LUMA (LUminometric Methylation Assay) -: A high throughput method to the analysis of genomic DNA methylation
LUMA (LUminometric Methylation Assay) -: A high throughput method to the analysis of genomic DNA methylation
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DOI：
10.1016/j.yexcr.2006.03.006
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发表时间：
2006-07-01
影响因子：
3.7
通讯作者：
Ekstrom, Tomas J.
中科院分区：
文献类型：
--
作者：
Karimi, Mohsen;Johansson, Sofia;Ekstrom, Tomas J.
基因组 DNA 甲基化的变化被认为是正常和病理细胞过程中的重要事件，有助于正常发育和分化以及癌症和其他疾病。在这里，我们报告了一种估计全基因组 DNA 甲基化的新方法，称为 LUminometric 甲基化测定 (LUMA)。该方法基于甲基化敏感限制性酶的组合 DNA 切割和焦磷酸测序 (TM) 的聚合酶延伸测定。该方法定量、重现性好、易于放大。由于不需要对基因组 DNA 进行初步修饰，例如亚硫酸氢盐处理，因此总测定时间仅为 6 小时。此外，该检测仅需要 200-500 ng 基因组 DNA，并包含内部对照，以消除起始 DNA 量变化的问题。 LUMA 的准确性和线性通过体外甲基化 lambda DNA 进行验证。此外，通过 LUMA 评估 DNA 甲基转移酶敲除细胞系中以及用 DNA 甲基转移酶抑制剂（5-AzaCytidine）处理后的 DNA 甲基化水平。 LUMA 测定可以提供一种有用的方法来分析各种生理和病理条件（包括癌症的病因、诊断和预后方面）的全基因组 DNA 甲基化。 (c) 2006 Elsevier Inc. 保留所有权利。
Changes in genomic DNA methylation are recognized as important events in normal and pathological cellular processes, contributing both to normal development and differentiation as well as cancer and other diseases. Here, we report a novel method to estimate genome-wide DNA methylation, referred to as LUminometric Methylation Assay (LUMA). The method is based on combined DNA cleavage by methylation-sensitive restriction enzymes and polymerase extension assay by Pyrosequencing (TM). The method is quantitative, highly reproducible and easy to scale up. Since no primary modification of genomic DNA, such as bisulfite treatment, is needed, the total assay time is only 6 h. In addition, the assay requires only 200-500 ng of genomic DNA and incorporates an internal control to eliminate the problem of varying amounts of starting DNA. The accuracy and linearity of LUMA were verified by in vitro methylated lambda DNA. In addition, DNA methylation levels were assessed by LUMA in DNA methyltransferase knock-out cell lines and after treatment with the DNA methyltransferase inhibitor (5-AzaCytidine). The LUMA assay may provide a useful method to analyze genome-wide DNA methylation for a variety of physiological and pathological conditions including etiologic, diagnostic and prognostic aspects of cancer. (c) 2006 Elsevier Inc. All rights reserved.