Sensitive non-radioactive dot-blot hybridization using DNA probes labelled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence.
A new labelling method for cloned DNA probes used in hybridization assays is described. The DNA insert of recombinant plasmid DNA was made partially single-stranded for the labelling reaction by a restriction enzyme digest, followed by a controlled exonuclease III incubation. A thiol-containing psoralen derivative was covalently bound through irradiation with UV-light to the remaining double-stranded region of the plasmid DNA. The psoralen-SH groups were labelled with a large number of metal chelators (diethylentriamine pentaacetic acid, DTPA) using poly-L-lysine as a macromolecular carrier. The main advantage of the labelling procedure is that a high degree of labelling is achieved without modification of the single-stranded DNA hybridizing sequences. The specific hybrids were labelled after filter hybridization with europium ions through the chelating groups of DTPA. The europium ions were quantitatively detected by time-resolved fluorometry. The sensitivity of the assay for target DNA detection was in the low picogram range, comparable to radioactively labelled DNA probes.
Oser
A
A
Mildred-Scheel-Labor für Krebszellforschung, Martinsried, FRG.
Roth
W K
WK
Valet
G
G
D002614:Chelating Agents; D004274:DNA, Recombinant / Q000032:analysis*; D005063:Europium; D011564:Furocoumarins; D008722:Methods; D009693:Nucleic Acid Hybridization; D010957:Plasmids
D002614:Chelating Agents; D004274:DNA, Recombinant; D011564:Furocoumarins; D005063:Europium