PCR short notes - Definition, Requirements, Steps and Applications

PCR: Polymerase Chain Reaction
• Invented by Kary Mullis 1983
• Received Nobel Prize in chemistry in 1993
Definition: An in-vitro DNA amplification technique that allows synthesizing millions of copies of the gene or DNA of interest from a single copy
• It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.

Requirements or Reaction components:

1. Target DNA - contains the sequence to be amplified.
2. Two oligonucleotide primers : binds to the 3’ OH end of both strands
3. dNTPs – deoxy nucleotide triphosphates: DNA building units.
4. Thermostable DNA polymerase (Taq polymerase)-enzyme that catalyse the reaction
5. Mg⁺⁺ ions - cofactor of the polymerase enzyme.
6. Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme.

Steps in PCR

A PCR cycle consist of 3 steps: Denaturation, Primer annealing and Elongation.
Step I: Denaturation
• Reaction mixture heated to 94⁰C for 1 minute.
• The temperature denatures dsDNA into two single strands by breaking H-bond between them
Step 2: primer annealing
• Cooled to 55⁰C for 1.5min
• Primer binds to the 3’ end of the target DNA at both strands-primer annealing
Step 3: Elongation by thermostable DNA polymerase
• At 72⁰ C for 1 min
• Addition of nucleotides by Taq polymerase
• Duration to complete a cycle ~4-5 min
• In each cycle the no. of DNAs gets doubled-2->4->8->16….exponentially
• A reaction usually consist of 30-35 cycle forming about million copies

Applications of PCR :New applications are created every day,
• To amplify DNA fragments isolated from organisms
• To propagate DNA for gene manipulation and construction of DNA libraries
• Used in sex determination of embryo
• In forensic science, in DNA fingerprinting
• PCR products can be used for mapping genes
• PCR products can be used as probes
• Used to detect genetic diseases
• PCR can be used to identify genotypes
• PCR can be used to sequence DNA directly.