ABT 104 Lecture 4: PCR Primer Design

• PCR: Polymerase chain reaction or in vitro replication of genetic material.
• Steps in PCR

  • Denaturation 94-95C
  • Annealing 54-55C
  • Extension 72C
• Primer length: affects specificity, melting temperature, and time of annealing.
• Optimal length for primers: 17-24 or 30 bases
• Shorter primers: used for non-specific PCR amplification
• Longer primers: Used for specific amplification but may contain secondary structures.
• What affects melting temperature: length, composition, and concentration of salt.
• Optimal melting temp: 55-65C
• Range of melting temp differences for FPrimer and RPrimer: 2-3C
• Optimal melting temp for primers with high GC content: 75-80
• Annealing temp: Should be 5C lower than melting temp
• How to avoid secondary structures: optimize annealing temperature.
• Site used for secondary structures: IDT OligoAnalyzer
• Gibbs Free Energy: Measure of spontaneity of reaction which is the energy needed to break a DNA secondary structure
• Maximum ^G: -9 kcal/mol
• What does repeats and runs cause: mispriming.
• Optimal number of runs: maximum of 4 base pairs
• Optimal number of repeats: Maximum of 4 dinucleotides
• GC Clamp: Presence of G or C in the last 4 bases of the 3' end
• Optimal GC content for clamp: Not more than 3
• Homology at the end: must be 100%
• Parameters of a good primer:

  • Length: 17-24 bases
  • Content: 40-60% GC
  • Melting temp difference: 2-3C
  • GC Clamp: Less than 3 GC
  • Homology: 100% at the 3' end
• Primer design softwares:

  Primer BLAST

  Primer3Plus

  PrimerX