Analysis of the factor V Leiden mutation using the READIT Assay

Background: A variety of methods exist for the detection of single-nucleotide polymorphisms (SNPs) present in amplified segments of genomic DNA. We show the application of a novel SNP scoring tool for analysis of the factor V Leiden mutation. Methods and Results: We have developed a novel method for analyzing SNPs. The luciferase-based technique, known as the READIT Technology (Promega Corp, Madison, WI), was used to analyze 510 residual human samples sent for factor V Leiden testing from three independent testing laboratories. A blinded retrospective analysis of the factor V Leiden mutation was used to determine the accuracy and throughput capabilities of the technology. One hundred percent concordance was observed between the READIT Assay and genotype assignments made in the testing laboratories. In addition, greater than 6 SDs of separation were observed between the means of wild-type and heterozygote sample populations. Repetitive sample measurements with representative wild-type, heterozygote, and mutant samples showed that greater than 9 SDs separated the means of heterozygote and homozygote sample populations. Confidence intervals based on the means of wild-type, heterozygote, and mutant sample populations were determined. Conclusion: perfect concordance using the READIT Assay showed its effectiveness as a SNP scoring tool. The design of the factor V READIT Assay was straightforward, requiring the design of two unmodified oligonucleotides that differ at the 3' penultimate position to form perfect hybrids with the wild-type or Leiden form of the factor V sequence. The use of previously published amplification primers and conditions minimized the time needed to optimize and validate the assay. The READIT Calculator supplied with the assay allowed automated genotype assignments and statistical analysis from the READIT Assay data. Confidence-interval analysis validated the ability to distinguish between wild-type, heterozygote, and mutant samples using the READIT Assay.