How PCR works Cold Spring Harbor Animation Animated .GIF

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How PCR works

• Cold Spring Harbor Animation
• Animated .GIF files
• Blackboard

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Review The structure of DNA
Unzipping
Antiparallel Strands
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How PCR works

• Cold Spring Harbor Animation
• PCR.EXE

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How PCR works

• Animated .GIF 1

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How PCR works

• Animated .GIF 2

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How PCR works

• Blackboard
• (Dave attempts to draw this out!)

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PCR Reaction Components

• Water
• Buffer
• DNA template
• Primers
• Nucleotides
• Mg ions
• DNA Polymerase

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PCR Reaction Water

• Water
• The medium for all other components.

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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

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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

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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 15-30 nt
• Conc 0.1 1.0 uM (pMol/ul)

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PCR Reaction Nucleotides

• Water
• Buffer
• DNA template
• Primers
• Nucleotides
• Added to the growing chain
• Activated NTPs
• dATP, dGTP, dCTP, dTTP
• Stored at 10mM, pH 7.0
• Add to 20-200 uM in assay

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PCR Reaction Magnesium

• Water
• Buffer
• DNA template
• Primers
• Nucleotides
• Mg ions
• Essential co-factor of DNA polymerase
• Too little Enzyme wont 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

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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

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PCR Reaction Components

• Water
• Buffer
• DNA template
• Primers
• Nucleotides
• Mg ions
• DNA Polymerase

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A Typical PCR Reaction
Component ml Sterile Water 38.0 10X
PCR Buffer 5.0 MgCl2 (50mM)
2.5 dNTPs (10mM each) 1.0 PrimerFWD (25
pmol/ml) 1.0 PrimerREV 1.0 DNA
Polymerase 0.5 DNA Template
1.0 Total Volume 50.0
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A Simpler PCR Reaction
Component ml Sterile Water 38.0 10X PCR
Buffer 5.0 MgCl2 (50mM) 2.5
dNTPs (10mM each) 1.0 PrimerFWD (25
pmol/ul) 1.0 PrimerREV 1.0 DNA
Polymerase 0.5 DNA Template
1.0 Total Volume 50.0
Component ml PREMIX 24.0 Buffer MgCl2
dNTPs DNA Polymerase Enhancers
Sterile Water Primers FWDRev 1.0 DNA
Template 25.0 Total Volume 50.0
PREMIXES CAN REDUCE THE NUMBER OF ITEMS ADDED TO
THE MIX
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Using a PCR Mastermix
Component 1X(ml) 20X(ml) Sterile Water 38.0 760
10X PCR Buffer 5.0 100 MgCl2 (50mM) 2.5 50
dNTPs (10mM each) 1.0 20 PrimerFWD (25
pmol/ul) 1.0 20 PrimerREV 1.0 20 DNA
Polymerase 0.5 10 DNA Template 1.0 -- Total
Volume 50.0 980
Aliquot 49 ml
Add DNA as last step
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Typical Thermal Cycler Conditions
1. Initial Denaturation 95C 4 min 2. DNA
Denaturation 95C 1 min 3. Primer
Annealing 65C 1 min 4. Primer Extension
72C 1 min 5. Go to step 2, repeat 29
more times 6. Hold at 4 C 7. End
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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

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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.
http//www.newscientist.com/hottopics/dinosaurs/ba
ckfrom.jsp
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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...
http//news.bbc.co.uk/hi/english/sci/tech/newsid_1
911000/1911535.stm
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End of PCR Introduction