Discovering Rare Variants by Use of Melting Temperature Shifts Seen in Melting Curve Analysis (Editorials‪)‬

Two reports in this issue of Clinical Chemistry describe novel mutations in thrombophilia genes (1, 2). The authors of the reports describe variants detected by the identification of a melting temperature ([T.sub.m]) different from the expected [T.sub.m] of the target mutation and from the [T.sub.m] of the wild-type sequence. The variants reported in this issue are A20218G in the prothrombin (factor II) gene and C1690T in the factor V gene. These cases add to the reported variants identified by fluorescent hybridization probes and melting analysis on the LightCycler. In addition to factor II and V variants, variants have been discovered similarly in genes coding for MTHFR and HFE (3, 4). Melting analyses, as performed on the LightCycler or similar instruments, can distinguish variants that lie within the region of the target probe. Differences in base composition, nucleotide position, and nearest-neighbor environments all affect [T.sub.m]. These differences are detected by monitoring fluorescence during an increase in temperature. Melting is visualized by a loss of fluorescence as the probes dissociate from the template. Differences in [T.sub.m] from the expected [T.sub.m] for wild-type and target mutations are indicative of a nontarget (a variant other than the mutation the assay was designed to detect) mutation or variant.