Analysing Nucleic Acids

Question 1

Describe in viva cloning

Answer 1

1. Replicon and target DNA is cut with the same restriction endonuclease
2. Mix together
3. Join using DNA ligase
4. Transform into host cell
5. Selective propagation - use antibiotic resistance marker so only cells that take up replicon survive
6. Isolate target DNA

Question 2

Describe type II restriction endonucleases

Answer 2

Enzymes that cut at a specific DNA sequence
Recognition sequence is 4-8bp long
Sticky or blunt end

Question 3

Describe electrophoresis

Answer 3

DNA is negatively charged so moves to the anode

Smaller and more negatively charged fragments move further

Question 4

What are hybridisation assays?

Answer 4

Target DNA is immobilised

Probes (single stranded) bind to target DNA aid complementary

Question 5

What is southern blotting?

Answer 5

Making electrophoresis fragments into immobilised hybridisation ones.
Can be washed with probes and then probes washed away
Can use photographic film to see where the bands are

Question 6

Southern blot
Northern blot
Colony blot
Tissue in situ
Chromosome in situ 
Reverse hybridisation

Answer 6

Southern blot - DNA target and DNA probe
Northern blot - RNA target and DNA probe
Colony blot - bacterial DNA target and DNA probe
Tissue in situ - RNA target and RBA probe
Chromosome in situ - chromosome target and DNA probe

Question 7

Energy needed to denature DNA probe depends on…

Answer 7

Length - longer strand = more energy
Base composition - more G-C = more energy
Chemical environment - lots of 1+ charges outside can stabilise 1- charge of DNA

Question 8

What dies stringency mean?

Answer 8

How specifically the probe binds to target

Question 9

Define melting temperature

Answer 9

The midpoint temperature of transition between double stranded to single stranded nucleic acids

Question 10

Hybridisation stringency is… and can be increased by …

Answer 10

The power to distinguish between related sequences

Increase temperature and decrease Na+

Question 11

Describe in vivo cloning

Answer 11

Denature DNA (95 degrees)
Anneal DNA with primer (50 degrees)
Extend primer with DNA polymerase (72 degrees)

Question 12

What to consider when designing a primer

Answer 12

Length - 20 nucleotides for specificity
Base composition - no tandem repeats = hairpins
Not complementary at 3’ end as primers join to each other instead

Question 13

What are DNA microarrays used for?

Answer 13

Used to determine expression profile or many different genes at the same time