VICTORIA BIANCA B.

ACORDA, RMT & JHON MARIO CALIGUIRAN, RMT

1
 OUTLINE

I. What is Polymerase Chain Reaction (PCR)?

II. Steps in PCR amplification.

III. Essential Components of PCR.

IV. Analysis of PCR Product.

V. Different types of PCR techniques.

VI. Applications of PCR.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 2

 I. WHAT IS POLYMERASE CHAIN REACTION
(PCR)?
KARY Milus-1983
Essentially DNA replication in
vitro.
Can copy segments of DNA
by up to a billion-fold in the
test tube, a process called
amplification.
This yields large amounts of
specific genes or other DNA
segments that may be used
for a range of applications in
molecular biology.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 3

 I. WHAT IS POLYMERASE CHAIN REACTION
(PCR)?

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 4

 I. WHAT IS POLYMERASE CHAIN REACTION
(PCR)? (CONTD.)
PCR does not actually copy whole DNA molecules
but amplifies stretches of up to a few thousand base
pairs (the target) from within a larger DNA molecule
(the template).

Artificially synthesized oligonucleotide primers are

used to initiate DNA synthesis but are made of DNA
(rather than RNA like the primers used by cells).

Uses the enzyme DNA polymerase, which naturally

copies DNA molecules.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 5

 II. STEPS IN PCR AMPLIFICATION
1. Denaturation
2. Annealing
3. Extension

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 6

 II. STEPS IN PCR AMPLIFICATION

1. Denaturation:
separation of a double-
stranded DNA template into
two single strands

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 7

 II. STEPS IN PCR AMPLIFICATION

1. Denaturation:

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 8

 II. STEPS IN PCR AMPLIFICATION
2. Annealing

hybridization of DNA
primers to the DNA
template strands.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 9

 II. STEPS IN PCR AMPLIFICATION

3. Extension
addition of nucleotides to the
primers by DNA polymerase

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 10

 II. STEPS IN PCR AMPLIFICATION

Initial Denaturation
Denaturation
Annealing
Extension
Final Extension

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 11

 II.MOLECULAR PRINCIPLES OF PCR

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 12

 KNOWLEDGE CHECK

Which statement about the PCR process is

correct?

A. Temperature is the main driving force for

the denaturation and annealing steps.
B. Denaturation occurs between 70°C-80°C
C. An enzyme is required for the denaturation
step or the annealing step to occur
D. Extension has to occur before the
annealing step

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 13

 III. ESSENTIAL COMPONENTS OF PCR
The following are the essential components of PCR:

10mM Tris at pH 8.3, 50 mM KCl,

1.5-2.5 mM MgCl2.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 14

 KNOWLEDGE CHECK

Which of the following will

cause a problem in PCR?

a. A reaction buffer
b.A template
c. PCR Inhibitors
d.Primers

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 15

 III. ESSENTIAL COMPONENTS OF PCR (CONTD.)
Thermal Cyclers / Thermocyclers / PCR Machine:
PCR reaction is carried out in 0.2-0.5 ml volume
thermocyclers. It heats and cools the reaction
tubes to achieve the temperature required.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 16

 IV. ANALYSIS OF PCR PRODUCT

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 17

 V. DIFFERENT TYPES OF PCR TECHNIQUES

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 18

 V. REVERSE TRANSCIPTION PCR (RT-PCR)

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 19

 V. REVERSE TRANSCIPTION PCR (RT-PCR)
One-Step RT-PCR Technique

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 20

 V. REVERSE TRANSCIPTION PCR (RT-PCR)
TWO-Step RT-PCR Technique

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 21

 KNOWLEDGE CHECK

Which statement below is true?

a. Primers are long DNA fragments

b. DNA polymerase enzyme can withstand
high temperatures
c. RT-PCR can only be performed in one-
step
d. Standard PCR allows accurate
quantification of the DNA template

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 22

 VI. APPLICATIONS OF PCR
1. Disease diagnosis:
a. Because a specific sequence can be amplified
greatly, much less clinical material is needed to
make a diagnosis.
b. PCR can be used to detect pathogens that are
difficult to culture.
c. PCR can be used for cancer diagnosis.
2. Forensics.
3. Matching donor and recipient tissues for organ
transplants.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 23

