PCR Primers

• Polymerase Chain Reaction (PCR) - Technique that produces millions of copies of pieces of DNA, screening of genetic and infectious diseases, the reconstruction of phylogenetic trees, and forensic analysis.

  • Developed by Kary B. Mullis in 1985
  • Nobel Prize in Chemistry, 1993
  • Japan Prize, 1993
• PCR Process

  • dsDNA -> Denaturation -> Annealing -> Extension
• Initial Denaturation

  • Number of Cycles: 1
  • Temperature (°C): 94
  • Time: 5 mins
• Denaturation

  • Number of Cycles:
  • Temperature (°C): 94
  • Time: 30 sec - 1 min
• Annealing

  • Number of Cycles: 25-35
  • Temperature (°C): 50-65
  • Time: 30 sec - 1 min
• Extension

  • Number of Cycles:
  • Temperature (°C): 72
  • Time: 30 sec - 1 min
• Final Extension

  • Number of Cycles: 1
  • Temperature (°C): 72
  • Time: 5 min
• PCR buffer - Maintains the pH of the solution
• dNTPS - Provide free nucleotides
• Taq polymerase - Synthesis of complementary strand
• MgCl2 - Co-factor of Taq pol
• Forward primer and Reverse primer - provide a “free” 3'-OH group to which the Taq pol can add dNTPs
• Sterile nanopure water - brings the solution to desired volume
• PCR primers - short fragments of single stranded DNA (15-30 nucleotides in length) that are complementary to DNA sequences that flank the target region of interest.
• Characteristics of good PCR primers

  • Length: 18-24 bp
  • GC content: 40-60%
  • Melting temperature (Tm): 50-60 °C
  • Acceptable Tm difference: 3-5  °C
  • 5' and 3' ends: 1-2 G/C
  • Complementary regions: none/absent
• Other Considerations in Primer Design
• Other Considerations in Primer Design

  1. Avoid regions of secondary structure and have a balanced distribution of GC-rich and AT-rich domains.

  2. Avoid runs of 4 or more of one base or dinucleotide repeats Ex.

  TGGGG or ATATATATAT

  3. Avoid intra-primer homology (more than 3 bases that complement

  within the primer or inter-primer homology (forward and reverse

  primer having complementary sequences. (self-dimers or primer

  dimers)
• Primer Design Considerations

  • Avoid regions of secondary structure
  • Have a balanced distribution of GC-rich and AT-rich domains
  • Avoid runs of 4 or more of one base or dinucleotide repeats
  • Avoid intra-primer homology (more than 3 bases that complement within the primer)
  • Avoid inter-primer homology (forward and reverse primer having complementary sequences)
  • Avoid cross homology (primer is homologous to other regions of the template strand)
• Undesirable primer sequences

  • TGGGG
  • ATATATATAT
• Self-dimers


  Complementary sequences within the primer
• Primer dimers


  Complementary sequences between the forward and reverse primers
• Cross homology


  Primer is homologous to other regions of the template strand, leading to amplification of other genes