You are on page 1of 30

Polymerase chain

reaction
Introduction
Used toamplify a single or a few copies of a
piece ofDNAacross several orders of magnitude,
generating thousands to millions of copies of a
particularDNA sequence.
PCR is now a common and often indispensable
technique used in medical and biological research
labs for a variety of applications.
These includeDNA cloningforsequencing, DNA-
based phylogeny, or functional analysis of genes ;
the diagnosis ofhereditary diseases ; the
identification ofgenetic fingerprints(used in
forensic sciencesandpaternity testing); and the
The method relies onthermal cycling, consisting
of cycles of repeated heating and cooling of the
reaction forDNA
meltingandenzymaticreplicationof the DNA.
Primers (short DNA fragments) containing
sequences complementary to the target region
along with aDNA polymerase (after which the
method is named) are key components to enable
selective and repeated amplification.
As PCR progresses, the DNA generated is itself
used as a template for replication, setting in
motion achain reaction in which the DNA
template isexponentially amplified.
Almost all PCR applications employ a heat-
stable DNA polymerase, such asTaq polymerase
(an enzyme originally isolated from the
bacteriumThermus aquaticus). This DNA
polymerase enzymaticallyassembles a new DNA
strand from DNA building-blocks, thenucleotides ,
by using single-stranded DNA as a template and
DNAoligonucleotides(also calledDNA primers),
which are required for initiation of DNA synthesis.
The vast majority of PCR methods usethermal
cycling, i.e., alternately heating and cooling the
PCR sample through a defined series of
temperature steps.
In the first step, the two strands of the DNA
double helix are physically separated at a high
temperature in a process calledDNA melting.
In the second step, the temperature is lowered
and the two DNA strands becometemplatesfor
DNA polymerase to selectively amplify the target
DNA.
The selectivity of PCR results from the use
ofprimersthat arecomplementaryto the DNA
region targeted for amplification under specific
thermal cycling conditions.
1. dNTPs
2. MgCl2
3. PCR buffer
PCR tube THERMOCYCLER
An older model three-temperature thermal cycler
Electrophoresis
Exercise : How to interpret the results you got ???