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7/27/2019 PCR Tips and Tricks
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©2008, Promega Corporation. All rights reserved.
PCR Tips and Tricks
David TampaPromega Field Applications Specialist
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Overview of Topics
• Background information on PCR
•PCR enzymes and systems.
• Troubleshooting PCR
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What is PCR?
PCR (Polymerase Chain Reaction)
• Target DNA
• Thermostable DNA polymerase• Two oligonucleotide primers
• Deoxynucleotide triphosphates (dNTPs)
• Reaction buffer
• Magnesium• Optional additives
È
Thermal Cycler
Components of the PCR Reaction:
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PCR Process
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PCR Process (continued)
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A Typical PCR Protocol
•
Materials:
sterile water
10X amplification buffer with 15mM MgCl2
10 mM dNTP
50 μM oligonucleotide primer 1
50 μM oligonucleotide primer 25 unit/μl Taq Polymerase
template DNA (1 μg genomic DNA, 0.1-1 ng plasmid DNA) in 10 μl
mineral oil (for thermocyclers without a heated lid
1. Combine the following for each reaction (on ice) in a 0.2 or 0.5 ml tube:
10X PCR buffer 10 μl
Primer 1 1 μl
Primer 2 1 μl
dNTP 2 μltemplate DNA and water 85.5 μl
Taq Polymerase 0.5 μl
2. Prepare a control reaction with no template DNA and an additional 10 μl of sterile water.
3. If the thermocycler does not have a heated lid, add 70-100 μl mineral oil (or 2 drops of silicone oil) to each reaction.
4. Place tubes in a thermal cycler preheated to 94 degrees C.
5. Run the following program:
94 degrees C 1 min55 degrees C 1 min or annealing temperature appropriate for particular primer pair
72 degrees C 1 min (if product is <500 bp), 3 min (if product is >500 bp)
for 30 cycles.
Program a final extension at 72 degrees C for 7 min.
Julie B. Wolf, UMBC
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©2008, Promega Corporation. All rights reserved.
Applications
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General Applications
PCR
Reverse
Transcription
(RNA to cDNA)
-Cloning Gene Products- Analyzing Splicing
- qPCR (Quanitation)
- Gene Product
Regulation (miRNA)
DNA
-Cloning Genes/gDNA- ChIP assays
- qPCR (Quanitation)
- methylation studies
-Mutagenesis
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PCR in the Scheme of Molecular Biology
Animal or Organism
ProteinRNA
Gene Expression Studies
(qRT-PCR, Arrays, Northerns
RPAs)
Cloning transcripts
(RT-PCR)
Mutation Analysis(RT-PCR, Arrays)
DNA
Gene StructiureStudies
(Southerns, ChIP)
Cloning regulatory sites
(reporter assays, Footprinting)
Mutation Analysis SNPs
(PCR, Arrays, sequencing)Genotyping
(PCR)
The “Code” The “Response” The “Function”
Protein Structure
(NMR, x-ray)
Protein Expression
(Protein Arrays)
Protein Modifications
(Westerns, MS)Mutation Analysis
(Enzyme assays, Binding Studies)
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Cloning with T-Vectors
pGEM-T vs. pGEM-T Easy
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©2008, Promega Corporation. All rights reserved.
PCR optimization
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PCR Optimization
What parameters can be optimized?
• Magnesium concentration
• Buffer considerations
• Primer design
• Enzyme concentration
• Enzyme choice
• Template considerations
• Cycle parameters
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Magnesium Concentration
Lane M, Promega's pGEM® DNA Markers; Lane 1, 0mM Mg2+; Lane 2, 0.5mM
Mg2+; Lane 3, 1mM Mg2+; Lane 4, 1.5mM Mg2+; Lane 5, 2mM Mg2+; Lane 6 2.5mM
Mg2+; Lane 7, 3mM Mg2+ and Lane 8, 3.5mM Mg2+.
Effects of magnesium concentration on PCR amplification: PCR was
performed using various concentrations of Mg2+ to demonstrate this
effect on the amplification of a 1.8kb target luciferase gene.
