Learn about the importance of determining chromatin accesibility for structure analysis, the shortcomings of traditional methods and our solution to help overcome these limitations.
Chromatin structure is a key determinant of gene regulation, influencing processes like transcription, replication, and DNA repair by affecting access of transcription factors (TFs), DNA methyltransferases (DNMTs), and other regulatory elements to DNA. In general, more condensed chromatin regions (heterochromatin) limit accessibility of DNA-binding proteins such as TFs and DNMTs, impeding their physiologic performance, whereas more open chromatin states (euchromatin) are considered transcriptionally active. Determining chromatin accessibility can provide insight into the biological roles that protein-DNA interactions play and the corresponding effects of chromatin state on these interactions.
The traditional method for determining chromatin accessibility involves nuclease (DNase I; MNase)-mediated digestion of chromatin DNA followed by qPCR, microarray, or sequencing analysis. However, DNA damage, the need for high input amounts, and challenges in optimizing enzyme parameters (e.g., concentration, digestion time) present several drawbacks.
As part of chromatin structure analysis, the figure above illustrates the methodology for determining chromatin accessibility.
To address these issues, EpiGentek developed the EpiQuik™ Chromatin Accessibility Assay Kit for rapid, convenient, and cost-effective assessment of chromatin accessibility. This complete set of optimized reagents allows for quantitative chromatin analysis and determination of heterochromatic or euchromatic states via a simple real-time PCR-based assay. During the protocol, chromatin from mammalian cell/tissue samples is first extracted and then digested with a nuclease mix. The DNA is subsequently purified, and the chromatin state of user-defined gene targets can be examined downstream by qPCR. Two sets of internal control primers included in the kit, each targeting a specified gene region in either euchromatin (accessible to and digested by the nuclease mix) or heterochromatin (inaccessible and undigested), can be used to ascertain digestion efficiency. qPCR will show either a large or an insignificant Ct shift between digested and undigested samples for euchromatin or heterochromatin, respectively.
The entire procedure, from starting sample to ready-to-use DNA for PCR, can be completed in as little as 1 hour and 30 minutes, minimizing nuclear damage and preserving chromatin structure. Additionally, low sample input amount (1×105 - 2×106 of cells or 2 - 40 mg of tissue per reaction) and single-/multi-reaction flexibility make the EpiQuik™ Chromatin Accessibility Assay Kit the ideal tool for your chromatin analysis investigations.
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