Sharp Andrew J
Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY, USA.
Methods Mol Biol. 2012;925:69-78. doi: 10.1007/978-1-62703-011-3_5.
I provide a protocol for DNA methylation profiling based on immunoprecipitation of methylated DNA using commercially available monoclonal antibodies that specifically recognize 5-methylcytosine. Quantification of the level of enrichment of the resulting DNA enables DNA methylation to be assayed for any genomic locus, including entire chromosomes or genomes if appropriate microarray or high-throughput sequencing platforms are used. In previous studies (1, 2), I have used hybridization to oligonucleotide arrays from Roche Nimblegen Inc, which allow any genomic region of interest to be interrogated, dependent on the array design. For example, using modern tiling arrays comprising millions of oligonucleotide probes, several complete human chromosomes can be assayed at densities of one probe per 100 bp or greater, sufficient to yield high-quality data. However, other methods such as quantitative real-time PCR or high-throughput sequencing can be used, giving either measurement of methylation at a single locus or across the entire genome, respectively. While the data produced by single locus assays is relatively simple to analyze and interpret, global assays such as microarrays or high-throughput sequencing require more complex statistical approaches in order to effectively identify regions of differential methylation, and a brief outline of some approaches is given.
我提供了一种基于使用可特异性识别5-甲基胞嘧啶的市售单克隆抗体对甲基化DNA进行免疫沉淀的DNA甲基化分析方案。对所得DNA富集水平的定量分析能够对任何基因组位点进行DNA甲基化检测，如果使用合适的微阵列或高通量测序平台，还能对整个染色体或基因组进行检测。在之前的研究（1, 2）中，我使用了与罗氏Nimblegen公司的寡核苷酸阵列进行杂交，这取决于阵列设计，能够对任何感兴趣的基因组区域进行检测。例如，使用包含数百万个寡核苷酸探针的现代平铺阵列，可以以每100 bp或更高的密度对几条完整的人类染色体进行检测，足以产生高质量的数据。然而，也可以使用其他方法，如定量实时PCR或高通量测序，分别对单个位点或整个基因组的甲基化进行测量。虽然单一位点检测产生的数据相对易于分析和解释，但诸如微阵列或高通量测序等全局检测需要更复杂的统计方法才能有效地识别差异甲基化区域，本文将给出一些方法的简要概述。
