Abstract
BackgroundMethylation 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.ResultsWe 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.ConclusionsThe method allows high recovery from low levels of bisulfite-treated cell-free DNA, enhancing the analysis sensitivity of methylation detection from cell-free DNA.
Highlights
Methylation analysis of cell-free DNA is a encouraging tool for tumor diagnosis, monitoring and prognosis
Analyzing methylation of cell-free DNA in plasma and other body fluids is a encouraging tool for cancer diagnosis, monitoring and prognosis
Cytosine is quickly converted to uracil, whereas 5-methylcytosine is only slowly changed to thymine
Summary
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. The recovery of bisulfite-converted DNA based on current methods is very poor for the methylation analysis of cell-free DNA. Analyzing methylation of cell-free DNA (cfDNA) in plasma and other body fluids is a encouraging tool for cancer diagnosis, monitoring and prognosis. Most of current methods for analyzing DNA methylation are based on bisulfite-mediated deamination of cytosine [5,6,7,8]. The recovery of bisulfite-converted DNA is usually very poor
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