PraxiLab: PCR conventional

Applications of the method:

Genetic engineering- PCR allows scientists to clone genes or

segments of genes for identification and analysis. PCR also allows
scientists to manipulate a gene that has already been identified.
Various modified PCR techniques allow scientists to hybridize two
separate genes or genes segments into one, delete or invert regions
of DNA, and alter single nucleotides to change the gene and its
encoded protein in a subtler way.
Diagnostics- Polymerase chain reaction (PCR) is a broadly applied
laboratory test for the diagnosis of a wide variety of central nervous
system (CNS) diseases, including genetic and autoimmune diseases,
malignant neoplasms, and infections. With its ability to detect
minute amounts of DNA or RNA contained in tissues or fluids, PCR
has improved the rapidity and accuracy of diagnosis, enhanced
understanding of pathogenesis, and helped identify infectious causes
for diseases previously considered idiopathic. In addition, PCR can be
performed on a variety of tissues preserved in different ways—even
archival specimens can be used to provide important epidemiological
information.
Forensic analysis- The polymerase chain reaction (PCR)
revolutionized the forensic individualization of biological material by
forming the basis of the so-called “genetic fingerprint”, which allows
for e.g. comparisons between stains found at a crime scene and a
suspect, or the identification of human remains. PCR based
genotyping approaches became indispensable tools in modern
forensic science, routine case-work, and paternity testing.
DNA isolation- The use of DNA isolation technique should lead to
efficient extraction with good quantity and quality of DNA, which is
pure and is devoid of contaminants, such as RNA and proteins.
Manual methods as well as commercially available kits are used for
DNA extraction. Various tissues including blood, body fluids, direct
Fine needle aspiration cytology (FNAC) aspirate, formalin-fixed
paraffin-embedded tissues, frozen tissue section, etc., can be used
for DNA extraction. To amplify a segment of DNA using PCR, the
sample is first heated so the DNA denatures, or separates into two
pieces of single-stranded DNA. Next, an enzyme called "Taq
polymerase" synthesizes - builds - two new strands of DNA, using the
original strands as templates.

Screenshots of at least 5 steps in the protocol, and why each step is important
towards the achievement of the desired outcome(s) of this method/technique .
Image Importance
of step

1. Adding
30µl of the
10x PCR
buffer
containing
MgCl
Most
manufactu
rers
include a
solution of
Magnesiu
m chloride
(MgCl2)
along with
the DNA
polymeras
e and a
10X PCR
buffer
solution.
The 10 X
PCR buffer
solutions
may
contain 15
mM
MgCl2,
which is
enough for
a typical
PCR
reaction,
or it may
be added
separately
at a
concentrati
on
optimized
for a
particular
reaction.
2. Adding 49
µl of the
master mix
to the
samples

Master mix
contains all
the
componen
ts for PCR
mix to
occur;
including
the
individual
building
blocks of
DNA
(nucleotide
s, or
dNTP's), a
special
buffer to
maintain
optimum
pH, salts,
and MgCl2.
The master
 mix
enables
researcher
s to set up
controls
and test
different
concentrati
ons of their
target DNA
or RNA
templates
without
having to
individually
add precise
amounts of
enzymes,
buffers,
cofactor
(usually
MgCl2),
water and
dNTP to
each
reaction
tube or
plate well.
3. Placing the
eppendorfs
in the
thermal
cycler
Thermocyc
lers, or
thermal
cyclers, are
instrument
s used to
amplify
DNA and
RNA
samples by
the
polymeras
e chain
reaction.
The
thermocycl
er raises
and lowers
the
temperatu
re of the
samples in
a holding
block in
discrete,
pre-
programm
 ed steps,
allowing
for
denaturati
on and
reannealin
g of
samples
with
various
reagents
4. Doing
multiplicati
on cycles
PCR steps
of
denaturati
on,
annealing,
and
extension
are
repeated
(or
“cycled”)
many
times to
amplify the
target
DNA.

Denaturati
on- The
initial
denaturati
on step is
carried out
at the
beginning
of PCR to
separate
the
double-
stranded
template
DNA into
single
strands so
that the
primers
can bind to
the target
region and
initiate
extension.
Complete
denaturati
on of the
input DNA
helps
ensure
efficient
amplificati
on of the
target
sequence
during the
first
amplificati
on cycle.
Furthermo
re, the high
temperatu
re at this
step helps
inactivate
heat-labile
proteases
or
nucleases
that may
be present
in the
sample,
with
minimal
impact on
thermosta
ble DNA
polymeras
es. When
using a
hot-start
DNA
polymeras
e, this step
also serves
to activate
the
enzyme,
although a
separate
activation
step may
be
recommen
ded by the
enzyme
supplier.
Annealing-
this stage
enables
the
primers to
attach to a
specific
location on
the single-
stranded
template
DNA by
way of
hydrogen
bonding
(the exact
temperatu
re depends
on the
melting
temperatu
re of the
primers
you are
using).
Primers
are single
strands of
DNA or
RNA?
sequence
that are
around 20
to 30 bases
in length.
Why is the
annealing
temperatu
re
important?
During the
annealing
phase of
PCR, the
reaction
temperatu
re needs to
be
sufficiently
low to
allow both
forward
and
reverse
primers to
bind to the
template,
but not so
low as to
enable the
formation
of
undesired,
non-
specific
duplexes
or
intramolec
ular
hairpins,
both of
which
reduce
reaction
efficiency.
Extension-
The
extension
step, also
referred to
as the
elongation
step, is the
PCR step in
which Taq
polymeras
e adds
nucleotide
s to the
annealed
primer.
The
process of
repeating
the
denaturati
on,
annealing
and
extension
steps of
PCR is
known as
PCR
cycling.
The
temperatu
re at this
step
depends
on the
DNA
polymeras
e used; Taq
polymeras
e has its
optimum
activity
temperatu
re at 75–80
°C and
commonly
a
temperatu
re of 72 °C
is used
with this
enzyme. At
this step,
the DNA
polymeras
e
synthesizes
a new DNA
strand
compleme
ntary to
the DNA
template
strand by
adding
dNTPs that
are
compleme
ntary 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
polymeras
e used and
on the
length of
the DNA
fragment
to amplify.

Adding 6 µl
of the
forward
primer
Forward
Primer is a
5. DNA
stretch
that
attaches to
the
antisense
 strand (-)
of the DNA
that runs
in 3' to 5'
direction.
The
primers
anneal to
the DNA
strand and
bring
about
amplificati
on. The
forward
primer is
compleme
ntary to
the strand
they bind
to.
Compare and contrast the methods and features of PCR
Conventional vs Real-Time PCR.