Fluorescence lifetime for on-the-fly multiplex detection of DNA restriction fragments in capillary electrophoresis.
This paper describes the first use of frequency-domain fluorescence lifetime for multiplex detection of DNA restriction fragments in capillary electrophoresis (CE). The fragments were labeled with monomeric intercalating dyes that can be excited by either the 488- or 514-nm line of an argon ion laser and have lifetimes in the range of 0.5-2.5 ns. We were able to achieve multiplex lifetime detection in the CE separation of a restriction fragment digest and a DNA size ladder in the same run, for fragments shorter than 700 bp. Different gel buffer systems, including a modified polyacrylamide gel and several tris-borate-EDTA/hydroxyethylcellulose (TBE/HEC) gels, were investigated for separation and detection of the dye-labeled DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a gel containing 1% high molecular weight (90,000-105,000) HEC and 0.3% low molecular weight (24,000-27,000) HEC in TBE buffer.
McIntosh
S L
SL
Department of Chemistry, P. M. Gross Chemical Laboratory, Duke University, Durham, North Carolina 27708, USA.
Nunnally
B K
BK
Nesbit
A R
AR
Deligeorgiev
T G
TG
Gadjev
N I
NI
McGown
L B
LB
D004247:DNA / Q000737:chemistry*; D019075:Electrophoresis, Capillary; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007364:Intercalating Agents; D013056:Spectrophotometry, Ultraviolet
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007364:Intercalating Agents; D004247:DNA