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  • Title: Energy transfer cassettes for facile labeling of sequencing and PCR primers.
    Author: Berti L, Xie J, Medintz IL, Glazer AN, Mathies RA.
    Journal: Anal Biochem; 2001 May 15; 292(2):188-97. PubMed ID: 11355850.
    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.
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