This slideshow contains information about polymerase chain reaction with their steps requirements stages and advantages...

1. POLYMERASE CHAIN
REACTION
PREPARED BY:-
PRANJAL SAXENA
HAYMANSHU PANDEY
B.PHARMA 6TH
SEM .
ORIENTAL UNIVERSITY

2. POLYMERASE CHAIN REACTION
• The polymerase chain reaction (PCR) is a technique widely used in molecular
biology. It derives its name from one of it key components, a DNA polymerase
used to amplify (ic replicate) a piece of DNA by in vitro enzymatic replication.
As PCR progresses, the DNA thus generated is itselfused as template for
replication. This sets in motion a chain reaction in which the DNA template is
exponentially amplified.
• With PCR it is possible to amplify a single or few copies of a piece of DNA
across several orders of magnitude generating millions or more copies of the
DNA piece possible to amplify a single or few copies of a piece of DNA across
several orders of magnitude, generating millions or more copies of the DNA
piece. PCR can be performed without restrictions on the form of DNA, and it
can be extensively modified to perform a wide array of genetic manipulations.

3. POLYMERASE CHAIN REACTION
• The conventional molecular cloning techniques may considered in vitro DNA-
amplifying tools. Interestingly, the latest development in the field of synthetic
DNA* has evolved an altogether new method for the rapid amplification of DNA
in vitro, broadly termed as the Polymerase Chain Reaction (PCR).
• In reality, PCR is capable of multiplying DNA molecules to the extent of a billion
fold in vitro, thereby giving rise to huge amounts of very specific genes
employed for various purposes, such as : cloning, sequencing or mutagenesis. In
short, PCR utilizes the enzyme DNA polymerase, which eventu- ally copies DNA
molecules.

4. HISTORY OF POLYMERASE CHAIN
REACTION:-
•PCR was invented in the 1984 as a way
to make numerous copies of DNA
fragments in the laboratory.
•The in vitro version of DNA Replication.
• " Beginning with a single molecule of
the genetic material DNA, the PCR can
generate 100 billion similar molecules
in an afternoon !"
Kary Mullis
Nobel Prize 1993

5. PRINCIPLES OF POLYMERASE CHAIN
REACTION
• The PCR technique copies the target DNA by performing repeated cycles each
ontaining the following three main steps :
• 1-A denaturation or melting step to separate the two strands of DNA, this
step requires very high temp 95C for 10-20 seconds.
• 2-The Annealing step, allowing the primers too bind to the complementary
sequences on the template DNA, this step requires the temp to be dropped to
50-60 C.
• 3. The Elongation step, once the primers are bound to the template the
synthesis of DNA can start, the temperature should be increased to 70c which is
the optimum temperature for the polymerase enzy me.

6. REQUIREMENTS FOR POLYMERASE CHAIN
REACTION:-
• The PCR requires the following:
1. DNA Template
• Any source that contains one more target DNA molecules to be amplified can
be taken as template.
2. Primers
A pair of oligonucleotides of about 180-30 nucleotides with similar G+C contents
act as primers. They direct DNA synthesis towards one another. The primers are
designed to annual on opposite strands of target sequence so that will be
extended toward each other by addition of nucleotides to their 3'ends.
3. Enzyme
The most common enzyme used in PCR is a thermostable

7. REQUIREMENTS........
1. Deoxynucleoside triphosphates
the building blocks from which the DNA poly merases synthesizes a new DNA strand.
2.Buffer solution
providing a suitable chemical environment for optimum activity and stability of the
DNA polymerase.
3. Taq polymerase
is the enzyme to manufacture the DNA copies. The PCR involves a cou ple of high
temperature steps so we use a heat resistant DNA polymerase, this is extracted
from heat resistant bacteria living in a hot springs at temperature up to 80°C, or
anot her DNA polymerase with a temperature optimum at around 70 °C.

