Polymerase chain reaction (PCR)

1
Search ?
? Biology
? Chemistry ? virtual labs ?
E-learning ? Physics
? News ? scientific facts
?
Virtual Labs of Physics, Chemistry and Biology
Praxilabs ? Home / Biology / What Are
The Three Basic Steps of Conventional PCR?
What Are The Three Basic Steps of Conventional
PCR? ? Nourhan Essam ? April 28, 2021 ?
Biology, virtual learning articles ? Comments
Offon What Are The Three Basic Steps of
Conventional PCR? ? 960 Views
Prraxiillabs The Viirrttu
Polymerase chain reaction (PCR) is now a common
and often indispensable technique used in
medical and biological research labs for a
variety of applications. These include DNA
cloning for sequencing, DNA-based phylogeny, or
functional analysis of genes, the diagnosis of
hereditary diseases, and the detection and
diagnosis of infectious diseases.
0000
0218
Recent Posts Cell Cycle Regulation Cyclins and
CDKs Breaking News PraxiLabs in All Universities
in Liberia! PraxiLabs Wins the UN SDGs Award in
Education 2021 PraxiLabs Simulations at Kings
College London Faculty of Nanotechnology for
Postgraduate Studies, Cairo University, a New
Success Partner with PraxiLabs
Categories ? Biology ? Chemistry ?
E-learning ? News
The wide range of applications of PCR has led to
an ever-growing list of variants of the
technique. We will focus on the conventional PCR
which is the most basic type of PCR reaction and
this question what are the three basic steps of
conventional PCR.
? Physics
? scientific facts
? Testimonials
? virtual labs

• Table of Contents
• About PCR
• What Is PCR (polymerase chain reaction)?
• What Is the Conventional PCR Principle?
• What Are the Components of a Conventional PCR
  Setup?
• How Does Conventional PCR Work?
• What Are the Three Basic Steps of Conventional
  PCR?
• Denaturation step
• Annealing step
• Extension/elongation step
• Applications of PCR
• PCR and Infectious Diseases
• PCR Analysis and COVID-19 Infection Detection
• Create Your Free Account and Try Our Simulation
  Conventional PCR

? virtual learning articles
Virtual Science Lab-Praxilabs
Prraxiillabs The Viirrttu
0000
0218
Our Latest Articles
Cell Cycle Regulation Cyclins and CDKs
Breaking News PraxiLabs in All Universities in
Liberia!
About PCR This technology is developed by Kary
Mullis. In 1993, Mullis was awarded the Nobel
Prize in Chemistry along with Michael Smith for
his work on PCR.
PraxiLabs Wins the UN SDGs Award in Education 2021
PraxiLabs Simulations at Kings College London
Faculty of Nanotechnology for Postgraduate
Studies, Cairo University, a New Success
Partner with PraxiLabs
In 1989, Science magazine selected PCR as the
major scientific development and Taq
polymerase, the enzyme essential to PCRs
success, as molecule of the year. The advent of
PCR meant that insufficiencies in the quantity
of DNA were no longer a limitation in molecular
biology research or diagnostic procedures. It is
indeed difficult to find publications in the
biological sciences that do not describe the
application of PCR in some or other way.
Our Categories
? Biology (7)
? Chemistry (5)
? E-learning (9)
What Is PCR (polymerase chain reaction)?
? News (10)
? Physics (8) ? scientific facts (4) ?
Testimonials (1) ? virtual labs (10) ?
virtual learning articles (28)
The polymerase chain reaction (PCR) is a test
tube system for DNA replication which allows a
target DNA sequence to be selectively amplified
several million folds in just a few hours. The
PCR achieves amplification of a predetermined
fragment of DNA, (the target which can, e.g., be
from 100 to 1000 bp long) What Is the
Conventional PCR Principle? The concept of DNA
amplification by PCR is simple and its impact has
been extraordinary. The chemistry involved in
PCR depends on the complementarities (matching)
of the nucleotide bases in the double-stranded
DNA helix. When a molecule of DNA is sufficiently
heated, the hydrogen bonds holding together the
double helix are disrupted and the molecule
separates or denatures into single strands. If
the DNA solution is allowed to cool, the
complementary base pairs can reform to restore
the original double helix. What Is the Minimum
Data Necessary Before a Typical PCR Reaction Can
be Used? In order to use PCR, the exact sequence
of nucleotides that flank (lay on either side of)
the area of interest (the target area that needs
to be amplified), must be known. This is the
absolute minimum data necessary before a typical
PCR reaction can be used. This data is necessary
for the design of PCR primers that are 5-3
oligonucleotides of about 20 nucleotides in
length. These are designed to be complementary to
the flanking sequences of the target area, as
mentioned previously. Thus, the researcher has to
either use previous data (known information of
sequences) or, if this is unavailable, determine
the sequence of these regions experimentally.
The two primers (primer pair) can then be
synthesized chemically and will then serve as
leaders or initiators of the replication
step. The key to the replication reaction is
that it is driven by a heat-stable polymerase
molecule that reads a template DNA in the 3-5
direction and synthesises a new complementary
template in the 5- 3 direction, using free
dideoxy nucleoside triphosphates (dNTPs
nucleotide bases) as building blocks. What Are
the Components of a Conventional PCR Setup? Two
primers (Forward and reverse). Taq
polymerase. Deoxynucleoside triphosphates
(dNTPs). Buffer solution. Distilled water.
Template DNA. How Does Conventional PCR
Work? PCR is a powerful technique that allows
exponential amplification of DNA sequences. A PCR
reaction needs a pair of primers that are
complementary to the sequence of interest.
Primers are extended by the DNA polymerase. The
copies produced after the extension, so called
amplicons, are re-amplified with the same
primers, thus leading to an exponential
amplification of the DNA molecules. After
amplification, gel electrophoresis is used to
analyse the amplified PCR products and this makes
conventional PCR time consuming, since the
reaction must finish before proceeding with the
post-PCR analysis. Real Time PCR overcomes this
problem, because of its ability to measure the
PCR amplicons at early states of the reaction as
they are accumulate in a Real Time Detection
mode, thus measuring the amount of PCR product
where the reaction is still in the exponential
phase (QPCR). What Are the Three Basic Steps of
Conventional PCR?

