Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Methyl-Cytosine-Based Immunoprecipitation for DNA Methylation Analysis

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In mammalian cells, DNA is methylated at the 5-position of cytosine, usually in the context of CpG dinucleotides, but in some instances, such as human embryonic stem (ES) cells, non-CpG cytosines are methylated (Lister et al. 2009). This modification leads to recruitment of proteins that selectively recognize and bind 5-methylcytosine (5mC). Taking advantage of the structural identity of 5mC, various affinity purificationbased protocols have been developed to enrich for either DNA that is modified by 5mC or proteins that recognize 5mC. In this protocol, an antibody against 5mC is used to immunoprecipitate the methylated DNA. Other 5mC immunoprecipitation methods use methyl-binding proteins to analyze DNA methylation patterns (Rauch and Pfeifer 2005, 2009). Interestingly, Huang et al. (2010) have shown that the antibody that recognizes 5mC can distinguish between 5mC and 5hmC (5-hydroxymethylcytosine). Thus, we are optimistic that an antibody will be isolated that uniquely recognizes 5hmC. The method in this protocol can be scaled up to perform genome-wide DNA methylation analysis. A flowchart outlining the major steps in this protocol is shown in Figure 1. Because immunoprecipitation is a straightforward procedure that does not require any prior modification of genomic DNA, several commercial kits are now available to perform the immunoprecipitation-based detection of DNA methylation. Some of these are described in Table 1.


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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