Project description:We have engineered thermostable KlenTaq DNA polymerase variants called RIII H20, RIV A8 and RIV D15 that produce error signatures specific for 5-methylcytosine (5mC) without prior chemical treatment of the DNA samples. These signatures are amplified during DNA amplicon library preparation and are detected by NGS. This method was applied to distinguish C from 5mC in DNA templates generated by PCR (unmodified and methylated using the CpG Methyltransferase (M.SssI)). Finally, RIV A8 was used to detect 5mC in HeLa human genomic DNA (native methylation and supplementary methylated using the CpG Methyltransferase (M.SssI))
