Centrifugation Techniques for Virus Isolation

Extraction of Nucleic Acids

• Phenol/chloroform used to remove proteins and hydrophobic components from the aqueous phase.
• Alcohol precipitation involves using 2.5 volumes of ethanol or 1 volume of isopropanol, along with high salt concentrations (e.g., 0.3 M sodium acetate) for DNA isolation.
• Desalting process employs 70% ethanol to eliminate low molecular weight hydrophilic contaminants.

Chromosomal DNA Isolation

• Chromosomal DNA can form large, viscous aggregates that are visible and can be spooled using a glass rod.
• Typically consists of very long strands, can fragment into 50-100 kb sizes, and is prone to shearing.
• To prevent shearing: Avoid small pipette tips and vigorous agitation.
• Gene diagnostics tools (e.g., PCR) remain effective with high-molecular-weight DNA despite shearing,

Circular DNA/Plasmids

• Plasmids exhibit diverse structures and conformations and are analyzed through DNA binding dyes like ethidium bromide (EtBr).
• EtBr intercalates into double-stranded DNA, affecting buoyant density based on DNA conformation.
• Plasmid purification can be achieved through isopycnic centrifugation in an EtBr-CsCl gradient, segregating particles based on density.

Cesium Chloride (CsCl) Density Gradient

• CsCl creates a linear density gradient during ultracentrifugation, enhancing separation efficiency.
• Macromolecules migrate to their equilibrium positions under sufficient ultracentrifugation forces without pre-forming the gradient.

Isolation of Viral DNA

• Virus particles are concentrated via centrifugation before proteolytic digestion or chemical breakdown.
• Employs multiple steps of sequential centrifugation at increasing g-forces to separate heterogeneous particles.
• Isopycnic and rate zonal centrifugation methods allow for further separation based on density, size, and shape of viral particles.

DNA Extraction Kits

• DNA extraction kits streamline nucleic acid isolation through standardized protocols.
• Nucleic acids are released using proteolytic enzymes and purified via affinity chromatography columns that preferentially bind negatively charged nucleic acids depending on salt and alcohol concentrations.
• RNA purification can occur simultaneously, and co-purified RNA can be subsequently digested with RNase.

RNA Isolation

• RNA isolation follows similar protocols to DNA extraction but requires specific precautions due to the high prevalence of RNases in cells, which can degrade RNA.

This quiz covers the methods of centrifugation, particularly ultracentrifugation, for isolating and purifying viral DNA and nucleic acids. It focuses on the use of cesium chloride and the enrichment of virus particles through sequential centrifugation processes. Test your knowledge on these essential laboratory techniques!