How PCR works

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Review: The structure of DNA

Antiparallel Strands

Unzipping

How PCR works

Cold Spring Harbor Animation

PCR.EXE

How PCR works

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How PCR works

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How PCR works

Blackboard(Dave attempts to draw this out!)

PCR Reaction Components

WaterBufferDNA templatePrimersNucleotidesMg++ ions DNA Polymerase

PCR Reaction: Water

Water The medium for

all other components.

PCR Reaction: Buffer

WaterBuffer 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

WaterBufferDNA template Contains region to be

amplified Any DNA desired Purity not required Should be free of

polymerase inhibitors

PCR Reaction: Primers

WaterBufferDNA templatePrimers

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

WaterBufferDNA templatePrimersNucleotides

Added to the growing chain Activated NTP’s dATP, dGTP, dCTP, dTTP Stored at 10mM, pH 7.0 Add to 20-200 uM in assay

PCR Reaction: Magnesium

WaterBufferDNA templatePrimersNucleotidesMg++ 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

PCR Reaction: Polymerase

WaterBufferDNA templatePrimersNucleotidesMg++ ionsDNA Polymerase

The enzyme that does the extension

TAQ or similar Heat-stable Approx 1 U / rxn

PCR Reaction Components

WaterBufferDNA templatePrimersNucleotidesMg++ ions DNA Polymerase

A Typical PCR ReactionComponent l

Sterile Water 38.0 10X PCR Buffer 5.0 MgCl2 (50mM) 2.5 dNTP’s (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 lSterile Water 38.0 10X PCR Buffer 5.0 MgCl2 (50mM) 2.5 dNTP’s (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 lPREMIX 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

Using a PCR MastermixComponent 1X(l) 20X(l)Sterile Water 38.0 760 10X PCR Buffer 5.0 100 MgCl2 (50mM) 2.5 50 dNTP’s (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 Aliquot49 l

Add DNAas last step

Typical Thermal Cycler Conditions1. Initial Denaturation 95°C 4 min2. DNA Denaturation 95°C 1 min3. Primer Annealing 65°C 1 min4. Primer Extension 72°C 1 min5. Go to step #2, repeat 29 more times6. Hold at 4 C7. End

Finally: Some Mis-Uses (malas aplicaciones) of PCRSince 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 DNAGM Crops

                                       

               

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 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.

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