PCR and DNA Sequencing

Question 1

What are the different components which go into the PCR mixture?

Answer 1

• The DNA template; can be initiated from 200 picograms.
• Primers: polymerase needs primers because it can only add bases to pre-existing strands. They determine the maximum yield of the product . The primer sequence needs careful design to ensure proper binding.
• Taq polymerase: binds the dNTPs to the template strand
• dNTPs: bound by taq polymerase to the template strand
• Buffer and salts: prevents denaturation of the DNA and are needed for hybridisation and extension.

Question 2

What is the initiation step in PCR?

Answer 2

The sample is heated between 94-96 degrees celcius for 30 sec-5 mins.

Ensures the DNA is fully suspended and denatures the hydrogen bonds.

Question 3

What is the denaturing phase of PCR?

Answer 3

The sample is heated between 94-98 ^oC for 30 seconds.

This denatures the DNA further, splitting the double-stranded DNA into single-stranded DNA.

Question 4

What is the annealing phase of PCR?

Answer 4

The sample is cooled and held at 50-64 ^oC for 30 seconds.

This binds the primers to the template strands. The temperature must be slightly less than the Tm of the primers.

Primers bind over complementary templates because of high concentration.

Polymerase may also bind but will not proceed.

Question 5

What is the extension phase of PCR?

Answer 5

The sample is heated back up to 72 degrees for 30 seconds, or 1 minute per 1000 base pairs.

This synthesises the complementary strand as taq polymerase is activated, causing it to bind the dNTPs to their complimentary base pairs on the template strand.

Question 6

How many cycles does the PCR go on for?

Answer 6

• 15-40 cycles.
• Each cycle doubles the concentration of DNA.
• Thirty repeats = 2³⁰ copies made
• If there are too few cycles, there will not be enough amplification.
• If there are too many cycles, there will not be enough dNTPs, which will lead to truncated products.

Question 7

What is the final extension phase for PCR?

Answer 7

The sample is held at 72 degrees for several minutes. This ensures all strands are finished and reduces the truncated products.

Lastly, the products are held at 4^oC until needed; best condition for storing products.

Question 8

What is a ‘primer dimer’ and when is it formed?

Answer 8

A primer dimer consists of primer molecules that have hybridised to eachother as their base pairs are complimentary to each other.

This is due to a poor choice of primer sequence, or too much primer being added.

Question 9

Why may there be no amplification?

Answer 9

• Primers are too concentrated
• dNTPs are degraded from freezing
• the template DNA has degraded
• Annealing temperature is too high.
• You forgot to add something to the PCR mixture,

Question 10

When may non-specific amplification occur?

Answer 10

• Contamination
• Annealing temperature is too low.

Question 11

Why may weak amplification occur?

Answer 11

• Concentration is too low.
• Not enough cycles
• Annealing time is too short.

Question 12

What is the role of dNTPs and ddNTPs in Sanger Sequencing?

Answer 12

dNTPs such as dATP, dTTP, dCTP and dGTP are monomers used in the synthesis of DNA.

ddNTPs such as ddATP, ddTTP, ddCTP and ddGTP are monomers used to terminate DNA synthesis

Question 13

What is the difference between ‘sequencing’ and ‘profiling’?

Answer 13

• Sequencing:
  
  • looks at single base-level sequence
  • Provides info about proteins expressed; useful for biomedical analysis
  • Relies on polymerase reactions.
• Profiling:
  
  • looks at allele-level differences
  • Information about familial relationships; useful for forensic analysis.
  • Also relies on polymerase reactions.