2.2 - DNA replication in vitro: Polymerase chain reaction (PCR)

Question 1

What are the four deoxyribonucleoside triphosphates (dNTPs) used in DNA synthesis are what are there roles?

Answer 1

dATP, dGTP, dCTP, dTTP.

DNA synthesis requires dATP, dGTP, dCTP, and dTTP as substrates.

For interest only: RNA synthesis requires ATP, GTP, CTP, and UTP.

Question 2

Why is the DNA heated to 94 degrees C in PCR?

Answer 2

When a double stranded DNA (dsDNA) molecule is heated to 94 C, the paired strands will separate by breakage of hydrogen bonding present between complimentary bases, resulting the single stranded DNA formation. This allows the primers access to the single stranded DNA templates.

Question 3

What happens at 72 C during PCR?

Answer 3

The polymerase extends the primer to form a nascent DNA strand. This process is repeated multiple times, and because each new strand can also serve as a template for the primers, the region of interest is amplified exponentially.

Question 4

What happens if annealing temperature is too high in PCR?

Answer 4

If the annealing temperature is too high, primers are unable to bind to the template.

Question 5

What is a polymerase chain reaction?

Answer 5

a laboratory technique that can replicate a targeted region of a DNA molecule many times

Question 6

What are the 3 main steps in PCR (in order)?

Answer 6

denature DNA, anneal primers, extend primers using DNA polymerase

Question 7

What is “amplifying” a DNA section?

Answer 7

to take a specific section of DNA and replicate it over and over

Question 8

You wish to amplify the fragment shown below. Which primer sequences would you use? (primer sequences are inside the brackets)

5’-(TCATCC)ATG…CAT(GTGACT)-3’

3’-(AGTAGG)TAC…GTA(CACTGA)-5’

Answer 8

5′-TCATCC-3′ and 5′-AGTCAC-3′

Question 9

What is the sequence of the amplified product of DNA at the end of the PCR experiment?

Primer:
5’ – CTTCGAT – 3’

DNA sequence:
3’ - AGCAATTGCGCATTTAGCTTCCAG - 5’

Answer 9

5’ - TCGTTAACGCGTAAATCGAAGGTC - 3’

Question 10

Does the primer bind to the top or bottom strand of the DNA sequence provided?

Primer:
5’ – CTTCGAT – 3’

5’ – GGTTCGTTAAATCGAAGCT– 3’
3’ – CCAAGCAATTTAGCTTCGA – 5’

Answer 10

top strand

Question 11

List 4 key components for PCR

Answer 11

• DNA template
• Primers
• dNTPs
• DNA Polymerase