NAGE 7

Question 1

Why analyse nucleic acids?

Answer 1

Personalised medicine.

HER2 overexpression leads to more aggressive breast cancer.

So can help tailor treatment by genome sequencing and identifying if a cancer is HER2 positive or not.

Question 2

Explain cell based DNA cloning.

Answer 2

1. Target sequence cut. Replicon (e.g. plasmid) cut. Both with SAME RE (ensures ends of the 2 DNA sequences are compatible).
2. Purified and mixed.
3. Mix and join fragments using DNA Ligase.
4. Transformation of recombinant DNA into host cells.
5. Selectively choose the individual cell colonies we want (e.g. by using antibiotics on bacteria).
6. Expand cell culture and isolate recombinant DNA.

Question 3

What is a replicon?

Answer 3

Sequence capable of independent replication (Eg plasmid)

Question 4

What are (Type 2) REs?

Answer 4

Enzymes which cleave DNA at specific recognition sequences. (usually 4-8bp palindromic sequences).

Produce blunt/sticky ends.

Question 5

How does electrophoresis work to separate DNA fragments.

Answer 5

1. DNA negatively charged due to phosphate backbone.
2. DNA moves towards anode when electric current applied.
3. Using a gel matrix means small fragments travel further than large fragments.
4. DNA can subsequently be isolated or transferred to a membrane for replication.

Question 6

Explain how hybridisation assays can be used.

Answer 6

1. Immobilise target DNA onto a solid support (e.g. nylon)
2. Support must readily bind single stranded nucleic acids.
3. Target DNA then hybridised with radioactively/fluorescently labelled probe.

Question 7

What is a southern blot?

Answer 7

Method of transferring electrophoresed fragments onto membrane that immobilises them.

Allows membrane to be washed with probes so probes can attach to complementary sequences.

Question 8

What does the energy required to break the H-bonds depend on?

Answer 8

1. Strand length
2. Base composition
3. Chemical environment - (monovalent cations stabilise DNA duplex by neutralising the charge of the phosphate backbone). Denaturants destabilise DNA duplex.

Question 9

What is stringency?

Answer 9

How specifically probes binds to target DNA.

Question 10

What is melting temperature defined as? (DNA )

Answer 10

Midpoint temperature of transition from double stranded to single stranded nucleic acids.

Question 11

Hybridisation stringency increases with?

Answer 11

1. Increasing temperature

2. Decreasing Na conc

Question 12

What are the 3 factors to consider for PCR Primer design?

Answer 12

1. Length - roughly 20 nucleotides
2. Base composition - avoid tandem repeats of nucleotides (can form hairpins)
3. 3’ end - avoid complementarity of bases at 3’ end - may cause primer dimers.

Question 13

What is a DNA microarray?

Answer 13

A collection of microscopic DNA spots, commonly representing single genes, on a solid surface.

Question 14

What are DNA microarrays used for?

Answer 14

Expression profiling.