Polymerase Chain Reactions

Question 1

What does polymerase chain reactions do?

Answer 1

• PCR makes more copies of a section of DNA.
• It amplifies DNA.

Question 2

What is PCR used in?

Answer 2

• Identifying viral RNA/DNA in a patient.
• DNA profiling (forensics).
• Ancient organisms.
• Epidemiology.
• Detecting and identifying mutations.
• Identifying cancerous mutations.
• Paternity testing.

Question 3

• What do primers do?
• How long are primers?
• How many primers used in PCR?

Answer 3

• Primers are used to select the target length of DNA.
• Each primer is 20-30 nucleotides long.
• In PCR 2 primers are used. 1 is complimentary to the target strand. 1 is complementary to the opposite strand.

Question 4

Stages in PCR.

Answer 4

1 - Denaturation - DNA heated to 94-6 degrees Celsius. DNA separates into single strands.
2- Annealing - Cool mixture to 68 degrees Celsius, primers anneal to the DNA by the complimentary-base pairing.
3- Elongation - Heat to 72 degrees Celsius to allow DNA polymerase to extend the primers and copies of the target DNA are synthesised using target strands as templates.
4 - Amplification - repeat stage 1 and 2.

Question 5

What are the DNA polymerase used in PCR known as?

Answer 5

Taq polymerase as it has a higher optimum temp at 72 degrees - so it won’t denature at 96 degrees Celsius.

Question 6

Names of stages of electrophoresis/ DNA profiling

Answer 6

1- Extraction - PCR - 3 stages
2- Digestion - restriction enzymes
3- Separating DNA - gel electrophoresis - southern blotting
4- Hybridisation - radioactive/fluorescent DNA probes (complimentary).
5- Evidence - X-ray/UV light.

Question 7

What happens in the extraction and digestion stage of DNA profiling.

Answer 7

• DNA cut into fragments.
• Restriction enzymes recognise palindromic sequences and cut (digest) the DNA at these sites.
• Leaving “sticky ends” which overhang.
• Each enzyme is specific to a sequence as it is complimentary to their active site.

Question 8

What happens in the electrophoresis stage of DNA profiling?

Answer 8

• small charged molecules (DNA) separated.
  1- Agarose gel in box with wells at one end.
  
  • Wells closest to negative electrode.
  • Buffer solution added to cover gel.
    2 - Loading dye added to each DNA sample.
  • A micropipette is used to add sample to each well.
    3 - electric current passed through gel.
  • DNA fragments negatively charged, so move to anode.
    4 - DNA is invisible
  • Can be stained and rinsed.
  • Add fluorescent which fluoresces in light.

Question 9

What does southern blotting do?

Answer 9

• Separate DNA into single strand using alkaline solution.
• Single strand fragments transferred onto nylon membrane/ nitrocellulose sheet and left overnight.

Question 10

Hybridisation stage of DNA profiling.

Answer 10

• DNA probes labelled with a fluorescent marker or radioactively.
• Probes added to DNA fragments and bind by annealing.
• Used to locate genes/ identify absence/ presence of an allele.