Comprehensive Overview of Polymerase Chain Reaction (PCR) Techniques and Applications

POLYMERASE CHAIN REACTION (PCR)
The polymerase chain reaction (PCR) is a technique which is
used to amplify the number of copies of specific DNA sequences
in order to produce enough DNA to be tested using various
method of detection.
This technique finds application in Diagnosis of infectious
disease, Diagnosis of Gene Mutation and in Forensics, the
identity of an individual or paternity.

The polymerase chain reaction (PCR)
was discovered by Kary Mullis in
1983.
Mullis's invention was a game-
changer for molecular biology, and
he was awarded the Nobel Prize in
Chemistry in 1993 for his work.
Discovery of PCR

• DNA (Deoxyribonucleic acid) is the nucleic acid that functions
as the original blueprint for the synthesis of proteins. DNA
contains the sugar deoxyribose, phosphates and a unique
sequence of the nitrogenous bases Adenine (A), Guanine (G),
Cytosine (C) and Thymine (T).
• Ribonucleic acid (RNA) is a nucleic acid which is directly
involved in protein synthesis. RNA contains the sugar ribose,
phosphates, and the nitrogenous bases Adenine (A), Guanine
(G), Cytosine (C), and Uracil (U).
• DNA and RNA share the nitrogenous bases A, G, and C. Thymine
is usually only present in DNA and uracil is usually only present
in RNA.
DNA & RNA

• The DNA, as we know, is double stranded, and the two strands
pair with one another in a very precise way.
• Each letter in a strand will pair with only one kind of letter across
it in the opposite strand i.e. A always pairs with T and C always
pairs with G. For example:
TTAACGGGGCCCAAA…………..TTTAAACCCGGGTTT would pair with
AATTGCCCCGGGTTT……………AAATTTGGGCCCAAA
BASICS OF POLYMERASE CHAIN
REACTION (PCR)

1. NUCLEIC ACID TEMPLATE (TEMPLATE DNA)
Template DNA is the sample DNA that contains the selected nucleic
acid sequence (DNA target region) that needs to be amplified.
The template must be DNA only: Genomic DNA (gDNA) or
complementary DNA (cDNA).
Reverse transcriptase polymerase chain reaction (RT-PCR) uses
RNAs as starting materials, but RNAs are primarily converted to
complementary DNA (cDNA) before amplification.
The template DNA must be highly pure, with an ideal quantity of 30
μg to 50 μg.
Optimization of the amount of template DNA required is necessary
because higher DNA concentration increases the possibility of non-
specific amplification whereas low amount of DNA leads to reduced
yield.

2. Taq DNA POLYMERASE
Taq DNA polymerases are enzymes that catalyse the synthesis
of complementary DNA strands by assembling the nucleotides
sequentially according to the template strand.
Taq DNA polymerase enzyme extracted from the bacterium
Thermus aquaticus, is the most widely and the best known
DNA polymerase used in PCR.
Taq DNA polymerase is thermally stable and continues its
activity after the repeated heating and cooling cycle. It is stable
up to 95°C and shows the most effective reaction at around
72°C to 78°C.
In a 50 L reaction mixture, around 1 to 2 units of Taq
polymerase is sufficient for amplification.

3. PRIMERS
Primers are artificially synthesized short single-stranded
sequences of oligonucleotides that are complementary to the
target nucleic acid sequence in the template DNA.
They are short sequences of around 15 to 30 bases that act as
starting point for DNA synthesis.
Types of PCR Primers:
A) The Forward primers are complementary to the antisense strand
(template strand from 3’ to 5’ direction), and are responsible for
the amplification of the antisense strand. They are also called 5’
primers.
B) The Reverse primers are complementary to the sense strand
(template strand from 5’ to 3’ direction) and are responsible for the
amplification of the sense strand. They are also called 3’ primers.

