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Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Chemical modifications of DNA bases have a major effect on the execution of the DNA code. The global amount of DNA modifications provides valuable information regarding various biological processes as well as for disease development. Therefore, development of simple and reliable methods to quantify these markers is of great importance. Here we describe in detail protocols for global quantification of DNA modifications. Specifically, we describe quantification of two types of epigenetic modifications, unmethylated CpGs and 5-hydroxymethylcytosine (5-hmC), and two types of DNA damage lesions, oxidation and UV-induced damage. All methods are based on utilizing enzymatic recognition for covalent binding of a fluorescent dye to the DNA modification. Up to 90 labeled DNA samples are then loaded on a custom multi-well slide, which is imaged by a conventional slide scanner. The global amount of the measured modification can be calculated by the obtained fluorescence intensity.

Reactive oxygen species (ROS) can induce DNA damages in cells. 8-Oxo-7,8-dihydroguanine (OG) is viewed as one of the most frequent oxidative modifications in human genomes. It was reported that OG was also capable to facilitate the G-quadruplex formation and participate in the transcription process. Thus, OG might have potential functions in regulating gene expression. To investigate the molecular mechanisms of OG on affecting gene expression in vivo, it is necessary to determine the location of OG in DNA beforehand. Herein, we characterized Bsu DNA polymerase (Bsu Pol) and Tth DNA polymerase (Tth Pol), which can faithfully incorporate dATP or error-prone incorporate dCTP when bypassing OG, respectively. Based on the different coding performance, we achieved single-base resolution analysis of OG in DNA, which offers a promising approach for high-throughput analysis of OG at genome-wide scale.

Abstract

Abstract

Abstract

Abstract

5-Formyluracil (5fU) and 5-formylcytosine (5fC) which are widely present in human genomic DNAs play significant roles in epigenetic functions and have attracted widespread attention in many related fields. Therefore, creating highly effective, selective, and easy-operating detection methods for these important natural existing DNA modifications is important not only to understand the fundamentals of physiological regulation, but also serve as the basis for the next generation of therapeutics used to improve human’s health. Within last decades, various methods have been developed to qualitatively and quantitatively detect these modifications. We describe in detail the protocols of fluorescence labeling methods for detection of 5fU and 5fC in DNA. The highly selective fluorescence “switch-on” specificity towards 5fU or 5fC separately enabled a high signal-to-noise ratio in qualitatively and quantitatively detecting 5fU or 5fC and it is not affected by the presence of other DNA modifications which also bear formyl groups. These protocols offer solutions to problems related to fast, convenient, cost-efficient, and easy-operating detection of 5fU or 5fC in complex samples.

Abstract

Abstract

5-Formylcytosine (5fC) is a recently identified DNA modification in mammalian genome, derived from 5-methylcytosine (5mC) by TET-mediated oxidation. It has been demonstrated to be a critical intermediate during active demethylation and may also be a stable epigenetic marker playing independent biological roles. To further understanding its complicated epigenetic functions, quantitative analysis methods with high selectivity and sensitivity for 5fC are urgently required. In this respect, how to distinguish 5fC from 5-formyluracil (5fU) to achieve higher accuracy remains challenging because the latter one is more reactive. We take advantage of the slight difference in structure between 5fC and 5fU and introduce a Wittig-initiated photocatalytic triple domino reaction to selectively switch on 5fC using a commercially available phosphorus ylide. Such a fluorescence-based detection approach is easy to operate and short time-consuming, but highly selective, enabling accurately qualitative and quantitative analysis of 5fC.