CYTOGENETICS LEC

BSMT 2 - 2ND SEMESTER

POLYMERASE CHAIN REACTION

Lesson 8: Polymerase Chain Reaction ○ It is time-consuming because you need to wait for cells to
Ms. Jamie Go replicate on an agar plate.

POLYMERASE CHAIN REACTION

PCR: APPLICATION

● An in vitro method for amplifying DNA segments that depends on
repeated cycles of the three main steps of the polymerase chain
reaction:
1. denaturation
2. primer annealing
3. DNA polymerase-directed DNA synthesis
● Rapid creation of a large quantity of product (amplicons) from
very small quantities of the DNA template.
ORIGIN AND HISTORY
Uses of PCR of genetic applications and forensics:
● Screen mutations involved in genetic disorders
● Detect bacteria and viruses in humans (ex. COVID-19
"SARS-CoV2", Swine flu "H1N1"), and pathogenic bacteria in
contaminated food (ex. E. coli, salmonella)
● Collection of DNA from extinct species (for biology field)
● Comparison of DNA among living species
● Forensic genetic applications (paternity testing/ DNA testing,
crime scene analysis, individual identification)
● Production of DNA segments for genome sequencing projects

● Developed by Kary Mullis in 1983 PCR: PRINCIPLE

● Kary Mullis first visualized the idea of amplifying DNA in vitro while
in the process of working through a mutation-detection method
● First successful amplification happened in a short fragment of the
Escherichia coli plasmid, pBR322
● First paper of practical application was the amplification of beta
globulin and analysis for diagnosis of patients with sickle cell
anemia
● PCR was called the method a "polymerase catalyzed chain
reaction" because DNA polymerase was the enzyme used to
drive the replication of DNA in a chain reaction.
● PCR has since become increasingly user-friendly, more
automated, and more amenable to use in a clinical laboratory.
○ We use this for the detection of the COVID-19
POLYMERASE CHAIN REACTION
● Classified under a larger group of molecular techniques known as
'Nucleic Acid Amplification Techniques' (NATS)
● NATS (examples); mnemonics: SPT or SPIT
PCR, Transcription-based amplification
Target Amplification
systems, Genome Amplification Methods
Ligase chain reaction, Strand displacement
Probe Amplification
amplification, QB Replicase
Branched DNA amplification, Hybrid capture
Signal Amplification assays, Cleavage-based amplification,
Cycling probe
TARGET AMPLIFICATION
● Involves making many copies of a specific DNA sequence.
● Comparable to growing cells in culture and allowing the cells to
replicate their nucleic acid as well as themselves for easier
visualization on an agar plate.
● This method of cloning DNA using vectors and host cells is
labor-intensive and time-consuming.
● Waiting for cells to replicate at detectable levels, it can take days to
weeks or months, whereas replicating the nucleic acid in vitro only
takes minutes to hours.
● Based on the principles of DNA replication in vivo, there is an
automated version of DNA replication, performed in a test tube
containing a total reaction volume of 20-50 ml
● Despite very small total reaction volume, a typical PCR reaction
produces millions of copies of a short, targeted segment of DNA
from the original DNA molecules.
○ Even with the small amount of reaction volume (20-50 ml),
the PCR can produce millions of copies of the targeted
segments of the DNA
PCR: COMPONENT
● Template
● Primers
● dNTPs (deoxyribonucleotide TriPhosphates)
● Thermostable DNA polymerase
● Buffer and Magnesium (Mg 2+)
TEMPLATE
● Contains the region of interest to be amplified
● Could be a DNA, RNA, cDNA, or PCR product
● For routine clinical analysis - 100 ng to 1 ug of DNA
● Lesser amounts for more defined template preparations (e.g.
cloned target DNA or product from previous amplification)
● Should be of good condition, free of contaminating proteins, and
without nicks or breaks that can stop DNA synthesis or cause
misincorporation of nucleotide bases.
PRIMERS
● Short sequences (~18-30 bp long) of complementary DNA
that anneals with the target sequence in the template
● Determines the specificity of the PCR
● Chemically manufactured on a DNA synthesizer
○ Most laboratories purchase primers from
commercial providers
● Primer sequences are submitted in text form, along with other
specifications (i.e. amount, degree of purification, and any
modifications such as biotin or fluorescent dyes).

