Nucleic Acids Research, Vol 25, Issue 14 2816-2822, Copyright © 1997 by Oxford University Press
LG Lee, SL Spurgeon, CR Heiner, SC Benson, BB Rosenblum, SM Menchen, RJ Graham, A Constantinescu, KG Upadhya and JM Cassel
We have synthesized a set of four energy transfer dyes and demonstrated
their use in automated DNA sequencing. The donor dyes are the 5- or 6-
carboxy isomers of 4'-aminomethylfluorescein and the acceptor dyes are a
novel set of four 4,7-dichloro-substituted rhodamine dyes which have
narrower emission spectra than the standard, unsubstituted rhodamines. A
rigid amino acid linker, 4-aminomethylbenzoic acid, was used to separate
the dyes. The brightness of each dye in an automated sequencing instrument
equipped with a dual line argon ion laser (488 and 514 nm excitation) was
2-2.5 times greater than the standard dye- primers with a 2 times reduction
in multicomponent noise. The overall improvement in signal-to-noise was 4-
to 5-fold. The utility of the new dye set was demonstrated by sequencing of
a BAC DNA with an 80 kb insert. Measurement of the extinction coefficients
and the relative quantum yields of the dichlororhodamine components of the
energy transfer dyes showed their values were reduced by 20-25% compared
with the dichlororhodamine dyes alone.
ARTICLES
New energy transfer dyes for DNA sequencing
PE Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA. leelg@perkin-elmer.com
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