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

Pathology Department, University of Utah School of Medicine, ARUP Laboratories, Salt Lake City, UT

Address for correspondence: ARUP Laboratories, Medical Directors, 500 Chipeta Way, Salt Lake City, UT 84108

The first 300 words of the full text of this article appear below.

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_m) different from the expected T_m of the target mutation and from the T_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_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_m from the expected T_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.

In contrast, assays that use restriction enzymes, allele-specific amplification, or hybridization probes at a single detection temperature may not detect a variant or may show an indeterminate genotype (3)(4). With these 3 types of assays, when one allele carries the variant and the opposite allele is a wild type, the variant allele may be undetected, and the genotype would be reported as wild type. If the variant is in conjunction with a target mutation on the opposite allele, the genotype could be reported as . . . [Full Text of this Article]

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