Lecture 6- Analysis of Nucleic Acids

Question 1

Give an of personalised medicine

Answer 1

HER 2 and Breast Cancer

• Over-expression of HER2 –> more aggressive breast cancer
• Drugs developed specifically for those with the HER 2 mutation (Herceptin)
• sequence genome, ID if cancer is HER 2 +ve, can provide targeted treatment

Question 2

Describe the process of cell-based DNA cloning (in vivo)

Answer 2

1) Construction of recombinant DNA molecules in vitro by:
- Cut target DNA and a replicon (plasmid) with restriction endonuclease –> ends of two DNA sequences are compatible
- Purify and mix
- Mix and join DNA fragments using DNA ligase
2) Transformation of the recombinant DNA into host cells (bacteria/ yeast)
3) Selective propagation of individual colonies on agar pal (using antibiotics)
4) Expansion of cell culture and isolation of recombinant DNA

Question 3

How do restriction enzymes work?

Answer 3

• Type II restriction endonucleases cleave DNA at specific recognition sequences
• 4-8bp Palindromic recognition site
• Blunt and sticky ends
• Bacterial defence system- RE cleave only unmethylated DNA not host which is methylated
• longer recognition site= less frequent occurrence

Question 4

How are DNA fragments separated?

Answer 4

Electrophoresis

• DNA is -vely charged –> moves towards +ve anode when an electrical field is applied
• DNA travels through porous agarose gel and separates based on size
• small fragments = less retarded = travel faster towards anode
• DNA transferred to nylon membrane to form a replica for hybridisation

Question 5

What is nucleic acid hybridisation?

Answer 5

• it’s a method for detecting specific nucleic acid sequences

Question 6

Describe the process of hybridisation

Answer 6

1) Carry out gel electrophoresis and get a nylon membrane replica
2) Design a probe complementary to the sequence you are looking for
- must be visualised –> radioactive or fluorescent so that you can ID
3) Denature the DNA on the nylon membrane to get ssDNA
4) This is hybridised with a solution of the labeled probe

Question 7

How do you denature probe DNA?

Answer 7

Heat the probe DNA to disrupt the hydrogen bonds holding the two strands together

Question 8

What factors influence the energy required to denature the probe DNA?

Answer 8

1) Strand length- longer strand= more H bonds to break
2) Base composition (A=T and C = - G)
3) Chemical environment- Na+ stabilises -ve charge of phosphates. Denaturants (urea/formamide) destabilise DNA

Question 9

Define stringency

Answer 9

How specifically the probe binds to the DNA. low stringency= some mismatch, high stringency= perfect base- pairing

Question 10

Define hybridisation stringency and what does it increase with?

Answer 10

• The power to distinguish between related sequences
  1) Increase in temp
  2) Decrease in Na+ conc

Question 11

Define melting temperature (Tm)

Answer 11

A measure of nucleic acid duplex stability

-midpoint temp of transition from ds to ss DNA

Question 12

Give an example of cell-free cloning (in vitro)

Answer 12

Polymerase Chain Reaction (PCR)- allows amplification of specific target DNA

Question 13

Describe the process of PCR

Answer 13

1) Design primers 15-25 nucleotides long, complementary to target DNA
2) Mix the primers, dnTPs, Taq polymerase and DNA
3) Denature the DNA (heat to 94C)
4) Anneal primers to ssDNA by lowering temp (50-60C)
5) Raise temp for Taq polymerase to add dNTPs 5’ -> 3’ and generate new strands (72*C)
6) Repeat cycle

Question 14

How are primers selected for PCR?

Answer 14

1) Length- around 20 nucleotides long= specific to target sequence
2) Base composition- avoid tandem repeats –> hair pins where it anneals onto itself
3) 3’ end- avoid complementarity –> primer dimers