eoe sub articles

EoE Sub Articles

1. Continuous Dilution Assembly of Mono-nucleosomes
<ol>
	<li>Generate and purify an approximately 400 bp DNA substrate that contains an off-centered Widom 601 nucleosome positioning sequence.&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: To limit the unwanted formation of di-nucleosomes, each &#8216;arm&#39; flanking the positioning sequence should not exceed ~150 bp.
	<ol>
		<li>Use plasmid pGEM3Z-601 along with the designed primers and amplify the substrate DNA using PCR. For the...

high-speed time-lapse atomic force microscopy to capture nucleosome dynamics

Source: Stumme-Diers, M. P., et al., <a href="https://app.jove.com/v/58820">Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging.</a>&#160;J. Vis. Exp.&#160;(2019)
In this video, we demonstrate high-speed time-lapse atomic force microscopy, AFM, imaging techniques to study the dynamics of nucleosome complexes. As the AFM tip scans across the surface of the nucleosome, it detects alterations in the nucleosome height and records the DNA unwrapping over time.

<img alt="Figure 1" src="/files/ftp_upload/21461/21461_Figure1.jpg" /> 
Figure 1:&#160;Schematic of the syringe pump used for microscale nucleosome assembly.&#160;The assembly mixture is positioned to be in contact with the end of the syringe needle. As the dilution buffer is delivered by the syringe pump to the assembly mixture the concentration of NaCl is decreased, promoting nucleosome assembly. This figure is adapted from Stumme-Diers&#160;et al.
<img alt="Fi...

High-Speed Time-Lapse Atomic Force Microscopy to Capture Nucleosome Dynamics

Source: Stumme-Diers, M. P., et al., Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging.&#160;J. Vis. ...

Nucleosomes &#8212; eukaryotic DNA repeating units &#8212; consist of DNA segments wrapped around histone protein cores. DNA unwrapping around the histone core is crucial for nucleosome dynamics.
To capture nucleosome dynamics, begin with a smooth, functionalized mica substrate mounted to an atomic force microscope, AFM, scanner stage. Overlay the substrate with a nucleosome-containing buffer.
The negatively charged nucleosome DNA interacts with positive charges on the functionalized mica, immobilizing them on the surface. Wash the immobilized mica substrate with a buffer to prevent the nucleosomes overcrowding.
Transfer the nucleosome-containing scanner stage to an imaging chamber containing a pre-mounted cantilever, with a tip at the edge and facing up toward the nucleosomes.
Fill the chamber with an imaging buffer. Align the laser beam onto the cantilever back to ensure accurate deflection measurements.
The AFM tip oscillates close to the fully wrapped nucleosomes and scans over the surface to measure the nucleosomes&#39; height at regular intervals.
Spontaneous DNA unwrapping increases nucleosome height, alternating the tip&#39;s oscillations and leading to cantilever deflection. This cantilever deflection shifts the reflected laser beam position, which is detected by a position-sensitive detector and produces topographic nucleosome images.
In AFM images, the histone core appears as a glowing blob with unwrapped DNA of increasing heights over time, indicative of nucleosome dynamics.

high-speed-time-lapse-atomic-force-microscopy-to-capture-nucleosome

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Microscopy Techniques

In this video, we demonstrate high-speed time-lapse atomic force microscopy, AFM, imaging techniques to study the dynamics of nucleosome complexes. As the AFM tip scans across the surface of the nucleosome, it detects alterations in the nucleosome height and records the DNA unwrapping over time.

Overview

Protocol

<img title="figure-representative results-25" alt="figure-representative results-25" src="/files/ftp_upload/21461/21461_Figure1.jpg" /> 
Figure 1:&#160;Schematic of the syringe pump used for microscale nucleosome assembly.&#160;The assembly mixture is positioned to be in contact with the end of the syringe needle. As the dilution buffer is delivered by the syringe pump to the assembly mixture the concentration of NaCl is decreased, promoting nucleosome assembly. This figure is adapted from Stumme-Diers&#160;et al.
<img alt="Fi...

High-Speed Time-Lapse Atomic Force Microscopy to Capture Nucleosome Dynamics

In This Article

Overview

Protocol

Representative Results