title: Whole Genome Methylation Scanning Based on phi29 Polymerase Amplification.
abstract: Identifying differences in DNA methylation is critical to understanding how epigenetics influences gene expression during processes such as development. Here, we propose a method that employs a single, methylation-sensitive restriction endonuclease of choice, to produce discrete pools of methylated and unmethylated DNA from the same sample. A pool of restriction fragments representing unmethylated regions of the genome is first obtained by digestion with a methylation-sensitive endonuclease. The restriction-digested DNA is then concatamerized in the presence of stuffer-adaptor DNA, which prevents interference from originally unmethylated DNA by blocking the ends of the restriction fragments. The concatamerized DNA is amplified by phi29 polymerase to remove methylation marks, and again digested with the same endonuclease to produce a pool of DNA fragments representing methylated portions of the genome. The two pools of DNA fragments thus obtained can be analyzed by end-sequencing or hybridization to a genomic array. In this report we detail a proof of concept experiment that demonstrates the feasibility of our method.
authors.affiliation: Australian Research Council Centre of Excellence for Integrative Legume Research, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, New South Wales, Australia.
authors.forename: R
authors.initials: R
authors.lastname: Brooks
authors.forename: R J
authors.initials: RJ
authors.lastname: Rose
authors.forename: M B
authors.initials: MB
authors.lastname: Sheahan
authors.forename: S
authors.initials: S
authors.lastname: Kurdyukov
mesh_terms: D001435:Bacteriophages / Q000201:enzymology*; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D018745:Genome, Plant; D046913:Medicago truncatula / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes