INTEGRATION HOST FACTOR (IHF) APPLIED FOR PARTIAL DIGESTION BY RESTRICTION ENDONUCLEASES IN LARGE DNA-MOLECULES (IARC PROCEDURE)

The IHF protein of Escherichia coli was successfully used in IHF-media ted Achilles' Heel Cleavage (IHF-AC) technique (Koob et al., 1988; Kur et al., 1992), and leads to the generation of very rare restriction s ites in large DNA molecules. The first step of this procedure is methy lation of DNA in the presence of IHF, when the overlapping ihf/restric tion sites are protected from methylation, and in the second step the DNA is cut by the cognate restriction enzyme. The aim of the present s tudy is to develop a very exact and reproducible procedure to obtain o nly a few well-defined cuts with the IHF-pre-treated DNA, depending on the variety of all parameters. This technique (IARC, i.e., IHF-assist ed rare cutters) employs the restriction enzyme and only one auxiliary protein (IHF). The advantage of the IARC procedure is that no methyla tion is required (as opposed to the IHF-AC method). Using the IARC app roach, the effects of various IHF concentrations were evaluated on cle aving the activity of the DraI, PacI, PmeI, and SwaI enzymes using DNA or phage lambda or the entire genomic 4.7-Mb DNA of E. coli. At low I HF concentrations only a few cut sites were eliminated by IHF binding, but at high IHF concentration, enzymes were able to cut in only one o r several specific sites.