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Buffer Considerations
Demonstration of buffer compatibility: A 360bp region of the human
alpha-1-anti-trypsin gene was amplified by PCR using Promega's Taq
DNA Polymerase, in Storage Buffer B or Storage Buffer A, incombination with the reaction buffer supplied by one of three vendors
[Promega (P), Vendor X or Vendor Y].
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Enzyme Choice
Comparison of Thermostable DNA Polymerases
Taq*/ AmpliTa
q®*
VentR®*
(exo-)
VentR®*/
Tli
Deep
VentRTM* Pfu* Pwo* UITmaTM*
5'-3'
Exonuclease
Activi ty
Yes No No No No No No
3'-5'
Exonuclease
Activi ty
No No Yes Yes Yes Yes Yes
Reverse
Transcriptase
Activi ty
Weak Weak n.a. n.a. n.a.. n.a. Weak
Resulting
DNA Ends
A
overhang
s
70%Blunt;
30%
Singlebase
overhangs
Blunt Blunt Blunt Blunt>95%
Blunt
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Enzyme Choice
• 3’ to 5’ Exonuclease Enzymes are called “ Proof
Readers”
• These enzymes have higher fidelity (better accuracy)
• Generally more expensive
• Not as good Strand Displacement as Taq
• Sometimes packaged in Mixes with Taq• No “ A” Tailing, create blunt ends
• Most more susceptible to 95°C Denaturation
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Cycle Parameters
• Annealing temperature- starting approximately 5°C below calculated Tm
• Extension time
- every 1kb of amplicon: 1 minute (Is it always true?)
• Number of cycles
- 25-40 cycles
• Hot start- limit the availability of one necessary reaction component
until reaching a higher temperature (>60°C)
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Touchdown PCR
• Start with High annealing temperature (~5° higher
than theoretical primer annealing temp Tm)
•Successive cycles lower annealing temp 0.5° or 1°
• 95° 1 min
• 65° 20 sec
• 72° 1min
Cycle 1
• 95° 1 min
• 63° 20 sec
• 72° 1min
Cycle 3
• 95° 1 min
• 64° 20 sec
• 72° 1min
Cycle 2
Etc…
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Template Considerations
• Amount:
1µg of 1kb dsDNA = 9.12 x 1011 molecules
1µg of pGEM® Vector DNA = 2.85 x 1011 molecules
1µg of lambda DNA = 1.9 x 1010 molecules
1µg of E. coli genomic DNA = 2 x 108 molecules
1µg of human genomic DNA = 3.04 x 105 molecules
• Quality:
Inhibitors of DNA polymerases: salts, guanidine, proteases,
organic solvents and SDS, etc.“ Spiking” Experiment
Ethanol precipitation of the nucleic acid sample
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Troubleshooting PCR
http://www.promega.com/guides/pcr_guide/070_22/promega.html
• Identify the symptoms
• Find possible causes
Discussion:
- Low yield or no amplification product
- Multiple, nonspecific amplification products
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PCR reactions with Taq tend to be very robust and tolerant of
different reaction conditions. Although many people can directly
substitute other commercial Taq Polymerases in their standard
Taq protocols, some have found it necessary to re-optimize their reactions.
GoTaq Troubleshooting
• Annealing temperature can be different to lower
• Vortex reaction buffers (weighting agents) and MgCl2
• May require an additional 0.5mM Mg with larger amplicons
• Mouse tail genotyping (add less DNA)
• Green/Red/Blue loading buffers can interfere with
•Fluorescent detection
• 5X and 2X buffers (not 10x)
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More Troubleshooting
• What else can go wrong?
Reaction size too small (not enough enzyme)
Reducing the size of the reaction can dilute the
template, enzyme and other components that can effect
reactions.