• PCR is based on three simple steps required for
  any DNA synthesis reaction
• Denaturation of the template into single strands.
• Annealing of primers to each original strand for
  new strand synthesis.
• Extension of the new DNA strands from the
  primers.
• These reactions may be carried out with any DNA
  polymerase and result in the synthesis of defined
  portions of the original DNA sequence. However,
  in order to achieve more than one round of
  synthesis, the templates must again be denatured,
  which requires temperatures well above those
  that inactivate most enzymes. Therefore, initial
  attempts at cyclic DNA synthesis were carried out
  by adding fresh polymerase after each
  denaturation step (1,2). The cost of such a
  protocol becomes rapidly prohibitive.
• Typically, PCR consists of a series of 2040
  repeated temperature changes, called cycles, with
  each cycle commonly consisting of 23 discrete
  temperature steps. The cycling is often preceded
  by a single temperature step at a high
  temperature (gt90 C) and followed by one hold at
  the end for final product extension or brief
  storage. The temperatures used and the length of
  time applied in each cycle depend on a variety
  of parameters. These include the enzyme used for
  DNA synthesis, the concentration of divalent
  ions and dNTPs in the reaction, and the melting
  temperature of the primers.

Flowchart of the three main steps of PCR Now we
will know What happens at each stage of
PCR Initialization step (only required for DNA
polymerases that require heat activation by
hot-start PCR) This step consists of heating
the reaction to a temperature of 9496 C (or 98
C if extremely thermostable polymerases are
used), which is held for 19 minutes. Denaturatio
n step This step is the first regular cycling
event and consists of heating the reaction to
9498 C for 2030 seconds. It causes DNA
melting of the DNA template by disrupting the
hydrogen bonds between complementary bases,
yielding single-stranded DNA molecules. Annealing
step The reaction temperature is lowered to
5065 C for 2040 seconds allowing annealing of
the primers to the single-stranded DNA
template. Extension/elongation step The
temperature at this step depends on the DNA
polymerase used Taq polymerase has its optimum
activity temperature at 7580 C and commonly a
temperature of 72 C is used with this enzyme.
At this step, the DNA polymerase synthesizes a
new DNA strand complementary to the DNA template
strand by adding dNTPs that are complementary to
the template in 5' to 3' direction, condensing
the 5'-phosphate group of the dNTPs with the
3'-hydroxyl group at the end of the nascent
(extending) DNA strand. The extension time
depends both on the DNA polymerase used and on
the length of the DNA fragment to amplify.
3 steps of PCR and temperatures
PraxiLabs 3D virtual biology laboratory provides
the PCR experiment, where you can conduct the
experiment in the virtual biology lab, which
provides science students and professors with a
more accurate understanding of PCR
meaning. Applications of PCR Because of the
great sensitivity, PCR has found popularity in a
wide range of applications Molecular biologists
use PCR in gene cloning and DNA sequencing. The
cellular cloning is one of the most remarkable
applications of PCR. It makes it possible to
isolate, that is to say, to purify a gene without
resorting to traditional methods of molecular
cloning which consist in inserting a DNA library
in a plasmid vector which is then used to
transform a bacterial strain whose clones after
selection are screened. A cellular cloning is
used when using PCR because it is useless to use
a cellular system (bacteria, yeast, and animal or
plant cell) to amplify the clone. The
identification of genetic fingerprints used in
forensic scientists which use PCR to connect
blood, saliva, or tissue left at the scene of a
crime to a suspect or victim. Clinical
geneticists use PCR to determine whether or not
potential parents might carry a genetic disease
that could be passed along to their
children. DNA-based phylogeny, or functional
analysis of genes. The diagnosis of hereditary
diseases. The detection and diagnosis of
infectious diseases. PCR and Infectious
Diseases Infectious diseases can be caused by
microbial pathogens, including agents of fungal,
protozoan, bacterial, clamydial, rickettsia and
viral nature. Despite many advances in
diagnostics and vaccinology, infectious diseases
still have devastating consequences for
agricultural economies, worldwide.
Three examples of devastation with regard to
animal husbandry since the 1990s include the
