Synchronous luminescence: a new detection technique for multiple fluorescent probes used for DNA sequencing.
A method fo nonradioactive DNA sequencing, which has been marketed commercially, uses four different fluorescent tags to label DNA fragments and fixed-wavelength excitation/fluorescence detection of the labels. This study presents an alternative method of detection based on synchronous luminescence (SL), in which both excitation and emission wavelengths are scanned simultaneously. This approach has proven in the past to have significant advantages over fixed-wavelength luminescence in the analysis and identification of fluorescent analytes. In this paper, the utility of synchronous excitation was investigated as a method for DNA sequencing with fluorescent tags. A laser-based SL instrument, recently developed in this laboratory, was used to resolve the spectra of a mixture of the dyes used in the fluorescence-based sequencing scheme. A preliminary limit of detection of 720 zeptomoles (10(-21) M) of fluorescein isothiocyanate dye in solution was achieved with this instrument. The results presented here suggest that the SL technique could result in an increase in sensitivity and a decrease in error rate of identification during DNA sequencing.
Stevenson
C L
CL
Health and Safety Research Division, Oak Ridge National Laboratory, TN 37831-6101.
Johnson
R W
RW
Vo-Dinh
T
T
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives; D003568:Cytidine Monophosphate; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D008163:Luminescent Measurements; D012235:Rhodamines; D017422:Sequence Analysis, DNA / Q000379:methods*; D013050:Spectrometry, Fluorescence; D014966:Xanthenes
D005456:Fluorescent Dyes; D012235:Rhodamines; D014966:Xanthenes; C032930:7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole; C009434:tetramethylrhodamine isothiocyanate; C034657:Texas red; D009327:4-Chloro-7-nitrobenzofurazan; D003568:Cytidine Monophosphate; D016650:Fluorescein-5-isothiocyanate