Polymerase Chain Reaction-Basics

1. Amplification of gene using PCR
Introduction:
Polymerasechain reaction (PCR) is a technique used in molecular biology to
amplify a single copy or a few copies of a segment of DNA into thousands to
millions of copies of a particular DNA sequence. The polymerase chain
reaction (PCR) was originally developed in 1983 by the American biochemist
Kary Mullis. PCR is very simple, inexpensive technique for characterization,
analysis and synthesis of specific fragments of DNA or RNA from virtually
any living organisms.
Principles:
PCR amplifies a specific region of a DNA strand (the DNA target).
PCR amplify DNA fragments of between 0.1 and 40 kilo base pairs (kbp).
A basic PCR set-up requires the following components and reagents:
 DNA template
 Deoxyribonucleoside triphosphates (dNTPs)
 PCR buffer
 Primers (forward and reverse)
 Bivalent Cations:
 Taq polymerase
DNA Template:
DNA template that contains the DNA target region (target gene) to
amplify
Primers:
Two DNA primers that are complementary to the 3' (three prime) ends of
each of the sense and anti-sense strands of the DNA target. Without primers
there is no double-stranded initiation. Specific primers that are
complementary to the DNA target region are designed (custom-made)

2. beforehand in a laboratory or purchased from commercial biochemical
suppliers.
Deoxynucleoside triphosphates (dNTPs):
Deoxynucleoside triphosphates, or dNTPs are the building blocks from
which the DNA polymerase synthesizes a new DNA strand
Buffer Solution:
Buffer solution providing a suitable chemical environment for optimum
activity and stability of the DNA polymerase.
Bivalent Cations:
Magnesium (Mg) or Manganese (Mn) ions; Mg2+ is the most common, but
Mn2+ can be used for PCR-mediated DNA mutagenesis, as a higher Mn2+
concentration increases the error rate during DNA synthesis
Taq Polymerase:
A thermally stable DNA polymerase originally isolated from the
thermophilic bacterium Thermus aquaticus, which resistinactivation during
denaturation temperatures and allows primer extension at high temperature.
Steps of polymerase chain reaction-PCR
 The reaction is commonly carried out in a volume of 10–200 μl in
small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler.
 To perform PCR, extracted sample (which contains target DNA
template) is added to a reaction tube containing primers, free
nucleotides (dNTPs), and Taq polymerase.
 The PCR mixture is placed in a PCR machine.
 PCR machine increases and decreases the temperature of the PCR
mixture in automatic, programmed steps which generates copies of the
target sequence exponentially.
Polymerase Chain Reaction (PCR) has three major steps.

3. 1. Denaturation
2. Annealing
3. Extension
Denaturation (strand separation):
Conversion of the double strand DNA molecule to single strand DNA. This
reaction is usually performed at 94o
C
Annealing (primer binding):

4. The base pairing of a single stranded primer to its complementary region of
the ss DNA molecule is known as annealing. The common choice of
temperature range for this reaction is 55-60o
C.
Extension (synthesis of new
DNA): The annealing of a
primer provides a free 3’-OH
group for synthesis of ds DNA
by thermostable DNA
polymerase using ss DNA as a
template. Extension is the
synthesis of DNA by a
thermostable DNA
polymerase using 3’-OH end
of a primer. It is done at 72o
C,
the optimal working
temperature for thermostable
DNA polymerase.
Once the first round is
completed, the process is
repeated by cycling backto the
first reaction temperature and
next round of denaturation,
annealing and extension is
started(an automatic process
in thermo cycler). This 3 steps
temperature cycle is repeated
approximately 30 times which
results exponential amplification of target gene sequence.

5. After PCR reaction is completed, the agarose gel electrophoresis is
performed to determine the following:
(i)Whether or Not a Product is formed
(ii)Whether or Not the Product Formed is Right Size
(iii)Whether or Not a Single Band of Right size is formed.
Applications of PCR
 Identification and characterization of infectious agents
 Direct detection of microorganisms in patient specimens
 Identification of microorganisms grown in culture
 Detection of antimicrobial resistance
 Investigation of strain relatedness of pathogen of interest
 Genetic fingerprinting (forensic application/paternity testing)
 Detection of mutation ( investigation of genetic diseases)
 Cloning genes
 PCR sequencing