emergence of the prion, bovine spongiform
encephalopathy (BSE) the huge outbreaks of
foot-and- mouth disease (FMD) in Europe and
avian influenza (AI) in Asia and elsewhere. A
great many of these infectious diseases can be
transmitted from vertebrate animals to man
(called zoonoses) where more than 200 such
zoonotic diseases are known. Infectious diseases
are typically transmitted through the skin or
eyes (direct contact, insect vectors, bite
wounds, and sexual contact). In other cases
agents are airborne and infect the epithelial
cells lining the respiratory tract from where
further systemic infection may proceed.
Additional sources of infectious microorganisms
are contaminated food and water with a route of
infection through the mouth and alimentary
tract, or through the respiratory
system. Laboratory diagnostic technology is
directed towards either the detection of the
presence or absence of a pathogen and its
subsequent identification and characterization
The detection of the pathological effect of, or
immunological response to, infection by a
particular pathogen. PCR represents an entirely
new technology. In vitro bacterial or viral
culture is widely used to isolate and multiply
pathogens, so that the organism itself, or its
antigens, can be more readily detected, by being
present in greater quantity and generally with
fewer contaminants. PCR technology permits the
same principle (i.e., in vitro amplification) to
be applied to the detection of specific
sequences of nucleic acid. There are enormous
benefits to this approach. The application of
PCR to disease diagnosis has been somewhat
restricted to laboratories with the required
facilities, equipment, funding, and expertise.
The procedure must be made in very clean
conditions since contamination with minute
amounts of extraneous DNA may produce false
positive results. PCR Analysis and COVID-19
Infection Detection One of the most important
applications of PCR now is the detection of
COVID-19 Infection. Recently, with the emergence
of COVID-19 virus, we often hear the term PCR
analysis. Which is the basic analysis currently
used to detect Infected people with
COVID-19. Through PCR analysis, scientists can
detect the presence of viruses that cause
infection, even when they are present in small
quantities in the body. This method contributes
to the diagnosis of transmissible viral
diseases, as well as to the identification of
mutations in various genetic disorders. For
more information, write our article PCR Analysis
COVID-19 Infection Detection Method PraxiLabs
Initiative Experiments PraxiLabs Virtual Lab of
Conventional PCR
PraxiLabs provides simulation of conventional PCR
test to make the students gain hands-on
experience of the principle and practice on
conventional polymerase chain reaction
(PCR). PCR should ideally be performed in a
dedicated clean area which is free from other
work involving DNA. Steps of the Experiment 1.
Set up the reaction wearing gloves at all times
and label the lids of the eppendorf. 2. Prepare
an amount of the Master Mix sufficient to 6
samples of the DNA by adding the special
reagents for the experiment in the master mix
tube. 243 ml of distilled water (40.56). 30 ml
of the 10X PCR buffer containing MgCl2 (56). 6
ml of the 10 Mm dNTPs (16). 6 ml of the Forward
primer (16). 6 ml of the Reverse primer
(16). 3 ml of the Taq DNA polymerase
(0.56). 3. Add 49 ml of the Master Mix you just
prepared to 1 ml of each DNA sample. 4. Place the
eppendorfs in the thermal cycler and carry out an
initialization denaturation at 94 degrees
Celsius for 5 minutes for one cycle. 5. Carry out
multiplication cycles, which is divided into
three consecutive steps Denaturation step at 94
degrees Celsius for 30 seconds, 30 cycles.
Annealing step at 50-65 degrees Celsius for 30
seconds, 30 cycles. Elongation step at 72
degrees Celsius for one minute, 30 cycles. 6.
Carry out final elongation at 72 degrees Celsius
for 10 minutes for one cycle. 7. Carry out the
final step in the experiment which is the final
hold at 4 degrees Celsius for an indefinite time
for one cycle. Video To Clarify The Steps of
Conventional PCR Simulation
PCR ((Pollymerrase Chaiin Reacttiion)) Viirrttu
Waattcchh llaatteerr SShhaarree
Watch on
Create Your Free Account and Try Our Simulation
Conventional PCR
About Nourhan Essam
Will Virtual Lab Simulations
Benefit from Using PraxiLabs
Previous
Next
PraxiLabs Organized a Webinar
The University of Jeddah Will
Replace Hands-On Real Labs?
Simulations in Their Courses
Powered by WordPress Designed by TieLabs
Copyright 2021, All Rights Reserved