Basic things you must know about a polymerase chain reaction

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.