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● A good pair of forward and reverse primers must have a melting
temperature (Tm) difference of ≤ 5℃ so that both will hybridize
optimally at the same annealing temperature
○ In designing primers, Tm can be adjusted by
increasing the length of the primers or by placing the
primers in areas with more of fewer Gs and Cs in
the template
● Nucleotide sequence orders of primers are designed using
genomic sequence available from the NCBI or other resources
PRIMER SEQUENCE
● Determines the accuracy of binding to its complementary
sequence and not to other sequences. The one in charge of
where to bind.
PCR Assay
A PCR assay requires forward and reverse primers
FORWARD hybridizes to the complementary strand just 5` to
PRIMER the sequences to be amplified.
REVERSE
hybridizes just 3` to the sequence to be amplified.
PRIMER
● Template DNA
● It was mentioned that the forward primer hybridizes the 5` in the
sequence while the reverse primer hybridizes the 3` of the DNA
template.
● The primer sequences are responsible for the accuracy of the
binding.
○ So they make sure that the pair doesn’t go to other
pairs.
○ G (Guanine) is always paired with C (Cytosine) and
A (Adenine) is always paired with T (Thymine).
TYPES OF PRIMERS
● First set of forward & reverse primers used
in a two-round PCR assay (nested-PCR)
OUTER
● Used in the first round amplification in order
PRIMERS
to amplify the larger fragment of the target
gene.
● Second set of primers used in a two-round
PCR assay (nested-PCR)
● Used in the second round amplification in
INNER PRIMERS order to amplify the shorter fragment of the
target gene.
● Positioned in between the forward and
reverse outer primers.
● Primers that can detect targets with
variable nucleotide sequence in selected
DEGENERATE
sites.
PRIMERS
● Used to detect mutations or others species
variants.
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dNTPs
● Deoxyribonucleated bases
● Equimolar mixture of the 4 deoxynucleotide triphosphates
(dNTPs) - dATP, dTTP, dGTP, dCTP - added to the synthesis
reaction in concentrations sufficient to support the exponential
increase of copies of the template
● Standard procedures require 0.1 to 0.5 mM concentrations of
each nucleotide
● Substituted or labelled nucleotides (i.e deaza dGTP) may be
included in the reaction for special applications.
● The concentration and purity of dNTP preparations affect the
efficiency of the PCR reaction
● A single solution containing all four dNTPs is better compared with
four separate nucleotide solutions, as it lowers pipetting errors
● Higher concentrations are recommended for storage because
storing dNTPs in lower concentrations results in hydrolysis to
dNDP and dNMP
○ dNDP and dNMP can also result from poor manufacturing
conditions or contamination with heavy metals
○ Results to PCR inhibition
THERMOSTABLE DNA POLYMERASE
● Tth polymerase - from the bacteria Thermus thermophilus
○ Also have reverse - transcriptase activity, so it can be used
in RT-PCR in which the starting material is an RNA template
● Proof-reading enzymes
○ Added to allow Taq or Tth polymerase to generate large
products over 30,000 bases in length
● Although thermally stable, Taq polymerase is still subject to loss of
activity under adverse conditions.
○ Vigorous agitations of the polymerase enzymes is not
recommended - results in mechanical shearing, altering of
the secondary and tertiary structure, and irreversible
denaturation
○ Mixing also introduces air to the liquid, infusing the air-liquid
interface with bubbles, which can cause damage
● Modified Versions of the Polymerase enzyme:
○ From the cloning of the genes to the coding for the
polymerases;
○ Stoffel fragment
○ At high temperatures, half-life is ~2x that of Taq
polymerase
○ Has a broader range of optimal MgCl2 concentration
(2-10mM) that Taq
○ Recommended for allele-specific PCR and for
amplification of region with high GC content.
PCR BUFFERS
● Provide optional conditions for enzyme activity
BUFFERS
● Affect the denaturing and annealing
temperature of the DNA and the enzyme activity
● Increase in salt concentration makes longer
Monovalent
DNA products denature more slowly than shorter
Cations
DNA products during amplification, shorter DNA
products are amplified preferentially
● Ex. Potassium chloride, Ammonium sulfate
● Affects the primer annealing and enzyme
activity
Divalent
Cations ● Requirements vary depending on other
components of the reaction mix
● Ex. Magnesium chloride
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 ACCESSORY COMPONENTS
Bovine serum ● Binds inhibitors and stabilizes enzyme
albumin
(10-100ug/mL)
Dithiothreitol ● Provides reducing conditions to enhance
(0.01 mM) enzyme activity
● Lower denaturing temperature of DNA with
Formamide
high secondary structure; increase availability
(1-10%)
for primer binding
● Reduce secondary structure to allow
Chaotropic
polymerase extension through difficult areas;
agents
contribute to enzyme stability
(1-10%)
● Ex. Triton X-100, glycerol, dimethyl sulfoxide
● Enzymes are usually supplies with buffers optimized by the ● Temperature is 50-70℃, 20-90 seconds
manufacturer. ● Allows the primers to pair
● Often, buffer and ingredients mixed with dNTPs and stored as (anneal/hybridize) with the
aliquots of a master mix. complementary regions of the DNA
● Dedicated master mixes will include primers, so only target template (denatured, ss-DNA)
sequences must be added. ● Primary considerations in selecting
annealing temperature:
○ Melting temperature (™) of primer
sequences
○ Primer length
○ Base composition of primers (GC-rich
ANNEALING primers are more thermally stable
than AT-rich primers)
○ Whether or not all bases in a primer
are complementary bases in the
target sequence

