PCR Tips and Tricks

David Tampa Promega Field Applications Specialist

2008, Promega Corporation. All rights reserved.

Overview of Topics
Background information on PCR PCR enzymes and systems. Troubleshooting PCR

What is PCR?
PCR (Polymerase Chain Reaction)
Components of the PCR Reaction:
Target DNA Thermostable DNA polymerase Two oligonucleotide primers Deoxynucleotide triphosphates (dNTPs) Reaction buffer Magnesium Optional additives

Thermal Cycler
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PCR Process

PCR Process (continued)

A Typical PCR Protocol

Materials: sterile water 10X amplification buffer with 15mM MgCl2 10 mM dNTP 50 M oligonucleotide primer 1 50 M oligonucleotide primer 2 5 unit/l Taq Polymerase template DNA (1 g genomic DNA, 0.1-1 ng plasmid DNA) in 10 l 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 l Primer 1 1 l Primer 2 1 l dNTP 2 l template DNA and water 85.5 l Taq Polymerase 0.5 l 2. Prepare a control reaction with no template DNA and an additional 10 l of sterile water. 3. If the thermocycler does not have a heated lid, add 70-100 l 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 min 55 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

Applications

2008, Promega Corporation. All rights reserved.

General Applications
DNA
Reverse Transcription (RNA to cDNA)

PCR
-Cloning Genes/gDNA - ChIP assays - qPCR (Quanitation) - methylation studies -Mutagenesis -Cloning Gene Products - Analyzing Splicing - qPCR (Quanitation) - Gene Product Regulation (miRNA)

PCR in the Scheme of Molecular Biology

Animal or Organism

The Code
DNA

The Response The Function

RNA
Gene Expression Studies (qRT-PCR, Arrays, Northerns RPAs) Cloning transcripts (RT-PCR) Mutation Analysis (RT-PCR, Arrays)

Protein
Protein Structure (NMR, x-ray) Protein Expression (Protein Arrays) Protein Modifications (Westerns, MS) Mutation Analysis (Enzyme assays, Binding Studies)

Gene StructiureStudies (Southerns, ChIP) Cloning regulatory sites (reporter assays, Footprinting) Mutation Analysis SNPs (PCR, Arrays, sequencing) Genotyping (PCR)

Cloning with T-Vectors

pGEM-T

vs.

pGEM-T Easy

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PCR optimization

2008, Promega Corporation. All rights reserved.

PCR Optimization

What parameters can be optimized?

Magnesium concentration Buffer considerations Enzyme choice Enzyme concentration Primer design Template considerations Cycle parameters

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Magnesium Concentration
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.

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+.
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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, in combination 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*
5'-3' Exonuclease Activity 3'-5' Exonuclease Activity Reverse Transcriptase Activity

VentR*
(exo-)

VentR*/ Tli

Deep VentRTM*

Pfu*

Pwo*

UITmaTM*

Yes

No

No

No

No

No

No

No

No

Yes

Yes

Yes

Yes

Yes

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 95C Denaturation

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Cycle Parameters

Annealing temperature
- starting approximately 5C 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 (>60C)
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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 Cycle 1 95 1 min 65 20 sec 72 1min 95 1 min 64 20 sec 72 1min 95 1 min 63 20 sec 72 1min

Cycle 2

Cycle 3

Etc

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Template Considerations
Amount: 1g of 1kb dsDNA = 9.12 x 1011 molecules 1g of pGEM Vector DNA = 2.85 x 1011 molecules 1g of lambda DNA = 1.9 x 1010 molecules 1g of E. coli genomic DNA = 2 x 108 molecules 1g 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
Identify the symptoms Find possible causes

Discussion: - Low yield or no amplification product - Multiple, nonspecific amplification products

http://www.promega.com/guides/pcr_guide/070_22/promega.html

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GoTaq Troubleshooting
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. 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 gDNA amplification

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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: Thermocycler Issue [MgCl2] Template/ Primer Cycling/Rxn Conditions Enzyme Reagents No Product X XX X X X XX XX XX XX X XX XX XX Cloning Issues Wrong Sequence NonSpecific

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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 slightly different 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 1530 bases - G+C content 4060% - 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 = 53.84C BioMath Calculators Restriction enzyme sites placed at the 5-ends of the primers for convenient cloning of PCR product. ( Digestion of Restriction 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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GoTaq Hot Start Polymerase

2008, Promega Corporation. All rights reserved.

Problems with some conventional amplifications

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 60-95C 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 Promegas 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 low temperatures Sequester primers Others Various ways to bind magnesium, polypeptide methods
Well talk more about these methods later
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Vendor Comparison
Vendor Invitrogen Product Platinum Taq DNA Polymerase HotStarTaq DNA Polymerase GoTaq Hotstart DNA Polymerase HotStart-ITTaq DNA Polymerase TaKaRa Taq Hot Start Version Hot-start Method Antibody Chemical Chemical Antibody Sequester primers Antibody

Applied Biosystems AmpliTaq Gold Qiagen Promega USB Takara GE Promega

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illustra Hot Start Master Sequester primers Mix TaqBead Hot Start Polymerase (M5661) Wax bead

Set up and cycling

Set up reactions at room temperature Put reactions in room temperature thermal cycler Cycling (Just like GoTaq DNA Polymerase!) Initial denaturation: 2 minutes at 94-95C (inactivates the antibodies and denatures DNA)

Denaturation: 94-95C for 30 seconds to 1 minute Annealing: optimize specifically for primer melting temperature Extension: 72-74C, 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 72-74C Refrigeration: 4C soak recommended

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Hot-start versus no hot-start

Note: Standard Taq is GoTaq DNA Polymerase (M300) Note: This is the hotstart 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 3end 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 95C for 5-15 minutes. Sequester primers: Limitation of amount of primers that can be used.

Antibody hot-start method does not have any of these problems.

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Troubleshooting
Customer switching from product using chemical modification method of hot-start has decreased yield with GoTaq Hot Start Polymerase. Possible Cause: Initial denaturation to activate GoTaq Hot Start Polymerase should be only 2 minutes at 94-95C! Do not use 515 minute denaturation used with chemically modified Taq DNA polymerases. Extended time at 94-95C will inactivate the polymerase. Comment: Use initial denaturation step of 2 minutes at 94-95C 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 GoTaq Conversion Campaign Guides: PCR Protocols & Reference Polymerases Tools: BioMath Calculators

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Wrap-Up Exercise (Contd)

The customer called in and said he got no PCR amplification. Here is information he provided: He is amplifying 500 bp from genomic DNA, using specific primers with Tm 66oC. He uses 60oC for annealing temperature,1.5 mM MgCl2 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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