Energy Transfer Cassettes for Facile Labeling of Sequencing and PCR Primers

✍ Scribed by Lorenzo Berti; Jin Xie; Igor L. Medintz; Alexander N. Glazer; Richard A. Mathies

• Publisher: Elsevier Science
• Year: 2001
• Tongue: English
• Weight: 469 KB
• Volume: 292
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2001.5069

✦ Synopsis

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.