4. DEOXYNUCLEOTIDE TRIPHOSPHATES (dNTPs)
Deoxynucleotide triphosphates (dNTPs) are artificially
synthesized nucleotides that act as building blocks of new
DNA strands.
There are 4 different dNTPs used in the PCR;
deoxyadenosine triphosphate (dATP), deoxyguanosine
triphosphate (dGTP), deoxythymidine triphosphate
(dTTP), and Deoxycytidine triphosphate (dCTP).
These four dNTPs are sequentially added to the annealed
primer by the DNA polymerase enzyme generating a new
strand of DNA complementary to the template strand.

5. PCR BUFFERS AND OTHER CHEMICALS
A PCR buffer is a solution containing chemicals that regulate
pH and provide essential ions, such as potassium (K+) and
magnesium (Mg2+), to support the DNA polymerase enzyme
and facilitate the PCR process.
Its primary functions are to maintain optimal reaction
conditions, ensure primer-template annealing, and increase
PCR specificity and performance by minimizing non-specific
amplification and reducing secondary DNA structures.

Key Components of a PCR Buffer:
Tris-HCl: A buffer to resist pH changes and maintain the
optimal pH range (usually 8.0 to 9.5) for the DNA polymerase.
Potassium Chloride (KCl): Provides essential potassium ions
(K+) that stabilize the DNA primer-template annealing
complex, reducing electrostatic repulsion and promoting
specific binding.
Magnesium Chloride (MgCl2): A critical cofactor for DNA
polymerase activity and DNA primer annealing.

PCR has three main stages, which involve a process of heating and
cooling:
Step 1, Denaturation: the double-stranded template DNA is heated,
which separates it into two single strands.
Step 2, Annealing: the temperature is lowered to enable the DNA primers
to attach to the template DNA.
Step 3, Extension: the temperature is raised again and the new strand of
DNA is made by the Taq polymerase enzyme.
These three stages are repeated 20-40 times, doubling the number of
DNA copies each time. This is called thermal cycling.
It’s carried out by a machine called a thermal cycler. Depending on the
speed of the machine, PCR can be performed in under and hour or up to a
few hours.
POLYMERASE CHAIN REACTION (PCR)

STEP 1, DENATURATION:
The reaction mixture is heated to 94-95⁰C, for between 15 and
30 seconds.
The high temperature causes the hydrogen bonds between the
bases in two strands of template DNA to break and the two
strands to separate.
This results in two single strands of DNA, which will act as
templates to produce the new copies of each strand of DNA.
It is important that the temperature is maintained at this stage
for long enough to ensure that the DNA strands have separated
completely.

STEP 2, ANNEALING:
The reaction is cooled to enable the primers to attach to a
specific location on the single-stranded template DNA by way of
hydrogen bonding.
The temperature depends on the characteristics of the primer,
but is usually between 50 and 65⁰C.
The two separated strands of DNA are complementary and run
in opposite directions (from one end – the 5’ end – to the other
– the 3’ end). As a result, there are two primers – a forward
primer and a reverse primer.
The annealing step usually takes about 10-30 seconds.

STEP 3, EXTENSION:
The heat is increased to 72⁰C to enable the new DNA to be
made by Taq DNA polymerase enzyme which adds DNA bases.
At 72 C, the Taq polymerase begins to build the
⁰
complementary strand. It attaches to the primer and then
adds DNA bases to the single strand one-by-one.
The result is a brand-new strand of DNA and a double-
stranded molecule of DNA.
The duration of this step depends on the length of DNA
sequence being amplified. It usually takes around one
minute to copy 1,000 DNA bases.

COMMON PCR TYPES:
CONVENTIONAL PCR: The basic method used to amplify specific DNA sequences.
REVERSE TRANSCRIPTASE PCR (RT-PCR): Used to detect and amplify RNA by
first converting it into complementary DNA (cDNA).
REAL-TIME PCR (qPCR): Monitors the amplification of DNA in real-time using
fluorescent markers, allowing for quantification of the target sequence.
MULTIPLEX PCR: A technique that amplifies multiple different DNA targets from a
single sample in one reaction using multiple primer sets.
NESTED PCR: Increases specificity and sensitivity by performing two sequential
rounds of PCR with two different sets of nested primers.
TYPES OF POLYMERASE CHAIN
REACTION (PCR)

