B: PCR Gene technology

Question 1

Process of PCR —

Answer 1

1. Primers are added to reaction in large excess over target DNA
2. Reactions contains: target DNA, forward + reverse primers, DNA precursors, thermostable DNA polymerase, appropriate buffer with Mg++.
3. Melt all double stranded DNA to single strand by heating to 94 degrees
4. Reduce temperature (55-65) to allow annealing of primers to target DNA. As primers are in excess, they will anneal to their complement faster
5. Change temperature to 70 for optimal DNA polymerase action so that hybridised primers can be extended.
6. Cycle is repeated about 30 times.

Question 2

Process of gel electrophoresis —

Answer 2

DNA is negatively charged so it will migrate to positive electrode in an electric field.
- Agarose or polyacrylamide can be used to make a gel matrix.
- DNA migration rate is directly related to size of DNA.
- Detection of PCR products is achieved by staining gel with fluorescent dye that binds strongly to double stranded and weakly to single stranded DNA.
- Different primers can be labelled with different fluorescent dyes allowing detection of different PCR products of same size.

Question 3

Principals underlying PCR

Answer 3

1. A primer hybridises to its complement if it’s present in a single stranded DNA
2. Primer hybridises to its complement at temperature below the Tm
3. DNA polymerase synthesizes new DNA in a 5’-3’ direction from the 3’ end of the primer (extends primer)
4. DNA polymerase used in PCR is thermostable –> can be heated and cooled without losing its activity
5. When a pair of primers facing each other on the upper and lower strands of a DNA target are extended by DNA polymerase, new copies of the upper and lower strands between the primers are synthesized and the new strands become templates for the primers. Everytime this is repeated, the number of DNA copies is doubled –> repeated cycles of extension by DNA polymerase amplifies the DNA between the primers exponentially.

Question 4

Explain how PCR can be used to diagnose Hereditary Haemochromatosis (PCR-RFLP)

Answer 4

• Design primers that flank 845G>A mutation –> these allow amplification of region of the HFE gene bearing the mutation site. Designed to amplify 140bp region
• Extract DNA from individual
• Amplify region of HFE gene encompassing mutation site using PCR + primers
• Incubate PCR products with restriction enzyme Rsal
• Analyse PCR treated products - normal allele = 140bp, mutated allele = 90/50bp

Question 5

Genetics of HH disease

Answer 5

PCR can be used to detect presence/ absence of C282Y mutation in DNA sample.
Gene is HFE gene, HFE gene encodes MHC protein HFE that binds beta-2 micro globulin. Plays a role in up regulating expression of hepcidin (hormone that down regulates entry of iron into blood).
Mutation changes single G base at position 845 to A which chances codon TGC to TAC which changes cysteine to tyrosine.