Lecture 22: PCR

Question 1

PCR requirements

Answer 1

1. ssDNA “template”
2. Primers:
   a. to generate primers, parts of the
   sequence must be known
   b. “Forward primer” - attaches to ‘bottom
   strand’ (parent strand). Left to right
   c. “Reverse primer” - attaches to ‘top
   strand’ (daughter strand). Right to left.

Question 2

What are the stages of PCR

Answer 2

1. “Denaturation” to turn dsDNA to ssDNA
   a. ~95C
2. “Annealing” of primer to ssDNA
   a. Temperature depends on length and
   sequence of primer
3. “Elongation” of DNA using DNA
   polymerase to add dNTPs

Question 3

How many dsDNAs are produced from one dsDNA

Answer 3

2

Question 4

How can we decide what we applify

Answer 4

By selecting our primers, we can amplify any particular region of a DNA template e choose

Question 5

How are PCR primers designed?

Answer 5

1. decide on annealing temp: usually 
   between 50-60C
2. Decide on time 
3. Elongation 72C
4. Decide on how long to run PCR. General 
    rule "1 minute = 1kb"
3.

Question 6

Why does the annealing temperature matter?

Answer 6

1. Determined by primer length and 
   sequence
2. High annealing temp (70C) = longer 
    primer
   a. If it was short, primer wouldn't 
       stably anneal as would melt off
3. TOO LOW a temperature the primer 
    would be able to anneal to incorrect 
    regions 
4. All primers have a "TM" (Temperature 
    Melting)
5. GENERAL RULE
   a. For every A&T = 2 
   b. For every G&C = 4
6. So, 20nt long primer with 10 T&A's 
    and 10 G&C's
   a. 10 x 4 = 40
   b. 10 x 2 = 20 
   c. TN = 60C - 5 = 55C

Question 7

what dictates primer length?

Answer 7

1. the GC and AT content
2. High AT % = longer primer as
   temperature needs to be high enough
   for efficient annealing
3. The more complex (the longer the
   template) the longer the primer
   a. therefore higher annealing
   temperature
   b. this is because there may be the same
   sequence in many places if too short

Question 8

What else can you do with PCR outside of ampification?

Answer 8

1. Can add more bases TO THE 5’ END
   of the primers, which will be
   incorporated
2. Deletion of a gene

Question 9

How can you merge two genes together using PCR?

Answer 9

1. Need one pair (forward and reverse) of
   primers to fit both of the genes
   2.