Universal SNP genotyping assay with fluorescence polarization detection
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 dete...
| Veröffentlicht in: | BioTechniques. - 1988. - 31(2001), 3 vom: 14. Sept., Seite 560, 562, 564-8, passim |
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| Weitere Verfasser: | , , , |
| Format: | Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2001
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| Zugriff auf das übergeordnete Werk: | BioTechniques |
| Schlagworte: | Comparative Study Journal Article Research Support, U.S. Gov't, P.H.S. Buffers Coloring Agents DNA-Binding Proteins Genetic Markers Indicators and Reagents EXO1 protein, human EC 3.1.- mehr... |
| Zusammenfassung: | 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 |
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| Beschreibung: | Date Completed 15.02.2002 Date Revised 28.09.2018 published: Print Citation Status MEDLINE |
| ISSN: | 1940-9818 |