Design principles of PCR primers - Database & Sql Blog Articles

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Experimental Procedure

The primers must be perfectly complementary to the target template sequence and should not bind to non-specific regions. It's essential to avoid the formation of stable dimers or hairpin structures between the primers themselves during DNA polymerization.

DNA Polymerization (Mismatch) must be carefully managed to ensure accuracy. When designing primers, consider the following key factors:

1. The length of a primer is typically between 15–30 base pairs, with 18–27 being the most commonly used range.

2. Primer sequences should not have high similarity within the template, especially at the 3' end. If the 3' end has multiple identical bases, such as GGG or CCC, it may increase the chance of mismatches and errors.

3. The base at the 3' end of the primer significantly affects the efficiency of Taq DNA polymerase. A mismatch at the last base, particularly when it's an A, can reduce amplification efficiency. Therefore, it's advisable to avoid using A at the 3' end of the primer.

4. The GC content in the primer sequence should generally be between 40–60%. Too high or too low can hinder the initiation of the reaction. Additionally, the GC content of the forward and reverse primers should be similar to maintain balance.

5. Another critical parameter is the melting temperature (Tm), which is the temperature at which 50% of the primer and its complementary sequence form a double-stranded DNA molecule. An optimal annealing temperature usually ranges from 55°C to 70°C. For best results, the Tm values of both primers should be as close as possible.

6. Primer dimer or hairpin formation can lead to PCR failure. It’s important to design primers that minimize these structures as much as possible.