A Simple Restriction Fragment Length Polymorphism-Based Method for Multiplex Testing of Thrombosis Risk Factors FV Leiden and F2 G20210A with Highly Sensitive Contamination Detection

ABSTRACT

Factor V (FV) Leiden and F2 G20210A are inherited genetic risk factors that are in the first line of laboratory tests for thromboembolic diseases. Their detection relies on PCR assays, which are subject to contamination, as well as pipetting error, when manually performed and require individual assays for each gene. In this article, we report an improved PCR and restriction endonuclease assay for the simultaneous detection of the FV Leiden and F2 G20210A variants, based on multiplex amplification with fluorescent primers, digestion control, identity monitoring, and contamination tracking.