Abstract
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.
Original language | English (US) |
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Pages (from-to) | 188-197 |
Number of pages | 10 |
Journal | Analytical Biochemistry |
Volume | 292 |
Issue number | 2 |
DOIs | |
State | Published - May 15 2001 |
Keywords
- Capillary electrophoresis
- DNA diagnostics
- DNA sequencing
- Energy transfer primers
- Fluorescent labeling
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology