Polymerase Chain Reaction (PCR)

Introduction to PCR

• Definition: PCR (Polymerase Chain Reaction) is a technique to amplify DNA segments.
• Enzyme Involved: Requires polymerase enzyme (DNA or RNA polymerase).

Goal of PCR

• Amplification: To produce a higher concentration of a specific DNA segment for experimental purposes.

Steps Involved in PCR

1. Denaturation

   • Temperature: Heated to 94°C for 1 minute.
   • Effect: Separates complementary DNA strands.
   • Consideration: Melting temperature (Tm) of DNA varies by species due to GC content.

2. Annealing

   • Temperature: Reduced to 54°C for 45 seconds.
   • Primers: Oligonucleotide sequences (forward and reverse primers) bind to the DNA strands flanking the region of interest.
   • Primer Requirements: Must have >60% sequence complementarity with the target DNA.

3. Extension

   • Temperature: Increased to 72°C for 2 minutes.
   • Nucleotide Addition: DNA polymerase adds dNTPs to synthesize the new DNA strand.

PCR Cycle Overview

• One Cycle: Denaturation → Annealing → Extension.
• Repeat Cycles: Typically 20-40 cycles for significant amplification.

Key Components of PCR Reaction

• Master Mix: Contains host DNA, primers, Taq polymerase, and dNTPs.
• Taq Polymerase: Heat-resistant polymerase derived from Thermus aquaticus.
• Thermocycler: Machine that controls temperature variations during PCR cycles.

Importance of Temperature and Timing

• Criticality: Altering the temperatures can have a significant impact on product yield.
• Heating and Cooling: Essential for each step to function correctly.

Mechanism of DNA Replication in PCR

• Denaturation: Achieved using heat instead of helicase and other enzymes.
• Polymerization: Achieved with Taq polymerase, which is heat-resistant.

PCR Optimization Factors

1. Buffers and Salts:

   • KCl concentration affects primer binding.
   • Magnesium chloride concentration is crucial for enzyme activity.

2. Primer Design:

   • Length (18-30 bp), GC content (40-60%), and specific design to avoid self-complementarity.

3. Cycling Conditions:

   • Initial denaturation, annealing temperature adjustments (<5°C of Tm).
   • Cycle number typically ranges from 30-40.

Limitations and Effects

• Pletcher Effect: After ~30 cycles, Taq polymerase efficiency decreases, leading to a plateau in product yield.

Applications of PCR

• Molecular Biology: Key in molecular identification, sequencing, genetic engineering, mutation screening, prenatal diagnosis, and more.
• Key Techniques: Used in various techniques such as PFG, RFLP, and in the Human Genome Project.

Conclusion

• PCR is a fundamental technique in molecular biology with widespread applications.