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, I and MTHFR genotypes identified in the NanoChip system on 225 samples Were compared with genotypes from LightCycler(R) 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. (C) 2003 American Association for Clinical Chemistry.