Cytosine methylated DNA synthesized by Taq polymerase used to assay methylation sensitivity of restriction endonucleaseHinfl

Abstract

We have studied the resistance of cytosine methylated DNA to digestion by the restriction endonuclease Hlnft, using a simple PCR procedure to synthesize DNA of known sequence In which every cytosine is methylated at the 5 position. We find that Hinfl cannot digest cytosine methylated DNA at the concentrations normally used in restriction digests. Complete digestion is possible using a vast excess of enzyme; under these conditions, the rate of Hinfl digestion for cytosine methylated DNA is at least 1440-fold slower than for unmethylated DNA. The presence of an additional methylated cytosine at the degenerate position internal to the recognition sequence does not appear to increase the resistance to Hinft digestion. We also tested Hhall an isoschizomer of Hinfl, and found that it is completely inactive on cytosine methylated DNA. The procedure we have used should be of general applicability in determination of the methylation sensitivities of other restlction enzymes, as well as studies of the effects of methylation on gene expression in direct DNA transfer experiments.