Topic 7: PCR

Question 1

PCR +ex (2)

Answer 1

a method of amplifying DNA
Ex: You have one copy of Gene X we can use PCR to amplify our DNA sequence: make billions of copy

Question 2

What is the purpose of PCR? (3)

Answer 2

1. Detect the presence or absence of the gene (If sample does not have gene X, it wont amplify it)
2. Getting enough DNA copies to perform sequencing, enabling us to detect a mutation
3. Cloning and genetic engineering

Question 3

Components needed to do PCR (4)

Answer 3

1. Template DNA
2. DNA primers
3. Dntps (3 phosphate)
4. DNA polymerase (aka taq polymerase)

Question 4

DNA primers in PCR:

+ALWAYS

Answer 4

• Approx 20 nucleotides long with a free 3’-OH group
  ALWAYS written 5’-3’

Question 5

Forward primers extend

Answer 5

left-right

Question 6

Reverse primer extend

Answer 6

Extend right to left

Question 7

reverse compliment always refer to

Answer 7

reverse primer

Question 8

PCR steps

Answer 8

1. Denaturation: ~95 degrees, breaks apart the 2 strands of the DNA template (H bond broken)
2. Annealing: 55-65 degree, primers bind at the appropriate locations on the template based on base complementarity
3. Synthesis: Taq polymerase catalyzes elongation by adding nucleotides to the 3’ end of th primer

Question 9

What determines the correct annealing temperature to use in a PCR reaction? (2)

+how is that determined

Answer 9

• Depends on the melting temperature of the primers
• The melting temperature is the temp at which ~50% of primer molecules are bound and 50% are not

Question 10

What determines the melting temperature of the primer? (2)

Answer 10

• GC richness: more GC give higher melting temp as its bonds is held by 2 H-Bond.
• Length of the primers: the longer the primer the higher the melting temp

Question 11

Our annealing temp should be —– than the melting temp of the primers because—-

Answer 11

1. lower
2. to encourage the binding you dont want to break it apart

Question 12

Forward and reverse primer have the same

Answer 12

melting temp

Question 13

Primers are incooporated inside the

Answer 13

desired PCR products

Question 14

Rules and trends for forward and reverse primer trends (3)

Answer 14

1. the primer sequence are included in the PCR product
2. The forward primer looks the same as the top strand
3. the reverse primer is the reverse compliment of the top strand

Question 15

We start seeing the exact PCR sequence in

Answer 15

cycle 2