Detection and identification of base alterations within the region of factor V leiden by fluorescent melting curves

Background: Factor V Leiden (G1691A) is a common cause of inherited thrombo sis. In fluorescent melting curve analysis, the Leiden mutation is distingu ished from the wild-type by a decrease in melting temperature (Tm) of a wil d-type probe. Because Tm depends on the type and position of the mismatch, other base alterations, such as the recently described base alteration A169 2C, should be distinguishable from the true Leiden mutation. Methods and Results: Of 2,100 samples tested for the factor V Leiden mutati on using a wild-type probe, 200 heterozygous or homozygous mutant samples w ere further tested using a Leiden probe. The Tm of the A1692C base alterati on was 1,5 degrees C greater than the Leiden mutation with the wild-type pr obe and 8 degrees C less with the Leiden probe. One sample was heterozygous for a new base alteration G1689A with a Tm 0.8 degrees C greater than the Leiden mutation with the wild-type probe, and 10 degrees C less with the Le iden probe. Tm estimates from fluorescence melting curve analysis have intr a-assay standard deviations of approximately 0.1 degrees C, Conclusions: Fluorescence melting curve analysis can distinguish between se quence alterations with Tms differing by less than 1 degrees C. This is the first demonstration of a widely applicable technique that can significantl y increase the specificity of hybridization techniques without the need for sequencing.