Primers not specific
Magnesium, Magnesium, Magnesium
Magnesium is imortant cofactor thought to help stabilize
DNA structure. In general more is needed for gDNAamplification
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Yet More Troubleshooting
• Thermocyclers can loose calibration over time,
• Some blocks on thermocyclers do not support “fast cycling”
protocols
• Older thermocyclers need mineral oil layer over reactions
• Bad nucleotides
• Nucleotides are liable to freeze/thaw cycles and higher
temperatures
• DNA contaminants
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How Do I know what to Troubleshoot?
Problem: No
Product
Cloning
Issues
Wrong
Sequence
Non-
Specific
Thermocycler
Issue
X
[MgCl2] XX
Template/
Primer
X XX XX XX
Cycling/Rxn
Conditions
X X XX
Enzyme X XX XX
Reagents XX
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Control Reaction
• The best and most efficient way to troubleshoot reactions
Work out conditions with readily avalible control so future
troubleshooting is easy
• Plasmids work as control templates but can have slightlydifferent conditions compared to gDNA or cDNA
• For qPCR a standard curve can help with absolute
quantitation
• You can later “spike” control reactions to analyze
components
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Primer Design
• General Consideration:
- Length ranging from 15–30 bases
- G+C content 40–60%
- Avoid internal secondary structure- The 3´-ends of the primers should not be complementary
- Avoid three G or C nucleotides in a row near the 3´-end
- Ideally, both primers should anneal at the same temperature
• Melting Temperature (Tm) calculation:Tm = 81.5 + 16.6 x (log10[Na+]) + 0.41 * (%G+C) – 675/n
( [K+] = [Na+]; n = number of bases in the oligonucleotide)
e.g. calculate the Tm of a 22mer oligonucleotide with 60% G+C in 50mM KCl:
Tm = 81.5 + 16.6 x (log10[0.05]) + 0.41 * (60) – 675/22 = 53.84°C
BioMath Calculators
• Restriction enzyme sites placed at the 5´-ends of the primers for convenient
cloning of PCR product.
( Digestion of Restr iction Sites Close to the End of Linear DNA:
http://www.promega.com/guides/re_guide/chaptwo/2_6.htm )
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Primer Design
• Other Considerations:
Salt conditions when calculating Tms
Primer Dimers/Hairpins
“A” and “T” ends Restriction sites built in?
Extra base overhangs?
Antisense sequence
GC or AT rich regions
Purity of primers (desalted, HPLC)
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Primer Design Tools
• http://www.idtdna.com/SCITOOLS/scitools.aspx
• http://www.promega.com/biomath/
• http://frodo.wi.mit.edu/primer3/
• http://www.ncbi.nlm.nih.gov/tools/primer-blast/
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©2008, Promega Corporation. All rights reserved.
GoTaq® Hot Start Polymerase
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Problems with some conventional amplif ications
With some amplifications have:
• Nonspecific priming
• Primer dimer formation
This can lead to:
• Secondary products and lack of specificity
• Lower yields
• Lower sensitivity
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How to eliminate some of these problems?
Hot-Start PCR
What is hot-start PCR?• In hot-start PCR the amplification reaction is rendered inactive
until the temperature reaches 60-95°C eliminating or minimizing
the binding of the primers to each other or at non-homologous
locations in the target DNA.
How?
• Separate reaction components until reactions reach high
temperature
• Inactivate components until reactions reach high temperature
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Commercialized hot-start methods
• Embed Taq DNA polymerase or Mg in Wax
Promega’s TaqBead™ Hot Start Polymerase (M5661)
• Antibody inhibition of Taq DNA polymerase
This is method used for GoTaq® Hot Start Polymerase
• Chemical modification of Taq DNA polymerase to inactivate at lowtemperatures
• Sequester primers
• Others… Various ways to bind magnesium, polypeptide methods
We’ll talk more about these methods later…
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Vendor Comparison
Vendor Product Hot-start Method
Invitrogen Platinum® Taq DNA
Polymerase
Antibody
Applied Biosystems AmpliTaq Gold®
ChemicalQiagen HotStarTaq® DNA
Polymerase
Chemical
Promega GoTaq® Hotstart DNA
Polymerase
Antibody
USB HotStart-IT™Taq DNA
Polymerase
Sequester primers
Takara TaKaRa Taq™ Hot
Start Version
Antibody
GE illustra Hot Start Master
Mix
Sequester primers
Promega TaqBead™ Hot Start
Polymerase (M5661)
Wax bead
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Set up and cycling
• Set up reactions at room temperature
• Put reactions in room temperature thermal cycler
• Cycling (Just l ike GoTaq® DNA Polymerase!)
