CONTENTS

Introduction
Principle of PCR
Components of PCR
Types of PCR
Steps of PCR
1.denaturation
2.annealing
3.elongation
4.analysis with electrophoresis
Aplication of PCR
Limitations of PCR
Introduction:
PCR or Polymerase Chain Reaction is a technique used in molecular
biology to create several copies of a certain DNA segment. This technique
was developed in 1983 by Kary Mullis, an American biochemist. PCR has
made it possible to generate millions of copies of a small segment of DNA.
This tool is commonly used in the molecular biology and biotechnology

What is PCR?

Studying isolated pieces of DNA is nearly impossible. Large amounts of a

sample of DNA are necessary for molecular and genetic analyses.
Sometimes called molecular photocopying, conventional polymerase chain
reaction (PCR) is a technique used to amplify (replicate) trace amounts of
DNA and RNA from a sample. A PCR thermal cycler is used to produce
the large amounts required for research.
PCR is based on using the ability of DNA polymerase to synthesize new
strand of DNA complementary to the offered template strand. Because
DNA polymerase can add a nucleotide only onto a preexisting 3'-OH
group, it needs a primer to which it can add the first nucleotide. This
requirement makes it possible to delineate a specific region of template
sequence that the researcher wants to amplify. At the end of the PCR
reaction, the specific sequence will be accumulated in billions of copies
(amplicons)
The PCR process can be used for a wide variety of laboratory and clinical
applications and purposes. Forensic labs use it to analyze DNA samples
from a crime scene. Clinical health care labs use it to diagnose patients
infected from a virus. Pharmaceutical research labs use it to analyze and
duplicate DNA and RNA samples for use in the manufacturing of drugs and
vaccines.

PCR requirements needed

Four components (reagents or chemicals) are needed for the PCR process:

 A DNA or RNA sample (from saliva, blood, hair, skin scraping, etc.)
 DNA primers: short single-stranded DNA that promotes synthesis of
a complementary strand of nucleotides
 DNA polymerase: an enzyme that aids in the synthesis of a
complementary strand of DNA
 Nucleotide solution mix containing adenine (A), thymidine (T),
cytosine (C), and guanine (G) used to build duplicate DNA strands
 Principle of PCR
The PCR technique is based on the enzymatic replication of DNA. In PCR,
a short segment of DNA is amplified using primer mediated enzymes. DNA
Polymerase synthesises new strands of DNA complementary to the
template DNA. The DNA polymerase can add a nucleotide to the pre-
existing 3’-OH group only. Therefore, a primer is required. Thus, more
nucleotides are added to the 3’ prime end of the DNA polymerase.

Components Of PCR
Components Of PCR constitutes the following:
DNA Template
The DNA of interest from the sample DNA that contains the target
sequence. At the beginning of the reaction, high temperature is
applied to the original double-stranded DNA molecule to
separate the strands from each other.
DNA Polymerase
Taq Polymerase is used. It is thermostable and does not denature at very high
temperatures. DNA polymerase
- a type of enzyme that synthesizes new strands of DNA complementary to the
target sequence. The first and most commonly used of these enzymes
isTaqDNA polymerase (fromThermis aquaticus), whereasPfuDNA
polymerase (fromPyrococcus furiosus) is used widely because of its
higher fidelity when copying DNA. Although these enzymes are subtly
different, they both have two capabilities that make them suitable for
PCR: 1) they can generate new strands of DNA using a DNA template
and primers, and 2) they are heat resistant.

Oligonucleotide Primers- These are the short stretches of single-stranded

DNA complementary to the 3’ ends of sense and anti-sense strands The
polymerase begins synthesizing new DNA from the end of the primer.
Nucleotides (dNTPs or deoxynucleotide triphosphates)
- single units of the bases A, T, G, and C, which are essentially "building
blocks" for new DNA strands
.
Deoxyribonucleotide triphosphate– These provide energy for
polymerization and are the building blocks for the synthesis of DNA. These
are single units of bases.
Buffer System– Magnesium and Potassium provide optimum conditions
for DNA denaturation and renaturation. It is also important for fidelity,
polymerase activity, and stability.
1.
 RT-PCR
Reverse Transcription PCR) is PCR preceded with conversion of sample
RNA into cDNA with enzyme
reverse transcriptase

