Detection of 5mC in DNA templates generated by PCR and HeLa human genomic DNA by linear PCR using KTq DNA polymerase variants
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)) Overall design: DNA templates generated by PCR (unmodified and methylated using the CpG Methyltransferase (M.SssI)) and HeLa human genomic DNA (native methylation and supplementary methylated using the CpG Methyltransferase (M.SssI)) were linear amplified by RIII H20, RIV A8 and RIV D15 (genomic DNA only by RIV A8). Respective ssDNA product was used to prepare DNA amplicon libraries which were analyzed by NGS to detect 5mC by reading an increased error rate.