Advanced PCR

1
Advanced PCR

• Dave Palmer, Byotix, Inc.

2
Advanced PCR

• PCR of Plant Material
• Multiplex PCR
• Modifications to Standard PCR
• PCR Troubleshooting

3
Plant DNA Extraction

• Rapid 3-Step Extraction Method
• Freeze
• TPS
• 100 mM Tris pH 9.5
• 1 M KCl
• 10 mM EDTA
• Heat 95C

D. Thomson and R. Henry, 1995, Single-step
protocol for preparation of plant tissue for
analysis by PCR, BioTechniques, 19394-400
4
How PCR works

• Cold Spring Harbor Animation
• PCR.EXE

5
Review The structure of DNA
Helix
Complementary Base Pairing
6
Multiplex PCR What

• PCR using several primer pairs SIMULTANEOUSLY
• Typically generates a product band for each
  primer pair

7
Multiplex PCR Why

• Detect several genes at once
• eg. transgenic plant screen
• Internal controls
• VERY important
• Tells you how well the PCR reaction worked
• Reduces false negatives

8
Multiplex PCR How

• Same as regular PCR
• Care in primer design
• Much greater chance of primer-dimers
• Annealing temperatures must be close
• Much greater chance of artifacts

9
A Typical PCR Reaction
Component ml 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
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A Typical Multiplex PCR Reaction
Component ml Sterile Water 34.0 10X PCR
Buffer 5.0 MgCl2 (50mM) 2.5
dNTPs (10mM each) 1.0 Primer1FWD
1.0 Primer1REV 1.0 Primer2FWD
1.0 Primer2REV 1.0
Primer3FWD 1.0 Primer3REV
1.0 DNA Polymerase 0.5 DNA Template
1.0 Total Volume 50.0
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Multiplex PCR Example

• Three primer pairs
• Effect, Marker, and Internal Control genes
• Control is smallest fragment, also brightest band
• Effect is largest fragment, faintest band

Effect Gene
Marker Gene
Control Gene
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Multiplex PCR Example

• Three primer pairs

Effect Gene
Marker Gene
Control Gene
Which are transgenic?
13
Multiplex PCR

• Problem
• If the control gene product is the BRIGHTEST in a
  set, then its very difficult to tell the
  difference between weak PCR reactions and
  nontransgenics.
• Solution
• Redesign primers.

Effect Gene?
Marker Gene?
Control Gene
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Multiplex PCR Example

• Three primer pairs
• Control, marker, and effect genes
• Control gene fragment is largest and (almost)
  faintest
• Effect gene is smallest and brightest

Control Gene
Marker Gene
Effect Gene
Primers
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Multiplex PCR Example

• Three primer pairs

Control Gene
Marker Gene
Effect Gene
Primers
Which are transgenic?
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Other Types of PCR

• Different templates
• Nested PCR
• RT-PCR
• Unusual protocols
• iPCR
• Real-time PCR

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PCR Troubleshooting

• The effect of each component

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PCR Reaction Components

• Water
• Buffer
• DNA template
• Primers
• Nucleotides
• Mg ions
• DNA Polymerase
• Extras

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PCR Reaction Components

• Water

• Purity
• Contamination
• Amplification Products

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PCR Reaction Components

• Buffer

• Must match polymerase
• Typically contain KCl and Tris
• Can vary over a slight range
• Not much difference in range from 0.8 X to 2.0 X
• Primer efficiency reduced outside this range

http//info.med.yale.edu/genetics/ward/tavi/p06.ht
ml
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PCR Reaction Components

• DNA template

• Amount of DNA present
• Less DNA means more cycles
• Complexity of DNA
• Eg. plasmid vs. whole genome
• Purity
• Interfering factors, eg. enzymes, salts
• Degradation
• PCR more forgiving of degraded DNA
• Contamination
• Amplification products
• Presence of poisons
• Eg. EDTA which scavenges Mg

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PCR Reaction Components

• Primers

• Age
• Number of freeze-thaws
• Contamination
• Amount
• Can vary over a wide range (50X)
• 100-500 nM typical
• Too low low amplification
• Too high low amplification

http//info.med.yale.edu/genetics/ward/tavi/p05.ht
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PCR Reaction Components

