On-the-fly fluorescence lifetime detection of labeled DNA primers.
The first application of frequency-domain, on-the-fly fluorescence lifetime detection to the detection, identification and resolution of fluorescent labeled oligonucleotide primers in capillary electrophoresis is presented. These studies, which are directed towards the development of four-decay DNA sequencing, employed two common sequencing primers, SP6 and M13/pUC. The primers were each tagged by a derivative of either fluorescein or BODIPY. Fluorescence emission maxima and lifetimes of the free dyes and the labeled primers were determined both in batch mode and on-the-fly in capillary electrophoresis. Fluorescence intensity and lifetime electropherograms were extracted from dynamic lifetime data that were acquired at 0.1-s intervals during the CE separation. Lifetimes were recovered using either conventional non-linear least-squares analysis or the self-modeling maximum entropy method, which does not require prior knowledge of the system. Based on migration time and fluorescence lifetime, peaks could be detected and identified, and co-eluting peaks could be resolved. Interference from background impurities and scattered light was greatly reduced by the combination of physical separation and lifetime resolution.
Li
L C
LC
Department of Chemistry, P.M. Gross Chemical Laboratory, Duke University, Durham, NC 27708-0346, USA.
He
H
H
Nunnally
B K
BK
McGown
L B
LB
D017931:DNA Primers / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000379:methods
D017931:DNA Primers