EB3302 Cell and Molecular Laboratory

Factors affecting PCR

amplifications and the
troubleshooting
Regina Ho Chang Lin 00013655
Loo Jun Mae 00013428
Loo Jia Hao 00013255
Woon Wei Khong 00013254
What is PCR?
Polymerase Chain Reaction
A technique used to multiply a specific region of
DNA in vitro

(CSH, n.d.); (ThermoFisher, n.d.) Components needed in PCR

Buffer
MgCl2
dNTP
Primers
DNA sample
DNA polymerase
Pre-Denaturation
94C for 3-5 mins
Denaturation
94C for 30s
Annealing
Depends on the melting temperature of
primer 5 C
Targeted strand 1kbp = 30s
Targeted strand > 1kbp = 1min
Extension
72 C
Targeted strand 1kbp = 30s
Targeted strand > 1kbp = 1min
Final Extension
72 C for 3-5mins
Incubation
4 C for
 Factors that affecting
PCR amplification
1. Primer Length
- The longer primer used, the higher
2. Base Composition of Primer
annealing temperature needed
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as thetargeted
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Aviod long than(A+T)
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-- The longer the targeted DNA, the lower contain
4. - The best
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designedis primer
bestSequence 40%-60% of (G+C)
is between
efficiency the PCR is
- 18-30bp
The primer should not have self
5. Denaturation
- The GC rich region Time and
in targeted DNA may
complementary region, might lead to hairpin
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Temperature denaturation
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- The
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should not C for 3-5mins
- If the temperature
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primertaken is too long, the DNA template will
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degrade
References List
Cold Spring Harbour Laboratory, CSH. (n.d.). Biology Animation
Laboratory: Polymerase Chain Reaction. [Online]. Retrieved on 5th April
2017 from: https://www.dnalc.org/resources/animations/pcr.html
ThermoFisher Scientific. (n.d.). PCR. [Online]. Retrieved on 5th April
2017 from: https://www.thermofisher.com/my/en/home/life-
science/pcr.html
Annealing temperature and time
Reaction is cooled to 50-65C.
If annealing temperature is too high, pairing between the primers and
template DNA will not take place and the PCR will fail
Low temperature to enable hybridization between primer and
template
high temperature to prevent amplification of non-target sites
Ideal annealing temperature is 56-61 degree and time is 30sec/kbp
Melting temperature
Depends on the length and GC content of the primer
The goal in choosing an annealing temperature is to balance the need
for stable pairing between primer and template (better at lower
temperatures) with maximizing specific pairing (higher temperatures
destabilize imperfectly matched primer-template duplexes, reducing
non-specific priming).
Extension temperature and time
Heat is increased to 72C to enable the new DNA to be made by a
special Taq DNA polymerase enzyme which adds DNA bases.
Taq DNA polymerase is an enzyme of thermophile bacteria
Stable at high temperature above 80 C
72C is the optimum temperature for the Taq polymerase to build the
complementary strand.
The duration of this step depends on the length of DNA sequence
being amplified but usually takes around one minute to copy 1,000
DNA bases (1Kb).
Concentration of different reagents
Mg concentration - 0.5 and 5.0 mM
dNTP - Concentrations of 200-250M of each dNTP recommended
for Taq polymerase in reactions containing 1.5 mM MgCl2.
Increasing concentration of dNTPs over 200 mM is not advisable
because it has two deleterious effects
Increase polymerase errors and dNTPs bind magnesium
PCR buffer - contains 50mM KCl and works well for amplification of
segments of DNA >500bp in length
Raising the KCl concentration to ~70-100mM often improves the yield of
shorter DNA segments.
Number of cycles
To produce a band visible on a gel depends largely on the starting
concentration of the target DNA template.
The number of cycles required for amplification depends on the
number of copies of template DNA present at the beginning of the
reaction and the efficiency of primer extension and amplification.
Ideal number of cycles is 25-35.
 PCR
A M P L I F I C AT I O N