Types of PCR
PCR is of the following types:
Real-time PCR
In this type, the DNA amplification is detected in real-time with the help of
a fluorescent reporter. The signal strength of the fluorescent reporter is
directly proportional to the number of amplified DNA molecules.
Nested PCR
This was designed to improve sensitivity and specificity. They reduce the
non-specific binding of products due to the amplification of unexpected
primer binding sites.
Multiplex PCR
This is used for the amplification of multiple targets in a single PCR
experiment. It amplifies many different DNA sequences simultaneously.
Quantitative PCR
It uses the DNA amplification linearity to detect, characterize and quantify
a known sequence in a sample.
Arbitrary Primed PCR
It is a DNA fingerprinting technique based on PCR. It uses primers the
DNA sequence of which is chosen arbitrarily.
PCR Steps

PCR process steps

The PCR process has 4 steps:collection, preparation, amplification,

and post PCR clean-up. The PCR machine steps happen in the
amplification step. It begins with a segment of a DNA sample placed in
a suitable tube along with the reagents and chemicals listed above. The
tube is placed into the PCR machine or thermal cycler. The thermal
cycler takes the solution through a 3-step process: denaturation,
annealing, and extension.
The PCR involves three major cyclic reactions:
Denaturation
Denaturation occurs when the reaction mixture is heated to 94 ℃ for about
0.5 to 2 minutes using a thermal cycler. The heat breaks breaks the
hydrogen bonds between the two strands of DNA and converts it into a
single-stranded DNA.
The single strands now act as a template for the production of new strands
of DNA. The temperature should be provided for a longer time to ensure
the separation of the two strands.

Annealing
The reaction temperature is lowered to 54-60℃ for around 20-40 seconds.
Here, the primers bind to their complementary sequences on the template
DNA. At this point, the nucleotides (A, T, C, G) from the added
mixture solution will pair with the individual separated strands of
DNA that resulted from the heating process
Primers are single-strand sequences of DNA or RNA around 20 to 30 bases
in length.
They serve as the starting point for the synthesis of DNA.
At this point, the nucleotides (A, T, C, G) from the added mixture
solution will pair with the individual separated strands of DNA that
resulted from the heating process
The two separated strands run in the opposite direction and consequently
there are two primers- a forward primer and a reverse primer.

Elongation
Once joined together, they form a new complementary strand of DNA
(termed extension of the DNA)
At this step, the temperature is raised to 72-80℃. Thus, a new duplicate
double-stranded DNA molecule has been formed from each of the
single strands of the original sample molecule. The temperature cycles
from 95°C to 50 to 60°C. The cycle is then repeated about 35 to 40
times using the thermal cycler which automatically repeats the heating
and cooling cycles of the process. .. Resulting DNA sequence is doubled
each time the heating/cooling cycle is conducted by the cycler. Thus,
what started out as a single short segment of DNA from one sample can
be amplified to form millions of copies after 35 doubling cycles.

The bases are added to the 3’ end of the primer by the Taq polymerase
enzyme.
This elongates the DNA in the 5’ to 3’ direction. The DNA polymerase adds
about 1000bp/minute under optimum conditions.
Taq Polymerase can tolerate very high temperatures. It attaches to the
primer and adds DNA bases to the single strand. As a result, a double-
stranded DNA molecule is obtained.
Step 4 - Analysis with Electrophoresis

Once the PCR process is completed, the resulting amplified (replicated)

segments generated can then be compared to other nucleotide segments
from a known source. The PCR-generated nucleotide sequences are
then placed next to known nucleotide sequences from humans,
pathogens, or other sources in a separating gel. Electrical current is
then run through the gel, and the various nucleotide sequences within
the gel form bands that resemble a ladder, according to their electrical
charge and molecular size. This is termed gel electrophoresis. Bands or
ladder-like steps that migrate to the same levels in the gel show identity
of nucleotide sequences. This method is one of the most popular ways
PCR tests are completed.

PCR Temperature Cycle

Flow chart of the three main steps of PCR with the PCR temperature
cycle, number of cycles, and total program length.
This process provides a new duplicate double-stranded DNA molecule
formed from each of the single strands of the original sample molecule.
This three-step process is done 35 to 40 times to amplify the DNA/RNA
into millions of duplicate segments
Applications of PCR
The following are the applications of PCR :

Medicine

 Testing of genetic disease mutations.

 Monitoring the gene in gene therapy.
 Detecting disease-causing genes in the parents.
 Gene mutation detection: PCR has helped detect gene mutations.
 Clinical microbiology: PCR has helped diagnose infectious diseases
by detecting pathogen DNA signatures.

Forensic Science

 Identifying the criminal from millions of people.

 Paternity testing: PCR has helped with paternity testing.
 Genetic fingerprinting: PCR has helped with genetic fingerprinting
 Forensic investigations: PCR has helped identify criminals from tiny
amounts of DNA left at crime scenes.

Research and Genetics

 Compare the genome of two organisms in genomic studies.

