Expand™ High FidelityPLUS PCR System Troubleshooting

Product No. EHIFIP-RO

Troubleshooting

ELEVATED ERROR RATES

The typical error rate (fidelity) of Taq DNA Polymerase, determined by the lacI-based PCR fidelity assay, ranges from 1 × 10-5 to 2.5 × 10-5. Roche has determined an error rate of 1.8 × 10-5 (i.e., on average every second base in 100,000 bases is misincorporated per template duplication). For the Expand High FidelityPlus PCR System, there is a six-fold lower error rate than for Taq DNA Polymerase, corresponding to approx. 0.4 × 10-5.

Note that these error rates are determined under ideal conditions, using relatively low cycle numbers and optimized salt concentrations (for a detailed description how we determine error rates of DNA polymerases used in PCR, see the following Biochemica Newsletter article Frey, B. & Suppmann, B. (1995) Biochemica 2, 8-9.

Furthermore, there are a number of parameters which can negatively influence PCR fidelity:

  • Organic solvents like DMSO, ethanol, phenol (the latter two maybe residues from DNA isolation)
  • Too long denaturating phase
  • Too many cycles
  • Too high enzyme concentration
  • Too high concentration of dNTPs
  • Impure or degraded template DNA
  • Non-optimal Mg2+ concentration
  • Non-optimal pH
  • Suboptimal primer synthesis

The fidelity of Taq DNA Polymerase has been shown to increase fivefold by decreasing the MgCl2 concentration from 2.5 to 1.5 mM. In addition, buffer composition, pH value, nucleotide and polymerase concentration, and cycling conditions have to be optimized for each particular enzyme to achieve lowest error rate as possible (e.g., it was previously shown that error rate of one proofreading enzyme could be increased by raising pH from 7.5 to 9.1, while error rate of another enzyme increased dramatically; a similar observation was made for Mg2+ concentration).

The most important measures are to verify the quality of template DNA and to perform a titration of MgCl2 to find the optimum concentration for fidelity. For high fidelity, DMSO or other organic solvents should be avoided, whenever possible. Perform the PCR reaction with a moderate number of cycles by increasing at the same time the input template DNA amount to achieve a sufficient sensitivity.

When the highest possible fidelity is required for your application, use a pure proofreading polymerase, such as PwoSuperYield DNA Polymerase, which has an 18-fold lower error rate than Taq DNA polymerase and allows amplification of up to 3 kb fragments.

Materials

     

EXPAND is a trademark of Roche

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