The polymerase chain reaction (PCR) is an in vitro technique used to amplify specific DNA sequences. It involves repeated cycles of heating and cooling of the DNA sample to separate the DNA strands and allow for replication by DNA polymerase. Three main steps in each cycle are denaturation to separate the strands, annealing of primers to the target DNA, and extension of the new strands. PCR can generate millions of copies of target DNA from a very small sample and is used for a variety of applications including disease diagnosis, DNA fingerprinting, and molecular cloning.

1. LIFE OF BIOCHEMISTRY
POLYMERASE CHAIN REACTION
(PCR)
BY:
HARISH K

2. DEFINITION:
• It is other wise called DNA AMPLIFICATION.
• It’s invitro techniques.(Cell free amplificator techniques)
• Techniques used to generating large quantities of specific DNA from very
minute amount of sample DNA.
• Billion or Million of multiple identical copies of DNA interest in rapidly.

3. DISCOVERY:
• This PCR techniques is first discovered by KARRY MULLIS in 1984.
• He got a noble prize in 1993.

4. PROCESS OF PCR:
• There are three steps involving PCR:
1. Denaturation
2. Renaturation Or Annealing
3. Synthesis or Extension

5. REQUIREMENTS:
1. Target DNA (100 to 35,000 Base pair).
2. Two primers (17 to 30 nucleotides).
3. Four deoxyribonucleotide(d ATP, d GTP , d CTP , d TTP).
4. Taq DNA polymerase.

6. PROCESS OF PCR:
DENATURATION:
• Sample DNA is melting at 91 to 95°C at 1minute.
• Two standards are separated.
• This process is allow for 15 to 30 second.

7. RENATURATION OR ANNEALING:
• Sample mixture to cool slowly about 55°C.
• The primer base pair with the complementary reagion flanking target DNA
. Stand .This process is called Annealing or Renaturation.
• Primer serve as the starting point of DNA synthesis.
• Two primer are completely to the two direction of ends 5’ to 3’ and 3’ to 5’direction
forward and Reverse primer respectively.
• This process is takes about 10 to 30 seconds.

8. DENATURATION & RENATURATION:

9. SYNTHESIS OR EXTENSION:
• It is a final step of PCR techniques.
• Heat allowed for above 72°C.
• Taq DNA polymerase enzymes are added to this step.
• Taq DNA polymerase are activated due to synthesis of DNA in forward and
Reverse primers with One minute to produced 1000 DNA base pair at a time.
• 2minutes to produce 2000 Base pairs.Time is slightly increased due to
increase the synthesis of new base .
• The reaction can be stopped by raising the temperature about 95°C.

11. TAQ DNA POLYMERASE:
• It’s key role in synthesizing and amplifying new strands of DNA, Taq
DNA Polymerase is essential to Polymerase Chain Reaction (PCR).
• It is Thermostable in 95°C.
• It is involved in Synthesis or Extension step of PCR.
• Taq DNA polymerase from thermophilic bacterium ,Thermus
aquaticus.Taq Polymerase is added at the end because it used to be in
small amount as mentioned earlier and it used to be sensitive to pH.

12. IMPORTANT POINTS:
• 25 to 45 cycle are continuously repeated to multiple copies are synthesis in a
short period.
• Each cycle as three stages.
• New standard of DNA is refer to the Long primer .This will be used as the
second cycle.

13. VARIOUS TYPES OF PCR:
1. Nested PCR.
2. Anchored PCR.
3. Asymmetric PCR.
4. Inverse PCR.
5. Real time PCR.
6. Amplifief fragment length polymorphism.
7. Reverse transcription PCR (RT PCR) It is Used for Detecting the Corona
virus.Transcripts RNA to DNA.

14. APPLICATION OF PCR:
1. Classification of organisms
2. Genotyping
3. Molecular archeology (hair,blood,nail,semen and etc..)
4. Mutagenesis
5. DNA finger printing
6. Drug Discovery
7. Human genome project
8. Bioinfermatics
9. Detecting the pathogens
10.Genetic matching and etc….

15. CLINICAL DIAGNOSIS:
• PCR amplify human genes to check for mutations.
• To amplify microbial genes in sample.
• Detecting and characteristic of pathogens in infective condition.
• The PCR can be detect a single pathogen,a group of closely related pathogens.ex:TB
• Number of unrelated organism in a single run.(Multiple PCR).
• Prenatal diagnosis of inherited disease..

16. ADVANTAGES OF PCR:
1. The speed and easy diagnosis.
2. Identify quantities of organism.
3. Diagnosis not depends on microscope.
4. Accurately detect the pathogens.
5. Used for forensics .

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