Evaluation of electronic microarrays for genotyping factor V, factor II, and MTHFR.

BACKGROUND Genetic risk factors associated with venous thrombosis include mutations in the factor V (Leiden), factor II (prothrombin), and methylenetetrahydrofolate reductase (MTHFR) genes. We evaluated a method using electronically addressable microarrays for the detection of mutations in these genes that have been associated with vascular disease. METHODS The NanoChip Molecular Biology Workstation (Nanogen) uses electronic microarrays for mutation detection. Factor V, factor II, and MTHFR genotypes identified in the NanoChip system on 225 samples were compared with genotypes from LightCycler assays (Roche). We determined within- and between-cartridge signal and ratio variation and analyzed the effect of additional mutations at or near the detection area used for the NanoChip assays. RESULTS Genotypes determined for all three mutations on the NanoChip platform were in complete concordance with LightCycler results. Within-cartridge signal variation as measured by the CV of fluorescence signals was <10% for each allele when present. The within-cartridge CV for heterozygous mutant/wild-type ratios was <8.5%, and between-cartridge CV was <18%. A dilution study showed that results could be obtained in this assay with 6 ng of nucleic acid per PCR, the lowest input tested. The presence of additional sequence variations near the expected mutations can produce equivocal or discrepant results. CONCLUSIONS Mutation detection using the NanoChip Molecular Biology Workstation was accurate and reproducible for the three assays evaluated.