PCR

• Polymerase chain reaction was invented by Kary Mullis

• The idea was to develop a process by which DNA could be artificially multiplied through repeated
cycles of duplication driven by an enzyme called DNA polymerase
• DNA polymerase occurs naturally in living organisms. In cells it functions to duplicate DNA when
cells divide in mitosis and meiosis. Polymerase works by binding to a single DNA strand and creating
the complementary strand.
PRINCIPLE OF PCR

• The double stranded D N A is denatured to separate into two individual strands .

• Each stands is then allowed to hybridized with a primer.
• The primer- template duplex is used for DNA synthesis [the enzyme DNA polymerase].
• Three steps involved are,
Denaturation,
Renaturation,
Elongation
Synthesis.

PCR, as currently practiced, requires several basic components. These components are:

• DNA template, which contains the region of the DNA fragment to be amplified
• Two primers, which determine the beginning and end of the region to be amplified (see following
section on primers)
 • Taq polymerase (or another durable polymerase), a DNA polymerase, which copies the region to be
amplified
• Deoxynucleotides-triphosphate, from which the DNA Polymerase builds the new DNA
• Buffer, which provides a suitable chemical environment for the DNA Polymerase
• The PCR process is carried out in a thermal cycler. This is a machine that heats and cools the reaction
tubes within it to the precise temperature required for each step of the reaction. To prevent evaporation
of the reaction mixture (typically volumes between 15-100µl per tube), a heated lid is placed on top of
the reaction tubes or a layer of oil is put on the surface of the reaction mixture.

DNA polymerase

• The double-stranded DNA was separated into two single strands by heating it to 94°C (201°F).
• At this temperature, however, the DNA polymerase used at the time was destroyed, so the enzyme
had to be replenished after the heating stage of each cycle.
• The original procedure was very inefficient, since it required a great deal of time, large amounts of
DNA polymerase.
• Later, this original PCR process was greatly improved by the use of DNA polymerase taken from
thermophilic bacteria grown in geysers at a temperature of over 110°C (yellow stone national forest )
• The DNA polymerase taken from these organisms is stable at high temperatures and, when used in
PCR, does not break down when the mixture was heated to separate the DNA strands. Since there was
no longer a need to add new DNA polymerase for each cycle, the process of copying a given DNA
strand could be simplified and automated.
• One of the first thermostable DNA polymerases was obtained from Thermus aquaticus and was called
"Taq."
• A disadvantage of Taq is that it sometimes makes mistakes when copying DNA, leading to mutations
• Polymerases such as Pwo or Pfu, obtained from Archaea, have roofreading mechanisms (mechanisms
that check for errors)
• Combinations of both Taq and Pfu are available nowadays that provide both high processivity (fast
polymerization) and high fidelity (accurate duplication of DNA)

PRIMERS

5’ 3’

3’ 5’
• The DNA fragment to be amplified is determined by selecting primers.
• Primers are short, artificial DNA strands — generally from 15 to 40 nucleotides
• With a melting temperature between 55°C and 65°C
• .Primers that are too short would anneal at several positions on a long DNA template, which would
result in non-specific copies
• Melting temperatures that are too high, i.e., above 80°C, can cause problems since the DNA
polymerase is less active at such temperatures

Deoxynucleotides triphosphates dCTP, dGTP, dATP,dUTP

 Target DNA
5’ 3’

5’
Taq polymerase
5’

3’ 5’

Target DNA:
The targets in PCR are the sequences of DNA on each end of the region of interest, which can be a
complete gene or small sequence.

Promoters and inhibitors:

Addition of proteins such as bovine serum albumin (BSA) enhances PCR by protecting the enzyme DNA
polymerase

Steps…
1. The double stranded DNA is denatured to separate into two individual strands. Above 94°C it
denatures or “melts” resulting in two single stranded DNA strands.
2. Each strand is then allowed to hybridize with a primer renaturation. Annealing temp 30-600 c
(TA), primers hybridize or "bind" to it.
3. Polymerase binds →primer Addition of nucleotides like…….forming a chain Process → one
direction. A new strand is formed called as “ AMPLICON”
4. Since the Taq polymerase works best at around 75 degrees C, the temperature of the vial is
raised to 72-75 Degrees Celsius. The DNA polymerase recognizes the primer and makes a
complementary copy of the template which is now single stranded. Approximately 150
nucleotides/ sec.

Verification of the PCR product on gel.

 ladder is a mixture of fragments with known size to compare with the PCR fragments.

 Lane 1 → PCR fragment - approx 1850 bp.

 Lane 2 and 4 → fragments- approx 800 bp.
 Lane 3 → no product formed, so the PCR failed.
 Lane 5 → multiple bands are formed because one of the primers fits on different places.

APPLICATION OF PCR:

PCR in clinical diagnosis :

1. Prenatal diagnosis of inherited diseases. E.g.; sickle cell anemia, β – Thalassemia.

2. Diagnosis retro viral infections.
3. Diagnosis of bacterial infections. E.g.; Tuberculosis
4. Diagnosis of cancers .E.g.; Cervical cancer
5. Sex determination of embryos.
  PCR in DNA sequencing :
 PCR in gene manipulation and expression studies :
 PCR in comparative studies of genomes :
 PCR in forensic medicines :
 PCR in comparison with gene cloning :

Applications of PCR

The HIV-1 specimen is plasma collected in EDTA that must be separated from the cells within 6 hours

Heparin cannot be used as an anticoagulant because it inhibits PCR.

Extraction of DNA
The anticoagulant tube with the patient’s blood sample should be centrifuged to separate it into the
layers of plasma, Buffy coat, and the RBCs.

The Buffy coat is used for the extraction because it contains WBCs, which are nucleated and possess the
DNA.

Extract and discard plasma, taking care not to remove the Buffy coat.

Carefully extract 200µl of Buffy coat from each sample and place in designated tube

Add 25µl of protease to each tube.

Add 200µl of lysis buffer to each tube

Vortex each tube for 15 sec. to ensure proper mixing.

Incubate each tube for 10 min. at 56°C.

Add 210µl of ethanol, vortex and then centrifuge again.

The column portion is inserted into a new tube and washed twice, each time 500µl of buffer is used to
elute substances adsorbed to the column that are not DNA.

Next, 100µ L of eluting buffer is added.

• This is incubated for 5 minutes at 25°C, and then centrifuged

• The elute is kept this time because it contains the DNA.