Whole genome methylation scanning based on phi29 polymerase amplification |
| |
Authors: | R Brooks R J Rose M B Sheahan S Kurdyukov |
| |
Institution: | 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. |
| |
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. |
| |
Keywords: | |
本文献已被 PubMed SpringerLink 等数据库收录! |
|