Chemical Modification-Assisted Bisulfite Sequencing (CAB-Seq) for 5-Carboxylcytosine Detection in DNA

• Type of Material: Article
• Authors: Xingyu Lu, University of Chicago; Chun-Xiao Song, University of Chicago; Keith Szulwach, Emory University; Zhipeng Wang, University of Chicago; Payton Weidenbacher, University of Chicago; Peng Jin, Emory University; Chuan He, University of Chicago
• Language: English
• Date: 2013-06-26
• Publisher: American Chemical Society
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2013 American Chemical Society.
• Final Published Version (URL): http://dx.doi.org/10.1021/ja4044856
• Title of Journal or Parent Work: Journal of the American Chemical Society
• ISSN: 0002-7863
• Volume: 135
• Issue: 25
• Start Page: 9315
• End Page: 9317
• Grant/Funding Information: This work was supported by National Institutes of Health HG006827 (C.H.), NS079625, and HD073162 (P.J.).
• Keywords: TET; TDG; CELLS; Physical Sciences; REVEALS; 5-FORMYLCYTOSINE; Chemistry; CHEMISTRY; 5-METHYLCYTOSINE; Science & Technology; Chemistry, Multidisciplinary; GLYCOSYLASE; THYMINE DNA; 5-HYDROXYMETHYLCYTOSINE
• Research Categories: Biology, Genetics; Chemistry, Biochemistry

Abstract

5-Methylcytosine (5mC) in DNA can be oxidized stepwise to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the TET family proteins. Thymine DNA glycosylase can further remove 5fC and 5caC, connecting 5mC oxidation with active DNA demethylation. Here, we present a chemical modification-assisted bisulfite sequencing (CAB-Seq) that can detect 5caC with single-base resolution in DNA. We optimized 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)-catalyzed amide bond formation between the carboxyl group of 5caC and a primary amine group. We found that the modified 5caC can survive the bisulfite treatment without deamination. Therefore, this chemical labeling coupled with bisulfite treatment provides a base-resolution detection and sequencing method for 5caC.