Energy Transfer Cassettes for Facile Labeling of Sequencing and PCR Primers
| Autor: | Igor L. Medintz, Alexander N. Glazer, Jin Xie, Lorenzo Berti, Richard A. Mathies |
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| Rok vydání: | 2001 |
| Předmět: |
Energy transfer
Biophysics Color Biology Polymerase Chain Reaction Polymorphism Single Nucleotide Biochemistry Fluorescence DNA sequencing Capillary electrophoresis Multiplex Disulfides Molecular Biology Chromatography High Pressure Liquid DNA Primers Fluorescent Dyes Electrophoresis Capillary Sequence Analysis DNA Cell Biology Amplicon Combinatorial chemistry Acceptor Molecular biology Fluorescent labelling Spectrometry Fluorescence Energy Transfer |
| Zdroj: | Analytical Biochemistry. 292:188-197 |
| ISSN: | 0003-2697 |
| Popis: | Fluorescence energy transfer (ET) primers and terminators are the reagents of choice for multiplex DNA sequencing and analysis. We present here the design, synthesis and evaluation of a four-color set of ET cassettes, fluorescent labeling reagents that can be quantitatively coupled to a thiol-activated target through a disulfide exchange reaction. The ET cassette consists of a sugar-phosphate spacer with a FAM donor at the 3′-end, an acceptor linked to a modified T-base at the 5′-end of the spacer and a mixed disulfide for coupling to a thiol at the 5′-end. The acceptor dye emission intensities of ET labeled primers produced in this manner are comparable to commercial ET primers. The utility of our ET cassette-labeled primers is demonstrated by performing four-color capillary electrophoresis sequencing with the M13(-21)forward primer and by generating and analyzing a set of single-nucleotide-polymorphism-specific PCR amplicons. |
| Databáze: | OpenAIRE |
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