Cyanine dyes with high absorption cross section as donor chromophores in energy transfer primers.
Energy transfer (ET) fluorescent primers are significantly superior to single dye-labeled primers for DNA sequencing and multiplex genetic analyses (Ju, J., Glazer, A. N., and Mathies, R. A. (1996) Nature Med. 2, 246-249). We describe here ET primers in which a donor chromophore with a large absorption cross section but a low fluorescence quantum yield is exploited to increase the Stokes-shifted fluorescence emission of acceptor dyes. The new ET primers have 3-(epsilon-carboxy-pentyl)-3'ethyl-5,5'-dimethyloxacarbocyanine (CYA; epsilon M488nm 142,000 M-1 cm-1) at the 5' -end as a common energy donor, and fluorescein or rhodamine derivatives (FAM, R6G, TAMRA, and ROX), attached to a modified thymidine 10 bases away within the primer sequence, as acceptors. With 488-nm excitation, the fluorescence emission intensity of these four ET primers is 1.4- to 24-fold stronger than that of the corresponding primers labeled only with the single acceptor dye. When compared with the corresponding ET primers with a fluorescein derivative (FAM; epsilon M488nm 60,000 M-1 cm-1) as donor, the fluorescence emissions of primers with CYA as donor and FAM, R6G, TAMRA, and ROX as acceptors are respectively 0.8-, 1.0-, 1.7-, and 1.7-fold as intense. The low fluorescence quantum yield of the CYA donor resulted in distinct fluorescence signals for the DNA-sequencing fragments with much lower crosstalk between the four detection channels than that seen with ET primers based on a FAM donor. With single-stranded M13mp18 DNA as the template, the CYA ET primers provided DNA sequences on a four-color capilary sequencer with 100% accuracy in the first 500 bases.
Hung
S C
SC
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Ju
J
J
Mathies
R A
RA
Glazer
A N
AN
D002232:Carbocyanines / Q000737:chemistry; D002855:Chromatography, Thin Layer; D004396:Coloring Agents / Q000737:chemistry; D019075:Electrophoresis, Capillary; D004735:Energy Transfer; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D017422:Sequence Analysis, DNA / Q000379:methods; D013050:Spectrometry, Fluorescence; D013054:Spectrophotometry, Atomic
D002232:Carbocyanines; D004396:Coloring Agents; D005452:Fluoresceins; D005456:Fluorescent Dyes; C024098:6-carboxyfluorescein