title: A novel methyl-binding domain protein enrichment method for identifying genome-wide tissue-specific DNA methylation from nanogram DNA samples.
abstract: BACKGROUND
  Growing evidence suggests that DNA methylation plays a role in tissue-specific differentiation. Current approaches to methylome analysis using enrichment with the methyl-binding domain protein (MBD) are restricted to large (≥1 μg) DNA samples, limiting the analysis of small tissue samples. Here we present a technique that enables characterization of genome-wide tissue-specific methylation patterns from nanogram quantities of DNA.
  
  
  RESULTS
  We have developed a methodology utilizing MBD2b/MBD3L1 enrichment for methylated DNA, kinase pre-treated ligation-mediated PCR amplification (MeKL) and hybridization to the comprehensive high-throughput array for relative methylation (CHARM) customized tiling arrays, which we termed MeKL-chip. Kinase modification in combination with the addition of PEG has increased ligation-mediated PCR amplification over 20-fold, enabling >400-fold amplification of starting DNA. We have shown that MeKL-chip can be applied to as little as 20 ng of DNA, enabling comprehensive analysis of small DNA samples. Applying MeKL-chip to the mouse retina (a limited tissue source) and brain, 2,498 tissue-specific differentially methylated regions (T-DMRs) were characterized. The top five T-DMRs (Rgs20, Hes2, Nfic, Cckbr and Six3os1) were validated by pyrosequencing.
  
  
  CONCLUSIONS
  MeKL-chip enables genome-wide methylation analysis of nanogram quantities of DNA with a wide range of observed-to-expected CpG ratios due to the binding properties of the MBD2b/MBD3L1 protein complex. This methodology enabled the first analysis of genome-wide methylation in the mouse retina, characterizing novel T-DMRs.
authors.affiliation: Department of Ophthalmology, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287, USA. smerbs@jhmi.edu.
authors.forename: Verity F
authors.initials: VF
authors.lastname: Oliver
authors.forename: Jun
authors.initials: J
authors.lastname: Wan
authors.forename: Saurabh
authors.initials: S
authors.lastname: Agarwal
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Zack
authors.forename: Jiang
authors.initials: J
authors.lastname: Qian
authors.forename: Shannath L
authors.initials: SL
authors.lastname: Merbs