Control of partial digestion combining the enzymes dam methylase and MboI.
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 MboI, both recognizing the sequence 5'-GATC-3' but MboI 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 Km of the dam methylase was determined to be 115 ng DNA/microliters indicating a variance in fragment sizes generated at low DNA-concentrations. This effect is minimized above 150 ng/microliters. 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.
Hoheisel
J D
JD
Imperial Cancer Research Fund, Lincoln's Inn Fields, London, UK.
Nizetic
D
D
Lehrach
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D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D007700:Kinetics; D008780:Methyltransferases / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000302:isolation & purification; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D004247:DNA; D008780:Methyltransferases; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases