1. How PCR works Cold Spring Harbor Animation Animated .GIF files
Blackboard

2. Review: The structure of DNA
Unzipping
Antiparallel Strands

3. How PCR works
Cold Spring Harbor Animation
PCR.EXE

4. How PCR works
Animated .GIF #1

5. How PCR works
Animated .GIF #2

6. How PCR works
Blackboard
(Dave attempts to draw this out!)

7. PCR Reaction Components
Water
Buffer
DNA template
Primers
Nucleotides
Mg++ ions
DNA Polymerase

8. PCR Reaction: Water
Water
The medium for all other components.

9. PCR Reaction: Buffer Water Buffer
Stabilizes the DNA polymerase, DNA, and nucleotides
500 mM KCl
100 mM Tris-HCl, pH 8.3
Triton X-100 or Tween

10. PCR Reaction: Template DNA
Water
Buffer
DNA template
Contains region to be amplified
Any DNA desired
Purity not required
Should be free of polymerase inhibitors

11. PCR Reaction: Primers Water Buffer DNA template Primers
Specific for ends of amplified region
Forward and Reverse
Annealing temps should be known
Depends on primer length, GC content, etc.
Length nt
Conc 0.1 – 1.0 uM (pMol/ul)

12. PCR Reaction: Nucleotides
Water
Buffer
DNA template
Primers
Nucleotides
Added to the growing chain
Activated NTP’s
dATP, dGTP, dCTP, dTTP
Stored at 10mM, pH 7.0
Add to uM in assay

13. PCR Reaction: Magnesium
Water
Buffer
DNA template
Primers
Nucleotides
Mg++ ions
Essential co-factor of DNA polymerase
Too little: Enzyme won’t work.
Stabilizes the DNA double-helix
Too much: DNA extra stable, non-specific priming, band smearing
Used at 0.5 to 3.5 uM in the assay

14. PCR Reaction: Polymerase
Water
Buffer
DNA template
Primers
Nucleotides
Mg++ ions
DNA Polymerase
The enzyme that does the extension
TAQ or similar
Heat-stable
Approx 1 U / rxn

15. PCR Reaction Components
Water
Buffer
DNA template
Primers
Nucleotides
Mg++ ions
DNA Polymerase

16. A Typical PCR Reaction Component ml Sterile Water 38.0
10X PCR Buffer
MgCl2 (50mM)
dNTP’s (10mM each)
PrimerFWD (25 pmol/ml) 1.0
PrimerREV
DNA Polymerase
DNA Template
Total Volume

17. A Simpler PCR Reaction
Component ml
Sterile Water
10X PCR Buffer
MgCl2 (50mM)
dNTP’s (10mM each)
PrimerFWD (25 pmol/ul) 1.0
PrimerREV
DNA Polymerase
DNA Template
Total Volume
Component ml
PREMIX 24.0
Buffer
MgCl2
dNTP’s
DNA Polymerase
“Enhancers”
Sterile Water
Primers FWD+Rev 1.0 DNA Template 25.0
Total Volume 50.0
PREMIXES CAN REDUCE THE NUMBER OF ITEMS ADDED TO THE MIX

18. Using a PCR Mastermix Aliquot 49 ml Add DNA as last step
Component 1X(ml) 20X(ml)
Sterile Water
10X PCR Buffer
MgCl2 (50mM)
dNTP’s (10mM each)
PrimerFWD (25 pmol/ul)
PrimerREV
DNA Polymerase
DNA Template
Total Volume
Aliquot
49 ml
Add DNA
as last step

19. Typical Thermal Cycler Conditions
1. Initial Denaturation 95°C 4 min
2. DNA Denaturation °C 1 min
3. Primer Annealing °C 1 min
4. Primer Extension °C 1 min
5. Go to step #2, repeat 29 more times
6. Hold at 4 C
7. End

20. Finally: Some Mis-Uses (malas aplicaciones) of PCR
Since PCR is extremely sensitive, it is highly prone to giving “false positive” results.
Forensic and medical applications use strict protocols to minimize chance of errors.
Universities, working without standardized protocols, have frequently made incredible “discoveries”, only to find out later that it was all due to PCR artifacts.
Ancient DNA
GM Crops
                                                      

21. MisUses of PCR: Ancient DNA
According to Richard Thomas and his colleagues at London's Natural History Museum, writing in the August issue of Trends in Ecology and Evolution: 'It is highly unlikely that geologically ancient DNA survives in any fossil material so far studied.' The researchers spent three years attempting to extract DNA from insects entombed in amber. 'We worked with many more samples than the total number of published reports of success,' says Thomas. The result: complete failure. Not a single strand of prehistoric DNA to be seen.
When an organism dies, its tissues immediately begin to break down, creating a soup of enzymes, water and oxidative molecules that cut DNA strands into smaller and smaller fragments. As time passes, the amount of DNA remaining in the tissue steadily diminishes, and eventually disappears entirely.

22. MissUses of PCR: GM Crops
An intense scientific debate has opened up over whether genetically modified (GM) crops in Mexico have contaminated wild maize (corn).
Last November, the magazine Nature published data from two authors who said they had detected DNA from GM plants in wild crops growing in a remote area.
The criticisms of Quist and Chapela concentrate on their use of a method known as i-PCR, inverse polymerase chain reaction - a technique used to amplify samples to sufficient levels so that they can be more easily studied.
...the conclusion of the original paper "seems to be based on an artefact arising from the i-PCR [Quist and Chapela] used...

23. “End” of PCR Introduction