title: Energy transfer cassettes for facile labeling of sequencing and PCR primers.
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.
authors.affiliation: Department of Chemistry, University of California, Berkeley 94720, USA.
authors.forename: L
authors.initials: L
authors.lastname: Berti
authors.forename: J
authors.initials: J
authors.lastname: Xie
authors.forename: I L
authors.initials: IL
authors.lastname: Medintz
authors.forename: A N
authors.initials: AN
authors.lastname: Glazer
authors.forename: R A
authors.initials: RA
authors.lastname: Mathies
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003116:Color; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D019075:Electrophoresis, Capillary; D004735:Energy Transfer; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D013050:Spectrometry, Fluorescence
chemical_list: D017931:DNA Primers; D004220:Disulfides; D005456:Fluorescent Dyes