 VI. APPLICATIONS OF PCR (CONTD.)

4. Basic Research:
a. Comparison of sequence homology of conserved
genes in different organisms.
b. In developmental biology, PCR is very sensitive
and can be used to examine which genes are
turned on in early development (which mRNAs
are made).
c. May replace cloning as the amplification method
of choice to obtain large amounts of material for
sequencing.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 24

 PCR TROUBLESHOOTING

I. Low or no amplification.

II. Nonspecific amplification or smears.

III. Sequence errors within PCR products.

IV. Sequence errors at termini of PCR products.

V. False positive amplification.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 25

 I. LOW OR NO AMPLIFICATION
Possible causes Recommendations
DNA templates
Minimize shearing and nicking of DNA during isolation. Evaluate template
DNA integrity by gel electrophoresis, if necessary.
Poor integrity
Store DNA in molecular-grade water or TE buffer(pH 8.0) to prevent
degradation by nucleases.
Follow manufacturer recommendations stringently when using purification kits
to isolate template DNA. Consult the user manual and troubleshooting guides
to mitigate poor DNA quality.
Ensure that no residual PCR inhibitors such as phenol, EDTA, and proteinase
K are present if following chemical or enzymatic DNA purification protocols.
Low purity
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, which display high
tolerance to common PCR inhibitors carried over from soil, blood, plant
tissues, etc.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 26

 I. LOW OR NO AMPLIFICATION (CONTD.)
Possible causes Recommendations
Examine the quantity of input DNA and increase the amount if necessary.
Insufficient
Choose DNA polymerases with high sensitivity for amplification.
quantity
If appropriate, increase the number of PCR cycles.
Choose DNA polymerases with high processivity, which display high affinity
for DNA templates and are more suitable to amplify difficult targets.
Complex targets
(e.g., GC-rich or Use a PCR additive or co-solvent to help denature GC-rich DNA and
secondary sequences with secondary structures.
structures)
Increase denaturation time and/or temperature to efficiently separate double-
stranded DNA templates.
Check amplification length capability of the selected DNA polymerases. Use
DNA polymerases specially designed for long PCR.
Choose DNA polymerases with high processivity, which can amplify long
Long targets 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.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 27

 I. LOW OR NO AMPLIFICATION (CONTD.)

Primers

Review primer design. Use online primer design tools when appropriate.
Problematic Ensure that the primers are specific to the target of interest.
design
Verify that the primers are complementary to the correct strands of the target
DNA.

Aliquot primers after resuspension and store properly.

Old primers
Reconstitute fresh primer aliquots, or obtain new primers if necessary.

Optimize primer concentrations (usually in the range of 0.1–1 μM).

Insufficient
quantity For long PCR and PCR with degenerate primers, start with a minimum
concentration of 0.5μM.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 28

 I. LOW OR NO AMPLIFICATION (CONTD.)

Other Reaction Components

Use hot-start DNA polymerases to prevent degradation of primers by the

3’→5’ exonuclease activity of proofreading DNA polymerases. Hot-start DNA
Inappropriate polymerases also increase yields of the desired PCR products by eliminating
DNA polymerase nonspecific amplification.
Alternatively, set up PCR on ice, or add DNA polymerase last to the reaction
mixture.

Choose DNA polymerases with high sensitivity for amplification.

Review recommendations on the amount of DNA polymerase to use in PCR,
Insufficient
and optimize as necessary.
quantity of DNA
polymerase 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.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 29

 I. LOW OR NO AMPLIFICATION (CONTD.)

Other Reaction Components

Optimize Mg2+ concentration for maximum PCR yields. The presence of

EDTA, other metal chelators, or atypically high concentrations of dNTPs may
Insufficient Mg2+ require a higher Mg2+ concentration.
concentration
Check the DNA polymerase’s preference for magnesium salt solutions. For
example, Pfu DNA polymerase works better with MgSO4 than with MgCl2.

Review the recommended concentrations of PCR additives 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.
Excess PCR
additives or co- Increase the amount of DNA polymerase, or use DNA polymerases with high
solvents 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).