• Nucleotides

• 20-400 uM works well
• Too much can lead to mispriming and errors
• Too much can scavenge Mg
• Too low faint products
• Age
• Number of freeze-thaws
• Just 3-5 cycles is enough to make PCRs not work
  well
• Dilute in buffer (eg. 10mM Tris pH 8.0 to prevent
  acid hydrolysis)
• Contamination

http//info.med.yale.edu/genetics/ward/tavi/p13.ht
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PCR Reaction Components

• Mg is an essential cofactor of DNA polymerase
• Amount can vary
• 0.5 to 3.5 uM suggested
• Too low Taq wont work
• Too high mispriming

• Mg ions

http//info.med.yale.edu/genetics/ward/tavi/p14.ht
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PCR Reaction Components

• Bottom Line
• All components work over a wide range.
• Need to avoid contamination.
• Optimization by trial-and-error.

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PCR Reaction Components

• DNA Polymerase

• Thermostable?
• Activity declines with time at 95C
• Matches buffer?
• Age
• Contamination
• Concentration Typically 0.5 to 1.0 U/rxn

http//info.med.yale.edu/genetics/ward/tavi/p12.ht
ml
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PCR Reaction Components

• Extras

• Proprietary or added by user
• Glycerol, DMSO
• Stabilize Taq, decrease secondary structure
• May help or hurt, depending on primers
• Typically already in the Taq stock
• BSA
• Frequently helps, doesnt hurt
• Betaine
• Useful for GC-rich templates

http//info.med.yale.edu/genetics/ward/tavi/p16.ht
ml
http//taxonomy.zoology.gla.ac.uk/rcruicks/additi
ves.html
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PCR Cycling Parameters

• Denaturation Temp
• Annealing Temp
• Extension Temp
• Time
• Number of Cycles
• Reaction Volume
• Odd Protocols

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PCR Cycling Parameters

• Denaturation Step

• Must balance DNA denaturation with Taq damage
• 95C for 30 - 60s typically is enough to denature
  DNA
• Taq loses activity at high temps
• Half-life at 95C 40 min
• Half-life at 97.5C 5 min

http//info.med.yale.edu/genetics/ward/tavi/p08.ht
ml
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PCR Cycling Parameters

• Annealing Step

• Most critical step
• Calculate based on Tm
• Often does not give expected results
• Trial-and-Error
• Almost always must be done anyway
• Too hot no products
• Too cool non-specific products
• Gradient thermocyclers very useful
• Typically only 20s needed for primers to anneal

http//info.med.yale.edu/genetics/ward/tavi/p08.ht
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PCR Cycling Parameters

• Extension Step

• Temperature typically 72C
• Reaction will also work well at 65C or other
  temps
• Time (in minutes) roughly equal to size of the
  largest product in kb
• Polymerase runs at 60bp/s under optimum
  conditions
• Final long extension step unnecessary

http//info.med.yale.edu/genetics/ward/tavi/p08.ht
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http//info.med.yale.edu/genetics/ward/tavi/p10.ht
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PCR Cycling Parameters

• Number of Cycles

• Number of source molecules
• gt100,000 25-30
• gt10,000 30-35
• gt1,000 35-40
• lt50 20-30 fb. nested PCR
• Do not run more than 40
• Virtually no gain
• Extremely high chance of non-specific products
• Best optimized by trial-and-error

http//info.med.yale.edu/genetics/ward/tavi/p08.ht
ml
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PCR Cycling Parameters

• Reaction Volume

• Doesnt affect PCR results as long as volume is
  within limits.
• Heated lid important.
• 5ul, 20ul, 100ul all work.
• Slightly higher yield with lower volumes.

http//info.med.yale.edu/genetics/ward/tavi/p03.ht
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PCR Cycling Parameters

• Odd Protocols

• Hot-Start PCR
• Taq is added last
• Touchdown PCR
• Annealing temp is progressively reduced

35
Adventures in PCR

• Theres a fly in my primer!
• The protocol that never worked.

36
Theres A Fly In My Primer!

• This happened when we were first developing PCR
  methods.
• Transgene-specific primers not available for
  testing.
• Needed primers to test DNA extraction protocol,
  multiplexing.
• Synthesized NS-series primers
• Pastrik, 2000
• A plant-specific primer set suitable for potato
  amplification.

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Theres A Fly In My Primer!

