HIGH-SENSITIVITY MAPPING OF METHYLATED CYTOSINES
HIGH-SENSITIVITY MAPPING OF METHYLATED CYTOSINES
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DOI：
10.1093/nar/22.15.2990
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发表时间：
1994-08-11
影响因子：
14.9
通讯作者：
FROMMER, M
中科院分区：
文献类型：
--
作者：
CLARK, SJ;HARRISON, J;FROMMER, M
对DNA甲基化及其在发育、衰老和癌症基因控制中的潜在作用的理解一直受到缺乏敏感方法的阻碍，这些方法仅能从少量DNA中解析出准确的甲基化模式。我们现在已经开发出一种基因组测序技术，它能够使用从不到100个细胞中提取的DNA来检测任何目标序列的两条链上的每一条甲基化的胞嘧啶。在这种方法中，在5-甲基胞嘧啶保持不反应的条件下，亚硫酸氢钠被用来将单链DNA中的胞嘧啶残基转化为尿嘧啶残基。转化后的DNA用特异的引物扩增并测序。序列中剩余的所有胞嘧啶残基代表基因组中先前甲基化的胞嘧啶。所描述的工作确定了最大限度地提高变性、亚硫酸氢盐转化和扩增效率的程序，以允许从生殖细胞和早期发育阶段容易获得的少量基因组DNA中的单个基因的甲基化图谱。
An understanding of DNA methylation and its potential role in gene control during development, aging and cancer has been hampered by a lack of sensitive methods which can resolve exact methylation patterns from only small quantities of DNA. We have now developed a genomic sequencing technique which is capable of detecting every methylated cytosine on both strands of any target sequence, using DNA isolated from fewer than 100 cells. In this method, sodium bisulphite is used to convert cytosine residues to uracil residues in single-stranded DNA, under conditions whereby 5-methylcytosine remains non-reactive. The converted DNA is amplified with specific primers and sequenced. All the cytosine residues remaining in the sequence represent previously methylated cytosines in the genome. The work described has defined procedures that maximise the efficiency of denaturation, bisulphite conversion and amplification, to permit methylation mapping of single genes from small amounts of genomic DNA, readily available from germ cells and early developmental stages.