Rapid automated simultaneous screening of (G1691A) Factor V, (G20210A) prothrombin, and (C677T) methylenetetrahydrofolate reductase variants by multiplex PCR using fluorescence scanning technology.

ABSTRACT

The Factor V Leiden mutation (G1691A), and mutations in the prothrombin (G20210A) and 5,10-methylenetetrahydrofolate reductase (C677T) genes are common hereditary risk factors associated with venous thrombosis. The aim of this study was to develop an automated, PCR-based genotyping assay for rapid simultaneous screening of these three mutations. We adapted multiplex PCR, using primer modifications to introduce cleavage sites for restriction endonucleases into the fragments bearing each of the mutations. The three mutations were analyzed in a single tube by fluorescence scanning. An internal digestion control was introduced to prevent false-negative results due to incomplete digestion or a total lack of digestion. DNA fragment analysis was carried out using an automated capillary electrophoresis instrument (ABI310). This reliable, efficient, easy-to-use assay can be applied to specimens from large clinical trials and epidemiological surveys.