title: 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.
authors.affiliation: Washington University School of Medicine, St Louis, MO, USA.
authors.forename: T M
authors.initials: TM
authors.lastname: Hsu
authors.forename: X
authors.initials: X
authors.lastname: Chen
authors.forename: S
authors.initials: S
authors.lastname: Duan
authors.forename: R D
authors.initials: RD
authors.lastname: Miller
authors.forename: P Y
authors.initials: PY
authors.lastname: Kwok
mesh_terms: D000469:Alkaline Phosphatase; D000483:Alleles; D001483:Base Sequence; D002021:Buffers; D004396:Coloring Agents; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry; D005090:Exodeoxyribonucleases; D005454:Fluorescence Polarization; D005819:Genetic Markers; D005838:Genotype; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D007202:Indicators and Reagents; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
chemical_list: D002021:Buffers; D004396:Coloring Agents; D004268:DNA-Binding Proteins; D005819:Genetic Markers; D007202:Indicators and Reagents; C114331:EXO1 protein, human; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; D000469:Alkaline Phosphatase; D045643:DNA Repair Enzymes