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Nucleic Acids Research Advance Access originally published online on November 28, 2006
Nucleic Acids Research 2007 35(1):e4; doi:10.1093/nar/gkl955
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Nucleic Acids Research, 2007, Vol. 35, No. 1 e4
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Control of carry-over contamination for PCR-based DNA methylation quantification using bisulfite treated DNA

Reimo Tetzner, Dimo Dietrich and Juergen Distler

Epigenomics AG, Kleine Praesidentenstraße 1 10178 Berlin, Germany

*To whom correspondence should be addressed. Tel: +49 30 24345100; Fax: +49 30 24345299; Email: distler{at}epigenomics.com

Received July 5, 2006. Revised October 24, 2006. Accepted October 24, 2006.

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.

The authors wish to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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