PCR Fundamentals

Polymerase

• Taq polymerase: a thermostable DNA polymerase isolated from the thermophilic bacterium Thermus aquaticus
• Characteristics:
  • Heat-stable, allowing for high-temperature DNA synthesis
  • Highly processive, synthesizing long stretches of DNA
  • Lacks proofreading activity, resulting in higher error rates

Amplification

• PCR amplification: the process of generating millions of copies of a target DNA sequence
• Steps:
  1. Denaturation: DNA is heated to separate strands
  2. Annealing: primers bind to target sequence
  3. Extension: DNA synthesis occurs, facilitated by polymerase
• Amplification efficiency: influenced by factors such as primer specificity, template quality, and cycling conditions

Primers

• Definition: short, synthetic DNA sequences (18-24 nucleotides) complementary to target sequence
• Functions:
  • Bind specifically to target sequence, initiating DNA synthesis
  • Define the region to be amplified
• Primer design considerations:
  • Specificity: minimize non-specific binding
  • Melting temperature (Tm): ensure consistent binding
  • Primer-dimer formation: minimize secondary structure

qPCR (Quantitative PCR)

• Definition: a variant of PCR that monitors amplification in real-time
• Key features:
  • Uses fluorescent dyes or probes to detect amplicon formation
  • Enables quantitation of initial template concentration
  • Higher sensitivity and specificity compared to traditional PCR

Cycling Conditions

• Temperature cycling: a series of temperature changes facilitating PCR amplification
• Typical cycling conditions:
  • Denaturation (94-96°C, 15-30 seconds)
  • Annealing (50-65°C, 15-60 seconds)
  • Extension (72°C, 30-60 seconds)
• Optimization: adjustments to cycling conditions can improve amplification efficiency and specificity

Learn about the basics of Polymerase Chain Reaction, including Taq polymerase, PCR amplification, primer design, and qPCR. Understand the steps, characteristics, and optimization of PCR techniques.