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231222s2001 xx ||||| 00| ||eng c |
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|a pubmed25n0383.xml
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|a (DE-627)NLM114666881
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|a (NLM)11570500
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|a DE-627
|b ger
|c DE-627
|e rakwb
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|a eng
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| 100 |
1 |
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|a Hsu, T M
|e verfasserin
|4 aut
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| 245 |
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|a Universal SNP genotyping assay with fluorescence polarization detection
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|c 2001
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|a Text
|b txt
|2 rdacontent
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|a ohne Hilfsmittel zu benutzen
|b n
|2 rdamedia
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|a Band
|b nc
|2 rdacarrier
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|a Date Completed 15.02.2002
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|a Date Revised 28.09.2018
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|a published: Print
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|a Citation Status MEDLINE
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|a 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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|a Comparative Study
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|a Journal Article
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|a Research Support, U.S. Gov't, P.H.S.
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|a Buffers
|2 NLM
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|a Coloring Agents
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|a DNA-Binding Proteins
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|a Genetic Markers
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|a Indicators and Reagents
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|a EXO1 protein, human
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|a EC 3.1.-
|2 NLM
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|a Exodeoxyribonucleases
|2 NLM
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|a EC 3.1.-
|2 NLM
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|a exodeoxyribonuclease I
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|a EC 3.1.11.1
|2 NLM
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|a Alkaline Phosphatase
|2 NLM
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|a EC 3.1.3.1
|2 NLM
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|a DNA Repair Enzymes
|2 NLM
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|a EC 6.5.1.-
|2 NLM
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| 700 |
1 |
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|a Chen, X
|e verfasserin
|4 aut
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| 700 |
1 |
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|a Duan, S
|e verfasserin
|4 aut
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| 700 |
1 |
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|a Miller, R D
|e verfasserin
|4 aut
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| 700 |
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|a Kwok, P Y
|e verfasserin
|4 aut
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| 773 |
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|i Enthalten in
|t BioTechniques
|d 1993
|g 31(2001), 3 vom: 14. Sept., Seite 560, 562, 564-8, passim
|w (DE-627)NLM012627046
|x 0736-6205
|7 nnns
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| 773 |
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|g volume:31
|g year:2001
|g number:3
|g day:14
|g month:09
|g pages:560, 562, 564-8, passim
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|d 31
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|e 3
|b 14
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|h 560, 562, 564-8, passim
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