Colasanti, J., Sundaresan, V. (January 1991) Cytosine methylated DNA synthesized by Taq polymerase used to assay methylation sensitivity of restriction endonuclease HinfI. Nucleic Acids Res, 19 (2). pp. 391-4.
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Abstract
We have studied the resistance of cytosine methylated DNA to digestion by the restriction endonuclease HinfI, using a simple PCR procedure to synthesize DNA of known sequence in which every cytosine is methylated at the 5 position. We find that HinfI 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 HinfI 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 HinfI digestion. We also tested HhaII, an isoschizomer of HinfI, 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 restiction enzymes, as well as studies of the effects of methylation on gene expression in direct DNA transfer experiments.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | Base Sequence Cytosine/*chemistry DNA/*chemistry DNA-Directed DNA Polymerase/*chemistry Deoxyribonucleases, Type II Site-Specific/*chemistry Electrophoresis, Polyacrylamide Gel Gene Amplification Methylation Molecular Sequence Data Plasmids Polymerase Chain Reaction Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Taq Polymerase |
| Subjects: | bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > DNA methylation Investigative techniques and equipment > cloning > PCR Investigative techniques and equipment > assays > cloning > PCR bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > restriction enzyme |
| CSHL Authors: | |
| Communities: | CSHL labs |
| Depositing User: | Matt Covey |
| Date: | 25 January 1991 |
| Date Deposited: | 13 Jan 2016 17:00 |
| Last Modified: | 08 Nov 2017 16:46 |
| PMCID: | PMC333607 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/32065 |
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