Two for Typing: Homogeneous Combined Single-Nucleotide Polymorphism Scanning and Genotyping

Abstract

More than 20 years after the first in vitro amplification of DNA by PCR, this technique is firmly established for diagnostic purposes in many clinical laboratories. PCR technology is an exciting field that has continually advanced. Evolutionary steps have been, for example, the transition from conventional cycling to rapid-cycling (1), from post-PCR to real-time product analysis (2), and from gel-based to probe-based PCR product identification (3) and genotyping with energy transfer probes (4). One of the many possible applications of PCR in the clinical laboratory is genotyping. In this issue of Clinical Chemistry , Zhou et al., from Carl Wittwer’s group (5), contribute another innovation to this field. Their study reports a homogeneous technique based on simultaneous high-resolution melting of a whole PCR product and an unlabeled probe in the presence of saturating fluorescent DNA dye. It appears as the logical continuation of underlying previous work. The use of fluorescence resonance energy transfer probe pairs for genotyping by analysis of the melting curve of a probe covering the mutation site was followed by hundreds of published applications since its first description (4). Single-nucleotide polymorphisms (SNPs) under a probe affect probe melting. The degree of destabilization depends on the type of SNP itself and on the bases that lie next to the SNP. By using a nearest-neighbor model, the melting temperatures of matched and mismatched probes can be predicted …