  In the phylogenetic analysis of DNA from any source such as fossils.
 Analysis of gene expression.
 Gene Mapping
 DNA databasing: PCR has helped with DNA databasing for forensic
identification.
 DNA barcoding: PCR has helped with DNA barcoding of species.
 Environmental microbial diversity: PCR has helped with
metagenomics studies of environmental microbial diversity.
 Food adulteration: PCR has helped identify DNA and RNA
adulterants in food.
 Food safety: PCR has helped with food safety

Polymerase chain reaction (PCR) has several disadvantages, including:

 Contamination
PCR can be susceptible to contamination from other DNA or RNA sources, or from the
environment. This can lead to misleading data interpretation.
 Cost
PCR can be expensive.
 Lack of novel information
PCR can only amplify specific DNA sequences, so it can't detect novel DNA sequences.
 Inhibitors
Inhibitors in the sample, like heme from blood, can disrupt the PCR cycle and reduce the
process's sensitivity.
 Errors in amplification
The DNA polymerase used in PCR can make errors, which can lead to mutations in the generated
fragment.
 Primer binding
Primers can bind non-specifically to similar sequences on the template DNA, which can alter the
specificity of the PCR product.
 One-step PCR
One-step PCR is less sensitive than two-step PCR because the reaction conditions are a
compromise between the two combined reactions.
 Multiplex PCR
Multiplex PCR may have lower amplification efficiency than singleplex PCR reactions.
 False results
PCR can produce false-positive or false-negative results.
 Limited detection space
PCR may have limited detection space for simultaneously identifying multiple species, virulence
factors, or drug resistance

PCR cannot be used to amplify unknown targets. Prior information about

the target sequence is necessary to design the primers. DNA polymerases
are prone to error, which potentially causes mutations in PCR product
 Limitations of PCR and RT-PCR

The PCR reaction starts to generate copies of

the target sequence exponentially.
Only during the exponential phase of
the PCR reaction is it possible to
extrapolate back to determine the
LIMITATIO
starting quantity of the target
NS OF PCR
sequence contained in the sample.
Because of inhibitors of the
polymerase reaction found in the
sample, reagent limitation,
accumulation of pyrophosphate
molecules, and self-annealing of the
accumulating product, the PCR
reaction eventually ceases to amplify
target sequence at an exponential rate
and a "plateau effect" occurs, making
the end point quantification of PCR
products unreliable. This is the
attribute of PCR that
makesRealQuantitative RT-PCRso
necessary.
Limitations of PCR and RT-PCR

The PCR reaction starts to generate copies of

the target sequence exponentially.
Only during the exponential phase of
the PCR reaction is it possible to
extrapolate back to determine the
starting quantity of the target
sequence contained in the sample.
Because of inhibitors of the
polymerase reaction found in the
sample, reagent limitation,
accumulation of pyrophosphate
molecules, and self-annealing of the
accumulating product, the PCR
reaction eventually ceases to amplify
target sequence at an exponential rate
and a "plateau effect" occurs, making
the end point quantification of PCR
products unreliable. This is the
attribute of PCR that makesReal-Time
Quantitative RT-PCRso necessary.
Limitations of PCR and RT-PCR

The PCR reaction starts to generate copies of

the target sequence exponentially.
Only during the exponential phase of
the PCR reaction is it possible to
extrapolate back to determine the
starting quantity of the target
sequence contained in the sample.
Because of inhibitors of the
polymerase reaction found in the
sample, reagent limitation,
LIMITATIONS OF PCR

 PCR can be susceptible to contamination from other DNA or RNA

sources, or from the environment. This can lead to misleading data
interpretation.
 Lack of novel information
 PCR can only amplify specific DNA sequences, so it can't detect
novel DNA sequences.
 Inhibitors in the sample, like heme from blood, can disrupt the PCR
cycle and reduce the process's sensitivity.
 Errors in amplification
 The DNA polymerase used in PCR can make errors, which can lead
to mutations in the generated fragment.
 Primer binding
 Primers can bind non-specifically to similar sequences on the
template DNA, which can alter the specificity of the PCR product.
 One-step PCR
 One-step PCR is less sensitive than two-step PCR because the
reaction conditions are a compromise between the two combined
reactions.
 Multiplex PCR
 Multiplex PCR may have lower amplification efficiency than single
plex PCR reactions.
 False results
 PCR can produce false-positive or false-negative results.
 Limited detection space
 PCR may have limited detection space for simultaneously
identifying multiple species, virulence factors, or drug resistance
REFERENCE

Coleparmer.com
Byjus.com
Ncbi.nlm.nih.gov
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