Sidebar: Bisulfite Treatment for the Detection of DNA Methylation
Sodium bisulfite can deaminate or convert cytosine in DNA into uracil, but does not affect 5-methylcytosine. Bisulfite treatment of DNA is a prerequisite for DNA-methylation analysis for many epigenetics-based studies involving methylation profiling and the quantification of methylation status.
However, analytical procedures involving bisulfite-treated DNA are subject to variability due to DNA degradation, incomplete conversion, and low yields of DNA.
A team of scientists from Zymo Research investigated the procedure of bisulfite treatment of DNA paying particular attention to the chemistries involved in the process and to conversion rates to limit variability between samples and improve upon conventional methods.
They report that conventional bisulfite DNA conversion chemistries could be improved without the levels of DNA degradation typically resulting from incubation of reaction mixtures at high temperature and nonphysiological pH. Essential to this process was prohibiting the overconversion of 5-methylcytosine into uracil that can occur in some situations and reaction conditions.
The researchers found that the bisulfite conversion process could be simplified and the variability between treatments kept to a minimum by coupling heat denaturation with the conversion process and by using in-column desulphonation to clean and purify the converted DNA. This new method was found to yield an average of >80% recovery of input DNA with >99% cytosine to uracil conversion.
The method has been specifically designed to accommodate (in addition to purified DNA) biological fluids, cells, or tissue directly as the input material. This makes its application for FFPE and LCM-derived samples particularly well suited, according to the Zymo Research investigators.