● Temperature reaction lowered to between

68-75℃, 10-60 seconds
○ Temperature depends on
Components of PCR are: Primer, Nucleotides, Taq Polymerase, and the optimal temperature of
DNA. enzyme DNA polymerase
● Where DNA synthesis occurs
● DNA polymerase synthesizes a copy of
MAJOR STEPS IN PCR the template DNA by adding nucleotides to
1. Denaturation the hybridized primers
EXTENSION ● DNA polymerase replicates the template
2. Annealing
DNA by simultaneously extending the
3. Extension primers on both strands of the template

Additional PCR Steps:

1. Reverse Transcription (RT step or cDNA synthesis)
2. First/Second round amplification

● Double stranded DNA to be cloned is

denatured into single strands by heating
90-96℃ for 20-60 seconds
○ High temperature disrupts hydrogen
bonds between complementary base
PRINCIPLES OF PCR
pairings ● PCR is a “chain reaction” because it involves a chain of
● Initial denaturation step is lengthened for
reactions in series where each cycle is a round of amplification.
DENATURATION genomic or other large DNA template
fragments ● At the end of the 3 steps, or one cycle denaturation-primer
● Subsequent denaturation can be shorter annealing-primer extension, one copy of ds-DNA has been
● DNA can come from many sources: replicated into two ds-DNA (ds means double stranded)
genomic DNA, mummified remains, fossils, ● Returning to the denaturing temperature starts the 2nd cycle, with
clinical specimens (nasopharyngeal swab, the end result being a doubling in the number of ds-DNA
blood), forensic samples (dried blood,
molecules again
semen, hair)
● Results to millions of copies of the original region defined by the
primer sequences at the end of the PCR program.

