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Nucleic Acids Research, 1988, Vol. 16, No. 3 1181-1196
© 1988

Articles

Sensitive non-radioactive dot-blot hybridization using DNA probes labelled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence

Andreas Oser, Willy K. Roth1 and Günter Valet

Mildred-Scheel-Labor für Krebszellforschung D-8033 Martinsried, FRG 1Abteilung Virusforschung, Max-Planck-Institut für Biochemie D-8033 Martinsried, FRG

Received August 10, 1987. Revised November 27, 1987. Accepted December 16, 1987.

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.


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