Molecular Techniques

Question 1

Polymerase Chain Reaction

Answer 1

Denaturation:
- Heated to 95 degree celsius
- Hydrogen bonds between complementary bases break, separating double stranded DNA into single stranded DNA

Annealing of Primers:
- Cooled to 65 degree celsius
- Primers cbp with complementary sequences at 3’ end of target sequences in the single-stranded DNA
- Primers provide a free 3’ -OH end for Taq polymerase to add dNTPs to elongate a new strand of DNA

Primer Extension:
- Heated to 72 degree celsius
- Taq polymerase synthesises the rest of the new strand of DNA by adding dNTPs to 3’ -OH ends of both primers, by catalysing formation of phosphodiester bonds between adjacent dNTPs

Question 2

Gel Electrophoresis

Answer 2

1. When placed in an electric field, negatively charged DNA travels towards the postively charged anode.
2. DNA fragments of different molecular sizes can be separated based on their rate of migration through the gel
3. This is because size of DNA fragments is inversely proportional to the rate of migration through the gel.
4. Larger DNA fragments encounters more resistance, moving a shorter distance through the gel, and are found nearer to the wells

Question 3

DNA hybridisation

Answer 3

1. Gel slab is placed on top of sponge, soaked in alkaline solution, a sheet of nitrocellulose membrane followed by a stack of paper towels is placed on top of the gel.
2. Alkaline solution is drawn upwards through the gel, denaturing double stranded DNA into single stranded DNA, which is then drawn upwards onto the nitrocellulose membrane and binds to the membrane
3. Nitrocellulose membrane is removed and incubated with single stranded radioactive DNA probes, which hybridises via complementary base pairing to target sequences.
4. Excess unhybridises probes are removed and washed off
5. Autoradiography can be carried out by placing a photographic X-ray film over the nitrocellulose membrane and radioactivity in the bound probes will expose the film to form an image that corresponds with the bands that have hybridised with the probes.

Question 4

Advantages of PCR

Answer 4

1. Millions of copies of DNA can be obtained in a relatively short amount of time
2. Capable of amplifying sequences from minute amounts of target DNA
3. The use of thermostable Taq polymerase which can withstand high temperatures without denaturation allows PCR to be automated, and eliminates the need to replace it after every cycle
4. Easy to set and use a thermal cycler to carry out PCR

Question 5

Limitations of PCR

Answer 5

1. Too sensitive as a minute amount of contaminant DNA with a sequence complementary to the primers can be amplified along with target DNA, leading to a misleading result
2. There is infidelity of DNA replication as Taq polymerase has no proofreading function