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   Home  »  Epigenetic Resources  »  Latest Innovations in Studying DNA-Protein Interactions 
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Latest Innovations in Studying DNA-Protein Interactions


Interactions between DNA and proteins are of great biological significance as they govern numerous physiological processes, including DNA replication and recombination, gene transcription, chromosome segregation, signal transduction, and epigenetic silencing.  For studying DNA-protein interactions, the analytical method of choice should ideally ensure that:

  • True target protein-enriched regions are reliably identified, particularly from limited cell samples.
  • Enriched DNA contains minimal background.
  • Mapping of DNA-protein binding regions with minimal bias and at high resolution.
chip kits

Chromatin Immunoprecipitation

Chromatin immunoprecipitation, or ChIP, is one of the most popular methods employed to study protein-DNA interactions.  ChIP can be used to identify the specific genomic DNA region that binds a protein of interest, such as a modified histone or a transcription factor, in living cells.

A typical ChIP protocol involves the following sequential steps: in vivo cross-linking of cells; chromatin extraction; DNA shearing, typically by sonication; protein-DNA immunoprecipitation with an antibody against a protein of interest; cross-link reversal; and DNA purification.  The enriched DNA fragments can then be analyzed downstream by techniques like PCR or NGS.  The main limitations of this method are that it requires a large amount of input material, cells, or tissue to produce a strong enough signal over background noise, as well as the use of cross-linking during an initial fixation step.  Additionally, the majority of ChIP methods are considerably time-consuming, are labor-intensive, or have low throughput. 

ChIP Advancements

More advanced methods like ChIP-exo and ChIPmentation allow for reduced cell numbers and increased resolution.  ChIP-exo provides high-resolution mapping, but it is time-consuming and necessitates an ample supply of input cells.  ChIPmentation uses transposase and sequencing-compatible adaptors to enable the integration of ligation during the ChIP process, but it follows a traditionally slow (2-day) ChIP procedure and cannot achieve high-resolution mapping.

You May Also Want to Read:

  • Analysis Shows Aspirin May Inhibit Lung Cancer Via TAZ/PD-L1 Axis
  • Enhancing Resolution and Reducing Noise: How CUT&RUN and CUT&Tag Are Changing the Game
  • STAT5 and BATF Promote Chromatin Accessibility in Helper T Cells

Cleavage Under Target & Release Using Nuclease (CUT&RUN) and Cleavage Under Target & Tagmenation (CUT&TAG) were recently developed for mapping protein-DNA interaction with limited biological materials and have significantly improved mapping resolution.  However, they are both offered at a high cost.  CUT&RUN needs expensive PA/Mnase fusion protein that has significant A/T sequence bias, causing the target protein-interacted DNA region profiles to be seriously affected by the level of MNase digestion.  CUT&TAG displays the same digestion bias and is less specific due to off-target accessibility of chromatin caused by the Tn5 transposase enzyme.

What’s (Epi)Next?

To address these issues, EpigenTek has developed two new techniques, EpiNext CUT&RUN Fast and EpiNext CUT&Tag In-Place Sequencing, for enriching protein-bound DNA and mapping genome-wide protein-DNA interactions.  These innovative approaches combine the advantages of ChIP-exo, ChiPmentation, and CUT&RUN with the fastest procedures in convenient and affordable all-in-one kits for reliably identifying true target protein-enriched regions and achieving high-resolution mapping.  Both techniques utilize a unique nucleic acid cleavage enzyme mix, which has low sequence bias, to simultaneously fragment chromatin and cleave/remove unbound DNA sequences in both ends of the target protein/DNA complex in situ without affecting DNA occupied by the target protein, thereby minimizing immunocapture/sequencing background.  With these techniques, library DNA generation can be achieved in just a few hours, starting from as little as 500 of cells or 50 ng of chromatin.
These kits have the following advantages and features:

  • Fast and streamlined procedure, taking only 3 hours to complete for nucleic acid enrichment and only 5-6 hours for enrichment plus library prep.
  • No sonication, which eliminates the optimization and reproducibility issues associated with this step.
  • No fixation step for the ChIP-related kits, which can otherwise cause issues with epitope recognition by the antibody.
  • Fragmentation and immunocapture are both processed in the same single tube, which minimizes sample loss and allows for a lower input amount.
  • Cleavage and removal of unbound nucleic acid occur in situ, minimizing the number of steps in the procedure, and the nuclease mix used exhibits low sequence bias.
  • Nucleic acid is cut very close to the antibody binding site.  This allows for enrichment of shorter nucleic acid fragments, resulting in lower background levels and higher resolution mapping of target protein-DNA interaction regions.

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Suggested Reads:

Brain Tumor Study Investigates TRDMT1's Influence on DNA Methylation in Glioblastoma
Exploring DNA Methylation with Bisulfite Sequencing
Study Reveals Histone H3K9me2 & H3K79me3 as Potential Guardians Against Cancer
Comparison of Methods for Quantification of Global DNA Methylation
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