| Popis: |
Cytosine methylation of DNA in mammals has been associated with both physiological and pathological changes in gene-expression. DNA treatment with methylation sensitive and/or dependent restriction enzymes, followed by PCR amplification is a widely used approach to test CpG methylation. Recently, restriction endonuclease MspJI has been proposed as a promising tool for epigenetic analyses. In this paper, we have tested MspJI as a tool for detecting CpG methylation on mammalian genomic DNA. For this experiment mouse genomic sequences harboring or lacking CpG sites were 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 MspJI treatment reduced the amplification of genomic regions either containing or lacking of CpG motifs. In addition, treatment of bona fide non-methylated (in vitro amplified) DNA samples definitely demonstrated that MspJI shows significant activity against non-methylated DNA. These results show that star activity can be an important concern when using MspJI, even under standard conditions. Therefore, we conclude that (in contrast to classical restriction enzymes), careful case by case evaluation of reaction conditions is mandatory for optimizing the usefulness of MspJI in epigenetic studies. |
