks of end-product contamination and sample tracking errors. The specific detection of mutations at codon 3500 of the apo B gene on the LightCycler? is a rapid and reliable method that is ideally suitable for typing both small and large numbers of samples.
Clinical Chemical Laboratory Medicine, Walter de Gruyter
Print ISSN: 1434-6621
Volume: 38, 07/2000
Pages: 667 - 671