• First Experiment
• DNA extraction and multiplexing
• Worked great!
• Second Experiment
• To confirm first results
• Great, but ghost bands in controls. No cause
  for alarm.
• Implemented contamination controls.

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Theres A Fly In My Primer!

• Third Experiment
• To determine optimum number of cycles
• Contamination in controls, blamed on DNA on
  kimwipes.
• Contamination perfectly matched NS band.
• Four Experiment
• Clear NS bands in control lanes!
• Controls only H2O samples.
• What the heck is going on?!

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Theres A Fly In My Primer!

• Fifth Experiment
• Designed to determine where in DNA extraction
  process the contamination was getting in
• Get leaf gt Freeze leaf gt Add TPS gt Cook extract gt
  Spin extract gt Add DNA to PCR Tube
• Contamination was all across the board, including
  in UNOPENED PCR TUBES.

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Theres A Fly In My Primer!

• Other Key Observations
• Another primer pair (PHY) failed to work during
  this period of time.
• One morning the freezer door was slightly open
  didnt shut properly.
• Primers were diluted in H2O (no preservative such
  as EDTA)
• NS primer pair binds to rDNA genes, common across
  many species (not just potato).

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Theres A Fly In My Primer!

• SO... What Happened?
• One or more of the reagent tubes became
  contaminated. Likely primers.
• Contaminating organism was able to multiply a few
  cycles while the freezer door was open.
• Suspect contaminant was an oomycete fungus has
  similar NS gene.
• Extraordinary sensitivity of PCR led to detection
  of the NS gene in the contaminating reagent!

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Theres A Fly In My Primer!

• Moral of the Story
• PCR is VERY sensitive!
• Contamination with template DNA can come from the
  most unlikely source!

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The Protocol That Never Worked

• Early PCR method development
• We were setting up our PCR lab
• Method for DNA extraction and PCR supplied by lab
  in Holland
• Hey, its always best to use a protocol that
  works for someone else, right?
• Tried method
• Total failure. No bands at all.

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The Protocol That Never Worked

• Read about PCR troubleshooting.
• Changed Conditions
• More cycles. Less cycles.
• Hotter. Cooler.
• Known good primers.
• More Mg.
• New nucleotides.
• New polymerase.
• All failed.

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The Protocol That Never Worked

• Needed Known Good primers and DNA to properly
  test the PCR
• Went down the road to USDA-Albany to borrow some
  known good template DNA.
• Had primers known to work on potato DNA
  synthesized.
• IT WORKED!
• So That Was A Hint
• Must be something to do with DNA extraction
• Either not enough DNA, or some other problem...

46
The Protocol That Never Worked

• Immediately requested original paper that the DNA
  extraction protocol was based on.
• Learned a LOT from reading original paper!
• Authors examined effects of all sorts of
  conditions
• Freezing, temp, salt, EDTA, time, etc.
• Varying any condition by up to 50 didnt
  substantially affect results.
• Therefore we couldnt have screwed up the
  extraction.
• Comment Addition of EDTA requires a
  compensatory increase in the concentration of
  MgCl2.

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The Protocol That Never Worked

• Quick Calculations
• TPS buffer is 10 mM EDTA
• We added 1.0 ul of extract, therefore 10 nmol
  EDTA.
• The protocol called for 1.0 ul of 50 mM MgCl2,
  therefore 50 nmol Mg.
• One EDTA molecule can bind two Mg ions.
• Therefore, the TPS buffer was immediately
  scavenging almost half of the Mg we were
  adding!!!!

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The Protocol That Never Worked

• Quick Experiment
• Run a Mg gradient.
• No amplification with 1.0, 1.5 ul MgCl2
• Good amplification with 2.0 and 2.5 ul!
• The problem was all in the magnesium
• Actually, tried this earlier, but just happened
  not to try enough the first time...
• Never did determine why the Holland lab didnt
  have problems.

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The Protocol That Never Worked

• Moral of the Story
• Just because a protocol works for someone else is
  NO GUARANTEE it will work for you!
• Dont expect things to work out the first time
  out.
• Consider the troubleshooting to be a learning
  process youll learn SO much MORE if things go
  wrong and you have to figure out why!
• Its ALWAYS a good idea to go to the original
  source of the method!
• In this case, it was the original scientific
  paper which had the key to the answer.

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