Whole genome methylation profiling by immunoprecipitation of methylated DNA.
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.
Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY, USA. andrew.sharp@mssm.edu
Andrew J
AJ
Sharp
D000818:Animals; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D019175:DNA Methylation / Q000235:genetics*; D018392:Genomic Imprinting; D023281:Genomics / Q000379:methods*; D047468:Immunoprecipitation / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis
D004247:DNA