DNA Hybridisation

Question 1

How are DNA probes used?

Answer 1

Denature dsDNA, with pH>7 or temp. Add ssDNA labelled probe. Cool or pH, some of the ssDNA labelled probe is incorporated when reannealing. Identify labelled DNA using photographic film

Question 2

Why would you use southern blotting?

Answer 2

DNA: investigate gene structure, expansions, mutations, variations

Question 3

What are the stages of southern blotting?

Answer 3

1. restriction enzymes.
2. DNA gel electrophoresis.
3. alkaline = denatures to ssDNA.
4. Transfer to membrane
5. Hybridisation of complementary probe to detect specific DNA.
6. Stain with ethidium bromide and view under UV

Question 4

What is the function of northern blotting and the stages?

Answer 4

uses DNA to detect RNA

1. RNA isolated
2. Separated on gel electrophoresis
3. Transferred to nylon membrane
4. Hybridisation with DNA probe
5. Stain with ethidium bromide and view under UV

Question 5

What is the function of western blotting and the stages?

Answer 5

NOT DNA HYBRIDISATION

detects proteins by Abs labelled with enzymes

1. Protein electrophoresis
2. Transfer to nitrocellulose replica
3. Binding of primary Ab
4. Binding of enzyme linked secondary Ab
5. Visualisation with substrate (colour)

Question 6

What is the Sanger chain termination method?

Answer 6

allows us to work out the nucleotide sequence of DNA

Question 7

How does the Sanger chain termination method work?

Answer 7

ddNTPS terminates chain growth due to only H on 3rd carbon = no phosphodiester bond forms. 4 separate tubes each with dATP, dCTP, dGTP, dTTP and one ddNTPs. Wherever C/T/G/A needs to be added in the sequence there is a chance that: A d?TP will be used and the chain will continue growing OR A dd?TP will be used and stop further growth. we will see a mixture of new DNA molecules produced of different length depending on where the dd?TP is incorporated. Primer added. Gel run using separate lanes for each reaction tube