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Polymerase chain reaction

(PCR)
Sample collection (blood ,tissue ,swabs..etc)
Pre-PCR techniques

DNA extraction

DNA measurement

PCR

Electrophoresis
ost-PCR techniques

Sequencing blotting RFLP Hybridization

DATA analysis and documentation


Introduction
PCR is a highly sensitive technique that takes a
specific sequence of DNA of small amounts and
amplifies it to be used for further testing.
It was invented in 1983 by Dr.kary Mullis, for
which he received the Noble prize in chemistry in
1993.
PCR is based on using the ability ofDNA
polymeraseto 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
aprimer 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).
PCR is an exponentially in vitro progressing.
Components of PCR reaction
(1)DNA template
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
(2)DNA
from each other. 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 (fromThermus
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:-
(A)They can generate new strands of DNA using a
DNA template and primers.
(B)They are heat resistant.
Thermus aquaticus
Is a microbe found in 176F hot springs in
Yellow Stone National Forest.
(3)Primers
Oligonucleotides that define the sequence to be
amplified.
Primers range from 15 to 30 nucleotides, are
single stranded DNA, and are used for the
complementary building blocks of the target
sequence.
The optimal primer size is usually between 18
(4)Deoxynucleotide
and 28 bases.
triphosphates
(dNTPs
Single units of the bases A, T,) G, and C, which
are essentially "building blocks" for new DNA
strands.
(5)Magnesium ions
A necessary cofactor for DNA polymerase
activity.
(6)Buffer solution
Maintains pH and ionic strength of the reaction
solution suitable for the activity of the enzyme
PCR Reaction in a Test Tube PCR Machine
PCR cycles
(1)Denaturing:- 94- 95C.
(2) Primer Annealing:- 55- 65C.
(3)Extension of DNA:- 72C.
(1)Denaturing
Heated above the melting point of the two
complementary strands of the template DNA,
which allows the strands to separate.

Raise temperature to
94oC to separate the
duplex form of DNA into
single strands
(2)Annealing
Annealing is the process of allowing two
sequences of DNA to form hydrogen bonds.
The temperature is lowered (cooling the DNA to
55C)
which allows the primers to bind to the specific
and complementary DNA sequence to be
amplified.
5 TTAACGGCCTTAA . . . TTTAAACCGGTT 3 DNA
strand
AATTGCCGGAATT . . . . . . . . . .> forward primer
reverse primer <. . . . . . . . . . AAATTTGGCCAA
DNA t d 3 TTAACGGCCTTAA strand 3 . . .
TTTAAACCGGTT 5
Annealing temperature
Tm = 4 (G + C) + 2 (A + T)
(3)Extension
The DNA polymerase is able to extend the
primers by adding nucleotides to the developing
DNA strand.
Extend primers raise temp to 72oC, allowing Taq
pol to attach at each priming site and extend a
new DNA strand
Repeat the Denature, Anneal, Extension steps at
their respective temperatures.
Template DNA

Denature (heat to
95oC)
Denaturing

Lower temperature to
56oC Anneal with
primers 1
cycle
Template DNA

Increase temperature to
72oC DNA polymerase +
dNTPs

Elongation
DNA copies vs Cycle number
2500000

2000000
DNA copies

1500000

1000000

500000

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Cycle number