An optimized rapid bisulfite conversion method with high recovery of cell-free DNA

作者：Yi, Shaohua^*; Long, Fei; Cheng, Juanbo; Huang, Daixin

来源：BMC Molecular Biology, 2017, 18(1): 24.

DOI：10.1186/s12867-017-0101-4

• Bisulfite conversion
• Rapid deamination
• High recovery
• Cell-free DNA
• Methylation analysis
• droplet digital PCR

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 degrees 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.

• 出版日期2017-12-19
• 单位华中科技大学