Activity on non-methylated DNA limits the use of endonuclease MspJI for epigenetic analyses

JEREZ,. M.B.; JURI AYUB M.

The All Results Journal: Biology

The All Results Journal

Año: 2018 vol. 9 p. 1 - 6

Cytosine methylation of DNA in mammals has been associated with both physiological and pathologicalchanges in gene-expression. DNA treatment with methylation sensitive and/or dependent restriction enzymes, followed byPCR amplification is a widely used approach to test CpG methylation. Recently, restriction endonuclease MspJI has beenproposed as a promising tool for epigenetic analyses. In this paper, we have tested MspJI as a tool for detecting CpGmethylation on mammalian genomic DNA. For this experiment mouse genomic sequences harboring or lacking CpG siteswere selected. The extent of degradation was evaluated by PCR using primers flanking the chosen genomic regions.Digestion of mouse genomic DNA, in combination with end-point and real-time PCR reactions, revealed that MspJItreatment reduced the amplification of genomic regions either containing or lacking of CpG motifs. In addition, treatmentof bona fide non-methylated (in vitro amplified) DNA samples definitely demonstrated that MspJI shows significant activityagainst non-methylated DNA. These results show that star activity can be an important concern when using MspJI, evenunder standard conditions. Therefore, we conclude that (in contrast to classical restriction enzymes), careful case by caseevaluation of reaction conditions is mandatory for optimizing the usefulness of MspJI in epigenetic studies.