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-mediated Achilles' Heel Cleavage (IHF-AC) technique (Koob et al., 1988; Kur et al., 1992), and leads to the generation of very rare restriction sites in large DNA molecules. The first step of this procedure is methylation of DNA in the presence of IHF, when the overlapping ihf/restriction sites are protected from methylation, and in the second step the DNA is cut by the cognate restriction enzyme. The aim of the present study is to develop a very exact and reproducible procedure to obtain only a few well-defined cuts with the IHF-pre-treated DNA, depending on the variety of all parameters. This technique (IARC, i.e., IHF-assisted rare cutters) employs the restriction enzyme and only one auxiliary protein (IHF). The advantage of the IARC procedure is that no methylation is required (as opposed to the IHF-AC method). Using the IARC approach, the effects of various IHF concentrations were evaluated on cleaving the activity of the DraI, PacI, PmeI, and SwaI enzymes using DNA of phage lambda or the entire genomic 4.7-Mb DNA of E. coli. At low IHF concentrations only a few cut sites were eliminated by IHF binding, but at high IHF concentration, enzymes were able to cut in only one or several specific sites.
Kur
J
J
Department of Microbiology, Technical University of Gdańsk, Poland.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D005821:Genetic Techniques; D016680:Genome, Bacterial; D034982:Integration Host Factors; D008969:Molecular Sequence Data
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D034982:Integration Host Factors; D004262:DNA Restriction Enzymes