Allele polymorphism analysis in coagulation factors F2, F5 and folate metabolism gene MTHFR by using microchip-based multiplex real time PCR

Abstract

Single nucleotide polymorphism (SNP) genotyping methods are widely used for the detection of hereditary thrombophilias caused by genetic defects in the coagulation system. The hereditary thrombophilias are frequently associated with higher incidences of point mutations in hemostasis (F2 20210G>A, F5 1691G>A) and folate metabolism (MTHFR 677C>Т, MTHFR 1298A>C) genes. Moreover, the combination of gene abnormalities in F2 or/and MTHFR with F5 Leiden mutation leads to increased risk of developing thrombosis. Thus, simultaneous detection of the multiple gene mutations in a sample has important clinical relevance. The microchip-based multiplex real time PCR for estimation of allele specific polymorphism in hemostatic and folate metabolism genes presented here has a high efficiency and may be used for laboratory diagnosis. The optimized protocol for estimation of 4 different types of genetic polymorphisms allowed PCR to be performed with minimal quantity of DNA template and PCR reagents including Taq polymerase and a short-term thermocycling.

A duplex polymerase chain reaction‐restriction fragment length polymorphism for rapid screening of methylenetetrahydrofolate reductase gene variants: Genotyping in acute leukemia

“…Thus, a number of molecular approaches targeting genomic DNA for identification of both polymorphisms have also been developed (Table ). These include different types of conventional PCR, real‐time PCR, and DNA sequencing . All of these techniques are fast and elegant, but they require expensive equipment.…”