Mono- and bis-intercalating dyes for multiplex fluorescence lifetime detection of DNA restriction fragments in capillary electrophoresis.
The use of novel intercalating dyes as labels in DNA restriction fragment analysis by capillary electrophoresis with frequency-domain fluorescence lifetime detection is described. The dyes, including one mono-intercalating dye with three positive charges and three bis-intercalating, homodimeric dyes with four positive charges, were excited by the 488 nm line of an argon ion laser and exhibited lifetimes in the range of 1-3 ns. The separations were performed using a gel containing 1% high-molecular-weight (HMW) hydroxyethylcellulose (HEC) (90,000-105,000) and 0.3% low-molecular-weight (LMW) HEC (24,000-27,000) in Tris-borate-EDTA buffer (TBE). Multiplex lifetime detection of mixtures of dye-labeled DNA restriction fragment digests and size standard fragments was achieved. Compared to previous results obtained with several mono-intercalating dyes of lesser charge (McIntosh, S. L., Nunnally, B. K., Nesbit, A. R., Deligeorgiev, T. G., Gadjev, N. I., McGown, L. B., Anal. Chem. 2000, 72, 5444-5449), the present dyes provided a wider range of lifetimes and better lifetime discrimination in multiplex detection. There was no evidence of dye exchange during the capillary electrophoresis experiment.
Department of Chemistry, P. M. Gross Chemical Laboratory, Duke University, Durham, NC 27708, USA.
Sara L
SL
McIntosh
Todor G
TG
Deligeorgiev
Nikolai I
NI
Gadjev
Linda B
LB
McGown
D001562:Benzimidazoles / Q000737:chemistry; D004247:DNA / Q000032:analysis*; D053938:DNA Fragmentation; D004262:DNA Restriction Enzymes; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D007364:Intercalating Agents / Q000737:chemistry; D015394:Molecular Structure; D011804:Quinolines / Q000737:chemistry
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D011804:Quinolines; C470471:dye 50; C470469:dye 55; C470468:dye 57; C470470:dye 58; D004247:DNA; D004262:DNA Restriction Enzymes