Direct sensing of 5-methylcytosine by polymerase chain reaction.

Joos Aschenbrenner,Markus Wieland,Andreas Marx,Hüsnü Topal,Matthias Drum

doi:10.1002/anie.201403745

Joos Aschenbrenner, Markus Wieland + Show 3 more

Open Access

https://doi.org/10.1002/anie.201403745

Copy DOI

Abstract

The epigenetic control of genes by the methylation of cytosine resulting in 5-methylcytosine (5mC) has fundamental implications for human development and disease. Analysis of alterations in DNA methylation patterns is an emerging tool for cancer diagnostics and prognostics. Here we report that two thermostable DNA polymerases, namely the DNA polymerase KlenTaq derived from Thermus aquaticus and the KOD DNA polymerase from Thermococcus kodakaraensis, are able to extend 3′-mismatched primer strands more efficiently from 5 mC than from unmethylated C. This feature was advanced by generating a DNA polymerase mutant with further improved 5mC/C discrimination properties and its successful application in a novel methylation-specific PCR approach directly from untreated human genomic DNA.

Highlights

The epigenetic control of genes by the methylation of cytosine resulting in 5-methylcytosine (5mC) has fundamental implications for human development and disease
Methylated cytosines are found as symmetrical 5-methylcytosine (5mC) of the dinucleotide CpG within promoter regions,[2] in which 75 % are methylated throughout the mammalian genome
The most common method to obtain single-nucleotide resolution is based on treatment of the sample with bisulfite, resulting in the conversion of cytosine to uracil whereas 5mC remains unchanged;[14] this is followed by DNA sequencing or PCR amplification

Summary

Introduction

The epigenetic control of genes by the methylation of cytosine resulting in 5-methylcytosine (5mC) has fundamental implications for human development and disease. We found that discrimination between the mispaired C and 5mC templates is observed with KOD exo-, albeit to a lower extent than with KlenTaq (47 % extension of the A-primer when paired with 5mC template as compared to 34 % with C template; Figure 1 d).

Results

Conclusion