New dye-labeled terminators for improved DNA sequencing patterns.
We have used two new dye sets for automated dye-labeled terminator DNA sequencing. One set consists of four, 4,7-dichlororhodamine dyes (d-rhodamines). The second set consists of energy-transfer dyes that use the 5-carboxy-d-rhodamine dyes as acceptor dyes and the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein as the donor dye. Both dye sets utilize a new linker between the dye and the nucleotide, and both provide more even peak heights in terminator sequencing than the dye-terminators consisting of unsubstituted rhodamine dyes. The unsubstituted rhodamine terminators produced electropherograms in which weak G peaks are observed after A peaks and occasionally C peaks. The number of weak G peaks has been reduced or eliminated with the new dye terminators. The general improvement in peak evenness improves accuracy for the automated base-calling software. The improved signal-to-noise ratio of the energy-transfer dye-labeled terminators combined with more even peak heights results in successful sequencing of high molecular weight DNA templates such as bacterial artificial chromosome DNA.
Rosenblum
B B
BB
PE Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA. rosenbbb@perkin-elmer.com
Lee
L G
LG
Spurgeon
S L
SL
Khan
S H
SH
Menchen
S M
SM
Heiner
C R
CR
Chen
S M
SM
D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry; D004735:Energy Transfer; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D015394:Molecular Structure; D012235:Rhodamines / Q000737:chemistry*; D017422:Sequence Analysis, DNA / Q000379:methods*
D004269:DNA, Bacterial; D005452:Fluoresceins; D005456:Fluorescent Dyes; D012235:Rhodamines; C024098:6-carboxyfluorescein; C045132:4-carboxyfluorescein