{"title":"An optimized rapid bisulfite conversion method with high recovery of cell-free DNA","authors":"Shaohua Yi, Fei Long, Juanbo Cheng, Daixin Huang","doi":"10.1186/s12867-017-0101-4","DOIUrl":null,"url":null,"abstract":"<p>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.</p><p>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.</p><p>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.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"18 1","pages":""},"PeriodicalIF":2.9460,"publicationDate":"2017-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-017-0101-4","citationCount":"14","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"BMC Molecular Biology","FirstCategoryId":"1085","ListUrlMain":"https://link.springer.com/article/10.1186/s12867-017-0101-4","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 14
Abstract
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.
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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