TROUBLESHOOTING

PRESENTER
LOO JIA HAO
WO O N W E I K H O N G
 LOW OR NO AMPLIFICATION

No Band
DNA
Primers
templates

Possible Causes

Other Thermal
Faint Band reaction cycling
components conditions
DNA TEMPLATES
Possible Cause Recommendation
Poor integrity Minimize shearing and nicking of DNA during isolation - evaluate template DNA
integrity by gel electrophoresis, if necessary.
Store DNA in molecular-grade water or TE buffer (pH 8.0) to prevent
degradation by nucleases.
Low purity Ensure that no residual PCR inhibitors such as phenol, EDTA, and proteinase K
are present if following chemical or enzymatic DNA purification protocols.
Re-purify, or precipitate and wash DNA with 70% ethanol, to remove residual
salts or ions (e.g., K+, Na+, etc.) that may inhibit DNA polymerases.
Choose DNA polymerases with high processivity (display high tolerance to
common PCR inhibitors carried over from soil, blood, plant tissues, etc.)
Insufficient quantity Examine the quantity of input DNA and increase the amount if necessary.
Choose DNA polymerases with high sensitivity for amplification.
If appropriate, increase the number of PCR cycles.
DNA TEMPLATES (CONTD)
Possible Cause Recommendation
Complex targets Choose DNA polymerases with high processivity (display high affinity
(e.g., GC-rich or secondary for DNA templates and are more suitable to amplify difficult targets)
structures) Use a PCR additive or co-solvent to help denature GC-rich DNA and
sequences with secondary structures.
Increase denaturation time and/or temperature to efficiently separate
double-stranded DNA templates.
Long targets Check amplification length capability of the selected DNA polymerases
and use DNA polymerases specially designed for long PCR.
Choose DNA polymerases with high processivity (which can amplify
long targets in a shorter time)
Reduce the annealing and extension temperatures to help primer
binding and enzyme thermostability.
Prolong the extension time according to amplicon lengths.
PRIMERS
Possible Cause Recommendation
Problematic design Review primer design (Use online primer design tools when appropriate)
Ensure that the primers are specific to the target of interest.
Verify that the primers are complementary to the correct strands of the target
DNA.
Old primers Aliquot primers after resuspension and store properly.
Reconstitute fresh primer aliquots, or obtain new primers if necessary.
Insufficient quantity Optimize primer concentrations (usually in the range of 0.11 M).
For long PCR and PCR with degenerate primers, start with a minimum
concentration of 0.5 M.
OTHER REACTION COMPONENTS
Possible Cause Recommendation
Inappropriate DNA Use hot-start DNA polymerases to prevent degradation of primers by the
polymerase 35 exonuclease activity of proofreading DNA polymerases.
Alternatively, set up PCR on ice, or add DNA polymerase last to the reaction
mixture.
Insufficient quantity of Choose DNA polymerases with high sensitivity for amplification.
DNA polymerase Review recommendations on the amount of DNA polymerase to use in PCR,
and optimize as necessary.
Increase the amount of DNA polymerase if the reaction mixture contains a
high concentration of an additive (e.g., DMSO, formamide) or inhibitors from
the sample sources.
Insufficient Optimize Mg2+ concentration for maximum PCR yields
Mg2+concentration Check the DNA polymerases preference for magnesium salt solutions (For
example, Pfu DNA polymerase works better with MgSO4 than with MgCl2 )
OTHER REACTION COMPONENTS
(CONTD)
Possible Cause Recommendation
Excess PCR additives Review the recommended concentrations of PCR additives or co-solvents and
or co-solvents use the lowest possible concentration when appropriate.
Adjust the annealing temperatures, as high concentrations of PCR additives or
co-solvents weaken primer binding to the target.
Increase the amount of DNA polymerase, or use DNA polymerases with high
processivity.
Consider using an additive or co-solvent specifically formulated for a given
DNA polymerase (e.g., GC Enhancer supplied with Invitrogen Platinum
DNA polymerases).
dUTP or modified Ensure that the selected DNA polymerases are able to incorporate the
nucleotides in reaction modified nucleotides.
mix Optimize the ratio of the modified nucleotide to dNTP to increase PCR
efficiency.
Nonhomogeneous Mix the reagent stocks and prepared reactions thoroughly to eliminate density
reagents gradients that may have formed during storage and setup.
THERMAL CYCLING CONDITIONS
Possible Cause Recommendation
Suboptimal Optimize the DNA denaturation time and temperature
denaturation
Suboptimal annealing Optimize the annealing temperature stepwise in 12C increments, using
a gradient cycler when available (The optimal annealing temperature is usually 3
5C below the lowest primer Tm)
Adjust the annealing temperature when a PCR additive or co-solvent is used.
Use the annealing temperature recommended for a specific DNA polymerase in
its optimal buffer. Annealing temperature rules for primer sets can vary between
different DNA polymerases.
Suboptimal extension Select an extension time suitable for the amplicon length.
Reduce the extension temperature (e.g., to 68C) to keep the enzyme active
during amplification of long targets (e.g., >10 kb).
Use DNA polymerases with high processivity for robust amplification even with
short extension times.
Suboptimal number of Adjust the number of cycles (generally to 2535 cycles) to produce an adequate
PCR cycles yield of PCR products. Extend the number of cycles to 40 if DNA input is fewer
than 10 copies.
 SEQUENCE ERRORS WITHIN
PCR PRODUCTS
Possible Cause
Low fidelity of DNA polymerase
Excess Mg2+concentration
Unbalanced dNTP concentrations
High number of cycles
Recommendation
Use DNA polymerases with exceptionally high fidelity to generate
PCR fragments for downstream applications such as cloning,
sequencing, and site-directed mutagenesis.
Review Mg2+ concentrations and reduce as necessary. Excessive
concentrations favor misincorporation of nucleotides by DNA
polymerases.
Ensure equimolar concentrations of dATP, dCTP, dGTP, and dTTP
in the reaction. Unbalanced nucleotide concentrations increase
the PCR error rate.
Reduce the number of cycles without drastically lowering the
yield of the desired PCR products. High numbers of cycles
increase the incorporation of mismatched nucleotides.
Increase the amount of input DNA when appropriate to avoid
running an excessive number of cycles.
 SEQUENCE ERRORS WITHIN
PCR PRODUCTS (CONTD)
Possible Cause Recommendation
UV-damaged DNA Use a long-wavelength UV (360 nm) light box to visualize
fragments in gels, and limit the illumination time as much as
possible.
If using a short-wavelength (254312 nm) light box, limit the UV
illumination to a few seconds and keep the gel on a glass or plastic
plate.
Alternatively, use dyes with longer-wavelength (less damaging)
excitation to visualize the DNA.
Sequencing error Sequence both DNA strands to verify the reliability of sequencing
results. Use duplicate samples when appropriate.
Non-specific amplification or smears
 DNA Templates
Possible Recommendations
causes
Excess DNA Lower the quantity of DNA based on review of optimal amount of DNA input
input

