VAL34LEU POLYMORPHISM DETECTION BY REAL TIME PCR ASSAY USING FLUORESCENCE RESONANCE ENERGY TRANSFER ON ROTOR-GENE 6000

Abstract

Factor XIII (FXIII) Val34Leu is the most importantpolymorphism of the A subunit in factor XIII gene. The aim of the presentstudy was to develop an efficient method based on real time PCR withFluorescence resonance energy transfer (FRET) detection and melting curveanalysis for the detection of Val34Leu polymorphism.Specific primers were used to amplify the relevant fragment of the factorXIII-A gene and fluorescence resonance energy transfer hybridizationprobes were used for detection in a Rotor-Gene Q 5Plex HRM platform.Melting temperature (Tm) for the wild type was at 68.8, while Tm forhomozygote mutant was at 63.8; heterozygote demonstrated both peaks.Our results showed that primers and probes propoesd in this studydemonstrated a high specificity to identify wild type , heterozygous andhomozygous mutantgenotypes.Due to the increasing molecular diagnosis in developing countries and theimportance of identifying polymorphisms, this real time PCR assay is ofgreat importance. An important advantage of this approach is the highsensitivity and specificity.