Unexplained DNA Melting Behavior in a Genotyping Assay

Abstract

A common methionine/valine polymorphism at codon 129 of the prion protein gene modulates disease susceptibility and phenotypic variability of human prion diseases (1)(2). All patients with the new variant of Creutzfeldt-Jakob disease (vCJD) were homozygous for methionine at codon 129 (3). We developed a genotyping assay that uses rapid-cycle PCR and melting point analysis of fluorogenic hybridization probes, but we encountered an unexplained artifact of melting behavior that may lead to misinterpretation when using this method. Asymmetric PCR allowed reliable genotyping results. Primers (5′-CACAGTCAGTGGAACAAG-3′ and 5′-GTACACTTGGTTGGGGT-3′; GenBank accession no. HSU29185, positions 25735–25752 and 25935–25919, respectively) and probes (anchor probe, 5′-CCGAAATGTATGATGGGCCTGCTCAT-fluorescein-3′; detection probe, 5′-LC-Red640-CACTTCCCAGCACGTAGCC-phosphate-3′; GenBank accession no. HSU29185, positions 25874–25849 and 25846–25828, respectively) were designed using a beta version of the LightCycler probe design software (Ver. 0.99.11; Roche Molecular Biochemicals). This software uses recommended methods for probe design (4) and calculates melting temperatures ( T ms) by a nearest-neighbor method. PCR reactions were carried out in a final volume of 10 μL in glass capillaries (Roche Molecular Biochemicals). Each reaction mixture contained 5 pmol of forward primer, different amounts of reverse primer (0.5–5 pmol), 2 pmol each of anchor and detection probe, 3 mmol/L MgCl2, 1 μL of LightCycler-FastStart DNA Master Hybridization Probes (which includes reaction buffer, …