PCR & RT PCR .

Basic Molecular Biology
PCR & RT PCR
PRASHANT YADAV
M.Sc. Microbiology
Source - https://reach.cdc.gov/course/basic-molecular-biology-
module-4-polymerase-chain-reaction-pcr-and-real-time-pcr

Polymerase chain reaction or PCR is one of the
most powerful tools in molecular biology. It
was developed in 1983 by an American
biochemist, Kary Mullis. This innovative
technology won Mullis the Nobel Prize in
Chemistry in 1993.
PCR allows amplification of a specific section
of DNA from a DNA template, generating
millions of copies of the DNA fragment within
a matter of hours. The technique is commonly
used in research, forensic, and clinical
laboratories for a wide variety of applications
including cloning, mutagenesis, genotyping,
and diagnosis of diseases.
PCR- Polymerase chain reaction

Workflow in Molecular Biology Laboratories
The molecular diagnostic workflow begins with sample collection, where a sample is
identified and collected for diagnostics, properly following laboratory guidelines.
The sample then goes into nucleic acid extraction, where the nucleic acid is isolated
and purified from the sample.
Then the target or nucleic acid of interest is amplified in the nucleic acid
amplification stage.
And finally the target is detected using a variety of methods such as PCR product
detection, microarrays, or sequencing in the final stage of Detection.

PCR (Safety Precutation)
Standard laboratory safety practices must be followed
to avoid contact with potential biological hazards and
prevent sample contamination. When performing
nucleic acid extraction, you must wear personal
protective equipment (PPE), such as safety eyewear,
gloves, and laboratory coats.
Refer to the material safety data sheet (MSDS) for
complete safety information regarding reagents used in
nucleic extraction procedures.
.

PCR & Molecular Diagnostic
The evolution of PCR completely
transformed medical diagnosis. Variations
of PCR methods, such as Reverse
Transcription PCR (RT-PCR) and Real-Time
PCR (qPCR), have been developed to
efficiently amplify and quantitatively
analyze DNA or RNA from biological
specimens.
Novel PCR-based diagnostic tests allow
detection of infectious diseases, genetic
variations and mutations, cancer
biomarkers, and blood disorders with
faster turnaround time and vastly
improved sensitivity and accuracy

The Principle of PCR
PCR employs DNA polymerase to add a nucleotide
to the 3' end of a pre-existing short DNA fragment
called a primer to generate new DNA
complementary to the template strand. Basic steps in
PCR include:
•Denaturation
•Annealing
•Extension
These steps are performed at different temperatures
and repeated in multiple cycles, hence the
name "chain reaction.“
Since successful PCR requires reliable heating and
cooling steps, a thermocycler or PCR machine is
used. This apparatus allows programmable
temperature changes that simplify PCR execution.
For more information about DNA replication by DNA
polymerase,

Denaturation: separation of a double-
stranded DNA template into two single
strands
For DNA synthesis to occur, the DNA
template must be single-stranded. As we
learned from Module 1, unwinding of
DNA in the cells is accomplished by the
energy-dependent DNA enzyme
called helicase. However, PCR is
performed in vitro, where the enzyme and
its cofactors are absent.
Denaturation

In the absence of an enzyme, heat energy can be used to break the interactions
between the two DNA strands. During the heat denaturation step, high
temperature (92°C - 95°C) is applied to the template to disrupt the hydrogen
bonds between complementary bases and hydrophobic stacking interactions, as
well as separate the double helix structure of DNA. This step generally lasts 15
- 30 seconds.

Annealing: hybridization of DNA primers to the DNA
template strands.
In this step, the primers bind to the target DNA sequences
within the template strands in a sequence-specific
manner, following the rules of base-pair
complementarity.
For annealing to occur, the temperature has to be
lowered. Temperature is based on the melting
temperature (Tm) of the primer, where Tm is the
temperature at which 50% of the primer binds
specifically to the template.
In general, the optimum annealing temperature is about
3° – 5 °C below the Tm (typically 50°- 60°C). This step
generally lasts 15 - 60 seconds.
If the temperature is too high, it prevents stable
hybridization.
If the temperature is too low, it leads to non-specific
hybridization.
Annealing

Extension: addition of nucleotides to the primers by
DNA polymerase
Once primers bind to the templates, a DNA
polymerase reads the template sequences and adds
complementary nucleotides to the 3' ends of the
primers, generating longer DNA strands.
During this step, the temperature is adjusted to the
optimum temperature of the polymerase. The
optimum temperature of a widely used
enzyme, Thermus aquaticus or Taq DNA
polymerase, is in the range of 70 - 80°C. The activity
of Taq polymerase is drastically lower at 25°C (room
temperature).
The extension time depends both on the DNA
polymerase used and the length of the target DNA
sequence to amplify. Under optimum conditions,
Taq polymerase extends 1000 bases in 1 minute.
The newly synthesized PCR products from this step
can also serve as templates for the next round of
amplification.
Extension

