Quantification of polymerase chain reaction products in agarose gels with a fluorescent europium chelate as label and time-resolved fluorescence spectroscopy.
We have 5'-end-labeled one polymerase chain reaction (PCR) primer with the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). After performing PCR in the presence of another unlabeled primer, we separated the 362 bp PCR product with 2% low melting point agarose gel electrophoresis. The gel was then immersed into a Eu3+ solution. During soaking, Eu3+ diffuses into the gel and associates with BCPDA to form a fluorescent complex of long fluorescence lifetime. This complex can be quantified by scanning the gel with a time-resolved fluorometric reader. Because BCPDA and Eu3+ are not fluorescent by themselves, background signals are very low. The detection limit was about 5 ng of DNA. We have also shown that the BCPDA-labeled product could be blotted and detected on the membrane by using an anti-BCPDA antibody. These two technologies may find applications other than in PCR, e.g. in fluorescence-based DNA sequencing and in solution hybridization.
Chan
A
A
Department of Clinical Biochemistry, Toronto Hospital, Ontario, Canada.
Diamandis
E P
EP
Krajden
M
M
D015139:Blotting, Southern; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis*; D004996:Ethidium; D005456:Fluorescent Dyes; D010618:Phenanthrolines; D016133:Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling
D005456:Fluorescent Dyes; D010618:Phenanthrolines; C054206:4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid; D004247:DNA; D004996:Ethidium