title: Control of carry-over contamination for PCR-based DNA methylation quantification using bisulfite treated DNA.
abstract: In this study, we adapted the well known uracil DNA glycosylase (UNG) carry-over prevention system for PCR, and applied it to the analysis of DNA methylation based on sodium bisulfite conversion. As sodium bisulfite treatment converts unmethylated cytosine bases into uracil residues, bisulfite treated DNA is sensitive to UNG treatment. Therefore, UNG cannot be used for carry-over prevention of PCR using bisulfite treated template DNA, as not only contaminating products of previous PCR, but also the actual template will be degraded. We modified the bisulfite treatment procedure and generated DNA containing sulfonated uracil residues. Surprisingly, and in contrast to uracil, 6-sulfonyl uracil containing DNA (SafeBis DNA) is resistant to UNG. We showed that the new procedure removes up to 10,000 copies of contaminating PCR product in a closed PCR vessel without significant loss of analytical or clinical sensitivity of the DNA methylation analysis.
authors.affiliation: Epigenomics AG, Kleine Praesidentenstrasse 1, 10178 Berlin, Germany.
authors.forename: Reimo
authors.initials: R
authors.lastname: Tetzner
authors.forename: Dimo
authors.initials: D
authors.lastname: Dietrich
authors.forename: Juergen
authors.initials: J
authors.lastname: Distler
mesh_terms: D003110:Colonic Neoplasms / Q000235:genetics; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000737:chemistry; D005260:Female; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D013447:Sulfites / Q000737:chemistry*; D014498:Uracil / Q000737:chemistry; D051981:Uracil-DNA Glycosidase
chemical_list: D004273:DNA, Neoplasm; D013447:Sulfites; D014498:Uracil; D004247:DNA; D051981:Uracil-DNA Glycosidase; C009279:sodium bisulfite