PCR Tips and Tricks

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



2

Overview of Topics

• Background information on PCR

•PCR enzymes and systems.

• Troubleshooting PCR



3

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:



4

PCR Process



5

PCR Process (continued)



6

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




Applications



8

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



9

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)



10

Cloning with T-Vectors

pGEM-T vs. pGEM-T Easy




PCR optimization



12

PCR Optimization

What parameters can be optimized?

• Magnesium concentration

• Buffer considerations

• Primer design

• Enzyme concentration

• Enzyme choice

• Template considerations

• Cycle parameters



13

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.



14

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].



15

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



16

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



17

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)



18

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…



19

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



20

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



21

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)



22

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



23

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



24

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



25

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



26

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 )



27

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)



28

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/




GoTaq® Hot Start Polymerase



30

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



31

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



32

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…



33

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



34

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



35

Hot-start versus no hot-start

Note: Standard Taq is

GoTaq® DNA Polymerase(M300)

Note: This is the hot-

start model for QC test



36

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)



37

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.



38

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.



39

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



40

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



41

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



42

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)



43

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?

Documents

PCR Tips and Tricks