Paper - Review

DOI: 10.1038/nprot.2012.012

Abstraction

DNA methylation
← epigenetic marker (← that has a (crucial role) in many biological processes)

The functional consequences (← of DNA methylation)
→ a genome-wide analysis

⭐ MeDIP-seq: Methylated DNA immunoprecipitation-sequencing
→ balances (accuracy & genome coverage & resolution & cost)
→ combining MeDIP & (massively parallel DNA sequencing)
→ Good specificity (> 0.97) & enrichment (> 100-fold)

Introduction

DNA methylation
→ (post-replicative maintenance) & (de novo addition of methyl group) to DNA

✒ Mammalian
→ DNA methylation occurs is dichotomous: CG & non-CG
→ mCG: hemi-methylation ← on a single strand
→ mCG is temporary state (← between cell division & enzymatic action (DNMT1))

mC (← ∋ mCHG & mCHH)
→ mC methylation is asymmetrical
→ is only present ∃! in significant quantities (← in stem cell)
→ affect accessibility (← of certain transcription factor-binding sites)
∴ determine the activity (← of stem cell-specific transcriptional networks)

hmC
→ is much less ↓ abundant
→ is generated (← by the TET family)
→ part of mechanism (← which earmarks certain regions)

CpG islands
← DNA methylation at CG-rich sites
→ are found in gene promoter regions
→ is associated (← with transcriptional repression)

Protocol development

MeDIP
→ is targeting (the vast majority of the methylome)
→ can detect (methylated cytosines) (← in both mC & mCG contexts)
∵ Antibodies (← used for MeDIP) have equal specificity (← against mC & mCG)

MeDIP-seq
← combining MeDIP (← with NGS)
→ provides high-quality methylomes (→ at 100- to 300- bp resolution)
∴ MeDIP-seq was used (→ to generate the 1st methylome of mammalian genome)

Comparisons with other methods

Current sequencing-based methods (← for methylome analysis) can be classified into two groups: 1) bisulfite conversion-based methods 2) enrichment-based methods

✒ Bisulfite conversion-based methods
← include (whole-genome bisulfite sequencing) & (RRBS: reduced representation bisulfite sequencing)
→ are still considered to be (the gold standard) (← of DNA methylations analysis)
→ permit quantification (← at single-base resolution)

✒ Enrichment-based technology
→ MBD-seq (← which used the methyl-binding proteins MBD2 & MBD3L1)
→ uses the methyl-binding domain of MECP2

Limitations of the approach

Limitation of MeDIP-seq
→ genomic resolution & methylated DNA recovery

✒ Genomic resolution
→ MeDIP-seq provides a 150- to 200- bp resolution
→ concomitant (← with sequence inset size)
→ single-base pair resolution is inherently desire

✒ Methylated DNA recovery
→ is affected by mC/mCG density
→ is only for methylated portion of the genome

Application of the method

Optimized MeDIP-seq protocol
→ has the advantage of requiring low-input DNA
∴ MeDIP-seq suitable for studies (← involving minute clinical samples)

The protocol is optimized down to 50 ng of genomic input DNA

⭐ MeDIP-seq showing excellent levels of (specificity & enrichment) achieved from 50 ng DNA concentration
Enrichment is insufficient (← for cost-effective MeDIP-seq) without❌ further optimization

High-quality MeDIP-seq data

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