PCR

Question 1

What does it mean if fragments of DNA are AMPLIFIED?

Answer 1

• Clone the DNA fragment to make large quantities.

Question 2

2 different methods of amplifying DNA fragments

Answer 2

• In vivo: in living organism.
• In vitro: in a lab.

Question 3

What is the “in vitro” method of amplifying a DNA fragment?

Answer 3

• PCR
• Polymerase chain reaction.
• Done on automated machine.

Question 4

Equipment required for PCR.

Answer 4

• Thermocycler.
• DNA fragment.
• DNA polymerase - spec. Taq polymerase.
• Primers.
• Free DNA nucleotides.

Question 5

Why do we use Taq polymerase in PCR?

Answer 5

• Taken from bacteria.
• Heat-resistant, won’t denature at high temperatures.

Question 6

What are primers?

Answer 6

• Short sequences of single-stranded DNA.
• Complementary to DNA base sequences at start/ end of DNA fragment.

Question 7

Name three steps involved in PCR. Temp at each stage?

Answer 7

1.) Denaturation 95⁰C
2.) Annealing 55⁰C
3.) Synthesis 72⁰C

Question 8

What happens in this step of PCR? Temp?

Denaturation.

Answer 8

• Thermocycler temp increased to 95⁰C
• Hydrogen bonds in DNA break –> splits DNA into 2 single-stranded template strands.

Question 9

True or False

First step of PCR: “denaturing” involves enzymes being denatured.

Answer 9

• False.
• Involves hydrogen bonds in DNA breaking, splits DNA into 2 single-stranded template strands.

Question 10

What happens in this step of PCR? Temp?

Annealing.

Answer 10

• Temp of thermocycler decreased to 55⁰C so primers can attach to start/ end of DNA base sequence of DNA fragment.
• Hydrogen bonds form between primers and comp DNA base sequences on DNA fragment.

Question 11

What happens in this step of PCR? Temp?

Synthesis

Answer 11

• 72⁰C: optimum temp for Taq polymerase.
• Free DNA nucleotides allign to comp base pairs on EACH template strand of DNA fragment.
• Taq polymerase joins free DNA nucleotides to make new strand to allign next to EACH template strand.

Question 12

At the end of one round of PCR, how many DNA fragments do you have? Single/ double stranded?

Answer 12

• 2 DOUBLE STRANDED DNA FRAGMENTS.

Question 13

What happens to no. of DNA fragments per cycle of PCR?

Answer 13

• No. of DNA fragments will double for each PCR cycle.

Question 14

3 advantages of PCR.

Answer 14

1.) Automated: more efficient.
2.) Rapid.
3.) Doesn’t require living cells like “in vivo”
- Quicker/ less complex techniques.

Question 15

Why is synthesis stage of PCR at 72⁰C?

Answer 15

• Optimum temp for Taq polymerase.

Question 16

Past paper 2 2021 concept.

Describe what PCR graph with number of molcules against number of PCR cycles would look like.

Answer 16

• Increase = very slow at first: MP1–> initially number of molecules doubling is low. (ie. 2,4,8,16.)
• MP2: Number of molecules doubles each cycle to produce exponential increase.