Analysis of repetitive element DNA methylation by MethyLight.
Analysis of repetitive element DNA methylation by MethyLight.
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DOI：
10.1093/nar/gki987
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发表时间：
2005
影响因子：
14.9
通讯作者：
Laird PW
中科院分区：
文献类型：
--
作者：
Weisenberger DJ;Campan M;Long TI;Kim M;Woods C;Fiala E;Ehrlich M;Laird PW
重复元件代表了人类基因组的很大一部分，并包含了在正常人类出生后体细胞组织中发现的大部分CpG甲基化。这些序列中DNA甲基化的缺失可能是全球低甲基化的主要原因，这种低甲基化是已研究的大部分人类癌症的特征。基因组中5-甲基胞嘧啶的含量与临床结果、饮食历史、生活方式等之间的关系引起了广泛的兴趣。然而，目前还没有一种高通量、准确和容易获得的技术，甚至可以应用于石蜡包埋的组织DNA。在这里，我们报告了定量甲基化和非甲基化重复序列的发展，即ALU和LINE-1序列以及着丝粒卫星α(Satα)和伴随着丝粒卫星2(SAT2DNA)序列。高效液相色谱检测Alu、SAT2和LINE-1重复序列的甲基化水平与全球DNA甲基化显著相关，联合检测Alu和SAT2甲基化水平与全球DNA甲基化水平高度相关。这些甲基光检测仅依赖于实时聚合酶链式反应，并为全球DNA甲基化分析提供替代标记。我们还描述了一种新的设计策略，用于开发甲基化不依赖于甲基化的甲基化控制反应，该反应基于一条链上通过进化脱氨而耗尽的CpG二核苷酸的Alu序列。我们表明，一个这样的基于铝的反应提供了一个极大的改进的DNA检测的正常化应用中，并不容易受到标准化错误引起的癌症相关的非整倍体和拷贝数变化。
Repetitive elements represent a large portion of the human genome and contain much of the CpG methylation found in normal human postnatal somatic tissues. Loss of DNA methylation in these sequences might account for most of the global hypomethylation that characterizes a large percentage of human cancers that have been studied. There is widespread interest in correlating the genomic 5-methylcytosine content with clinical outcome, dietary history, lifestyle, etc. However, a high-throughput, accurate and easily accessible technique that can be applied even to paraffin-embedded tissue DNA is not yet available. Here, we report the development of quantitative MethyLight assays to determine the levels of methylated and unmethylated repeats, namely, Alu and LINE-1 sequences and the centromeric satellite alpha (Satα) and juxtacentromeric satellite 2 (Sat2) DNA sequences. Methylation levels of Alu, Sat2 and LINE-1 repeats were significantly associated with global DNA methylation, as measured by high performance liquid chromatography, and the combined measurements of Alu and Sat2 methylation were highly correlative with global DNA methylation measurements. These MethyLight assays rely only on real-time PCR and provide surrogate markers for global DNA methylation analysis. We also describe a novel design strategy for the development of methylation-independent MethyLight control reactions based on Alu sequences depleted of CpG dinucleotides by evolutionary deamination on one strand. We show that one such Alu-based reaction provides a greatly improved detection of DNA for normalization in MethyLight applications and is less susceptible to normalization errors caused by cancer-associated aneuploidy and copy number changes.
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影响因子：
8.8
作者：
Florl, AL;Steinhoff, C;Müller, M;Seifert, HH;Hader, C;Engers, R;Ackermann, R;Schulz, WA
通讯作者：
Schulz, WA
影响因子：
4.8
作者：
Kondo, Y;Issa, JPJ
通讯作者：
Issa, JPJ
影响因子：
14.9
作者：
EHRLICH, M;GAMASOSA, MA;GEHRKE, C
通讯作者：
GEHRKE, C
影响因子：
--
作者：
Ehrlich, M;Hopkins, NE;Sawyer, J
通讯作者：
Sawyer, J
影响因子：
3.6
作者：
Jackson, K;Yu, MC;Ehrlich, M
通讯作者：
Ehrlich, M