Factor V Leiden and
prothrombin G20210A are clinically relevant genetic risk factors for
venous thrombosis. Analysis for both mutations is increasingly being performed on patients exhibiting
hypercoagulability. The goal of the current study was to evaluate the performance of primer-engineered multiplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for the simultaneous detection of
factor V Leiden and
prothrombin G20210A. Primer-engineered multiplex PCR-RFLP methods for the detection of
factor V Leiden and
prothrombin G20210A from the medical literature were reviewed. A modified method was optimized in which both mutations generate HindIII RFLPs and the
prothrombin amplicon contains an invariant HindIII recognition site to assess the completeness of
endonuclease digestion. Digested amplification products were analyzed by
agarose gel electrophoresis in a single gel lane and visualized by
ethidium bromide. Primer-engineered multiplex PCR-RFLP was used to analyze 205 human genomic
DNA samples whose
factor V Leiden genotypes had been previously determined by MnlI PCR-RFLP. Complete concordance for
factor V Leiden genotypes was observed between the two methods in the 205-sample cohort comprising 139 wild-type, 62 heterozygous mutant, and four homozygous mutant individuals. For
prothrombin G20210A, primer-engineered multiplex PCR-RFLP identified 196 wild-type and nine heterozygous mutant individuals in the 205-sample cohort. To independently verify
prothrombin genotypes, the nine heterozygous mutants and an additional 11 wild-type patient samples (representing 10% of patient samples) were subjected to
DNA sequencing. Complete concordance was observed between
DNA sequencing and primer-engineered multiplex PCR-RFLP results. In further validation, 123 of the
DNA samples consisting of four heterozygous mutant and 119 wild type individuals were genotyped with the Invader Assay for
Factor II (
prothrombin G20210A). Results showed 100% concordance between the Invader Assay and primer-engineered multiplex PCR-RFLP. A primer-engineered multiplex PCR-RFLP based on single
restriction endonuclease digestion has been evaluated and shown to simultaneously and accurately detect
factor V Leiden and
prothrombin G20210A mutations. The method is robust and readily adaptable to the clinical molecular diagnostic laboratory.