一种优化的亚硫酸氢盐快速转化无细胞DNA的高回收率方法

IF 2.946 Q3 Biochemistry, Genetics and Molecular Biology BMC Molecular Biology Pub Date : 2017-12-19 DOI:10.1186/s12867-017-0101-4
Shaohua Yi, Fei Long, Juanbo Cheng, Daixin Huang
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引用次数: 14

摘要

游离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。由于血浆中游离DNA的含量极低,甲基化分析的敏感性是一个非常重要的问题。目前大多数DNA甲基化分析方法都是基于亚硫酸亚硫酸盐介导的胞嘧啶脱氨作用在胞嘧啶和5-甲基胞嘧啶中的差异。然而,基于现有方法的亚硫酸盐转化DNA的恢复对于无细胞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,并获得了高的无细胞DNA回收率。采用快速脱氨和碱性脱硫相结合的方法,在硅胶柱上提纯DNA。采用微滴数字PCR技术对亚硫酸氢盐处理DNA的转化效率和回收率进行了研究。优化后的反应在30分钟内完成了胞嘧啶的完全转化。最低70?°C,亚硫酸处理的无细胞DNA的回收率约为65%,高于目前的方法。该方法允许从低水平亚硫酸处理的游离DNA中获得高回收率,提高了从游离DNA中进行甲基化检测的分析灵敏度。
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An optimized rapid bisulfite conversion method with high recovery of cell-free DNA

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.

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来源期刊
BMC Molecular Biology
BMC Molecular Biology 生物-生化与分子生物学
CiteScore
4.80
自引率
0.00%
发文量
0
审稿时长
>12 weeks
期刊介绍: BMC Molecular Biology is an open access journal publishing original peer-reviewed research articles in all aspects of DNA and RNA in a cellular context, encompassing investigations of chromatin, replication, recombination, mutation, repair, transcription, translation and RNA processing and function.
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