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. 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. 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.

中文翻译：

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一种优化的快速亚硫酸氢盐转化方法，具有高无细胞 DNA 回收率。


无细胞 DNA 的甲基化分析是肿瘤诊断、监测和预后的一个令人鼓舞的工具。由于血浆中存在微量的游离 DNA，甲基化分析的灵敏度非常重要。目前大多数 DNA 甲基化分析方法都是基于胞嘧啶和 5-甲基胞嘧啶之间亚硫酸氢盐介导的胞嘧啶脱氨基作用的差异。然而，基于现有方法的亚硫酸氢盐转化的DNA的回收率对于游离DNA的甲基化分析来说非常差。我们优化了亚硫酸氢盐转化关键步骤的快速方法，并具有高无细胞 DNA 回收率。将快速脱氨步骤和碱性脱磺化与硅胶柱上的 DNA 纯化相结合。通过液滴数字 PCR 研究了亚硫酸氢盐处理的 DNA 的转化效率和回收率。反应的优化导致在 70 °C 下 30 分钟内完成胞嘧啶转化，亚硫酸氢盐处理的无细胞 DNA 的回收率约为 65%，高于现有方法。该方法可以从低水平的亚硫酸氢盐处理的游离 DNA 中获得高回收率，从而提高游离 DNA 甲基化检测的分析灵敏度。