Rapid Detection of the 46C → T Polymorphism in the Factor XII Gene, a Novel Genetic Risk Factor for Thrombosis, by Melting Peak Analysis Using Fluorescence Hybridization Probes

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Abstract
Factor XII (FXII) level is an important intermediate phenotype associated with thrombotic disease. The 46C → T transition in the exon 1 of the Factor XII (F12) gene is a significant, prevalent, and independent genetic risk factor for thrombotic disease. It is also associated with interindividual variation of plasma FXII zymogen levels. The aims of this study were to develop a rapid, reproducible, and easy method for 46C → T genotyping and to compare its reliability with the classical endonuclease digestion methodology. DNA samples from 100 subjects were genotyped for the 46C → T transition using the classical endonuclease digestion method with Sfna I. The genotypes of three of them (each with a different 46C R T genotype) were confirmed by direct sequencing analysis. We then set out to construct a LightCycler PCR protocol to detect the 46C → T polymorphism. This protocol was designed to combine a rapid-cycle polymerase chain reaction (PCR) with an allele-specific fluorescent probe melting for mutation detection. In the three sequenced samples, as well as in the remaining 97, the LightCycler PCR procedure unambiguously resulted in the same genotype previously observed by sequencing and endonuclease digestion. Characteristic fluorescent curves were obtained for each genotype; the first derivative of these curves had a maximum at an apparent hybridization temperature (Tm) that was specific for each probe/allele duplex. The whole process took less than 40 min. Thus, if this method is used with a rapid DNA extraction, the genotypes would be obtained within 60 min after receiving a blood sample. In conclusion, the technique presented allows for easy, reliable, and rapid detection of this polymorphism, and is suitable for typing both small and large numbers of DNA samples.