Image of exponential amplification by PCR.
From one copy of double-stranded DNA,
each template strand generates one strand of
PCR product in the first cycle resulting in
two copies of double-stranded DNA.
In the second PCR cycle, all four strands
serve as a template for generation of four
additional strands of PCR product.
This results in four copies of double-stranded
DNA. The number of DNA molecules
continues to double after the following
rounds of PCR amplificatio
PCR CYCLING

Although PCR occurs in three basic steps, a typical standard
PCR protocol contains five steps as follows:
Initial Denaturation
to ensure complete denaturation of the template
Denaturation
Annealing Repeat for 25-35 cycles
Extension
Final Extension
to fill in the incomplete newly synthesized PCR products
Under ideal conditions that allow 100% amplification
efficiency, the amount of DNA target is doubled at each
cycle, leading to exponential amplification of the DNA
fragment.

A DNA template is a particular DNA strand containing the target sequence to
amplify. When preparing the template, take precautions to eliminate or
minimize the amount of PCR inhibitors.
Note: Substances, such as phenol, ethanol, detergents or EDTA, that have
adverse effects on PCR are called PCR inhibitors. PCR inhibitors prevent the
amplification of nucleic acids thereby leading to false negative results, which is
a significant drawback of PCR.
PCR inhibitors can originate from the sample or may be introduced during
sample processing or nucleic acid extraction.
STANDARD PCR
DNA TEMPLATE-

PCR relies on the ability of DNA polymerase to
synthesize a new DNA strand from a DNA template.
When the nucleic acid of interest is RNA, an extra
step is required initially to generate a complementary
DNA (cDNA) template from the RNA. This step is
called reverse transcription, accomplished by an
enzyme called reverse transcriptase.
The cDNA produced from this step then serves as a
template for PCR amplification by DNA polymerase.
The PCR analysis of RNA is therefore referred to
as reverse transcription PCR or RT-PCR.
RT-PCR is very sensitive and commonly used to
detect and quantify messenger RNA (mRNA)
Image of reverse transcription PCR which is
used when the nucleic of interest is RNA. RNA
is first used to generate cDNA by reverse
transcriptase. The cDNA is then used as a
template for PCR amplification by DNA
polymerase
REVERSE TRANSCRIPTION- PCR

RT-PCR can be performed as either a one-step
or a two-step reaction.
One-Step RT-PCR: The reverse transcription
occurs in the same tube as the PCR
amplification.
Advantages:
Simple and fast setup
High reproducibility
Lower possibility of contamination
Recommended uses:
Analysis of large numbers of sample
One-Step RT-PCR
Image of two Eppendorf tubes demonstrating a one-step RT-PCR process where
reverse transcription and PCR amplification occur in the same tube.
The first tube on the left contains RNA. An arrow to the right of the first tube
indicates reverse transcription and PCR amplification leading to the second tube on
the right.
The second tube on the right contains PCR products.

RT-PCR can be performed as either a one-step or a two-step reaction.
Two-step RT-PCR:
The reverse transcription and the PCR amplification occurs in separate
tubes.
Advantages:
Multiple PCRs from one RT reaction
Flexibility for primers and PCR conditions
Long-term storage of cDNA
Recommended uses:
Analysis of multiple targets in one sample.
Two-step RT-PCR
Image of three Eppendorf tubes
demonstrating a two-step RT-
PCR process where reverse
transcriptase and PCR
amplification occur in separate
tubes.
The first tube on the left
contains RNA. An arrow to the
right of the first tube indicates
reverse transcription leading to
the second tube in the middle
that contains cDNA.
An arrow to the right of the
middle tube indicates PCR
amplification leading to the third
tube on the right which contains
PCR products.

For standard PCR, amplified products are
traditionally detected by gel electrophoresis
after PCR cycling is completed. The PCR
products resolved on the gel are visualized by
applying a stain such as ethidium bromide.
Standard PCR provides only a semi-
quantitative estimation of the abundance of the
PCR product. It does not allow accurate
quantification of the amount of DNA template
originally input into the reaction.
For more information about gel electrophoresis,
ANALYSIS OF PCR PRODUCT

As stated earlier, the end-point detection of
products from traditional PCR only allows
qualitative measurements of the PCR products.
Real-time PCR or quantitative PCR (qPCR) is a
variation of PCR developed to quantitatively
determine the amount of target DNA or cDNA
nucleic acid. The same principle of traditional PCR
amplification is applied, but PCR products are
detected at each cycle in "real-time" as the
reaction progresses using a thermocycler with
fluorescence detection capability.
Note: Real-time PCR (qPCR) should not be
confused with RT-PCR (reverse transcription
PCR). Real-time PCR of RNA is referred to as
real-time RT-PCR or
qRT-PCR.
Real-time PCR or quantitative PCR (qPCR)