This defined as a normal PCR
process. Here the primers bind
specifically to each other with 2 DNA
strands.
All that needed for the PCR process
are PCR tubes, Taq DNA polymerase,
buffer, and target DNA, primers. The
whole process takes place within 35-
40 minutes repeatedly.
This is viewed by gel electrophoresis
technique. 2% agarose gel is used
with ethidium bromide dye for
analysing the samples.
CONVENTIONAL PCR

Real-time PCR also called quantitative PCR (qPCR), in which amplification
and simultaneous quantitation of a target DNA is done in the same PCR
machine, using commercially available fluorescence-detecting
thermocyclers.
Fluorescent dyes specifically label DNA of interest, and the amount of
fluorescence generated is proportional to the quantity of DNA present.
The two most commonly used real-time PCR methods are SYBR green (a
dye that binds to double-stranded DNA but not to single-stranded DNA,
and, when so bound, fluoresces) and TaqMan probes, respectively.
SYBR Green uses a fluorescent dye that binds non-specifically to all
double-stranded DNA, while TaqMan probes use a fluorogenic probe that
binds only to a specific target sequence via hybridization.
REAL-TIME PCR

Reverse Transcriptase PCR (RT-PCR) amplifies target
RNA. Addition of reverse transcriptase (RT) enzyme
prior to PCR makes it possible to amplify and detect
RNA targets.
Reverse transcriptase enzyme transcribes the
template RNA and forms complementary DNA
(cDNA).
Single-stranded cDNA is converted into double-
stranded DNA using DNA polymerase. These DNA
molecules can now be used as templates for a PCR
reaction.
Reverse transcription and PCR amplification can be
performed as a two-step process in a single tube or
with two separate reactions. In both cases, RNA is
first reverse-transcribed into cDNA, which is then
used as the template for PCR amplification.
REVERSE TRANCRIPTASE (RT) PCR

Multiplex PCR is a variant of PCR method
in which more than one target sequence
are amplified using multiple sets of
primers within a single PCR mixture.
Multiplex PCR technique detects
different pathogens in a single sample,
this enables amplification of several gene
segments at the same time, instead of
specific test runs for each.
Multiplex PCR is a space, time, and cost-
effective method for genetic analyses
that need to be repeated many times
MULTIPLEX PCR

Figure 1: Schematic presentation of an example for location of
samples and controls on a 96 well plate for the ABI 7500.
Rows A-H; columns 1-12= layout of the 96 well plate
S1; S2; S3; S4= Master mix and samples 1-4
PC= Master mix and positive control
NC= Master mix and negative control

Nested PCR is a modification of PCR intended to
reduce non-specific binding in products due to the
amplification of unexpected primer binding sites.
Nested polymerase chain reaction involves two sets
of primers, used in two successive runs of PCR.
The target DNA undergoes the first run of PCR with
the first set of primers, shown in green.
The product from the first reaction undergoes a
second run with the second set of primers, shown in
red.
The first set of primers also called “outer primers”
amplify a large fragment of the gene which is used
as a template in the second round of PCR that
targets a smaller region of the amplicon using the
second set of primers also known as “inner primers
or nested primers.”
NESTED PCR

The BioFire FilmArray principle is based on an
automated, two-stage nested multiplex PCR
process within a single, self-contained FilmArray
pouch, which automates nucleic acid extraction,
amplification, and detection of multiple pathogens
from a clinical sample.
A hydration solution is injected into the pouch,
rehydrating freeze-dried reagents for sample
preparation and nested multiplex PCR.
 During the first stage, primers amplify all targets,
and the second stage uses specific primers to
selectively amplify only the target DNA from the
first stage, resulting in melt curve analysis to
confirm the presence of pathogens.
BIOFIRE FILMARRAY

Comprehensive Overview of Polymerase Chain Reaction (PCR) Techniques and Applications

Comprehensive Overview of Polymerase Chain Reaction (PCR) Techniques and Applications