Nucleic Acids Research, Vol 24, Issue 6 1144-1148, Copyright © 1996 by Oxford University Press
J Ju, AN Glazer and RA Mathies
DNA primer sets, labeled with two fluorescent dyes to exploit fluorescence
energy transfer (ET), can be efficiently excited with a single laser line
and emit strong fluorescence at distinctive wavelengths. Such ET primers
are superior to single fluorophore-labeled primers for DNA sequencing and
other multiple color-based analyses [J. Ju, C. Ruan, C. W. Fuller, A. N.
Glazer and R. A. Mathies (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351].
We describe here a novel method of constructing fluorescent primers using a
universal ET cassette that can be incorporated by conventional synthesis at
the 5'-end of an oligonucleotide primer of any sequence. In this cassette,
the donor and acceptor fluorophores are separated by a polymer spacer (S6)
formed by six 1',2'-dideoxyribose phosphate monomers (S). The donor is
attached to the 5' side of the ribose spacer and the acceptor to a modified
thymidine attached to the 3' end of the ribose spacer in the ET cassette.
The resulting primers, labeled with 6-carboxy-fluorescein as the donor and
other fluorescein and rhodamine dyes as acceptors, display well-separated
acceptor emission spectra with 2-12-fold enhanced fluorescence intensity
relative to that of the corresponding single dye-labeled primers. With
single- stranded M13mp18DNA as the template, a typical run with these ET
primers on a capillary sequencer provides DNA sequences with 99% accuracy
in the first 550 bases using the same amount of DNA template as that
typically required using a four- color slab gel automated sequencer.
ARTICLES
Cassette labeling for facile construction of energy transfer fluorescent primers
Department of Chemistry, University of California, Berkeley 94720 USA.
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