How PCR works

Cold Spring Harbor Animation Animated .GIF files Blackboard

Review: The structure of DNA

Unzipping Antiparallel Strands

How PCR works

Cold Spring Harbor Animation

PCR.EXE

How PCR works

Animated .GIF #1

How PCR works

Animated .GIF #2

How PCR works

Blackboard (Dave attempts to draw this out!)

PCR Reaction Components

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

PCR Reaction: Water

Water

The medium for all other components.

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

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

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)

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

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

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

PCR Reaction Components

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

A Typical PCR Reaction

Component l Sterile Water 38.0 10X PCR Buffer 5.0 MgCl2 (50mM) 2.5 dNTPs (10mM each) 1.0 PrimerFWD (25 pmol/l) 1.0 PrimerREV 1.0 DNA Polymerase 0.5 DNA Template 1.0 Total Volume 50.0

A Simpler PCR Reaction

Component l
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

PREMIXES CAN REDUCE THE NUMBER OF ITEMS ADDED TO THE MIX

PREMIX 24.0 Buffer MgCl2 dNTPs DNA Polymerase Enhancers Sterile Water Primers FWD+Rev 1.0 DNA Template 25.0 Total Volume 50.0

Using a PCR Mastermix

Component 1X(l) 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 20X(l) 760 100 50 20 20 20 10 -980

Aliquot 49 l

Add DNA as last step

Typical Thermal Cycler Conditions

1. 2. 3. 4. 5. 6. 7. Initial Denaturation 95C DNA Denaturation 95C Primer Annealing 65C Primer Extension 72C Go to step #2, repeat 29 more Hold at 4 C End 4 min 1 min 1 min 1 min times

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

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/backfrom.jsp

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_1911000/1911535.stm

End of PCR Introduction