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Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
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The HELP-Based DNA Methylation Assays.

John M Greally1

  • 1Department of Genetics, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Ave., Bronx, NY, 10461, USA. John.greally@einstein.yu.edu.

Methods in Molecular Biology (Clifton, N.J.)
|December 11, 2017
PubMed
Summary
This summary is machine-generated.

DNA methylation survey assays, like the HELP-tagging assay, offer affordable genome-wide analysis. This chapter details the latest HELP-tagging method and its extension for 5-hydroxymethylation quantification.

Keywords:
CpG dinucleotideCytosine methylationEpigeneticEpigenomeMassively parallel sequencing

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Area of Science:

  • Genomics
  • Epigenetics
  • Molecular Biology

Background:

  • Restriction enzymes are crucial for DNA methylation assays, enabling genome representation through methylation-dependent or insensitive enzyme approaches.
  • Genome-wide DNA methylation survey assays remain vital due to their cost-effectiveness compared to comprehensive sequencing methods.
  • Existing methods include reduced representation bisulphite sequencing (RRBS), Illumina microarrays (HumanMethylation450K, EPIC), and HELP-based assays.

Purpose of the Study:

  • To describe the latest iteration of the HELP-tagging assay, incorporating Illumina Tru-Seq adapters.
  • To introduce the extension of the HELP-tagging assay for quantifying 5-hydroxymethylation (HELP-GT assay).

Main Methods:

  • Utilizing restriction enzymes for genome representation in DNA methylation analysis.
  • Employing the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay methodology.
  • Transitioning from microarray-based reporting to massively parallel sequencing for HELP assays.
  • Implementing Illumina Tru-Seq adapters in the latest HELP-tagging assay.

Main Results:

  • The latest HELP-tagging assay is detailed, leveraging Illumina Tru-Seq adapters for enhanced DNA methylation analysis.
  • The development of the HELP-GT assay is highlighted, enabling the quantification of 5-hydroxymethylation.
  • Massively parallel sequencing has been adopted for HELP assays, improving upon previous microarray-based reporting.

Conclusions:

  • The HELP-tagging assay represents an advancement in affordable, genome-wide DNA methylation analysis.
  • The HELP-GT assay expands the utility of HELP-based methods to include 5-hydroxymethylation.
  • These evolving HELP assays provide valuable tools for epigenetic research, bridging the gap between cost and comprehensiveness.