DNA hybridization assay using ATTOPHOS, a fluorescent substrate for alkaline phosphatase.
A fluorometric procedure for the detection of DNA-DNA hybrids is described. The procedure involved the detection of probe-bound alkaline phosphatase with the fluorescent substrate ATTOPHOS. This substrate is converted to ATTOFLUOR by alkaline phosphatase and fluoresces strongly at 550 nm when excited with a wavelength of 440 nm. DNA hybridization assays were performed both with dilutions of purified target plasmid DNA (pSE9 or PBR322) and whole bacterial cells. Streptavidin-alkaline phosphatase conjugates were added to react with bound probe. Fluorometric assays, as well as colorimetric assays, using 5-bromo-4-chloro-3-indolylphosphate + nitroblue tetrazolium for alkaline phosphatase activity were performed. The fluorescence of the substrate was measured at time intervals, and the slope of the regression line calculated. A slope four times greater than that of background was considered positive. One hundred femtograms or 2.2 x 10(4) molecules of homologous DNA were detected with the fluorescent assay as compared with 10,000 femtograms or 2.2 x 10(6) molecules of homologous DNA with the colorimetric assay. Similar results were obtained with whole cells. Approximately 1 x 10(3) homologous cells were detected fluorometrically and 1 x 10(5) cells were detected colorimetrically. Based on these results, we conclude that, in our hands, the DNA hybridization assay described here using ATTOPHOS as the substrate for alkaline phosphatase is a very sensitive assay for the detection of DNA-DNA hybrids.
Cano
R J
RJ
Biological Sciences Department, California Polytechnic University, San Luis Obispo 93407.
Torres
M J
MJ
Klem
R E
RE
Palomares
J C
JC
D000469:Alkaline Phosphatase / Q000378:metabolism; D003124:Colorimetry; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis; D004274:DNA, Recombinant / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism / Q000528:radiation effects; D005470:Fluorometry; D009344:Neisseria gonorrhoeae / Q000235:genetics; D009693:Nucleic Acid Hybridization; D009943:Organophosphorus Compounds / Q000378:metabolism / Q000528:radiation effects; D010777:Photochemistry; D010957:Plasmids; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D005456:Fluorescent Dyes; D009943:Organophosphorus Compounds; C075487:AttoPhos; D000469:Alkaline Phosphatase