Karimi Mohsen, Johansson Sofia, Stach Dirk, Corcoran Martin, Grandér Dan, Schalling Martin, Bakalkin Georgy, Lyko Frank, Larsson Catharina, Ekström Tomas J
Departments of Clinical Neuroscience, Karolinska Institutet, Karolinska University Hospital-Solna, SE-171 76 Stockholm, Sweden.
Exp Cell Res. 2006 Jul 1;312(11):1989-95. doi: 10.1016/j.yexcr.2006.03.006. Epub 2006 Apr 19.
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. 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.
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，对正常发育、分化以及癌症和其他疾病都有影响。在此，我们报告一种估计全基因组DNA甲基化的新方法，称为荧光甲基化检测法（LUMA）。该方法基于甲基化敏感限制性内切酶的联合DNA切割和焦磷酸测序的聚合酶延伸检测。该方法具有定量、高度可重复且易于扩大规模的特点。由于不需要对基因组DNA进行诸如亚硫酸氢盐处理等初步修饰，整个检测时间仅为6小时。此外，该检测仅需要200 - 500 ng基因组DNA，并包含一个内部对照以消除起始DNA量不同的问题。通过体外甲基化的λDNA验证了LUMA的准确性和线性。此外，通过LUMA评估了DNA甲基转移酶敲除细胞系以及用DNA甲基转移酶抑制剂（5 - 氮杂胞苷）处理后的DNA甲基化水平。LUMA检测可能为分析包括癌症的病因、诊断和预后等多种生理和病理状况下的全基因组DNA甲基化提供一种有用的方法。
