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Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads

Nature Protocols volume 7, pages 340–350 (2012)Cite this article

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Abstract

We describe a method for the efficient and selective identification of DNA containing the 5-hydroxymethylcytosine (5-hmC) modification. This protocol takes advantage of two proteins: T4 β-glucosyltransferase (β-gt), which converts 5-hmC to β-glucosyl-5-hmC (β-glu-5-hmC), and J-binding protein 1 (JBP1), which specifically recognizes and binds to β-glu-5-hmC. We describe the steps necessary to purify JBP1 and modify this protein such that it can be fixed to magnetic beads. Thereafter, we detail how to use the JBP1 magnetic beads to obtain DNA that is enriched with 5-hmC. This method is likely to produce results similar to those of other 5-hmC pull-down assays; however, all necessary components for the completion of this protocol are readily available or can be easily and rapidly synthesized using basic molecular biology techniques. This protocol can be completed in less than 2 weeks and allows the user to isolate 5-hmC-containing genomic DNA that is suitable for analysis by quantitative PCR (qPCR), sequencing, microarray and other molecular biology assays.

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Figure 1: Overview of 5-hmC enrichment mediated by β-gt and JBP1.
Figure 2: Workflow for JBP1 purification and cross-linking to magnetic beads.
Figure 3: Workflow for JBP1-mediated pull-down of 5-hmC–containing DNA.
Figure 4: Results from high-throughput sequencing of JBP1-enriched DNA.

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Acknowledgements

This work was funded by the Norwegian Research Council and the Norwegian Cancer Society.

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Author notes

  1. Adam B Robertson and John Arne Dahl: These authors contributed equally to this work.

Authors and Affiliations

  1. Centre for Molecular Biology and Neuroscience, Institute of Medical Microbiology, Oslo University Hospital Rikshospitalet, Oslo, Norway

    Adam B Robertson, John Arne Dahl, Rune Ougland & Arne Klungland

  2. Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway

    Arne Klungland

Authors
  1. Adam B Robertson
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  2. John Arne Dahl
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  4. Arne Klungland
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Contributions

A.B.R., J.A.D. and A.K. initiated the project. A.B.R., J.A.D. and A.K. designed the study. A.B.R., J.A.D. and R.O. designed and conducted the experiments. A.B.R. drafted the manuscript and all the authors participated in writing and editing the manuscript.

Corresponding author

Correspondence to Arne Klungland.

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Competing interests

The authors have been given a provisional patent for the method described in this protocol.

Supplementary information

Supplementary Note 1

Test oligonucleotide sequences and qPCR primers (DOC 31 kb)

Supplementary Note 2

Sequence of pET28a-JBP1 (DOC 42 kb)

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Robertson, A., Dahl, J., Ougland, R. et al. Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads. Nat Protoc 7, 340–350 (2012). https://doi.org/10.1038/nprot.2011.443

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