Polymerase chain reaction (PCR) is a technique used to amplify DNA sequences. It involves repeated cycles of heating and cooling of the DNA sample to cause DNA replication. Each cycle doubles the number of copies of the target DNA sequence. Key steps in PCR include DNA denaturation to separate strands, annealing of primers to the strands, and elongation of new strands by a polymerase enzyme. Factors like primer concentration, temperature, and number of cycles influence the exponential amplification of the target DNA sequence. PCR has many applications in research, forensics, and medicine.

1. Polymerase Chain Reaction
By Umair Rasool Azmi

2. Introduction
• A noble prize winner technique developed by Kary Mullis in 1983.
• Polymerase chain reaction (PCR) is a technique used for detecting,
quantifying and amplifying DNA sequence(s).

3. Working Principle
• By using different temperature and time combinations, duplicate
copies of a given nucleotide sequence are made
• DNA is Unwinded by using high temperature
• Primers and a polymerase enzyme are attached
• Nucleotides are added with the help of polymerase enzyme

4. Requirements
• Template DNA sequence
• Reaction buffer (pH8-8.5)
• Mgcl2
• Deoxyribonucleotide triphosphate (dNTP’s)
• Forward and reverse primers
• Taq polymerase enzyme
• Distilled water
• All the materials are mixed in a single PCR tube

5. Procedure
• PCR involves three steps:
1. Denaturation
2. Annealing
3. Elongation
• These step are carried out in a machine called as
thermocycler which controls the temperature during the
whole reaction.

6. 1. Denaturation
• Temperature is 94ºC
• Hydrogen bonds between double strands are broken and DNA is
unwinded

7. 2. Annealing
• Temperature is 50-60ºC
• At this stage Primers are attached or binded with DNA strands.

8. 3. Elongation
• Temperature is 72ºC
• At this temperature, Taq polymerase starts adding nucleotides in
new strands

9. Different temperature and time combinations
in PCR cycle(s)
• Initial denaturation at 94ºC for 5 mins
Consequent cycles (approx. 30-40 cycles)
• Denaturation: 94ºC for 30 secs
• Annealing: 55ºC for 30 secs
• Extension: 72ºC for 45 secs
• Final extension at 72ºC for 5 mins
• Holding Temperature 4ºC

10. Exponential Amplification in PCR

11. Factors Affecting PCR
• Mgcl2 concentration: optimum concentration 1.5-5mM
• dntp’s concentration: optimum concentration 20-200µM
• Enzyme concentration: optimum concentration 1-2.5 units
• Primer concentration: optimum concentration 0.1-0.5µM
• Template concentration: optimum concentration <2ng
• PCR program not set properly
• Inhibitors in the reaction e.g. chloroform, EDTA, SDS, Ethanol etc.

12. Uses
• For DNA amplification
• DNA Sequencing or Genotyping
• For detection of specific sequences in
Molecular Research- DNA Fingerprinting, Cloning, Identification of
Mutations etc.
Medical Research- Drug Discovery, Identification of Pathogens etc.