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 30

 I. LOW OR NO AMPLIFICATION (CONTD.)

Other Reaction Components

Ensure that the selected DNA polymerases are able to incorporate the
dUTP or modified modified nucleotides.
nucleotides in
reaction 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
reagents density gradients that may have formed during storage and setup.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 31

 I. LOW OR NO AMPLIFICATION (CONTD.)

Thermal Cycling Conditions

Optimize the DNA denaturation time and temperature. Short denaturing times
Suboptimal and low temperatures may not separate double-stranded DNA templates well.
denaturation On the other hand, long denaturation times and high temperatures may
reduce enzyme activity.

Optimize the annealing temperature stepwise in 1–2°C increments, using a

gradient cycler when available. The optimal annealing temperature is usually
3–5°C below the lowest primer Tm.
Suboptimal
Adjust the annealing temperature when a PCR additive or co-solvent is used.
annealing
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.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 32

 I. LOW OR NO AMPLIFICATION (CONTD.)

Thermal Cycling Conditions

Select an extension time suitable for the amplicon length.

Reduce the extension temperature (e.g., to 68°C) to keep the enzyme active
Suboptimal
during amplification of long targets (e.g., >10 kb).
extension
Use DNA polymerases with high processivity for robust amplification even
with short extension times.

Suboptimal Adjust the number of cycles (generally to 25–35 cycles) to produce an

number of PCR adequate yield of PCR products. Extend the number of cycles to 40 if DNA
cycles input is fewer than 10 copies.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 33

 II. NONSPECIFIC AMPLIFICATION OR SMEARS
Possible Causes Recommendations

DNA templates

Excess DNA Review the optimal amounts of DNA input. Lower the quantity to reduce the
input generation of nonspecific PCR products.

Degraded DNA may appear as smears or lead to high background in gel

electrophoresis. Minimize shearing and nicking of DNA during isolation.
Poor 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
sequences (e.g., for DNA templates and are more suitable to amplify difficult targets.
GC-rich or Use a PCR additive or co-solvent to help denature GC-rich DNA and
secondary sequences with secondary structures. Increase denaturation time and/or
structures temperature to efficiently separate double-stranded DNA templates.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 34

 II. NONSPECIFIC AMPLIFICATION OR SMEARS
(CONTD.)
Possible Causes Recommendations

DNA templates
Check amplification length capability of the selected DNA polymerases. Use
DNA polymerases specially designed for long PCR.
Choose DNA polymerases with high processivity, which can amplify long
Long targets 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.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 35

 II. NONSPECIFIC AMPLIFICATION OR SMEARS
(CONTD.)
Primers

Review primer design. Use online primer design tools when appropriate.
Verify that the primers are specific to the target, with minimal homology to
other regions in the template.
Ensure that the primers do not contain complementary sequences or
Problematic consecutive G or C nucleotides at the 3′ ends, to prevent primer-dimer
design 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.

Optimize primer concentrations(usually in the range of 0.1–1 μM). High

High quantity
primer concentrations promote primer-dimer formation.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 36

 II. NONSPECIFIC AMPLIFICATION OR SMEARS
(CONTD.)

Other Reaction Components

Excess DNA Review recommendations on the amount of DNA polymerase to use in PCR,
polymerase and decrease as necessary.

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

Review Mg2+ concentrations and lower as appropriate to prevent nonspecific

Excess Mg2+
PCR products. Optimize Mg2+ concentrations for each primer set and target
concentration
DNA.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 37

 II. NONSPECIFIC AMPLIFICATION OR SMEARS
(CONTD.)
Thermal Cycling Conditions

Increase denaturation time and/or temperature to efficiently separate DNA

Insufficient
when working with GC-rich templates and sequences with secondary
denaturation
structures.

Incorrect Use the annealing temperature recommended for a specific DNA polymerase
annealing in its optimal buffer. Annealing temperature rules for primer sets can vary
temperature between different DNA polymerases.

