Date post: | 05-Apr-2018 |
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8/2/2019 PCR History Pt2[1]
1/23
How PCR works
Cold Spring Harbor Animation
Animated .GIF files
Blackboard
8/2/2019 PCR History Pt2[1]
2/23
Review: The structure of DNA
Antiparallel Strands
Unzipping
8/2/2019 PCR History Pt2[1]
3/23
How PCR works
Cold Spring Harbor Animation
PCR.EXE
http://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXE8/2/2019 PCR History Pt2[1]
4/23
How PCR works
Animated .GIF #1
http://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXE8/2/2019 PCR History Pt2[1]
5/23
How PCR works
Animated .GIF #2
http://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXE8/2/2019 PCR History Pt2[1]
6/23
How PCR works
Blackboard
(Dave attempts to draw this out!)
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7/23
PCR Reaction Components
Water
Buffer
DNA templatePrimers
Nucleotides
Mg++ ionsDNA Polymerase
http://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXE8/2/2019 PCR History Pt2[1]
8/23
PCR Reaction: Water
Water The medium for all
other components.
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9/23
PCR Reaction: Buffer
Water
Buffer
Stabilizes the DNApolymerase, DNA, andnucleotides
500 mM KCl
100 mM Tris-HCl, pH 8.3 Triton X-100 or Tween
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10/23
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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11/23
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)
http://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXEhttp://var/www/apps/conversion/current/tmp/Impostazioni%20locali/Temporary%20Internet%20Files/Content.IE5/K969NPZ8/PCR.EXE8/2/2019 PCR History Pt2[1]
12/23
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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13/23
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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14/23
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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15/23
PCR Reaction Components
Water
Buffer
DNA templatePrimers
Nucleotides
Mg++ ionsDNA Polymerase
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16/23
A Typical PCR ReactionComponent l
Sterile Water 38.0
10X PCR Buffer 5.0
MgCl2 (50mM) 2.5
dNTPs (10mM each) 1.0
PrimerFWD (25 pmol/l) 1.0PrimerREV 1.0
DNA Polymerase 0.5
DNA Template 1.0
Total Volume 50.0
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17/23
A Simpler PCR Reaction
Component lSterile Water 38.0
10X PCR Buffer 5.0
MgCl2 (50mM) 2.5
dNTPs (10mM each) 1.0
PrimerFWD (25 pmol/ul) 1.0PrimerREV 1.0
DNA Polymerase 0.5
DNA Template 1.0
Total Volume 50.0
Component lPREMIX 24.0
Buffer
MgCl2
dNTPs
DNA PolymeraseEnhancers
Sterile Water
Primers FWD+Rev 1.0 DNA
Template 25.0
Total Volume 50.0
PREMIXES CANREDUCE THENUMBER OF
ITEMS ADDED TO
THE MIX
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18/23
Using a PCR Mastermix
Component 1X(l) 20X(l)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 20PrimerREV 1.0 20
DNA Polymerase 0.5 10
DNA Template 1.0 --
Total Volume 50.0 980 Aliquot
49 l
Add DNA
as last step
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19/23
Typical Thermal CyclerConditions
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 times6. Hold at 4 C
7. End
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20/23
Finally: Some Mis-Uses (malasaplicaciones) of PCR
Since PCR is extremely sensitive, it is highlyprone to giving false positive results.
Forensic and medical applications use strictprotocols to minimize chance of errors.
Universities, working without standardizedprotocols, have frequently made incrediblediscoveries, only to find out later that itwas all due to PCR artifacts.
Ancient DNA
GM Crops
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21/23
MisUses of PCR: Ancient DNA
http://www.newscientist.com/hottopics/dinosaurs/backfrom.jsp
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 inamber. '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 tobreak 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.
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22/23
MissUses of PCR: GM CropsAn intense scientific debate has opened up overwhether 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 remotearea.
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_1911000/1911535.stm
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23/23
End of PCR Introduction
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