——— Jenny Wang

1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?

• Phusion DNA Polymerase:
  • Derived from a Pyrococcus species, this enzyme combines high processivity (ability to synthesize long DNA strands) with proofreading activity (3’→5’ exonuclease). This reduces errors during PCR (~50x lower error rate than Taq polymerase).
  • Thermostability: Functions optimally at high temperatures (up to 98°C), enabling efficient amplification of GC-rich templates.
• dNTPs:
  • Deoxynucleotide triphosphates (dATP, dTTP, dCTP, dGTP) are substrates for DNA synthesis. Phusion Master Mix includes balanced concentrations to minimize misincorporation.
• Mg²⁺-Containing Buffer:
  • MgCl₂ is a cofactor for polymerase activity. The buffer maintains pH (~8.0) and ionic strength to stabilize primer-template binding.
• Enhancers (e.g., DMSO or betaine):
  • Disrupt secondary structures (e.g., hairpins) in templates with high GC content by reducing DNA melting temperature (Tm).
• Stabilizers (e.g., BSA or glycerol):
  • Bovine serum albumin (BSA) binds to contaminants (e.g., polyphenols in plant DNA) that inhibit polymerase. Glycerol prevents enzyme denaturation during freeze-thaw cycles.

2. What are some factors that determine primer annealing temperature during PCR?

• Primer Length:

  • Longer primers (e.g., 20–30 nt) have higher Tm due to more hydrogen bonds. Tm can be estimated using the formula:

    Tm=2°C×(A+T)+4°C×(G+C)Tm=2°C×(A+T)+4°C×(G+C).

• GC Content:

  • GC base pairs (3 H-bonds) stabilize duplexes more than AT pairs (2 H-bonds). Primers with >60% GC may require higher annealing temperatures.

• Salt Concentration:

  • High salt (e.g., 50 mM KCl in PCR buffer) stabilizes DNA duplexes by shielding phosphate backbone repulsion, raising Tm.

• Primer Concentration:

  • Excess primers increase nonspecific binding (e.g., primer-dimers); lower concentrations may require lower Tm.

• Additives:

  • DMSO (5–10%) disrupts base pairing, lowering Tm by ~5–10°C. Betaine (1–1.5 M) equalizes Tm differences in GC-rich regions.

3. PCR vs. Restriction Digest: Comparison

4. Why does the PvuII digest require CutSmart buffer?