Increase the annealing temperature to improve specificity. The optimal

annealing temperature is usually no less than 3–5°C below the lowest primer
Tm.
Low annealing
temperature Optimize the annealing temperature stepwise in 1–2°C increments, using a
gradient cycler when available.
Consider touchdown PCR to enhance specificity.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 38

 II. NONSPECIFIC AMPLIFICATION OR SMEARS
(CONTD.)

Thermal Cycling Conditions

Long annealing Shorten the annealing time to minimize primer binding to nonspecific
time sequences.

High extension Reduce the extension temperature 3–4°C to help the DNA polymerase’s
temperature thermostability, especially for long PCR.

Prolong the extension time when amplifying long DNA targets.

Insufficient
extension time Include a final extension step with sufficient time (5–15 minutes) to extend the
whole target.

High number of Reduce the number of cycles, without drastically lowering the yield of the
cycles desired PCR products, to prevent accumulation of nonspecific amplicons.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 39

 III. SEQUENCE ERRORS WITHIN PCR PRODUCTS
Possible Causes Recommendations

Use DNA polymerases with exceptionally high fidelity to generate PCR

Low fidelity of
fragments for downstream applications such as cloning, sequencing, and site-
DNA polymerase
directed mutagenesis.

Excess Mg2+ Review Mg2+ concentrations and reduce as necessary. Excessive

concentration concentrations favor misincorporation of nucleotides by DNA polymerases.

Unbalanced
Ensure equimolar concentrations of dATP, dCTP, dGTP, and dTTP in the
dNTP
reaction. Unbalanced nucleotide concentrations increase the PCR error rate.
concentrations

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 40

 III. SEQUENCE ERRORS WITHIN PCR PRODUCTS
(CONTD.)
Possible Causes Recommendations

Reduce the number of cycles without drastically lowering the yield of the
desired PCR products. High numbers of cycles increase the incorporation of
High number of mismatched nucleotides.
cycles
Increase the amount of input DNA when appropriate to avoid running an
excessive number of cycles.

Use a long-wavelength UV (360 nm) light box to visualize fragments in gels,

and limit the illumination time as much as possible.
UV-damaged If using a short-wavelength (254–312 nm) light box, limit the UV illumination
DNA 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.

Sequence both DNA strands to verify the reliability of sequencing results. Use
Sequencing error
duplicate samples when appropriate.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 41

 IV. SEQUENCE ERRORS AT TERMINI OF PCR
PRODUCTS

Possible Causes Recommendations

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

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

Contaminating Use molecular-grade, nuclease-free reagents in PCR setup. Set up reactions

nucleases on ice to keep the activity of possible contaminating exonucleases low.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 42

 IV. SEQUENCE ERRORS AT TERMINI OF PCR
PRODUCTS (CONTD.)
Possible Causes Recommendations

Use a long-wavelength UV (360 nm) light box to visualize fragments in gels,

and limit the illumination time as much as possible.
Problematic If using a short-wavelength (254–312 nm) light box, limit the UV illumination
primer design 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.

Low primer Sequence both DNA strands to verify the reliability of sequencing results. Use
quality duplicate samples when appropriate.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 43

 V. FALSE POSITIVE AMPLIFICATION
Possible Causes Recommendations

Avoid primers containing complementary and self-complementary sequences,

Problematic
which favor primer-dimer formation and self-oligomerization and their
primer design
subsequent amplification.

Crossover Avoid contamination of DNA in the work environment by following general

contamination recommendations for PCR setup.

Use pipette tips with aerosol barriers. Dedicate a separate work area and
Carryover decontaminate the area after each use.
contamination Follow PCR carryover control techniques such as dUTP incorporation with
UDG treatment.

POLYMERASE CHAIN REACTION AND TROUBLESHOOTING 44

 REFERENCES
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 REFERENCES (CONTD.)
ShahriarShahi, SepidehZununiVahed, NazaninFathi, SiminSharifi.
2018. Polymerase chain reaction (PCR)-based methods: Promising
molecular tools in dentistry. International Journal of Biological
Macromolecules, Volume 117: 983-992. ISSN 0141-8130,
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PCR Troubleshooting Guide. Thermo Fisher Scientific.
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Roux, K. H. (2009).Optimization and Troubleshooting
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