pcr history pt2[1]

8/2/2019 PCR History Pt2[1]

1/23

How PCR works

Cold Spring Harbor Animation

Animated .GIF files

Blackboard


2/23

Review: The structure of DNA

Antiparallel Strands

Unzipping


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.EXE


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.EXE


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.EXE


6/23

How PCR works

Blackboard

(Dave attempts to draw this out!)
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.EXE


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.EXE


8/23

PCR Reaction: Water

Water The medium for all

other components.
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.EXE


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
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.EXE


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.EXE


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
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.EXE


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
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.EXE


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


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


23/23

End of PCR Introduction
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pcr history pt2[1]

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