PCR Troubleshooting

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PCR Troubleshooting

• Dave Palmer, Bio-Rad

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How PCR works

• Cold Spring Harbor Animation
• PCR.EXE

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

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

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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
• Reduces false positives

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Multiplex PCR How

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

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

• Three primer pairs
• Control, resistance, and trait genes
• Control gene fragment is largest and (almost)
  faintest
• Trait gene is smallest and brightest

Control Gene
Resistance Gene
Trait Gene
Primers
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Multiplex PCR Example

• Three primer pairs

Control Gene
Resistance Gene
Trait Gene
Primers
Which are transgenic?
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Other Types of PCR

• Different templates
• Nested PCR
• RT-PCR (reverse-transcriptase)
• Different protocols
• iPCR
• Touchdown PCR
• 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
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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
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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
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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
• Even 92C for 1s can be enough
• 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
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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 mostly 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
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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

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Programming the Instrument

• Using right plastic?
• Using right seals?
• Heated lid enabled?

• Right protocol entered?
• Is power reliable?
• Proper reaction volume?

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Basic Experimental Design

• A well-designed experiment can keep you from ever
  getting into trouble!
• A poorly-designed experiment is asking for
  problems!!!!

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Basic Experimental Design

• Main point Always use CONTROLS
• Positive control
• So youll know what a successful result looks
  like.
• Negative control
• Lets you know if you have contamination.

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Experimental Design Controls
No positive or negative controls What does this
result mean??
U
Only a positive control How do we know the
result isnt due to contamination?
U

Both positive and negative controls Results can
be interpreted with confidence.
U
-

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Experimental Design Replication
Our unknown is definitely positive... but how
sure are we?
-

We ran the same sample three times. Is our
unknown really positive?
U
-

U
U
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Review

• Here are the key things to know

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Review 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
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Review Purpose of Temp Cycling

• Denaturing
• Annealing
• Extension

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Review Practical uses of PCR

• Disease detection
• Cloning
• Forensics
• Food quality control
• Paternity testing
• Identification

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Review Basic Experimental Design

• Unknown Samples
• Positive Controls
• Negative controls

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End of PCR Troubleshooting