Polymerase Chain Reaction Lecture

1. Meet Kary Mullis, inventor of the PCR
“Sometimes a good idea comes
to you when you are not looking
for it. Through an improbable
combination of coincidences,
naïveté, and luck mistakes, such
a revelation came to me one
Friday night in April, 1983, as I
gripped the steering wheel of
my car and snaked along a
moonlit mountain road into
northern California’s redwood
country.” Sci. Am. 1990 262:56-61, 64-5.

2. Denaturation
Annealing
3'
Extension
1 Cycle
5'
5'
5'
3'
3'
5'
5' 3'
3' 5'
5'
5'
3'
3'
3'
5'
5'
5'
3'
3'
3'
3'
The Polymerase
Chain Reaction

5. What you will do in lab next week:
Component Volume (L)
10X PCR buffer 5
MgCl2 2
dNTPs 1
Forward primer 1
Reverse primer 1
Template DNA* 1
Pfx Polymerase 0.5
Sterile dH2O 38.5
*During the first part of next week’s lab you will be
purifying genomic DNA from E. coli K-12.

6. Typical program, similar to the one that you will run:
1) 94°C for 2 min.
2) 94°C for 15 sec.
3) 55°C for 30 sec.
4) 68°C for 1.0 min per kb of amplified template*
5) Go to step 2 for 25 – 35 cycles
6) 68°C for 5 min. (may not do this step)
7) 4°C 
8) End cycle
*How many kb in the adhP gene?

7. Anatomy of a PCR Reaction
Denaturation Step
-Usually: 45 sec – 1 min at 94 °C
Primer Annealing Step (critical)
-Optimal if done at ~ 3 – 5 °C below Tm. Often is
done using temperature gradient, when several PCR
rxns are done in parallel at increasing temperatures.
Elongation Step
-The buffer is optimal for polymerase
-Depending on polymerase, 1 – 2 min for every kb of
synthesized DNA at 72 – 76 ° C. For Pfx
polymerase, 1 min/kb at 68 – 74 °C is optimal

8. Zen and the Art of Choosing the
Goldilocks Annealing Temperature
(Williamson and Rybicki, 1991: J Med Virol 33: 165-171).
http://www.mcb.uct.ac.za/pcroptim.htm

9. How long should a primer be?
Statistically, a unique, 16-bp sequence will appear
once in how many base pairs?

10. Primer Design: Some Guidelines
1) Primers should be ~18 – 22 bp long
2) Should have similar Tms, in the range of 52 – 65 °C
3) They should have ~40 – 60% GC content
4) Should have a “GC” clamp
5) Should have minimal secondary structure
6) Should have minimal repeats – e.g., ATATATAT
7) Should have minimal runs (no more than four) of
any one base
http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html

11. Secondary Structure
www.csd.uwo.ca/courses/CS661b/primerDesign.ppt

12. One formula (among many) for estimating the Tm
of primers
Tm = 4(G + C) + 2(A + T) °C
Using the above formula, what is the Tm for the
following primer?
5 -CACCATGGCGTATTGCAATCCGG-3 
http://www.mcb.uct.ac.za/pcroptim.htm

13. Which thermostable DNA polymerase should I use?
You will be using Platinum Pfx, which is claimed to
have a higher fidelity than Pfu.

14. http://molecool.wustl.edu/krolllab/PDFs/Pfu%20polymerase-Stratagene.pdf

15. But, from the Invitrogen
website we have the
following comparison:
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Nucleic-Acid-Amplification-