Polymerase Chain Reaction

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Polymerase Chain Reaction
Enzyme that copies DNA or RNA
A reaction that, once started, uses its own
product as a reactant thus the reaction feeds
itself leading to exponential growth
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Kary Mullis
Nobel Prize
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(No Transcript)
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How does PCR work?
Start (1 copy)
1. Melting (94, 30 sec)
2. Primer Annealing (45- 65, 30 sec)
3. Primer Extension (72, 1 min per kb)
Finish (2 copies)
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Exponential amplification
Start 1 copy
4th Cycle 16 copies 10th Cycle 1024
copies 20th Cycle 1.05 million copies 30th
Cycle 1.07 billion copies of copies 2n, (n
of cycles)
1st Cycle 2 copies
2nd Cycle 4 copies
3rd Cycle 8 copies
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What is PCR used for?

• The short answer just about anything with DNA
  (or RNA)
• Cloning into plasmids, etc.
• cDNA creation (cloning genes from mRNA)
• Mutagenesis
• DNA (or RNA) quantification
• Radioactive and non-radioactive labels
• Screening bacterial clones for a correct plasmid
• Genotyping
• DNA sequencing

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PCR Methods

• Conventional PCR
• Reverse-transcriptase (RT) PCR
• Real-time PCR

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Ingredients

• Reactants Final concentration
• PCR reaction buffer (10X) dilute 110
• Magnesium (MgCl2 or MgSO4) 1-5 mM (start with 2
  mM)
• dNTPs 0.2 mM each
• Upstream primer 0.5 uM
• Downstream primer 0.5 uM
• Polymerase 2.5 Units
• Template DNA 20-750 ng
• Mineral oil overlay?
• follow instructions that come with polymerase
• for plasmid DNA, use 20 ng per 100uL reaction
• for genomic DNA, use 100-750 ng per 100uL
  reaction

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Tweaking the reaction

• Magnesium
• Affects the specificity of primer binding to the
  template DNA and the efficiency of the reaction
  (higher Mg lower specificity higher
  efficiency)
• If you get little or no product, try repeating
  the reaction with a range of Mg
• Template DNA
• The purity of the template DNA will affect the
  reaction
• Too much DNA can inhibit the reaction usually
  less is more!
• Primer Annealing temperature
• Start out with this set a couple degrees lower
  than the melting temperature of your primers and
  adjust if necessary.
• If you dont get any product, try lowering the
  annealing temperature
• If you get non-specific bands, try raising the
  annealing temperature

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Polymerases
Taq old faithful, the original PCR polymerase
now comes in every possible flavor
High-fidelity stays on the DNA better and has
proofreading activity, so it amplifies longer
templates with less errors (up to 12 kb)
examples Taq HiFi, Pfu Turbo Hotstart
polymerase is inactive until heated to 94 this
helps to reduce non-specific amplification
(improvise this by not adding the polymerase
until the first cycle has reached 94)
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Primers

• How long do you need them to be?
• 4n chance that you will get a random match (n
  length of primer)
• 15 nt long 1 match per 1 billion base pairs
• 20 nt long 1 match per 1 trillion base pairs
  (human genome is 3 billion)
• Watch for primer homology and possible secondary
  structures
• Primer pairs should not complement each other,
  especially at the 3 end
• Stretches of 5 Gs will form a secondary
  structure also avoid repetitive sequences
• Make sure you design the primers in the right
  orientation!!!

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Designing primers
Melting/Annealing temperature shortcut G or C
4 A or T 2

• Count the of A/T and G/C in your primer
• Tm 2(AT) 4(GC) - 5
• So, a 20 bp primer with 50 GC will have a Tm of
  55 210 410 5 55

5-ACGATGCATGTAGCTACGTAGGtargetCGGGTACTGAACGGC
AAACCTAGTC-3 3-TGCTACGTACATCGATGCATCC.target
GCCCATGACTTGCCGTTTGGATCAG-5
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Controls

• Negative Control (VERY IMPORTANT)
• Add water instead of template DNA
• Confirms that your reagents are not contaminated
• Positive Control
• If possible, run a parallel reaction with
  something that you know the primers will amplify

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• Experiment
• The DHFR gene is present in hamster CHO cells.
  You would like to put the hamster DHFR gene in
  human Hela cells and confirm integration by PCR.
  
• You dont think your primers will amplify a
  target from Hela cells, but youd like to be
  sure.
• You have the DHFR gene from CHO cloned onto a
  plasmid.

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Case 1
DHFR (plasmid)
CHOK-1 DNA
Hela DNA
Water (no DNA)
Marker 1
Marker 2

• Problem

Solutions?
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Case 2
DHFR (plasmid)
CHOK-1 DNA
Hela DNA
Water (no DNA)
Marker 1
Marker 2

• Problem

Solutions?
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Case 3 screening clones
Positive Control
Clone 1
Clone 2
Clone 3
Clone 4
Clone 5
Water (no DNA)
Marker

• Problem

Solutions?
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General tips

• Set up your PCR reactions away
  from where you usually work
• Use sterile technique and wear gloves
• Filtered tips arent a bad idea
• Design primers with similar melting temperatures
• Add your template DNA last

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Reverse Transcriptase PCR

• Purpose
• To make cDNA from an RNA template
• Reverse Transcriptase copies a DNA product from
  an RNA template

RNA
Primer dNTPs Buffer
Reverse Transcriptase
RNA

Normal PCR reaction
Complementary DNA (cDNA)
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PCR applications DNA quantitation

• Conventional PCR
• Semi-quantitative at best
• You only measure the end product, which only
  gives you a rough idea of the amount of starting
  template
• Real-time PCR
• quantitative
• Measures the production of product as it is made
  using a fluorescent dye or fluorescent probe, so
  you can monitor the reaction in real-time and
  accurately measure the amount of starting template

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Real-time PCR How does it work?
(Unbound SyBR green)
(Bound SyBR green)
Melting (94)
Annealing (55)
Extension (72)
Product
Measure Fluorescence
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Real-time PCR
10 copies
100 copies
1000 copies
10,000 copies
100,000 copies
Crossing point
Unknown sample amplifies at cycle 14 what is the
starting copy ?
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Core facilities

• 740 Light Hall get everything you need for PCR
  right here, including primers, polymerases, and
  kits

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Helpful Web Links

• Tons of information about PCR, including working
  with a difficult template. (Includes information
  for beginners for setting up PCR and a
  troubleshooting guide, among other things.)
• www.roche-applied-science.com/sis/amplification/h
  ints/hints_01.htm
• A good place to order any kind of
  oligonucleotide
• www.idtdna.com (Integrated DNA Technologies)
• Website to help with primer design. (Everything
  from a calculator to help with dilutions to
  checking for primer dimers to designing a primer
  set for you.)
• www.idtdna.com/SciTools/SciTools.aspx

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Got Questions?

• Steven Gray
• Ellen Fannings lab, 2325 Stevenson Center
  (on the far side of MRBIII, on the 3rd floor)
• 343-5802
• Steven.j.gray_at_vanderbilt.edu