Chemical modification-assisted bisulfite sequencing (CAB-Seq) for 5-carboxylcytosine detection in DNA - PubMed (original) (raw)

. 2013 Jun 26;135(25):9315-7.

doi: 10.1021/ja4044856. Epub 2013 Jun 17.

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Chemical modification-assisted bisulfite sequencing (CAB-Seq) for 5-carboxylcytosine detection in DNA

Xingyu Lu et al. J Am Chem Soc. 2013.

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.

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Figures

Figure 1

Figure 1

Sanger sequencing results of unlabeled 76 mer 5caC (upper panel) and EDC-labeled 5caC (bottom panel) after bisulfite treatment. The original sequence and the sequence after bisulfite were shown above the corresponding peak. In the bottom panel, the labeled DNA was not further purified before bisulfite treatment. The small background peak of T comes from unlabeled cytosine, which reflects the labeling efficiency.

Scheme 1

Scheme 1. EDC-catalyzed chemical labeling of 5caC.a

_a_Designed labeling strategy for 5caC. The carboxyl group of 5caC in duplex DNA can be labeled by EDC-catalyzed coupling reaction to form biotin-S-S-5caC using biotin-S-S-amine.

Scheme 2

Scheme 2. Proposed deamination mechanism of 5caC under bisulfite treatment

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