Abstract 3025: Efficiency of bisulfite modification and DNA recovery in clinical samples with minute amounts of circulating DNA
Authors: Krause Hans , Bryzgunova Olga , Laktionov Pavel P. , Skvortsova Tatyana , Bondar Anna , Morozkin Evgeniy , Lebedeva Alena , Vlassov Valentin , Miller Kurt 
Published: June 2012

Abstract
In higher eukaryotes methylation of cytosine residues within CpG dinucleotides has profound effects on gene expression and hence for the regulation of mammalian development. Global changes in this reversible epigenetic event are a hallmark of cancer, too. Disruption of epigenetic processes can lead to altered gene function and finally to malignant cellular transformation. Tumor-associated DNA methylation at cytosine residues has been described for free DNA circulating (cirDNA) in the bloodstream of many different cancer specimens. Confirmation of these methylated sequences therefore provides a unique opportunity to develop cirDNA-based minimal-invasive assays for early cancer detection as well as other applications in clinical diagnostics.
Sodium bilsulfite modification of DNA turns epigenetic differences into genetic ones, in particular unmethylated cytosines are converted to thymidines (by uracil), whereas methylated cytosines (5mC) still pair as cytosines, thus allowing for a broad range of sensitive detection techniques even in the presence of excess amounts of unmethylated templates. Prevention of complete conversion and/or DNA loss during the modification reaction due to non-specific degradation and accompanying washing/elution steps is of paramount importance when analyzing minute amounts of cirDNA in bodily fluids of cancer patients.
This study evaluated three commercially available bisulfite modification kits with regard to procedural DNA loss as well as the efficacy of cytosine conversion to uracil. For efficacy estimation of DNA conversion, enzymatically generated fully methylated as well as unmethylted templates of the RARB gene promoter region (GenBank X56849) were analyzed by pyro-sequencing at 8 CpG sites. In addition, cirDNAs of healthy and prostate cancer patients were analyzed at this locus. Our data demonstrate that all commercial kits displayed similar efficacy in conversion of unmethylated cytosines, close to 99% despite of DNA concentration. However, when low concentrations of DNA were used, the basic level of genomic DNA methylation increased up to 11% depending from the position of the CpG site. Procedural DNA loss was calculated by methyl-independent Q-PCR for ubiquitously distributed LINE1 elements. EZ DNA Methylation-Gold Kit from Zymo Research provided the highest yield of DNA with a medium yield not lower than 86%. Efficacy of DNA recovery by this kit did not depend from initial DNA concentration. This observation is contrary to the results obtained with QIAGEN and Chemicon kits, respectively, that recovered no more than 20% starting material from high DNA input (500ng/probe) and only 2.7-5.8% for low DNA amounts (80ng/probe).
For situations where low DNA amounts are subjected to methylation assays that require bisulfite modification our data suggest that EZ DNA Methylation-Gold Kit from Zymo Research is the most appropriate tool to use.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3025. doi:10.1158/1538-7445.AM2011-3025