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Updated: Feb 16, 2026
Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution
Published on: February 24, 2015
An optimized rapid bisulfite conversion method with high recovery of cell-free DNA
Shaohua Yi1, Fei Long2, Juanbo Cheng2
1Department of Forensic medicine, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Rd, Wuhan, 430030, China. yishaoxia@126.com.
Background:
Methylation analysis of cell-free DNA is a encouraging tool for tumor diagnosis, monitoring and prognosis. Sensitivity of methylation analysis is a very important matter due to the tiny amounts of cell-free DNA available in plasma. Most current methods of DNA methylation analysis are based on the difference of bisulfite-mediated deamination of cytosine between cytosine and 5-methylcytosine. However, the recovery of bisulfite-converted DNA based on current methods is very poor for the methylation analysis of cell-free DNA.
Results:
We optimized a rapid method for the crucial steps of bisulfite conversion with high recovery of cell-free DNA. A rapid deamination step and alkaline desulfonation was combined with the purification of DNA on a silica column. The conversion efficiency and recovery of bisulfite-treated DNA was investigated by the droplet digital PCR. The optimization of the reaction results in complete cytosine conversion in 30 min at 70 °C and about 65% of recovery of bisulfite-treated cell-free DNA, which is higher than current methods.
Conclusions:
The method allows high recovery from low levels of bisulfite-treated cell-free DNA, enhancing the analysis sensitivity of methylation detection from cell-free DNA.
