DNA modifications are crucial biological processes that involve the chemical modification of bases or backbones in a DNA molecule to regulate its structure and function. These modifications play key roles in various biological processes such as maintaining normal cellular function, genomic imprinting, embryonic development, and tumorigenesis. While these modifications do not disrupt Watson-Crick pairing, they do influence DNA-protein interactions in the major groove of the double helix. Among them, DNA methylation is the predominant DNA modification, which is also a key focus of research in the field of epigenetics.
DNA methylation is the process of introducing a methyl group to the 5th carbon of cytosine in CpG dinucleotides, forming 5-methylcytosine (5mC). This modification is prevalent in eukaryotes like plants and animals, serving as the only form of DNA methylation in mammals. DNA methyltransferases (Dnmts) are essential for establishing and maintaining DNA methylation patterns in the genome. Dnmts are classified into two groups based on their structure and function. Dnmt1 is responsible for maintaining methylation states and facilitating de novo DNA methylation at non-CpG sites, while Dnmt3 enzymes (including Dnmt3a, Dnmt3b, and Dnmt3L) are primarily involved in de novo DNA methylation processes.
Fig.1 Mechanisms of DNA cytosine methylation in plants.1,3
DNA demethylation plays an important role in facilitating gene expression and reprogramming within cells. This process primarily occurs through the following two ways.
In dividing cells, Dnmt1 inhibition or dysfunction prevents newly added cytosines from being methylated, thus lowering the overall methylation level of the genome.
The ten-eleven translocation (TET) enzymes catalyze the conversion of 5mC to derivatives such as 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC). The derivatives are further excised and restored to unmodified cytosine by thymine DNA glycosylase (TDG) and base excision repair (BER), resulting in active DNA demethylation.
Bisulfite sequencing is a commonly utilized chemical method for detecting 5mC. In this reaction, sodium bisulfite deaminates cytosine bases into uracil, which can be distinguished from 5mC. The main approaches include site-specific bisulfite sequencing (BS), whole genome bisulfite sequencing (WGBS), and methylation capture sequencing (MC-seq).
Genomic DNA can be processed with methylation-sensitive restriction endonucleases, which distinguish sequences based on methylation status, e.g. Hpa II/Msp I (C↓CGG) and Sma I/Xma I (CCC↓CGG).
DIP analysis of DNA using a 5mC antibody enables efficient mapping locations of DNA methylation markers. This method is cost-effective, readily accessible, and easy to analyze compared to other techniques. DIP has proven effective in identifying different DNA modifications such as 5mC, 5hmC, 5fC, and 5caC. The success of DIP largely depends on the availability of antibodies that can identify the specific DNA modifications. As a prominent supplier of antibodies worldwide, Amerigo Scientific provides high-quality antibody products tailored for DNA methylation detection to support the advancement of your research endeavors.
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