A Modified Bisulfite Conversion Method for the Detection of DNA Methylation

Abstract

Aim: Our purpose is to improve the conventional procedures for bisulfite conversion used to detect 5-methylcytosine in DNA. Methods: Impacts of different bisulfite salts, bisulfite conversion temperature, antioxidants and denaturants on DNA conversion and degradation were assessed by methylation-sensitive melt curve analysis. The modified method was tested on different genes and the conversion efficiency was analyzed by bisulfite sequencing. Results: We developed a modified bisulfite conversion method that completes this process within 2 h. We demonstrate that high temperature denaturation is the major cause for DNA degradation, and the addition of ethylene glycol dimethyl ether is an effective way to accelerate the bisulfite conversion. The conversion efficiency is comparable to many other commercial kits. Conclusion: Our modified bisulfite conversion method is simple, cost efficient and less time consuming and is compatible with different genes and samples, thus has a great potential for the future research and clinical applications.

Keywords::

• bisulfite conversion
• DNA degradation
• DNA methylation
• ethylene glycol dimethyl ether (DME)
• sodium bisulfite

Acknowledgements

We would like to thank the First Affiliated Hospital of Fujian Medical University for providing the clinical samples needed for the study, Shaoli Cai and Zhang Lin for administrative assistance, Dr Daliang Li and the members of the Chen’s Laboratory for technical assistance and helpful discussion.

Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at:www.tandfonline.com/doi/full/10.2217/epi.2016.0174

Financial & competing interests disclosure

This work was supported by Fujian Provincial Health and Education Alliance Funds (WKJ-FJ-28), Fujian Key Laboratories Funds, and a Fujian Provincial Lingjun Scholarship to Qi Chen. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

Collection of all of the blood or tumor tissue samples was approved by the medical ethics committee of the First Affiliated Hospital of Fujian Medical University (Fujian Medical University affiliated medical ethics review permit No. (2013)23). Y Zheng in the Section for Hepatobiliary Pancreatic Surgery in our hospital intends to carry out the research on “The study on Reprimo gene methylation and gastric cancer early detection, and comprehensive evaluation”, which does not violate ethical principles, and agreed on the patient’s informed consent and on the premise to maintain the confidential information under the research program implementation.

Additional information

Funding

This work was supported by Fujian Provincial Health and Education Alliance Funds (WKJ-FJ-28), Fujian Key Laboratories Funds, and a Fujian Provincial Lingjun Scholarship to Qi Chen. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.