Methyl-sensitive restriction enzymes are those that can cut when the recognition site is either methylated or unmethylated depending on the enzyme, and are most effective when paired with an isoschizomer
(a restriction endonuclease that recognizes the same sequence), such as Hpall and MspI, respectively.
Isolation and characterization of BliAI, an isoschizomer
of ClaI from Bacillus licheniformis.
profiling of cytosine methylation: the help assay.
A, One percent agarose gel staining with ethidium bromide showing non-digested DNA (ND) and DNA digested with MspI or HpaII, which is an isoschizomer
of MspI methylation sensitive enzyme, at different media concentrations of 5-aza-2'-deoxycytidine (5-aza-CdR; 0, 0.5, 1.0, and 10 [micro]M).
Digestion with the Cfr9I, an isoschizomer
of SmaI, yielded identical PFGE profiles (Figure).
Briefly, this assay uses a methylation-insensitive restriction enzyme (MspI) along with its methylation-sensitive isoschizomer
(HpaII) to create 2 differentially digested DNA aliquots.
We used two different methods (Methods I and II) based on DNA digestion by HpaII (a methylation-sensitive enzyme) and MspI (a methylation-insensitive isoschizomer
enzyme) followed by real-time PCR amplification to quantify methylation.
Molecular typing of the PCR-positive samples was performed by using sequencing and restriction fragment length polymorphism analysis (27) by hydrolysis of the PCR products with the Tru9I restriction endonuclease (SibEnzyme, Novosibirsk, Russia) (isoschizomer
MseI) with subsequent electrophoresis in 15% polyacrylamide gel.