Poor Minimize shearing and nicking of DNA during isolation.

integrity Evaluate the integrity of the template DNA prior to PCR by gel electrophoresis, if necessary. Store DNA in
molecular-grade water or TE buffer (pH 8.0) to prevent degradation by nucleases.

Complex Choose DNA polymerases with high processivity, which display high affinity for DNA templates and are more
sequences suitable to amplify difficult targets.
(e.g., GC-rich Use a PCR additive or co-solvent to help denature complex sequences. Increase denaturation time and/or
or secondary temperature to efficiently separate double-stranded DNA templates.
structures)

Long targets Check amplification length capability of the selected DNA polymerases. Use DNA polymerases specially
designed for long sequences in PCR.
Choose DNA polymerases with high processivity, which can amplify long targets in a shorter time.
Reduce the annealing and extension temperatures to help primer binding and enzyme thermostability.
Prolong the extension time according to amplicon lengths.
 Primers
Possible Recommendations
causes

Problematic Review primer design. Use online primer design tools when appropriate. Verify that the primers are specific to
design the target, with minimal homology to other regions in the template.

Ensure that the primers do not contain complementary sequences or consecutive G or C nucleotides at the 3
ends, to prevent primer-dimer formation.

Avoid direct repeats in the primers to prevent misalignment in binding to the target. Consider longer primers
to enhance specificity.

Consider nested PCR to improve specificity.

High Optimize primer concentrations (usually in the range of 0.11 M). High primer concentrations promote primer-
quantity dimer formation.
 Other reaction components
Possible Recommendations
causes
Excess DNA Review recommendations on the amount of DNA polymerase to use in PCR, and decrease if necessary.
polymerase

Inappropriate Use hot-start DNA polymerases that have no activity at room temperature but are functional only after a high-
DNA temperature activation step, to enhance specificity. With non-hot-start DNA polymerases, set up PCR on ice to
polymerase keep enzyme activity low.

Excess Mg2+ Review Mg2+ concentrations and lower as appropriate to prevent nonspecific PCR products. Optimize Mg2+
concentration concentrations for each primer set and target DNA.
 Thermal cycling conditions
Possible causes Recommendations

Insufficient Increase denaturation time or temperature to efficiently separate DNA when working with GC-rich templates
denaturation and sequences with secondary structures.

Incorrect Use the annealing temperature recommended for a specific DNA polymerase in its optimal buffer.
annealing Increase the annealing temperature to improve specificity. The optimal annealing temperature is usually no
temperature less than 35C below the lowest primer Tm.

Long annealing Shorten the annealing time to minimize primer binding to nonspecific sequences.
time
High extension Reduce the extension temperature 34C to help the DNA polymerases thermostability, especially for long
temperature PCR.
Insufficient Prolong the extension time when amplifying long DNA targets.
extension time Include a final extension step with sufficient time (515 minutes) to extend the whole target
High number of Reduce the number of cycles, without drastically lowering the yield of the desired PCR products, to prevent
cycles accumulation of nonspecific amplicons.
 Sequence errors at termini of PCR products
Possible causes Recommendations

Problematic Avoid direct repeats within primer sequences, as multiple repeats may appear from sequence misalignment
primer design at the ends of the PCR products.

Low primer Order PCR primers with purification to remove nonfull-length DNA oligos, which are truncated at their 5
quality ends.

Contaminating Use molecular-grade, nuclease-free reagents in PCR setup. Set up reactions on ice to keep the activity of
nucleases possible contaminating exonucleases low.

UV-damaged Use a long-wavelength UV (360 nm) light box to visualize fragments in gels, and limit the illumination
DNA time as short as possible.
If using a short-wavelength (254312 nm) light box, limit the illumination to a few seconds and keep the
gel on a glass or plastic plate.
Alternatively, use dyes with longer-wavelength (less damaging) excitation to visualize the DNA.

Sequencing error Sequence both DNA strands to verify the reliability of sequencing results. Use duplicate samples when
appropriate.
 False positive amplification
Possible causes Recommendations

Problematic Avoid primers containing complementary and self-complementary sequences, which favor primer-dimer
primer design formation and self-oligomerization and their subsequent amplification.

Crossover Avoid contamination of DNA in the work environment by following general recommendations for PCR setup
contamination

Carryover Use pipette tips with aerosol barriers. Dedicate a separate work area and decontaminate the area after
contamination each use.

Follow PCR carryover control techniques such as dUTP incorporation with UDG treatment.