Polymerase Chain reaction (PCR)/KR/Pptx.

It is an invitro DNA Replication.
POLYMERASE CHAIN REACTION-PCR
This presentation highlights the major principles behind the PCR.

1. POLYMERASE
CHAIN REACTION
(PCR).

2. INTRODUCTION:
The polymerase chain reaction (PCR) is a
laboratory (in vitro )technique for
generating large quantities of a
specified DNA
Obviously,PCRis a cell free amplification
technique for synthesizing multiple
identical copies (billons) of any DNA
interest.
Developed in 1984, by KarryMullis.
It is considered as a basic tool for the
molecular biologist.

3. PRINCIPLE OF PCR :
The double stranded DNA of interest is denatured to separate into
two individual strands.
Each strand is then allowed to hybridize with a primer.
The primer-template duplex is used for DNA synthesis.
The common three steps involved in this techniques are:
DENATURATION
RENATURATION
ELONGATION
These are repeated agianand again to generate multiple forms of
target DNA.

4. MATERIALS REQUIRED:
DNA TEMPLATE:
The DNA of interest from the sample

5. DNA POLYMERASE:
Taqpolymerase is mostly
used in PCR.
It is thermostable and
does not denature at
very high temperature.
It was isolated from
bacterium Thermus
aquaticus.

6. OLIGONUCLEOTIDE PRIMERS:
These are the short stretches of single starandedDNA
complementary to the 3’ ends of sense and anti sense codons.
There are two primers,namelyforward and reverse primers.

7. DROXYRIBONUCLEOTIDE
TRIPHOSPATE:
They provide energy for
polymerization and are
building blocks for the
synthesis of DNA.
These are single unit
bases.

8. BUFFER SYSTEM:
Magnesium and potassium
provide optimum conditions
for DNA denaturation and
renaturation.
It is also important for fidelity,
polymerase activity and
stability.

9. PCR STEPS:
1.DENATURATION
Denaturation occurs when the reaction mixture
is heated to 95°C for about 1 minute.
This breaks the Hydrogen bond between two
strands of DNA and converts it into single
stranded DNA.
The single strands now act as a template for
the production of new strands of DNA.
The temperature should be provided for a long
time to ensure the separation of two strands.

10. 2.ANNEALING
The reaction temperature is lowered to
54-60°C for around 20-40 seconds.
Here,theprimers bind to their
complementary sequences on the
template DNA.
Primers are single strand sequence of
DNA or RNA around 20 -30 bases in
length.
They serve as the starting point for the
synthesis of DNA.
The two separated strands run in the
opposite direction and consequently
there are two primers-forward and
reverse primers.

11. 3.ELONGATION:
At this step,thetemperature is raised to 72-80°C .
The bases are added to the 3’ end of the Primer by the Taqpolymerase
enzyme.
This elongates the DNA in the 5’ –3’ direction .
The DNA polymerase adds about 1000 basepairsper minute under optimum
conditions.
Taqpolymerase can tolerate very high temperature.
It attaches to the primer and adds DNA bases to the single strand.
As a result,adouble stranded DNA molecule is obtained.
These three steps are repeated 20-40 times in order to obtain a
no.of.sequencesof DNA of interest in a very short time period.

13. APPLICATIONS:
IN MEDICINE:
Testing of genetic disease mutation
Monitoring the gene in gene therapy.
Detecting disease causing genes in
the parents.
IN FORENSIC SCIENCE
Used as a tool in genetic engineering.
Identifying the criminal from millions of people.
Paternity test

14. RESEARCH AND GENETICS:
Compare the genome of two organisms in genomic studies.
In the phylogenetic analysis of DNA from any source such as fossils.
Analysis of gene expression.
Gene mapping.

15. Overview of Application:

16. A PowerPoint presentation by :
R.KEERTHANA
B.SC MICROBIOLOGY
.