To optimize the detection efficiency of conventional PCR, in addition to optimizing the reaction system and primer design, attention should also be paid to fine-tuning the amplification program. Here are some key strategies:

1. Gradient PCR determines the optimal annealing temperature
The annealing temperature is crucial for amplification specificity. Annealing temperature can be quickly screened through gradient PCR (such as setting a gradient of 50 ° C-65 ° C) to avoid non-specific bands or amplification failures. If there is a significant difference in primer Tm values, "Touchdown PCR" can be attempted, which gradually reduces the annealing temperature from a higher temperature and prioritizes the amplification of highly specific products.

2. Optimize loop parameters
-Pre denaturation time: For templates with high GC content, the initial denaturation can be extended to 5-10 minutes to ensure DNA melting.
-Extension time: Adjust according to the length of the amplified fragment (usually calculated at 1kb/minute), but pay attention to differences in enzyme activity. For example, high fidelity enzymes may take longer.
-Cycle number: Conventional amplification recommends 25-35 cycles. Excessive cycling can increase the risk of primer dimers, and sensitivity and specificity can be balanced by diluting the template or reducing the number of cycles.

3. Use of additives
-DMSO or betaine: can alleviate the effects of complex secondary structures, especially suitable for long fragments or high GC templates (recommended final concentration 3-5%).
-Optimization of Mg ² ⁺ concentration: Mg ² ⁺ is a cofactor of Taq enzyme, and its concentration (usually 1.5-2.5mM) needs to be balanced with dNTPs. The optimal concentration can be determined through gradient experiments with intervals of 0.5mM.

4. Template quality and quantity
Avoid using templates that degrade or contain inhibitors. If the sample is environmental DNA, additional preprocessing steps (such as column purification) can be added. The recommended template size is between 1-100ng. Excessive template size may lead to non-specific amplification, while insufficient template size may require an increase in the number of cycles.

5. Comparison settings
Including positive control (known template), negative control (no template), and internal control (such as housekeeping gene) to exclude reagent contamination or system abnormalities.