Polymerase chain reaction (PCR) is a technique used to amplify a specific segment of DNA. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers that bind to the DNA region to be amplified, and nucleotides. Each cycle doubles the amount of target DNA. Key steps include DNA denaturation, primer annealing, and primer extension. PCR is a precise method for copying DNA and has many applications in research, forensics, disease detection and more.

1. Polymerase Chain
Reaction

2. CONTENT:
•INTRODUCTION
•Basic Requirements
•Steps Involved In PCR
•Advantages
•Application
•Conclusion

3. INTRODUCTION:
-Polymerase chain reaction(PCR) is a laboratory technique used
to make multiple copies of a segment of DNA.
-It is very precise and can be used to amplify,or copy,a specific
DNA target from a mixture of DNA molecules.
-PCR was discovered by Kary Mullis in 1985 and got Nobel prize in
1993.
-This is carried out in vitro technique.
-PCR amplification is achieved by using oligonucleotide
primers,which are short,single-stranded oligonucleotides that are
complementary to the region of known sequence.

4. Basic Requirements:
DNA Polymerase:- The DNA Polymerase used in PCR should be heat stable.
-The widely used DNA Polymerase is Taq DNA Polymerase.
-Taq DNA Polymerase is obtained from bacterium Thermus Aquaticus ,it is
thermostable up to 94°c with an optimum working temperature of 80°c.
Primers: -For PCR, two oligonucleotides primers are required which can
base pair with two strands of DNA on the two sides of specific region to be
amplified.
-This primers are about 18-30 nucleotides.
-The specificity of amplification depend on the extent to which the primers
can recogniseand bind to sequence other then the target DNA sequence.
Deoxy Nucleoside Triphosphate(dNTP):
-Four types of DNA precursors are required i.e d-ATP,d-CTP,d-GTP and d-TTP
which arecomplementary to the individual DNA strands of target DNA
segment.

5. Thermal Cycler:-
-It is an automated cycler.
-It has in-build microprocessor controlled temperature cycling needed for
PCR.
DNA Templates:-Is that particular DNA
Sequence which we want to copy.
-A reaction buffer is required to provide
a stable pH.Magnesium plays a vital role
in PCR reaction.Magnesium Chloride is
An essential cofactor that enhances the
Activity of Taq Polymerase.

6. -Steps Involved in PCR:-
1-Before starting PCR, the DNA segments or Gene to be amplified
should be isolated and purified.
2-Cyclical Amplification of DNA:-
-Before starting the reaction the solution taken in PCR tube
should contain desired volumes of Polymerase enzyme,target
DNA segment,four type of dNTP,RNA segment (primer) and
buffer solution.
-Then it is subjected to Thermal Cycler which involves three steps.

7. a)Denaturation process:-
-The reaction mixture is heated at 95°c for a short time (15-30
sec) to denature the target DNA into a single strands that will
act as template for DNA synthesis.

8. b)Primer Annealing:-
-The denatured solution is then allowed to cool and the primers in
the mixture recognize the
two strands and border the sequence to be amplified.
-This sequence are located at the 3’ ends of two strands of the
desired segments.
-The duration of annealing step is 1 min.

9. c)Primer Extension:-
-The annealed mixture is then incubated at 72°c for 5min.
-At this temperature ,the Taq Polymerase enzyme synthesis new strand of DNA
by adding dNTP from 3’OH end of the primer.
-The duration of primer extension is usually 2min at 72°c.

11. -Each PCR cycle follows these
three steps that are repeated.

12. Advantages:-
•Small amount of DNA is required per test.
•Result obtained more quickly – usually within 1 day.
•PCR is much more precise in determining the sizes of alleles –
essentials for some disorders.
•It is used throughout the field of molecular biology helping
researchers clone & sequence Gene for the detection of
mutations.
•It has more recently become a modality for detecting
microbial agents requiring only a small sample for analysis.

14. Conclusion:-
-It is a highly accurate and rapid method for duplicating genetic
material.
-The discovery of thermostable Polymerase enzymes he permitted the
automation of PCR, thus reducing the man power required to conduct
these experiments.
PCR is not only vital in clinical laboratory by amplifying small amounts of
DNA for STD Detection, but it also important for genetic predisposing for
defects such as Factor V
Leiden.
This technology can also be employed in law enforcement, genetic
testing of animal stocks and vegetable hybrids and drug screening
along with many more areas.

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