DNA analysis Flashcards
What charge does DNA have?
Negative.
Describe the steps in electrophoresis.
- The DNA preparation is run on a gel
- An electric current is applied across the gel and the DNA migrates to the positive electrode (anode).
- The gel is then visualised.
What are the 2 types of gel used in electrophoresis?
Agarose and acrylamide.
What is agarose?
A polysaccharide from seaweed.
Which type of gel gives a high resolution?
Acrylamide.
What is acrylamide?
A cross-linked polymer that is highly neurotoxic.
With agarose there is a need to estimate the sizes of the fragments as it is such low resolution. Describe how you would do this, give 3 steps.
- Run a marker collection, e.g. an RE digest where the fragments are known sizes, on a gel.
- Plot this digest on a graph: the y-axis is log size and the x-axis is distance travelled from the well.
- Use the standard curve to the size of unknown fragments based on how far they have travelled.
What chemical is essential for visualising an electrophoresis gel?
Ethidium bromide.
What is ethidium bromide (EB)?
A toxic dye.
How does EB work?
It intercalates with the DNA molecules, displaying increased fluorescence when intercalated than when free.
What wavelength of UV radiation is absorbed by the DNA and transmitted to EB?
254nm.
What wavelength of UV is re-emitted by EB?
590nm.
What spectrum is the wavelength of UV re-emitted by EB in?
Red-orange.
How is the gel visualised after exposure to UV?
With a transilluminator and polaroid film.
There are 3 states for the DNA in a plasmid. What are they?
- Supercoiled, the natural state.
- Relaxed, when the DNA has been nicked.
- Linear.
Which DNA state travels the furthest in electrophoresis?
Supercoiled.
Which DNA state travels the least in electrophoresis?
Linear.
What is Southern Blotting?
The hybridisation of a DNA probe to a DNA sample.
Describe the 6 major steps in Southern Blotting.
- Run a RE digest on a gel.
- Denature the DNA with NaCl
- De-purinate the DNA with HCl (optional)
- ‘Blot’ the gel to transfer the DNA fragments onto a membrane.
- Add a labelled probe in a buffer solution.
- Detect the probes (depends on which type of probe is used).
Why must the DNA be denatured in SB?
Only ssDNA can transfer to the membrane, the double-strands must be split apart.
What is the purpose of depurination in SB?
To remove the purine bases, making the DNA fragments smaller.
How does the blotting work?
By capillary action.
What is used as a membrane in SB?
Nitrocellulose or nylon.
If the probes were radioactive how would they be visualised?
By autoradiography.
Give 5 major uses of southern blotting.
- Checking for gene insertion
- Finding gene homologues across species
- Finding clones in DNA libraries
- Forensics - DNA fingerprinting
- Pre-natal screening using FISH (not strictly SB but same principle)
What is Northern Blotting?
Hybridisation of a DNA probe to an RNA sample.
What is the purpose of northern blotting?
Tells you which genes are being expressed in a tissue.
What happens to excess probes in SB and NB?
They are washed off by buffer solution.
