High-speed conversion of cytosine to uracil in bisulfite genomic Sequencing analysis of DNA methylation
High-speed conversion of cytosine to uracil in bisulfite genomic Sequencing analysis of DNA methylation
复制标题
DOI：
10.1093/dnares/11.6.409
复制
发表时间：
2004-12-31
期刊：
影响因子：
4.1
通讯作者：
Hayatsu, H
中科院分区：
文献类型：
--
作者：
Shiraishi, M;Hayatsu, H
亚硫酸氢盐基因组测序是一种广泛使用的分析 DNA 胞嘧啶甲基化的技术。通过用亚硫酸氢盐处理 DNA，胞嘧啶残基被脱氨基为尿嘧啶，同时 5-甲基胞嘧啶基本保持完整。随后的 PCR 和核苷酸序列分析可以明确确定胞嘧啶残基的甲基化状态。与目前实践的程序相关的一个主要警告是，需要 16-20 小时才能完成胞嘧啶向尿嘧啶的转化。在这里，我们报告说，通过在高温下使用高浓度的亚硫酸氢盐溶液，可以在更短的时间内实现胞嘧啶完全脱氨基为尿嘧啶。时程实验表明，用 9 M 亚硫酸氢盐在 90 摄氏度下处理 DNA 20 分钟或在 70 摄氏度下处理 40 分钟，DNA 中的所有胞嘧啶残基均转化为尿嘧啶。在这些条件下，大多数 5-甲基胞嘧啶保持完整。当来自细胞系的高分子量DNA（含有许多甲基化状态已知的基因）在上述条件下用亚硫酸氢盐处理并扩增和测序时，得到的结果与文献报道的结果一致。尽管在此过程中发生了一些 DNA 降解，但扩增所需的处理 DNA 量几乎等于传统亚硫酸氢盐基因组测序程序所需的量。这种新颖的方法提高了 DNA 甲基化分析的速度，预计将推动 DNA 科学的各个方面的发展。
Bisulfite genomic sequencing is a widely used technique for analyzing cytosine-methylation of DNA. By treating DNA with bisulfite, cytosine residues are deaminated to uracil, while leaving 5-methyleytosine largely intact. Subsequent PCR and nucleotide sequence analysis permit unequivocal determination of the methylation status at cytosine residues. A major caveat associated with the currently practiced procedure is that it takes 16-20 hr for completion of the conversion of cytosine to uracil. Here we report that a complete deamination of cytosine to uracil can be achieved in shorter periods by using a highly concentrated bisulfite solution at an elevated temperature. Time course experiments demonstrated that treating DNA with 9 M bisulfite for 20 min at 90 degrees C or 40 min at 70 degrees C all cytosine residues in the DNA were converted to uracil. Under these conditions, the majority of 5-methyleytosines remained intact. When a high molecular weight DNA derived from a cell line (containing a number of genes whose methylation status was known) Was treated with bisulfite under the above conditions and amplified and sequenced, the results obtained were consistent with those reported in the literature. Although some degradation of DNA occurred during this process, the amount of treated DNA required for the amplification was nearly equal to that required for the conventional bisulfite genomic sequencing procedure. The increased speed of DNA methylation analysis with this novel procedure is expected to advance various aspects of DNA sciences.