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 PHASES OF PCR AMPLIFICATION CURVE
1. Linear Ground ● Where PCR begins THERMAL CYCLERS
Phase ● Amplification not detectable
● Amplification becomes detectable
2. Exponential ● Doubling of products occurs every cycle
Phase ● Optimal point for analyzing data in qPCR
● Quantitation must be done at this phase
● The reaction components are being
consumed and the reaction is slowing.
3. Log linear
● Every succeeding cycle does not result in
Phase
doubling of PCR products anymore.
● Lowered PCR efficiencies
● The reaction is closed to stopping
(almost no amplification)
4. Plateau Phase
● Reagents are already depleted in the ● Aka thermocyclers
reaction
● Designed to rapidly and automatically ramp (change) to the
required incubation temperatures, holding at each one for
designated periods
● Early versions were designed as heater/coolers with
programmable memory to record the appropriate reaction
conditions
○ Limited memory available for recording the reaction
conditions and sample was limited.
○ Wax or oil (vapor barriers) – added to prevent
condensation of the sample on the tops of the tubes during
the temperature changes; made subsequent sample
handling difficult.
● Later models designed with heated lids that eliminated the need
for vapor barriers.
○ In the long run, they were able to create new models with heated lids
which eliminates the need to add wax or oil – thus, we don’t really
have to use vapor barriers anymore.

Illustration of the 4 phases in a polymerase chain reaction
1. First, we have the linear ground phase, this is where PCR
begins.
2. Next, exponential phase where amplification slowly becomes
detectable.
3. Next, we have the Log-linear phase where the reaction
components are being consumed and the reaction is slowing
down.
4. Lastly, the reaction of the log linear phase will slow down because
it is going to the plateau phase, where the reaction is close to
stopping – there is little to no more amplification and the reagents
in this phase are already depleted in the reaction – thus, slowing
down.
PCR: INSTRUMENTATION
● The first PCRs were performed using multiple water baths or
heat blocks set at the required temperatures for each of the steps
of the PCR cycle.
● Tubes were manually moved from one temperature to another.
● Before the thermostable enzymes were discovered, new enzyme
had to be added after each denaturation step.
● Availability of heat-stable enzymes greatly initiated the automation
of the whole PCR process.
○ Thankfully, with the availability of heat-stable enzymes, it became
more easier for the scientists or researchers to use the PCR test as
they did not have to add enzymes after each denaturation step.
● To accomplish PCR, an instrument must only manage temperature
according to a scheduled amplification program.
PCR: TROUBLESHOOTING
CONTAMINATION
● Common problem for running a PCR.
● Significant concern in “open-tube” methods that involve target
amplification and manipulation of the PCR product since
theoretically, PCR amplifies a single molecule.
○ Thus, contaminants are also amplified
○ Of great concern in medical or forensic laboratory where
results may be interpreted based on the presence,
absence, size, or amount of a PCR product.
● Major source of contamination: presence of PCR products from
previous amplifications.
○ Highly concentrated PCR product DNA can aerosolize
when tubes are uncapped and when the amplified DNA is
pipetted.
○ Perfect template for primer binding and amplification in a
subsequent PCR using the same procedures.
CONTAMINATION CONTROL
● Mainly directed toward eliminating PCR product from the setup
reaction.
● Both physical and chemical controls are performed.
● Physically separate the pre-PCR areas
from the post-PCR analysis areas by
PHYSICAL assigning separate rooms or using isolation
CONTAMINATION cabinets
CONTROL ● Equipment, including lab gowns and
gloves, and reagents should be dedicated
to either pre- or post-PCR

