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Chemical Modification-Assisted Bisulfite Sequencing (CAB-Seq) for 5-Carboxylcytosine Detection in DNA
- Lu, Xingyu, Song, Chun-Xiao, Szulwach, Keith, Wang, Zhipeng, Weidenbacher, Payton, Jin, Peng, He, Chuan
- Journal of the American Chemical Society 2013 v.135 no.25 pp. 9315-9317
- DNA, deamination, oxidation, proteins, thymine-DNA glycosylase
- 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.