Energy transfer cassettes for facile labeling of sequencing and PCR primers

Lorenzo Berti, Jin Xie, Igor L. Medintz, Alexander N. Glazer, Richard A. Mathies

Research output: Contribution to journalArticle

40 Scopus citations

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 languageEnglish (US)
Pages (from-to)188-197
Number of pages10
JournalAnalytical Biochemistry
Volume292
Issue number2
DOIs
StatePublished - May 15 2001

Keywords

  • Capillary electrophoresis
  • DNA diagnostics
  • DNA sequencing
  • Energy transfer primers
  • Fluorescent labeling

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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    Berti, L., Xie, J., Medintz, I. L., Glazer, A. N., & Mathies, R. A. (2001). Energy transfer cassettes for facile labeling of sequencing and PCR primers. Analytical Biochemistry, 292(2), 188-197. https://doi.org/10.1006/abio.2001.5069