Tetzner Reimo, Dietrich Dimo, Distler Juergen
Epigenomics AG, Kleine Praesidentenstrasse 1, 10178 Berlin, Germany.
Nucleic Acids Res. 2007;35(1):e4. doi: 10.1093/nar/gkl955. Epub 2006 Nov 28.
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.
在本研究中，我们采用了广为人知的用于PCR的尿嘧啶DNA糖基化酶（UNG）残留预防系统，并将其应用于基于亚硫酸氢钠转化的DNA甲基化分析。由于亚硫酸氢钠处理会将未甲基化的胞嘧啶碱基转化为尿嘧啶残基，经亚硫酸氢钠处理的DNA对UNG处理敏感。因此，UNG不能用于使用经亚硫酸氢钠处理的模板DNA进行PCR的残留预防，因为不仅先前PCR的污染产物，而且实际模板都会被降解。我们修改了亚硫酸氢钠处理程序，生成了含有磺化尿嘧啶残基的DNA。令人惊讶的是，与尿嘧啶不同，含6-磺酰基尿嘧啶的DNA（SafeBis DNA）对UNG具有抗性。我们表明，新程序在封闭的PCR容器中可去除多达10,000个污染性PCR产物拷贝，而不会显著降低DNA甲基化分析的分析或临床灵敏度。
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