PCR [Polymerase Chain Reaction]
• “Amplify” large quantities of DNA from small quantities
[billion fold amplification]
• Analyze single DNA fragments out of large complex mixture. [
Human genome mixture of 12 million 300bp fragments]
• Principle :
The method relies on thermal cycling, consisting of cycles of
repeated heating and cooling of the reaction for DNA melting and
annealing.
Primers (short DNA fragments) containing sequences
complementary to the target region along with a DNA 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 a chain reaction in which the
DNA template is exponentially amplified.
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Exponential amplification of DNA
Components of PCR reaction:
 Template DNA
 Primers
 Thermo-stable polymerase (Taq Polymerase )
 dNTP(Deoxynucleoside triphosphates (nucleotides
containing triphosphate groups), the building-blocks
from which the DNA polymerase synthesizes a new DNA strand.
(dATP, dTTP, dCTP, dGTP)
 PCR Buffer (mg++)
 Thermocyler
Detection of PCR products :
Running the sample on gel electrophoresis for separation according
to size.
RT PCR ( Reverse Transcriptase )
An application for conventional PCR for amplification of RNA
eg.(RNA viruses) by addition of the enzyme reverse transcriptase
before amplification to synthesize a DNA complementary to the
RNA template ..Then continue the amplification process.
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Applications of PCR
Detecting infectious agents
• PCR is extensively used in analysing clinical specimens for the
presence of infectious agents, including HIV, hepatitis (e.g
B,C).
• PCR has a great value in the early detection of HIV as it can
identify the DNA of the virus within human cells immediately
following infection, as opposed to the antibodies that are
produced weeks or months after infection.
Forensic analysis of DNA samples :
DNA fingerprinting by means of PCR has revolutionalized the
analysis of evidence from crime scenes.DNA isolated from a single
human hair, a tiny spot of blood, or a sample of semen is sufficient
to determine whether the sample comes from a specific individual .
Prenatal diagnosis and carrier detection of cystic fibrosis :
Cystic fibrosis is an autosomal recessive genetic disease resulting
from mutations in the cystic fibrosis transmembrane conductance
regulator (CFTR) gene. The most common mutation is a three-base
deletion that results in the loss of a phenylalanine residue from the
CFTR protein. Because the mutant allele is three bases shorter than
the normal allele, it is possible to distinguish them from each other
by the size of the PCR products obtained by amplifying that
portion of the DNA.
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