Genotyping single-nucleotide polymorphisms by the invader assay with dual-color fluorescence polarization detection

Background: The PCR-Invader assay is a robust, homogeneous assay that has b een shown to be highly sensitive and specific in genotyping single-nucleoti de polymorphism (SNP) markers. In this study, we introduce two changes to i mprove the assay: (a) we streamline the PCR-Invader method by assaying both alleles for each SNP in one reaction; and (b) we reduce the cost of the me thod by adopting fluorescence polarization (FP) as the detection method. Methods: PCR product was incubated with Invader oligonucleotide and two pri mary probes at 93 degreesC for 5 min. Signal probes corresponding to the cl eaved flaps of the primary probes [labeled with fluorescein and 6-carboxyte tramethy1rhodamine (TAMRA) dye] and Cleavase((R)) VIII enzyme (a flap endon uclease) were then added to the mixture. This reaction mixture was incubate d at 63 degreesC for 5 min. FP measurements were made with a fluorescence p late reader. Results: Eighty-eight individuals were genotyped across a panel of 10 SNPs, using PCR product as template, for a total of 880 genotypes. An average "n o call" rate of 3.2% was observed after first round of experiments. PCR pro ducts were remade in those samples that failed to produce any genotype in t he first round, and all gave clear-cut genotypes. When the genotypes determ ined by the PCR-Invader assay and template-directed dye-terminator incorpor ation assay with FP were compared, they were in 100% concordance for all SN P markers and experiments. Conclusions: The improvements introduced in this study make PCR-Invader ass ay simpler and more cost-effective, and therefore more suitable for high-th rough-put genotyping. (C) 2001 American Association for Clinical Chemistry.