A sensitive new method for rapid detection of abnormal methylation patterns in global DNA and within CpG islands
A sensitive new method for rapid detection of abnormal methylation patterns in global DNA and within CpG islands
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DOI：
10.1006/bbrc.1999.1187
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发表时间：
1999-09-07
影响因子：
3.1
通讯作者：
James, SJ
中科院分区：
文献类型：
--
作者：
Pogribny, I;Yi, P;James, SJ
为了评估全球DNA和CpG岛内DNA甲基化密度的变化，我们开发了一种简单的方法，该方法基于甲基化敏感的限制性内切酶的使用，该酶在DNA切割后留下5‘端鸟嘌呤，随后用放射性标记的[H-3]dCTP延伸单核苷酸。甲基化敏感的限制性内切酶HpaII和AciI在整个基因组中随机出现的CpG位点上有相对频繁的识别序列，BssHII是一种甲基化敏感的酶，类似地留下鸟嘌呤过剩，但识别序列是非随机的，主要发生在CpG岛内的未甲基化的CpG位点。这些酶的选择性使用可分别用于筛选全基因组甲基化和CpG岛甲基化状态的变化。限制性内切酶处理后，[H-3]dCTP与暴露的鸟嘌呤相对的掺入程度与未甲基化(切割)的CpG；位点数成正比。与现有方法相比，“胞嘧啶延伸分析”有几个优点，因为(A)放射性标记掺入与DNA的完整性无关，(B)甲基化检测不需要PCR扩增或DNA甲基酶反应，以及(C)它适用于ng数量的DNA。利用从人正常肝脏和人肝细胞癌中提取的DNA，通过检测到肿瘤DNA中全基因组低甲基化和CpG岛超甲基化的增加，证明了该方法的适用性。(C)1999年学术出版社。
To assess alterations in DNA methylation density in both global DNA and within CpG islands, we have developed a simple method based on the use of methylation-sensitive restriction endonucleases that leave a 5' guanine overhang after DNA cleavage, with subsequent single nucleotide extension with radiolabeled [H-3]dCTP. The methylation-sensitive restriction enzymes HpaII and AciI have relatively frequent recognition sequences at CpG sites that occur randomly throughout the genome, BssHII is a methylation sensitive enzyme that similarly leaves a guanine overhang, but the recognition sequence is nonrandom and occurs predominantly at unmethylated CpG sites within CpG islands. The selective use of these enzymes can be used to screen for alterations in genome-wide methylation and CpG island methylation status, respectively. The extent of [H-3]dCTP incorporation opposite the exposed guanine after restriction enzyme treatment is directly proportional to the number of unmethylated (cleaved) CpG; sites. The "cytosine-extension assay" has several advantages over existing methods because (a) radiolabel incorporation is independent of the integrity of the DNA, (b) methylation detection does not require PCR amplification or DNA methylase reactions, and (c) it is applicable to ng quantities of DNA. Using DNA extracted from normal human liver and from human hepatocellular carcinoma, the applicability of the assay is demonstrated by the detection of an increase in genome-wide hypomethylation and CpG island hypermethylation in the tumor DNA. (C) 1999 Academic Press.