Energy transfer cassettes for facile labeling of sequencing and PCR primers

Fluorescence energy transfer (ET) primers and terminators are the reagents of choice for multiplex DNA sequencing and analysis. We present here the de sign, synthesis and evaluation of a four-color set of ET cassettes, fluores cent labeling reagents that can be quantitatively coupled to a thiol-activa ted 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 lin ked 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 intensitie s of ET labeled primers produced in this manner are comparable to commercia l ET primers. The utility of our ET cassette-labeled primers is demonstrate d by performing four-color capillary electrophoresis sequencing with the M1 3(-21)forward primer and by generating and analyzing a set of single-nucleo tide-polymorphism-specific PCR amplicons. (C) 2001 Academic Press.