Quantification of nucleic acids on nitrocellulose membranes with time-resolved fluorometry.
We use a streptavidin-based macromolecular complex (SBMC) labelled with the europium chelate of 4,7-bis (chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) as a staining reagent for biotinylated DNA present on nitrocellulose filters. The fluorescent spots or bands obtained can either be observed under UV illumination, photographed by instant camera photography or quantified by using a specially designed instrument working as a high resolution time-resolved fluorometric scanner. The detection limit is approximately 10 pg of target DNA. Various experiments with use of biotinylated DNA probes hybridized to Southern transferred targets have shown that the new procedure is a useful versatile non-isotopic methodology for staining DNA on solid supports.
Christopoulos
T K
TK
Department of Clinical Biochemistry, Toronto Western Hospital, Ontario, Canada.
Diamandis
E P
EP
Wilson
G
G
D001426:Bacterial Proteins / Q000737:chemistry; D010582:Bacteriophage lambda / Q000235:genetics; D001710:Biotin / Q000737:chemistry*; D015139:Blotting, Southern; D003101:Collodion / Q000378:metabolism; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D015342:DNA Probes / Q000378:metabolism*; D004279:DNA, Viral / Q000032:analysis / Q000378:metabolism; D005470:Fluorometry / Q000295:instrumentation*; D010618:Phenanthrolines / Q000737:chemistry; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods*; D019809:Streptavidin
D001426:Bacterial Proteins; D015342:DNA Probes; D004279:DNA, Viral; D010618:Phenanthrolines; C054206:4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid; D001710:Biotin; D003101:Collodion; D004247:DNA; D019809:Streptavidin