Lecture 6: Purifying Nucleic Acids Flashcards
What are the two main characteristics of DNA which allow it to be purified from the rest of the cellular contents?
It’s very large size and it’s subcellular localisation in the nucleus.
What does a ribosome consist of?
60S subunit - 28S rRNA
40S subunit - 18S rRNA
Polysome - mRNA
Describe cell fractionation by centrifugation.
Repeated centrifugation at progressively higher speeds will fractionate homogenates of cells into their components. In general, the smaller the subcellular component, the greater is the centrifugal force required to sediment it.
At each speed in the centrifugation, at how many times gravity and for how long is the supernatant spun?
- low speed = 1000 times gravity for 10 minutes
- medium speed = 20,000 times gravity for 20 minutes
- high speed = 80,000 times gravity for 1 hour
- very high speed = 150,000 times gravity for 3 hours
What order do the subcellular contents separate?
- Low-speed centrifugation: pellet contains whole cells, nuclei, cytoskeletons
- Supernatant subjected to medium-speed centrifugation: new pellet contains mitochondria, lysosomes and peroxisomes.
- Supernatant subjected to high-speed centrifugation: new pellet contains microsomes and small vesicles
- Supernatant subjected to very-high-speed centrifugation: new pellet contains ribosomes, viruses, large macromolecules
Describe velocity sedimentation.
In velocity sedimentation, subcellular components sediment at different speeds according to their size and shape when layered over a dilute solution containing sucrose.
To stabilize the sedimenting bands against convective mixing caused by small differences in temperature or solute concentration, the tube contains a continuous shallow gradient of sucrose that increases in concentration towards the bottom of the tube (typically from 5% to 20% sucrose).
Following centrifugation, the different components can be collected individually most simply by puncturing the tube and collecting drops from the bottom, as illustrated here.
Describe equilibrium sedimentation.
In equilibrium sedimentation, subcellular components move up or down when centrifuged in a gradient until they reach a position where their density matches their surroundings.
Although a sucrose gradient is shown here, denser gradients, which are especially useful for protein and nucleic acid separation, can be formed from cesium chloride.
Following centrifugation, the different components can be collected individually most simply by puncturing the tube and collecting drops from the bottom, as illustrated here.
What charge are histones?
Positively charged. They can interact with the negative charges of the phosphate backbone of DNA.
Why are nucleic acids not denatured by organic solvents?
Nucleic acids are polyanionic, which means they are insoluble in organic solvents and so are not denatured by them.
Under which conditions are RNA backbones hydrolysed?
Alkaline conditions, due to the 2’ OH group on ribose being vulnerable to base catalysis.
Why are DNA strands denatured at high temperatures?
The hydrogen bonds holding the DNA strands together are insufficient at high temperatures and the strands separate.
What are nucleases?
Enzymes which degrade nucleic acids by hydrolysing phosphodiester bonds.
Deoxyribonucleases specifically catalyse the degradation of DNA
Ribonucleases specifically catalyse the degradation of RNA
These specific differences are exploited in separating DNA from RNA - c.f. Avery, McCarty and McLeod
How do hydrodynamic forces cause shearing of DNA?
Hydrodynamic forces cause different parts of the DNA molecule to move in different directions, pulling the molecule into two smaller pieces.
When purifying nucleic acids, what should the cell be treated with?
Reagents which denature the other cell components, such as proteins (using proteinase or chaotropic agents), which do not denature RNA or DNA:
- strong detergent, e.g. SDS
- minimal agitation
- chelating agents, e.g. EDTA (to complex divalent cations such as Mg2+ which nucleases require for activity)
- remove contaminating proteins by organic extraction, e.g. phenol or phenol/chloroform (phenol is a nasty chemical)
Describe the Qiagen hispeed plasmid isolation procedure, an example of anion exchange chromotography.
- Bacteria are grown, harvested and resuspended in buffer.
- Then subject to high salt, strong detergent and strong alkali. This denatures and precipitates the bulk of the macromolecules such as proteins, cell wall, genomic DNA.
- Bind the lysate to anion exchange resin in the hispeed tip, elute with salt and precipitate with isopropanol, then catch the precipitate on a filter, dry and elute with a small volume of TE.
