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Nucleic Acids Research, Vol 26, Issue 24 5728-5733, Copyright © 1998 by Oxford University Press

ARTICLES

Direct probing: covalent attachment of probe DNA to double-stranded target DNA

J Fujiwara and M Oishi
Biotechnology and Medical Engineering Division, Aisin Cosmos R&D Co. Ltd, 5-2-11 Sotokanda, Chiyoda-ku, Tokyo 101, Japan.

We report a novel procedure, which can be applied to probing of specific DNA, for covalently attaching probe DNA to complementary sequences in double-stranded target DNA. Employing hairpin-like oligonucleotide probes in combination with successive use of recA protein and DNA ligase, probes can be attached directly to target DNA molecules without dissociation of the DNA. The hairpin-like structure of the probes was designed so that the terminus of the probe oligonucleotide can be brought into close stereochemical proximity to the terminus of the complementary strand of target DNA for ligation. Because of the elimination of the DNA dissociation and subsequent hybridization (and washing) steps in the currently employed method, the probing process has become greatly simplified and more efficient and may lead to development of fully automated probing systems.
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