Lecture 8

Question 1

What is Northern hybridisation?

Answer 1

Allows us to identify RNA molecules containing specific sequences
Used to identify specific genes expressed in certain tissues

Question 2

What happens in Northern blotting?

Answer 2

mRNA is extracted from tissue and separated by size on agarose
mRNA is transferred directly to membrane
Tissues placed on RNA and they suck the RNA out of the gel and onto the membrane
DNA probe is labelled with specific sequence we want to find and it is hybridised to the mRNA molecule
RNA put under X-ray or fluorescent light to

Question 3

What is in-situ hybridisation?

Answer 3

Hybridisation without blotting, carried out on tissues of whole organisms
Identifies the cellular and tissue distribution of mRNA from specific genes
Each probe is labelled with different fluorescent molecules to make it easier to see

Question 4

What is DNA sequencing?

Answer 4

Uses DNA to copy single stranded DNA and uses dideoxy nucleotides which interrupts the ability of DNA polymerase to copy DNA

Question 5

What is dideoxynucleotide chain-termination?

Answer 5

Dideoxy nucleotides are added to DNA chains to prevent DNA polymerase continuing the extended chain because they do not have an OH group and so you cannot add another nucleotide

Question 6

What are the dideoxynucleotides?

Answer 6

ddATP
ddGTP
ddTTP
ddCTP

Question 7

What are the steps of PCR?

Answer 7

Denaturing: reaction heated to 95 degrees to denature DNA into single strands
Annealing: reaction decreases to around 50 degrees to allow primers to hybridise to DNA
Extension: reaction temperature is raised to 72 degrees to allow Taq polymerase to synthesise DNA

Question 8

What are the limitations of PCR?

Answer 8

To synthesise primers, there needs to be information of the DNA target sequence
Contamination from other sources can cause issues
Cannot amplify long sequences