About Methylated & Non-Methylated pUC19 DNA Set
The Methylated & Non-methylated pUC19 DNA Set consists of two control DNAs (methylated and non-methylated) along with a set of specifically designed primers that can be used in conjunction with the EZ DNA Methylation™, EZ DNA Methylation-Gold™, and EZ DNA Methylation-Direct™ kits from Zymo Research to assess the efficiency of bisulfite-mediated conversion of DNA. These plasmids can be used in conjunction with genomic DNAs to provide internal controls to assess bisulfite conversion efficiency or to produce known mixtures of methylated and non-methylated DNA for assay calibration.
The Non-Methylated pUC19 DNA is pUC19 that was isolated from a methylation-negative strain of bacteria (Dam¯, Dcm¯) and can be used as a negative control for DNA methylation analysis. The Methylated pUC19 DNA is pUC19 that has been isolated from the same strain and has been enzymatically methylated at all cytosine positions comprising CG dinucleotides by M.SssI methyltransferase2 (EC 18.104.22.168; Figure 1) and can be used as a positive control for DNA methylation analysis.
|Figure 1. M.SssI methytransferase methylates all cytosine residues in the double-stranded CpG context.|
The primer set herein has been designed to amplify a fragment of the supplied pUC19 DNAs following bisulfite treatment. The methylated cytosines, comprising CG dinucleotides in the Methylated pUC19 DNA remain unconverted following bisulfite treatment, whereas nonmethylated cytosines are converted into uracil and detected as thymine after PCR. The supplied pUC19 DNA has been linearized at position 2177 using ScaI endonuclease.