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 ● Your materials in the pre-PCR area and
post-PCR analysis area must always be
separated.
● Gloves should be changed if contaminated
areas are touched before proceeding with
the PCR setup.
● Positive airflow, airlocks, and more
extensive measures are needed
throughout laboratories that process large
numbers of samples and test for limited
number of amplification targets.
● Items can flow from the pre- to the
post-PCR area but not in the opposite
direction, without decontamination
UV Light - has been used to decontaminate
and maintain pre-PCR cares.
● Catalyzes ss- and ds- breaks in the DNA
that will then interfere with replication.
● Isolation cabinets are equipped with UV
light sources that are turned on for about 20
mins after use.
○ It uses UV light for
decontamination before it
proceeds to the PCR run.
● Effectiveness may be increased by addition
of psoralens to amplification products after
analysis
○ Psoralens - intercalate between
the bases of dsDNA, covalently
binds to the thymidines, uracils,
and cytidines in the DNA chain
in the presence of UV light; bulky
adducts of the psoralens
prevents denaturation and
amplification of the treated DNA.
● May not be most effective for nucleic acid
decontamination
● Efficiency of UV light decontamination
depends on wavelength, energy and
distance of light source.
● The lab tech must avoid skin or eyes
exposure to UV light this can harm us
human.
● Damage plexiglass and some plastics, may
affect lab equipment like pipette after
extended exposure
● Overhead UV room lights also requires
hazardous high intensity to damage DNA at
distances from ceiling to bench surface.
○ So diba, ang UV light daw kay maka
affect siya sa lab equipment
especially kanang mga glass if ma
prolong iyahang exposure to UV light.
10% bleach (7 mm sodium hypochlorite)
● Frequently wiping of bench tops, hoods, or
any surface that comes in contact with
specimen material with dilute bleach or
alcohol removes most DNA contamination
● In forensic work, gloves are wiped with
bleach and allowed to air-dry before
handling evidence or items that come with
CHEMICAL specimen or evidence.
CONTAMINATION Enzymatic method-duTP-UNG system
CONTROL (DeoxyUridine Triphosphate-
uracil-N-glycosylase)
● Involves substitution of dUTP for dTTP in
the PCR reagent master mix, incorporating
dUTP instead of dTTP into the PCR product
● dUTP does not affect the PCR products
in most applications
● Enzyme UNG (uracil-N-glycosylase) is
added to the reaction mix at start of
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PCR-degrades any nucleic acid containing
uracil, like contaminating PCR product from
previous reactions.
● Will not work with some types of PCR, like
nested PCR, because a second round of
amplification requires presence of
first-round product
● Used routinely in qualitative PCR (real-time
PCR) produces where contamination
control is essential because contaminant
will affect interpretation of the amount of
product.
PROBLEMS & TROUBLESHOOTING
● Fragments from mispriming will carry the
primer sequence and become a template
for subsequent cycles of amplifications;
takes components away from the intended
reaction
● For some procedures, these artefacts do
not affect interpretation of results and can
be ignored as long as reaction efficiency is
not compromised. For others, it may
interfere with proper interpretations of
results or with ensuing procedures
● Initially averted by good primer design and
optimal amplification conditions.
● But even under the best conditions,
Mispriming
misprimes can occur during preparation of
Aberrant Primer
the reaction mix due to Taq polymerase
Binding
having activity at room temperature.
○ While mixes are prepared and
transported to the thermocycler, the
primers and template are in contact
at 22-25 degree C.
○ Primers can bind sequences other
than their exact complements in the
target, and the low-level activity of
Taq polymerase will extend them,
leading to misprimed products that
are already present before
amplification program begins
○ Hot-start PCR - can be used to
prevent this type of mispriming.
Gel elution- resolving amplification products by
gel electrophoresis
● Direct way to obtain a clean PCR product
● Cut out pieces of gel containing desired
bands and elute PCR product
● Agarose gel slices can be digested with
enzymes, such as beta agarose, or by
incubation with iodine to release the
product DNA.
PCR Product
Clean-up
Illustration of the gel elution.
The gel containing DNA e sud then e centrifuge then
you add the supernatant alcohol then you can
separate DNA precipitate. This is how you do the gel
elution by gel electrophoresis.
Use of spin columns
● Amplifications free of PCR components are
prepared using spin columns or silica
beads
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 ● DNA binds to column, and the rest of
reaction components are rinsed away by
centrifugation
● DNA can then be eluted from the column
● Provide better recovery than gel elution.
○ So mas segregate DNA
precipitate if mag use tag
spin columns.

Illustration how spin columns work

Maka separate jud siya more DNA precipitate than gel
elution after centrifugation. Mas daghan siyag DNA
precipitate na separate

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