A new method for accurate assessment of DNA quality after bisulfite treatment

Abstract

The covalent addition of methylgroups to cytosine has become the most intensively researched epigenetic DNA marker. The vast majority of technologies used for DNA methylation analysis rely on a chemical reaction, the so-called ‘bisulfite treatment’, which introduces methylation-dependent sequence changes through selective chemical conversion of non-methylated cytosine to uracil. After treatment, all non-methylated cytosine bases are converted to uracil but all methylated cytosine bases remain cytosine. These methylation dependent C-to-T changes can subsequently be studied using conventional DNA analysis technologies.The bisulfite conversion protocol is susceptible to processing errors, and small deviation from the protocol can result in failure of the treatment. Several attempts have been made to simplify the procedure and increase its robustness. Although significant achievements in this area have been made, bisulfite treatment remains the main source of process variability in the analysis of DNA methylation. This variability in particular impairs assays, which strive for the quantitative assessment of DNA methylation. Here we present basic mathematical considerations, which should be taken into account when analyzing DNA methylation. We also introduce a PCR-based assay, which allows ab initio assessment of the DNA quality after bisulfite treatment and can help to prevent inaccurate quantitative measurement resulting from poor bisulfite treatment.

Cytosine methylation profiling of cancer cell lines

“…All bisulfite-based methylation analysis methods suffer from a considerable amount of measurement variability introduced by the chemical treatment of genomic DNA. To assess the degree of this inherent variability, we previously dissected the method into its four components and measured the variability for each step in the process (10). The results demonstrated that the greatest source of process-dependent variability is the bisulfite conversion reaction (SD ϭ 10-15%).…”

Comparative analysis of DNA methylation profiles in peripheral blood leukocytes versus lymphoblastoid cell lines

“…The overall process variability of bisulfite based PCR methods has been reported to result in standard deviations of the quantitative measurement of 5% to 8%. 30,31 The cut-off of 20% was selected to signify a greater than two standard deviation difference. To further exclude false positive results we limited our selection to genes in which at least two CpG units were found to be differentially methylated.…”

Assessing the analytic validity of molecular testing for Huntington disease using data from an external proficiency testing survey

“…This method, which converts cytosine to uracil has been shown to have some limitations, including incomplete conversion and degradation of DNA during treatment, which can result in problems during PCR. 8,9 In addition, this participant may have been separating alleles on an agarose gel (as described in the original publication) instead of an acrylamide gel, which could also be responsible for sizing errors. Based on these data, bisulfite PCR seems not to be an optimum method for sizing alleles.…”