title: Direct sensing of 5-methylcytosine by polymerase chain reaction.
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.
authors.affiliation: Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstrasse 10, 78457 Konstanz (Germany).
authors.forename: Joos
authors.initials: J
authors.lastname: Aschenbrenner
authors.forename: Matthias
authors.initials: M
authors.lastname: Drum
authors.forename: Hüsnü
authors.initials: H
authors.lastname: Topal
authors.forename: Markus
authors.initials: M
authors.lastname: Wieland
authors.forename: Andreas
authors.initials: A
authors.lastname: Marx
mesh_terms: D044503:5-Methylcytosine / Q000032:analysis*; D006367:HeLa Cells; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D044503:5-Methylcytosine
keywords: DNA methylation; DNA polymerases; enzyme engineering; polymerase chain reaction