Quantitative analysis of PCR products relies on an understanding of four major phases in
the PCR amplification curve:
Linear ground phase where PCR begins. The amplification is not detectable.
Early exponential phase where the exponential amplification becomes detectable. The
DNA amount doubles at each cycle. Quantitation of nucleic acid must be done at this
phase.
Log-linear phase where reaction slows down due to the depletion of reagent
components and lowered PCR efficiencies.
Plateau phase where the reaction components become limited and the reaction stops
Image of a sigmoidal curve of the four major phases in PCR. The
first phase is the linear ground phase where the line is still flat.
The second phase is the early exponential phase where the line
rises sharply. This phase is labeled with a star as quantitation of
nucleic acid must be done at this phase.
The third phase is the log linear phase where the line still goes up,
but not as steep as the early exponential phase. The last phase is the
plateau phase where the line starts to become flat again.
PCR amplification curve

The end-point detection of standard PCR measures PCR products at
the plateau phase. The data from this phase cannot be used to
accurately quantify the amount of template.
As we can see from the plot on the right, samples with different
amounts of template (1x, 10x dilution, and 100x dilution) all yield a
comparable amount of DNA at the plateau phase of PCR. The
amounts of the final PCR products do not correlate well with the
amounts of the template
Image of three sigmoidal curves of three PCR with
different template concentrations (1x, 10x dilution,
and 100x dilution).
All three reactions reach the plateau phase with
comparable amounts of DNA. The plateau phase is
highlighted indicating where the end-point detection
occurs.

However, as we can see from the same plot on the right, the sample with
100x dilution of the template (light blue) requires more amplification cycles
than the sample with 1x template (light green) before the PCR curve
becomes detectable in the early exponential phase.
Real-time PCR applies this technique in quantification of nucleic acid
Image of three sigmoidal curves of three PCR
with different template concentrations (1x, 10x
dilution, and 100x dilution).
The PCR curve from the sample with the
highest amount of the template (1x) rises before
the other samples. The template concentration
correlates with how soon the PCR curve rises.
The early exponential phase is highlighted
indicating where the data from the real-time
detection is used for quantitation.

The PCR curve from real-time PCR is typically plotted on a semi-logarithmic scale to
allow better visualization. The straight line relationship between the amount of DNA
and cycle number is observed in the early exponential phase and used in nucleic
acid quantitation.
Two important terms in nucleic acid quantitation by real-time PCR are:
Threshold = the point at which significant and specific amplification occurs and the
fluorescence signal rises above the background level.
Ct (Threshold cycle) = the cycle number at which the fluorescence signal crosses the
threshold
Image of a plot of the PCR curve on a semi-
logarithmic scale. The curve in the early
exponential phase appears as a straight line
rising up.
The log linear phase following the early
exponential phase is a curved region
indicating where the reaction slows down.
The plateau phase appears as a straight, flat
line at the top end of the curve.
RT PCR : Threshold & Ct

As mentioned earlier, a sample with a higher template concentration requires fewer
cycle numbers to reach the threshold than those with lower template
concentrations, and thus displays a lower Ct value. This means Ct inversely
correlates with amount of template in the sample.
The plot on the right demonstrates amplification of four samples with different
template concentrations. The green sample, which contains the highest amount of
the template gives a fluorescence signal that reaches the threshold before the other
samples, and therefore, displays the lowest Ct value.
Image of semi-logarithmic plots of
PCR from four different samples.
The samples with higher template
concentrations provide curves that
rise earlier and therefore have lower
Ct values.
Analysis of Nucleic Acid Quantity and Purity

Real-time PCR utilizes a fluorescence
detection system integrated into a
thermocycler to watch the progress of
the reaction at each cycle. A plethora
of fluorescence-based technologies
have been developed to support the
popularity of the assay. Two most
commonly used methods of detection
are:
Fluorescent dye-based detection
Fluorescent probe-based detection
Real-Time PCR
Monitoring DNAAmplification in Real time - PCR

This method utilizes a dye that emits
fluorescence when incorporating itself into
double-stranded DNA. The fluorescence signal
increases at each PCR cycle as more double-
stranded DNA molecules are generated. The
most common dye used is SYBR® Green I.
Since this method provides DNA detection in a
non-sequence specific manner, the fluorescence
signal may originate from non-specific PCR
amplification. Preliminary analysis should be
conducted to demonstrate that only the desired
PCR product is generated from the PCR
conditions used. Additional assays, such as a
melt curve analysis may be performed after RT-
PCR to identify non-specific amplification
Fluorescent dye-based detection

