CMB2000/L02 PCR Amplifying DNA

Question 1

Give 5 pros of PCR.

Answer 1

Sensitive
Specific
Cheap
Rapid
Robust

Question 2

Give the reactants and products of the DNA synthesis reaction.

Answer 2

3’ OH + 5’ 3P -> Phosphate backbone + 2P + H2O

Question 3

Give the 6 reactants required for PCR.

Answer 3

dsDNA template
2 Primers
Polymerase
dNTPs
Magnesium
Buffer

Question 4

Describe the 2 primers required for DNA synthesis. (3)

Answer 4

Small ssDNA molecule
6-30 bases
Chemically synthesised

Question 5

What is the role of polymerase?

Answer 5

Copies template
Extends from 3’ end of primer

Question 6

Give the 4 dNTPs.

Answer 6

dATP
dCTP
dGTP
dTTP

Question 7

Why is magnesium required for PCR?

Answer 7

Co-factor for DNA polymerase enzyme

Question 8

Describe the role of Taq polymerase.

Answer 8

Copies DNA accurately (1 mistake per 1 billion nucleotides)

Question 9

Give the 3 regions of Taq polymerase.

Answer 9

Synthesis
Proof reading
Primer removal

Question 10

Why can’t primers be too long or too short?

Answer 10

Long - hybridise too slowly
Short - won’t be specific

Question 11

What GC content should primers contain and why?

Answer 11

40-60% as they have stronger H bonding and aid stability
Should also start and end with 1-2 G/C pairs

Question 12

What should the melting temperature of primers be?

Answer 12

50-60C

Question 13

What is the relationship between the Tmax of 2 primers used in a PCR?

Answer 13

Should be within 5C of each other

Question 14

Define co-factor.

Answer 14

A non-protein component of the reaction that’s needed to enable activity of catalysis
Metallic or a compound

Question 15

Which enzyme does magnesium act as a cofactor for?

Answer 15

DNA polymerase

Question 16

What is the optimum pH for PCR?

Answer 16

8-9.5

Question 17

Describe the ideal buffer for PCR.

Answer 17

Tris HCL
KCl promotes annealing
May be replaced by ammonium sulphate which destabilises base pairing bonds

Question 18

Give the 3 stages of PCR.

Answer 18

Denaturation
Annealing
Elongation

Question 19

Describe the first cycle of PCR.

Answer 19

Boil dsDNA
Anneal primer to ssDNA
Extend with polymerase and dNTPs
Boil, anneal different primer, complementary to new DNA and other strand of original DNA
Extend with polymerase

Question 20

How many copies of DNA would be present after 30 cycles of PCR?

Answer 20

2^30
>1 billion

Question 21

What is the role of Nancy520?

Answer 21

Dye intercalates between base pairs

Question 22

How can PCR products be detected?

Answer 22

Run products of agarose gel
Use intercalating dye to stain DNA to determine size and yield

Question 23

What PCR products can be identified from agarose gel electrophoresis? (4)

Answer 23

Molecular weight markers
PCR products
Primers
Template