Initial denaturation: 2 minutes at 94-95°C (inactivates the antibodies and
denatures DNA)
Denaturation: 94-95°C for 30 seconds to 1 minute
Annealing: optimize specifically for primer melting temperature
Extension: 72-74°C, allow 1 minute for every 1kb to be amplified
Cycle number: 25-30 cycles but can go up to 40 cycles
Final extension: 5minutes at 72-74°C
Refrigeration: 4°C soak recommended
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Hot-start versus no hot-start
Note: Standard Taq is
GoTaq® DNA Polymerase(M300)
Note: This is the hot-
start model for QC test
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Applications
• Assume these applications will be the same for GoTaq® Hot
Start Polymerase and GoTaq® DNA Polymerase.
Clone into T-Vectors, enzyme has template independent addition of
single deoxadenosine to 3’end
Purify amplification products using Wizard SV Gel and PCR Clean-Up
System (A9281) and Wizard SV 96 PCR Clean-Up System (A9341)
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Problems with the other hot-start methods
• Wax Bead: The wax barrier is inconvenient. Not possible to
adjust component (Mg or polymerase) present in wax bead.
• Chemical modification: Initial denaturation step is 95ºC for 5-15
minutes.
• Sequester primers: Limitation of amount of primers that can be
used.
Antibody hot-start method does nothave any of these problems.
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Troubleshooting
• Customer switching from product using chemical modificationmethod of hot-start has decreased yield with GoTaq® Hot Start
Polymerase.
Possible Cause: Initial denaturation to activate GoTaq® Hot StartPolymerase should be only 2 minutes at 94-95°C! Do not use 5-15 minute denaturation used with chemically modified Taq DNApolymerases. Extended time at 94-95°C will inactivate thepolymerase.
Comment: Use initial denaturation step of 2 minutes at 94-95°C to
activate GoTaq® Hot Start Polymerase.
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Summary: Hot Start Advantages
• Hot-start gives improved specificity, sensitivity and yield for some
amplifications
• Set up reactions at room temperature
• Put reactions in room temperature thermal cycler
• Can let reactions sit at room temperature for at least 24 hours before the
cycling protocol is started. Good for robotics!
• No long initial denaturation step in cycling protocol
• Can optimize magnesium – the buffers do not contain magnesium
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Summary: GoTaq® Hot Start Advantages
And as always with GoTaq® DNA Polymerase…
• Direct load gels with Green GoTaq® Flexi Buffer
• Use Colorless GoTaq® Flexi Buffer when direct fluorescence or
absorbance readings are required
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Miscellaneous Reagents
• Nucleotides (dATP, dTTP, dGTP, dCTP, dUTP)* Use of dUTP to prevent DNA Cross-Contamination
(UNG: Uracil-N-Glycosylase)
•Betaine, DMSO, and other enhancers
• PCR Nucleotide Mix vs. dNTP mix
• Pipette Tips
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Online Technical Resources
• Technical Bulletins and Manuals
• Promega Notes Articles
• Frequently Asked Questions
• Product Applications Page
• Guides: PCR Protocols & ReferencePolymerases
• Tools: BioMath Calculators
• GoTaq Conversion Campaign
Wrap Up Exercise (Cont’d)
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The customer called in and said he got no PCR amplification.
Here is information he provided:
Wrap-Up Exercise (Cont d)
He is amplifying 500 bp from genomic DNA,
using specific primers with Tm 66oC. He
uses 60o
C for annealing temperature,1.5 mMMgCl2 final concentration, and 1.25 units
Gotaq DNA Polymerase. He followed the
PCR cycle conditions recommended in the
PCR Core System.
How would you help troubleshooting the situation?
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