Universal SNP Genotyping Assay with Fluorescence Polarization Detection

Abstract

The degree of fluorescence polarization (FP) of a fluorescent molecule is a reflection of its molecular weight (Mr). FP is therefore a useful detection methodfor homogeneous assays in which the starting reagents and products differ significantly in Mr. We have previously shown that FP is a good detection method for the single-base extension and the 5'-nuclease assays. In this report, we describe a universal, optimized single-base extension assay for genotyping single nucleotide polymorphisms (SNPs). This assay, which we named the template-directed dye-terminator incorporation assay with fluorescence polarization detection (FP-TDI), uses four spectrally distinct dye terminators to achieve universal assay conditions. Even without optimization, approximately 70% of all SNP markers tested yielded robust assays. The addition of an E. coli ssDNA-binding protein just before the FP reading significantly increased FP values of the products and brought the success rate of FP-TDI assays up to 90%. Increasing the amount of dye terminators and reducing the number of thermal cycles in the single-base extension step of the assay increased the separation of the FP values benveen the products corresponding to different genotypes and improved the success rate of the assay to 100%. In this study the genomic DNA samples of 90 individuals were typed for a total of 38 FP-TDI assays (using both the sense and antisense TDI primers for 19 SNP markers). With the previously described modifications, the FP-TDI assay gave unambiguous genotyping data for all the samples tested in the 38 FP-TDI assays. When the genotypes determined by the FP-TDI and 5'-nuclease assays were compared, they were in 100% concordance for all experiments (a total of 3420 genotypes). The four-dye-terminator master mixture described here can be used for assaying any SNP marker and greatly simplifies the SNP genotyping assay design.

Association of a haplotype in the promoter region of the interferon regulatory factor 5 gene with rheumatoid arthritis

“…Six of the SNPs were genotyped by multiplex minisequencing (fluorescence single-base extension) with the SNPstream Genotyping System (Beckman Coulter, Fullerton, CA) (36), and 2 SNPs (rs752637 and rs2304256) were genotyped using a homogeneous single-base extension assay with fluorescence polarization detection (FP-TDI; PerkinElmer, Emeryville, CA) (37). Four SNPs in the IRF-5 gene (rs729302, rs3757385, rs2004640, and rs3807306) were genotyped for replication in samples from the Dutch Early Arthritis Clinic study, using the SNPstream System as described above.…”

Dense linkage disequilibrium mapping in the 15q11–q13 maternal expression domain yields evidence for association in autism

“…44 FP-TDI analysis was performed using materials supplied in commercially available Acycloprime kits according to the manufacturer's published protocols (Perkin-Elmer Lifesciences, Boston, MA, USA) and as described elsewhere. 45 …”

SNP genotyping with fluorescence polarization detection

“…Conversely, the FP values of TAMRA-ddC are low and those of R6G-ddU are high for homozygous T individuals. As for heterozygous individuals, the FP values for both TAMRA-ddC and R6G-ddU are high because of significant incorporation of both dye-terminators [Hsu et al, 2001b].…”

“…The latter measure rescues the remaining one-third of the failed reactions by ensuring that the correct terminator is not used up completely in the reaction. Therefore, with minimal optimization, all SNPs can be genotyped by the FP-TDI assay to-date [Hsu et al, 2001b].…”

“…We have shown previously that fluorescence polarization (FP) is a good detection method in assays where the molecular weight of the starting fluorescent reagent changes significantly when converted into products. Indeed, FP detection has been shown to work well in the primer extension assay [Chen et al, 1999;Hsu et al, 2001b], in the 5′-nuclease (TaqMan ® ) assay [Latif et al, 2001], and in the Invader ® assay [Hsu et al, 2001a]. In this article, the use of FP detection and the three assays for which FP detection works well are described.…”