Control of Partial Digestion Combining the Enzymes Dam Methylase and Mbol

A method is described which allows the preparation of reproducible partial digests without previous establishment of the incubation conditions. It is based on a combined application of dam methylase and the restriction endonuclease Mbol, both recognizing the sequence 5'-GATC-3' but Mbol unable to cut the methylated site. Due to their competition for the same substrate the DNA is partially digested, with the size of the resulting fragments strongly dependent on the ratio of enzymes. The 1^ of the dam methylase was determined to be 115 ng DNA//J indicating a variance in fragment sizes generated at low DNA-concentrations. This effect is minimized above 150 ng/jtl. Any influence of digestion time is avoided, because the reaction runs until complete modification of all sites. The dependence on enzyme concentration and presence of agarose was checked. Knowledge of these parameters allows an accurate prediction of fragment sizes generated at different conditions. The technique was successfully used to construct libraries from different sources, in particular chromosome-specific libraries from small amounts of flow-sorted material. INTRODUCTION Partial digestion of DNA is a basic prerequisite for the construction of genomic libraries (e.g., 1) as well as restriction mapping of recombinant clones (2). To get fragments of an appropriate size range, the degree of digestion has to be carefully controlled. As carried out up to now (1), partial digestion strongly depends on the DNA's concentration and purity, the amount of restriction enzyme used, as well as the incubation time. Therefore, using either dilutions of the restriction enzyme or different time-intervals of incubation to achieve partial cleavage of the DNA, conditions have to be checked empirically for each DNA. A way of generating partial digests in a time and enzyme amount independent manner (3) uses ultraviolet irradiation to make sites unaccessable for restriction enzymes. Such DNA, however, can not be used for cloning purposes. As alternative we describe here a method developed to produce accurate partial digestions of any size. The partially cut DNA-fragments can be produced quite easily and digestion is easily controlled. This is particularly valuable, if the DNA-amount is limited, as for instance in the construction of genomic libraries from flow-sorted chromosomes or microdissected DNA. Our approach is based on the combined use of a DNA-methylase and a restriction endonuclease, both acting on identical recognition sites, with the latter enzyme not able to cut the methylated form. The competition for the substrate between both enzymes should result in a partial digestion of the DNA, with the frequency of cleavage dependent on the ratio of methylase to restriction nuclease (4). Since partial A/fcoI-digestion is extensively used for cloning purposes, we concentrated