Molecular biology: Conventional PCR techniques
Author: Prof Estelle Venter
Licensed under a Creative Commons Attribution license.
types of microbial species based on unique DNA sequences
which are specific to each species.
What is PCR and where can it be used
The polymerase chain reaction (PCR) is a test tube
system for DNA replication which allows a “target” DNA
sequence to be selectively amplified several millionfold in just a few hours. The PCR
achieves
What makes a PCR specific?
The specificity of a PCR is dependent on the sequence of the
primers. A primer is a short strand of nucleic acid
(oligonucleotide) that serves as a starting point for DNA or
amplification of a predetermined fragment of DNA, (the
RNA synthesis. In a PCR, two primers are used; these are
target; which can e.g. be from 100 – 1000 bp long) with
complementary to approximately 20 base pairs at the 3'
the apparent disadvantage that the sequences flanking
(three prime) ends of each of the two strands of the double-
the target region must be known, the latter precludes
stranded
the use of PCR from analysis of DNA regions that have
synthesized in a laboratory. This is why it is necessary to
not previously been studied by standard methods.
know the sequence (at least at the ends) of the target DNA
DNA
target.
PCR
primers
are
chemically
region to be amplified.
Location of PCR primers:
Method:
The PCR requires a pair of oligonucleotide primers, one
complementary to the (-) strand at one end of the target
sequence and the other complementary to the (+) strand at
the other end. The reaction takes place in repeated cycles of
three steps: denaturation, primer annealing and extension.
Because of its sensitivity, PCR has opened possibilities not
available to older molecular techniques.
PCR can, for
example, be used:
•
to study minute quantities of DNA,
•
to amplify DNA and RNA,
•
to detect mutations,
•
in an ELISA by immobilization of a PCR product on the
microtitre plate,
•
in reverse-line blotting by hybridization of a labelled
PCR product to oligonucleotide probes immobilized on a
membrane, and
•
in gene expression.
The PCR can be used to assist in the detection and
identification of micro-organisms on the basis of their DNA
sequences. It also allows for discrimination between different
During the denaturation step, the two strands of the target
DNA helix are unwound and separated by heating.
The
primers cannot bind to the DNA strands at high temperatures,
so the temperature is lowered for the annealing step, in which
the primers bind to their complementary bases on the now
single-stranded target DNA.
The temperature is usually
raised slightly for the extention (polymerization) step, during
which a thermostable DNA polymerase enzyme synthesizes
a new strand of DNA complementary to the template strand
starting from the primer. Repeated thermal cycling results in
exponential amplification of the target DNA.
A schematic summary of the PCR reaction