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

We report a novel procedure, which can be applied to probing of specific DN A, for covalently attaching probe DNA to complementary sequences in double- stranded target DNA. Employing hairpin-like oligonucleotide probes in combi nation with successive use of recA protein and DNA ligase, probes can be at tached directly to target DNA molecules without dissociation of the DNA. Th e hairpin-like structure of the probes was designed so that the terminus of the probe oligonucleotide can be brought into close stereochemical proximi ty to the terminus of the complementary strand of target DNA for ligation. Because of the elimination of the DNA dissociation and subsequent hybridiza tion land washing) steps in the currently employed method, the probing proc ess has become greatly simplified and more efficient and may lead to develo pment of fully automated probing systems.