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.
In an application note (“Perfecting Bisulfite Treatment for DNA Methylation Detection”), the scientists described the use of the EZ DNA Methylation-Direct™ kit for the recovery of bisulfite-treated DNA from a range of sample inputs. They noted that the kit and associated reagents were designed to achieve data consistency and mitigate the loss of DNA. According to the team, the technologies involved in the kit ensure consistent recovery of input DNA from as few as 10 cells or as little as ~50 pg DNA.
“This is facilitated through the fine-tuning of bisulfite conversion chemistries that enable the reaction to proceed to near completion, that is, 99.8% conversion of nonmethylated cytosine, while maintaining the integrity of methylated cytosine during the process,” says Marc Van Eden, Ph.D., vp business development and marketing, and a co-author of the application note.
Dr. Van Eden maintains that the key to the high recovery of converted DNA is the advanced column/plate design featured in the company’s kit.
“The Fast-Spin columns and plates ensure rapid desulphonation as well as high recovery of converted DNA. Elution of buffers from the column/plate matrices is complete, negating buffer carryover,” continues Dr. Van Eden. “Thus, eluted, bisulfite-converted DNA is pure and ready for analysis. Further, the columns allow DNA to be eluted in ultrasmall volumes (≥6 µL) for highly concentrated DNA if required.”