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：

---

一种优化的快速亚硫酸氢盐快速转化方法，可回收无细胞DNA。

无细胞DNA的甲基化分析是用于肿瘤诊断，监测和预后的令人鼓舞的工具。由于血浆中可用的微量无细胞DNA，甲基化分析的灵敏度非常重要。最新的DNA甲基化分析方法基于胞嘧啶和5-甲基胞嘧啶之间亚硫酸氢盐介导的胞嘧啶脱氨作用的差异。但是，基于当前方法的亚硫酸氢盐转化的DNA的回收率对于无细胞DNA的甲基化分析非常差。我们优化了亚硫酸氢盐转化关键步骤的快速方法，并实现了无细胞DNA的高回收率。快速脱氨步骤和碱性脱硫与硅胶柱上的DNA纯化结合在一起。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分钟内胞嘧啶完全转化，亚硫酸氢盐处理的无细胞DNA的回收率约为65％，这比目前的方法要高。该方法可以从低水平的亚硫酸氢盐处理过的无细胞DNA中获得较高的回收率，从而提高了从无细胞DNA中甲基化检测的分析灵敏度。