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PCR Primer


Strategies to Improve Results

Provided by

Guy Afseth

PCR File Applications Specialist
Perkin Elmer - Applied Biosystems

Given at Northwestern University
June 9th, 1997

Revised by

Northwestern University Biotechnology Laboratory Staff

  1. Introduction - PCR Primer
    1. Components
    2. Primer Design
      1. Primer Design - I
      2. Primer Design - II
      3. Melting Temperature, Tm
      4. Tm Calculations, Nearest Neighbor Analyses I
      5. Tm Calculations, Nearest Neighbor Analyses II
      6. Quantitating Nucleic Acids
      7. Calculating Oligonucleotide Molarity

    3. Reaction Conditions-Background
      1. Conditions Affecting Fidelity
      2. Reaction Parameters That Affect PCR Efficiency
      3. Conditions Affecting Fidelity

  2. Reaction Set Up
    1. Optimization
      1. Optimization of PCR Reactions - Why?
      2. Biochemical Reminder
      3. Buffer Optimization

    2. PCR Primer Concentrations
    3. Template
      1. PCR Product Copy Number Depends on Target Starting Copy Number
      2. Input Copy Number, Number of Cycles, and Product Mass

    4. Mg++
      1. Mg++ in PCR
      2. Mg++ Optimization

    5. Enzyme
      1. Enzyme Choice
      2. Different PCR Enzymes for Different Applications
      3. Different PCR Enzymes Mean Different PCR Buffers
      4. Optimum Mg++ Ranges Depend Upon Polymerase
      5. Effect of [Mg++] and [Enzyme] on Yield

  3. Other Reaction Parameters and Potential Artifacts
    1. The Plateau Effect
      1. The Plateau Effect - I
      2. Plateau Effect - II

    2. Other Artifacts
      1. Three-way competition in PCR
      2. XX Mispriming Models
      3. Pre-PCR Mispriming

    3. Solution to Artifacts: Hot Starts
      1. Hot Start Procedures To Increase PCR Specificity
      2. Manual Hot Start Theory
      3. Manual Hot Start Method
      4. AmpliWaxTM Hot Start Method
      5. Hot Start Enzymatic
      6. Activation Curve of AmpliTaq Gold
      7. Using a Pre-PCR Hold for Activation
      8. Manual Hot Start Versus AmpliTaqTM Gold

The End