Array-based methylation analysis using anti-5mC antibody

描述(申请人提供)：DNA甲基化的改变是与早期胚胎发育、细胞分化和包括乳腺癌和前列腺癌在内的许多类型癌症的进展相关的最显著的变化之一。在过去的几年里，我们一直在开发检测DNA甲基化变化的技术，目标是癌症诊断的潜在临床应用。我们建立的DNA微阵列MS-AFLP方法已被证明是检测基因组中Not1标志物上DNA甲基化变化的有用方法。然而，由于在生产基于该技术的DNA微阵列方面缺乏商业兴趣，其实用性一直受到限制。对Not1限制性内切酶识别甲基化区域的依赖也限制了对基因组的询问范围。考虑到这一点，我们建议，在这一探索性/开发性R21研究资助申请中，进行几个试点实验，以测试一种概念上新颖的方法检测DNA甲基化变化的可行性，该方法可以利用目前可用的DNA微阵列。与任何试验性研究一样，这个项目本质上是有风险的，因为我们还没有对这个概念进行实验测试。然而，如果证明可行，这种新方法可能会在分析DNA甲基化全球变化的全基因组检测方面发挥革命性的作用。在本申请中，我们提出了以下两个具体目标。1.检测抗5-甲基胞嘧啶(抗5mC)抗体对基于磁珠的甲基化和非甲基化DNA片段群分离的作用。我们将建立实验条件，在此条件下，使用抗5mC可以分离出富含甲基化和未甲基化DNA片段的两个不同组分。2.检验上述分离的甲基化和非甲基化DNA片段群体对于基于阵列的DNA甲基化差异检测的有效性。我们将对这两个组分的DNA进行荧光标记，并进行DNA微阵列杂交实验。我们将使用袋克隆微阵列、DNA片段微阵列和寡核苷酸微阵列来评估该方法的敏感性、特异性和重复性。

DESCRIPTION (provided by applicant): An alteration in DNA methylation is one of the most prominent changes associated with early embryo development, cellular differentiation, and progression of many types of cancers, including breast and prostate cancers. In the past several years we have been developing techniques to detect alterations in DNA methylation, targeting potential clinical applications for cancer diagnosis. The DNA Microarray MS- AFLP method that we developed has been proven to be useful in detecting DNA methylation alterations at the Notl landmarks in the genome. However, the utility has been limited due to the absence of commercial interest in producing the DNA microarrays based on the technique. The dependence on Notl restriction enzymes to identify the methylated regions has also restricted the scope of interrogation of the genome. Keeping this in mind, we propose, in this Exploratory/Developmental R21 Research Grant application, to perform several pilot experiments to test the feasibility of a conceptually novel method for the detection of alterations in DNA methylation that can utilize the DNA microarrays that are currently available. As with any pilot study, this project is risky in nature because we have not experimentally tested the concept yet. However, if proven feasible, the new method may achieve a revolutionary role in analyzing the genome- wide detection of global changes in DNA methylation. In this application, we propose the following two specific aims. 1. To examine the utility of anti-5-methylcytosine (anti-5mC) antibodies toward the magnetic bead-based separation of methylated and unmethylated DNA fragment populations. We will establish the experimental conditions under which 2 different fractions rich in methylated and unmethylated DNA fragments may be separated, using anti-5mC. 2. To examine the utility of methylated and unmethylated DNA fragment populations separated above toward the array-based detection of differences in DNA methylation. We will fluorescently label DNA from those 2 fractions and perform DNA microarray hybridization experiments. We will evaluate the sensitivity, specificity, and reproducibility of the method, using BAG clone microarrays, DNA fragment microarrays, and oligonucleotide microarrays.