In most practical molecular biological experiments, recognition of specific base sequences is mainly carried out by DNA-DNA hybridization which involves dissociation (denaturation) of target DNA molecules to expose base sequences, and subsequent hybridization of the dissociated DNA with single-stranded DNA or deoxyoligonucleotide probes. Along with PCR, DNA-DNA hybridization is still one of the most popular techniques in molecular biology and genetic engineering, and is the essential step in Southern hybridization for analysis of genomic DNA and in DNA cloning in which colony or plaque hybridization is often included. The current processes of DNA-DNA hybridization, however, are quite cumbersome and time-consuming as they include DNA dissociation, transfer of the dissociated DNA to membranes, lengthy incubation for DNA-DNA hybridization and subsequent washing of the membrane filters. Direct recognition of specific base sequences in double-stranded DNA molecules without DNA dissociation, if established, should greatly simplify most of the current procedures involving DNA-DNA hybridization and thus may open the way to fully-automated DNA probing as well as DNA cloning, both of which are now considered to be quite unrealistic.

Employing hairpin-like oligonucleotide probes in combination with successive use of RecA protein and DNA ligase, we have been able to demonstrate that deoxyoligonucleotide probes can be covalently attached to target DNA molecules without dissociation of the DNA, thus enabling direct recognition of specific base sequences in double-stranded DNA.

The procedure is summarized as follows. The oligonucleotide probe, which was specifically designed for this purpose, consists of two parts; a probing sequence complementary to the terminal sequence of the target DNA and a universal hairpin structure which is able to covalently link the probe to the target DNA. In the first step, the homologous sequence present at the terminus of target DNA is recognized and one of the strands is displaced by the complementary sequence of the oligonucleotide probe through a RecA protein-mediated reaction. Second, the probe is covalently bonded to the target DNA by DNA ligase. The hairpin-like structure of the probe makes it possible to bring the 3'-terminus of the probe oligonucleotide into close proximity with the 5'-terminus of the target DNA molecule for ligation. In the actual process, the labeled (or biotinylated) oligonucleotide probe is incubated with RecA protein, mixed with target DNA, and further incubated to form probe-target DNA-RecA protein complex. DNA ligase is then added to covalently link the probe to the target DNA. After the reaction, the proteins are removed and the products can then be subjected to further analyses such as gel electrophoresis to detect hybridized DNA fragment or streptavidin trapping for cloning. If necessary, one could employ probes with a restriction cutting site at the hairpin-like structure so that covalently attached probes can be removed from target DNA after the reaction. Theoretically, hairpin probes can also be designed to probe the complementary sequence at the same terminus or the other terminal sequence of the same fragment. We will show you the results of several actual experiments in which the procedure was applied to probing and cloning of DNA molecules as well as an alternative procedure modified from the original one described above.

This work was supported by the R & D Project of Innovative Technology for the Earth Program which is sponsored by NEDO (New Energy and Industrial Technology Development Organization).

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