8. STAGES OF POLYMERASE CHAIN REACTION
• There are three major steps of polymerase chain reaction are:-.
1. Denaturation
2. Primer Annealing
3. Extension
• DENATURATION:- During the denaturation step, the reaction cocktail (reaction
mixture) is exposed to high temperature, usually 94-95°C for 20 secs. This high
temperature will denature the DNA-meaning the hydrogen bond between the two
complementary strands melt, unraveling the DNA molecule and exposing the
nucleotide bases. The high temperature of the denaturing step has the added
advantage of denaturing proteins (inactivating them) and disrupting cells so that you
don't have to always start with purified DNA as your amplification template. You can
often amplify DNA directly from cell lysates or even whole cells.

9. • PRIMER ANNEALING
• During the second step of each cycle, the temperature is lowered to an annealing
temperature (50°-60° C), allowing binding (annealing) of the primers to their
complementary targets on the DNA template (one for each DNA strand). These are designed
to flank the desired target region of your DNA template and serve as the starting points for
DNA synthesis by the Taq polymerase. Each pair of primers will have a particular annealing
temperature determined by the length of the primers. Using the proper annealing
temperature for your primer set is essential for efficient and accurate amplification.
• EXTENSION
• The reaction cocktail is now brought to the optimum reaction temperature for Taq
polymerase (65 to 85°C). During this step, the Taq will bind to each DNA strand • and
"extend" from the priming sites (add nucleotides to synthesize a complementary strand of
the targeted DNA).

11. STAGES OF POLYMERASE CHAIN REACTION:-
The polymerase chain reaction (PCR) for amplifying specific DNA sequences have
been shown.
These six stages have been duly explained here under:
1. Stage A : The target genes (DNA – combinant form) if first heated affect the
separation of the strands of DNA ; secondly, a reasonably excess amount of two
oligonucleotide primers**, of which one is complementary strand, is added along
with DNA-polymerase;
2. Stage B : As the resulting mixture attains the ambient temperature, the excess
of primers relative to the target DNA makes sure that most target strands anneal
to a primer exclusively and not each other. In this way, the primer extension
ultimately gives rise to a copy of the original double- stranded DNA.

12. STAGES.......
3. Stage C:
Further follow up of three above mentioned steps sequentially viz ; heating, primer annealing
and primer extensions results into the formation of a copy of the original double-stranded DNA.
In other words, DNA polymerase extends the perimers employing the target strands template.
4. Stage D:
Another prime extension of the resulting product yields the second double-stranded DNA.
5. Stage E :
The end product obtained from the previous step is subjected to incubation for a suitable
duration ; and the resulting mixture is heated once again so as to separate the strands.
Subse- quently, the mixture is brought to the room temperature whereby the primers aptly
get hybridized with the complementary regions of newly synthesized DNA. Thus, the whole
process is repeated. In this particular instance, the two additional PCR-cycles give rise to 8
to 16 copies, respectively, of the origi- nal DNA sequence.

13. STAGES OF
POLYMERASE
CHAIN
REACTION

14. TARGET
GENES
STAGE F

15. 6. Stage F :
It represents a plot between the number of PCR cycles (along the X-axis) and
the copies of the target gene (along the Y-axis). The graphical illustration
depicts the effect of carrying out PCR cycles on a DNA preparation initially
having only 10 copies of a target gene. The resulting graph is semilogarithmic in
nature.

16. ADVANTAGES OF POLYMERASE CHAIN
REACTION:-
• PCR-technique has two cardinal advantages, namely :
• (a) Each and every cycle virtually doubles the content of the original target DNA, and
• (b) A 10° to 10* fold increase in the target sequence is actually achieved after a 20-30 PCR cycle
• Genetic fingerprinting :
• can uniquely discriminate any person from the entire population of the world. samples of DNA
can be isolated from a crime scene , and compared to that from suspects, or from a DNA
database of earlier evidence.
• Parental testing :
• an individual is matched with their close relatives. Less discriminating forms of DNA
fingerprinting. DNA compared with that from possible parents, siblings, or children. Similar
testing can be used to confirm the biological parents of an adopted (or kidnapped) child.