Q：Poor amplification efficiency: Low or no amplification
· Enzymes: Experiments have shown that PCR enzymes have certain preferences for DNA. If it is difficult to obtain ideal amplification with one enzyme, other PCR enzymes should be tried.
· Templates and primers: Ensure that templates and primers are with high quality and that they are not degraded. Besides, if the template contains a lot of inhibitors (such as plant genomic DNA), it is recommended to use a DNA polymerase displaying high tolerance to PCR inhibitors, or the template can be diluted by serial dilution to optimize the template input.
· Reaction components and conditions: Increasing the number of cycles, reducing the annealing temperature, or appropriately increasing Mg2+ concentrations can improve amplification efficiency, while these will lead to a decrease in specificity and fidelity. Optimization of the reaction components and conditions is required according to specific experimental requirements.
Q: Poor amplification specificity: Primer-dimers, nonspecific bands, smears, etc.
· Use hot-start DNA polymerases: Hot-start PCR enzymes have higher amplification efficiency and specificity than non-hot-start PCR enzymes.
· Optimize reaction components and conditions: Increase the annealing temperature to improve specificity effectively. Also, reducing the number of cycles and appropriately decreasing Mg2+ concentrations can improve specificity but will lower the amplification efficiency. Optimization of the reaction components and conditions is required according to the specific experimental requirements. Also, some special PCR can be tried, such as nested PCR, touchdown PCR and so on.
· If the PCR products will be used for downstream applications and the target DNA band exists, the target band can be isolated by gel extraction.
Q: Poor amplification fidelity: mutation, insertion, deletion, etc.
· Select high-fidelity DNA polymerases: Family B DNA polymerase, such as Pfu DNA polymerase, with 3′→ 5′exonuclease activity, corrects mismatched base pairs to ensure high fidelity. In addition, polymerase blends containing Family B DNA polymerases also have a high fidelity, which is lower than the fidelity of Family B DNA polymerase.
· Optimize PCR reaction components and conditions: Increasing template input or reducing number of cycles can decrease mutation rate, and improve fidelity.
· Confirm the cause of low fidelity: Sometimes low fidelity is not caused by PCR, which usually only leads to a few point mutations. If a large number of mutations, significant insertions, or deletions, etc. are found after sequencing, actually they are likely to come from other experimental steps, such as DNA recombination occurring in DNA replication in E. coli or contamination in sequencing. In this case, pick some clones again and then identify them by sequencing or replace for other bacterial strains directly.
· RT-PCR fidelity: Fidelity of reverse transcriptase is usually lower than that of DNA polymerase. For RT-PCR, the mutation rate of upstream reverse transcription is greater than that of downstream PCR. If there is any mutations, another reverse transcription can be performed or reverse transcriptase of higher fidelity can be selected.
Q: How to add "A" in Pfu enzyme amplification?
A: In a 10 µl reaction as follows: add purified PCR product, 0.2 μl of Taq DNA Polymerase, 0.5 μl of 10 mM dNTP (or 0.5 μl of 2.5 mM dATP) and 1 μl of 10xTaq Buffer. Incubate at 72°C for 5–15 minutes.