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POLYMERASE CHAIN REACTION & its APPLICATION (PCR)
CONTENTS
INTRODUCTION
DEFINITION
HISTORY
REQUIREMENT OF PCR
DNA TEMPLATE
PRIMER
Taq DNA POLYMERASE
DNTPs
BUFFER SOLUTION
PROMOTERS AND INHIBITOR
PRINCIPLE
PROCEDURE
DENATURATION
ANNEALING
EXTENSION
PCR CYCLE
PCR PHASES
VARIATION OF PCR
PCR Vs GENE CLONING
APPLICATIONS
LIMITATIONS
CONCLUSION
REFERENCE
, 2
1. INTRODUCTION:-
PCR has revolutionized recombinant DNA technology. It generates microgram (μg)
quantities of DNA copies (up to billion copies) of the desired DNA segment, in a few
hours. PCR is a chain reaction because newly synthesized DNA strands act as
templates for further DNA synthesis for about 25-35 subsequent cycles. Theoretically
each cycle doubles the amount of DNA amplified.
The PCR process has been completely automated and compact thermal cyclers are
available. The PCR process is carried out in-vitro. The vast majority of PCR methods
use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined
series of temperature steps. PCR is now a common and often indispensable technique
used in medical and biological research labs for a variety of applications. These
include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis
of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints
(used in forensic sciences and paternity testing); and the detection and diagnosis of
infectious diseases.
2. DEFINITION:-
“Polymerase Chain Reaction (PCR) is a cell free, rapid and sensitive amplification
technique for synthesizing multiple identical copies of any DNA.”
3. HISTORY:-
In 1971 Kleppe and co-workers first described a method using an enzymatic
assay to replicate a short DNA template with primers in vitro.
Polymerase chain reaction developed by Karry Mullis in 1985 and he was
awarded the Nobel Prize in Chemistry in 1993 for his invention.
In 1989 the scientist Lawyer introduces Taq DNA polymerase from
thermophilic bacterium, Thermus aquaticus.
4. REQUIREMENT OF PCR:-
The PCR reaction requires the following components:
DNA Template - The sample DNA that contains the target sequence to be amplified.
Taq DNA polymerase - A type of enzyme that synthesizes new strands of DNA
complementary to the target sequence. The first and most commonly used of these
enzymes is Taq DNA polymerase, it can withstand high temperature. Taq polymerase
POLYMERASE CHAIN REACTION & its APPLICATION (PCR)
CONTENTS
INTRODUCTION
DEFINITION
HISTORY
REQUIREMENT OF PCR
DNA TEMPLATE
PRIMER
Taq DNA POLYMERASE
DNTPs
BUFFER SOLUTION
PROMOTERS AND INHIBITOR
PRINCIPLE
PROCEDURE
DENATURATION
ANNEALING
EXTENSION
PCR CYCLE
PCR PHASES
VARIATION OF PCR
PCR Vs GENE CLONING
APPLICATIONS
LIMITATIONS
CONCLUSION
REFERENCE
, 2
1. INTRODUCTION:-
PCR has revolutionized recombinant DNA technology. It generates microgram (μg)
quantities of DNA copies (up to billion copies) of the desired DNA segment, in a few
hours. PCR is a chain reaction because newly synthesized DNA strands act as
templates for further DNA synthesis for about 25-35 subsequent cycles. Theoretically
each cycle doubles the amount of DNA amplified.
The PCR process has been completely automated and compact thermal cyclers are
available. The PCR process is carried out in-vitro. The vast majority of PCR methods
use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined
series of temperature steps. PCR is now a common and often indispensable technique
used in medical and biological research labs for a variety of applications. These
include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis
of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints
(used in forensic sciences and paternity testing); and the detection and diagnosis of
infectious diseases.
2. DEFINITION:-
“Polymerase Chain Reaction (PCR) is a cell free, rapid and sensitive amplification
technique for synthesizing multiple identical copies of any DNA.”
3. HISTORY:-
In 1971 Kleppe and co-workers first described a method using an enzymatic
assay to replicate a short DNA template with primers in vitro.
Polymerase chain reaction developed by Karry Mullis in 1985 and he was
awarded the Nobel Prize in Chemistry in 1993 for his invention.
In 1989 the scientist Lawyer introduces Taq DNA polymerase from
thermophilic bacterium, Thermus aquaticus.
4. REQUIREMENT OF PCR:-
The PCR reaction requires the following components:
DNA Template - The sample DNA that contains the target sequence to be amplified.
Taq DNA polymerase - A type of enzyme that synthesizes new strands of DNA
complementary to the target sequence. The first and most commonly used of these
enzymes is Taq DNA polymerase, it can withstand high temperature. Taq polymerase