Fluorescently-labeled oligonucleotide probes
detect specific DNA sequences. Fluorescent
signals can be generated by different
mechanisms.
TaqMan probes recognize a specific region
within the target DNA sequence. The probes
are labeled with a fluorophore at the 5' end and
a quencher at the 3' end. During extension, Taq
DNA polymerase reaches the 5' end of the
probe. The 5'-3' exonuclease activity of the
polymerase cleaves the probe, releasing the
fluorophore away from the quencher, thus
resulting in increased fluorescence
Fluorescent probe-based detection
Image of the mechanism of detection by TaqMan probe with a fluorophore on the 5’ end and a
quencher on the 3’ end. When the probe is intact, the fluorescence from the fluorophore is
quenched by the quencher.
During extension, Taq polymerase extends the primer and cleaves off the fluorophore that the 5’
end of TaqMan probe. The fluorophore is now released from the quencher, and the fluorescence
can be detected

Determination of nucleic acid amount by real-time
PCR can be done by absolute or relative
quantification.
Absolute quantification is when the amount of
the target nucleic acid is reported as a copy
number or a concentration.
This method requires a standard curve generated
by external standards of known copy numbers or
concentrations of the same nucleic acid sequence
(or same gene). The standard curve to the right is
a plot of Ct against log of amounts of standard.
A standard curve must be run on the same plate at
the same time of the samples being quantified. A
separate standard curve must be run each time
Image of a linear plot between Ct
values and log of amounts of an
external standard DNA. Ct values are
on the Y axis while the amounts of the
template DNA are on the X axis. The
standard curve declines from left to
right because lower amounts of
template DNA require more cycles for
the fluorescence signals to cross the
threshold
Quantification of Nucleic Acid by Real-Time PCR

Relative quantification where the amount of target nucleic acid is
reported comparative to the abundance of another gene.
Both the target nucleic acid and the control gene are amplified in the
same tube. The amplification efficiency of the control gene should be
comparable to that of the target nucleic acid.
In the graph to the right:
C Test: Cycle number for tested specimen
C Reference: Cycle number for reference gene
∆C: Cycle number difference between tested specimen and reference
gene
Image of two sigmoidal curves, one for the
tested specimen and the other for the reference
gene, plotted between cycle numbers and
fluorescence values.
Fluorescence values are on the Y axis while
the cycle numbers are on the X axis. The
curve increases from left to right as the cycle
numbers increase.
The ∆C identifies the cycle number difference
between the tested specimen and the reference
gene

As a result of differences in the primer annealing, different target sequences such as
Guanine and Cytosine content and PCR product size are not amplified with the same
efficiency. The standard selected for PCR quantitation should have an amplification
efficiency comparable to that of the target nucleic acid. The amplification efficiency
(E) of a gene is calculated from a standard curve as generated for absolute
quantification:
E = (10(-1/S) – 1) x 100 where S is the slope of the standard curve.
An efficiency of 1 means the amount of PCR product doubles at each cycle. In general,
an efficiency of less than 90% is derived from an actual PCR analysis due to experiment
limitations. Lower efficiency could indicate:
•PCR inhibitors in the sample
•Low enzyme quality
•Flaws in primer or probe design
•Inaccurate sample or reagent preparation

It is good practice to include controls when performing PCR to ensure quality of
the results. This can be done by removing or adding certain components, referred
to as negative or positive controls.
Types of Negative Controls
No template control (NTC) - is the reaction with no DNA template input, which
allows the identification of nucleic acid contamination in the reagents.
No reverse transcriptase control (NRT)- is the reaction with no reverse
transcriptase added, which allows detection of specific contaminating DNA in
RNA samples.
No amplification control (NAC) - is the reaction with no DNA polymerase added,
which allows observation of background fluorescence that is not a result of PCR
amplification

It is good practice to include controls when performing PCR to ensure quality
of the results. This can be done by removing or adding certain components,
referred to as negative or positive controls.
Types of Positive Controls
Positive controls are used to demonstrate that the PCR conditions, including the
primers used, are suitable for the analysis. For example, a known purified
extract of the target of interest. Serial dilutions of positive controls can also be
used to generate a standard curve for quantification.

FRET probes bind to the PCR product in a sequence-specific fashion. When two
fluorescence resonance energy transfer (FRET) probes bind to the targets in close
proximity, the energy transfer from the donor fluorophore to the acceptor
fluorophore takes place allowing emission of fluorescence from the acceptor. This
fluorescence signal is observed during the annealing step
Image of the mechanism of FRET probes
which are two oligonucleotides. One probe has
a donor dye at the 3’ end, and the other probe
has an acceptor on the 5’ end.
The two probes bind specifically to adjacent
sequences. When both probes bind to the target
DNA, energy transfer occur, and fluorescence
from the acceptor can be detected.

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