Prevention of PCR cross-contamination by UNG treatment of bisulfite-treated DNA.

Abstract

Amplification of sodium bisulfite-treated DNA is widely used to study DNA methylation. The proportion of methylated sequences of a specific DNA region in a sample can be determined by the analysis of PCR products or directly calculated from real-time PCR amplification of bisulfite-treated DNA. At the same time, PCR based methods always involve the risk of false positive or incorrect quantitative results due to the unintended reamplification of contaminating PCR products. The incubation of PCR reactions with Uracil-DNA Glycosylase (UNG) prior to the thermal cycling in combination with the use of dUTP in the PCR amplification is a commonly used technology to prevent such cross-contamination. Since sodium bisulfite treatment converts unmethylated cytosine bases into uracil residues, not only contaminating PCR products but also the converted template DNAs would be degraded as well. This chapter describes a modified bisulfite treatment procedure to generate sulfonated DNA enabling the application of UNG-based carryover prevention to DNA methylation analysis. The high efficiency of the decontamination procedure as well as the universal applicability of this simple method is shown.