A new method for accurate assessment of DNA quality after bisulfite treatment.
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.
Ehrich
Mathias
M
SEQUENOM Inc., 3595 John Hopkins Ct., San Diego, CA, 92121, USA. mehrich@sequenom.com
Zoll
Scott
S
Sur
Sudipto
S
van den Boom
Dirk
D
D018899:CpG Islands; D004247:DNA / Q000032:analysis / Q000592:standards*; D053938:DNA Fragmentation; D019175:DNA Methylation; D004591:Electrophoresis, Polyacrylamide Gel; D015233:Models, Statistical; D016133:Polymerase Chain Reaction / Q000379:methods*; D011336:Probability; D011786:Quality Control; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013447:Sulfites / Q000737:chemistry*
D013447:Sulfites; C005200:sodium metabisulfite; D004247:DNA