Issue 2, 2019

Bisulfite-free, single base-resolution analysis of 5-hydroxymethylcytosine in genomic DNA by chemical-mediated mismatch

Abstract

5-Hydroxymethylcytosine (5hmC) is known as one of the vital players in nuclear reprogramming and the process of active DNA demethylation. Although the development of whole-genome sequencing methods for modified cytosine bases has burgeoned, the easily operated gene-specific loci detection of 5hmC has rarely been reported. Herein, we present a single-base resolution approach, i.e., chemical-assisted mismatch sequencing (CAM-Seq), which, when combined with traditional oxidation and chemical labeling mediation, can be used for mapping 5hmC at base resolution. We employ chemical oxidation to transform 5hmC to 5-formylcytosine (5fC), followed by chemical labeling to induce C-to-T base changes owing to the fact that the loss of the exocyclic 4-amino group of labeled 5fC leads to C to T conversion and subsequent pairing with adenosine (A) in PCR. The feasibility of CAM-Seq is demonstrated in different synthetic oligonucleotide models as well as in part of the genome of 5hmC-rich mouse embryonic stem cells (mESCs). Moreover, the gene fragment containing 5hmC can be easily biotinylated after oxidation, showing high enrichment efficiency. Our method has the potential capability to map 5hmC in genomic DNA and thus will contribute to promoting the understanding of the epigenetic modification of 5hmC.

Graphical abstract: Bisulfite-free, single base-resolution analysis of 5-hydroxymethylcytosine in genomic DNA by chemical-mediated mismatch

Supplementary files

Article information

Article type
Edge Article
Submitted
26 Sep 2018
Accepted
11 Oct 2018
First published
11 Oct 2018
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2019,10, 447-452

Bisulfite-free, single base-resolution analysis of 5-hydroxymethylcytosine in genomic DNA by chemical-mediated mismatch

Y. Wang, X. Zhang, F. Wu, Z. Chen and X. Zhou, Chem. Sci., 2019, 10, 447 DOI: 10.1039/C8SC04272A

This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. You can use material from this article in other publications, without requesting further permission from the RSC, provided that the correct acknowledgement is given and it is not used for commercial purposes.

To request permission to reproduce material from this article in a commercial publication, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party commercial publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements