The Purification of Nucleic Acids Flashcards
Where is DNA found in eukaryotes and prokaryotes?
In eukaryotes it is found tightly packed in the nucleus.
In prokaryotes it is found in the cytoplasm with all the cell components.
What are the two facts about DNA that should be considered when purifying it.
The size of DNA - it is very large.
The subcellular location of the DNA.
Where in the cytoplasm can DNA be found? and what type of DNA is located here?
Mitochondria - mitochondrial DNA
Ribosomes: polysomes - mRNA
60S subunit - 28S rRNA
40S subunit - 18S rRNA
When centrifuging to cause cell fractionation what is the rule for the centrifugal force?
The smaller the sub cellular component the greater the centrifugal force is needed.
Repeated centrifugataion at progressively higher speeds will fractionate homogenates of cells into their components.
Give examples of low/medium/high and very high speeds of centrifuging?
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 - 15,000 times gravity for 3 hours.
How does sedimentation centrifuging separate subcellular components?
Subcellular components sediment at different speeds according to their size and shape when layered over a dilute solution containing sucrose (stabilizing sucrose gradient). The concentration of sucrose increases towards the bottom of the tube.
Describe equilibrium sedimentation.
Subcellular components move up or down when centrifuged in a gradient until they reach a position where their density matches their surroundings. The gradient can be sucrose or cesium chloride (gives a denser gradient - for large proteins).
What does the negative phosphate in nucleic acids cause it to be called?
Polyanions and not soluble in organic solvents (or denatured by them).
How is the negative charge of nucleic acids neutralised?
By association with cations or histones.
What does anion exchange technology rely on?
Specific interactions between negatively charged phosphates on the nucleic acid background and positively charged molecules on the surface of beads.
How does anion exchange technology work?
DNA binds to the molecules on the beads in the presence of low salt concentration. Contaminants can then be removed by a low/medium salt buffer washed. The purified DNA can be eluted using a high salt buffer.
Why should excessive vortexing and pipetting be avoided during DNA extraction?
Genomic DNA consists of very large DNA molecules which are fragile and can break easily.
Why should DNA be stored in TE buffer?
DNA is subject to acid hydrolysis when stored in water.
Why must care be taken when exploiting the susceptibility of RNA/DNA to bases?
Would disrupt hydrogen bonds between strands, causing strand denaturation.
