Design principles of PCR primers - Database & Sql Blog Articles

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Experimental Procedure

The experimental procedure involves designing and using primers that specifically bind to the target DNA sequence. Primers must be complementary to the template sequence and avoid binding to non-target sites. This ensures accurate amplification and prevents unwanted side reactions.

During DNA polymerization, if mismatches occur, they can lead to the formation of primer dimers or hairpin structures, which interfere with the PCR process. To prevent this, careful design of the primers is essential.

When designing primers, several key factors should be considered:

• 1. The length of the primer is typically between 15-30 base pairs, with 18-27 bp being the most commonly used range.
• 2. The primer sequence should not have high similarity to other regions within the same template, especially at the 3' end. High similarity can lead to mispriming and reduce specificity. Also, sequences with more than one consecutive G or C at the 3' end, such as GGG or CCC, may increase the chance of errors.
• 3. The base at the 3' end of the primer has a significant impact on the efficiency of Taq DNA polymerase. Mismatches at the last base are more likely to occur when it's an A. Therefore, it's advisable to avoid using A at the 3' end of the primer.
• 4. The GC content of the primer should generally be between 40-60%. Too high or too low can affect the annealing process. Additionally, the GC content of the forward and reverse primers should be similar to ensure balanced amplification.
• 5. The melting temperature (Tm) of the primer is another important parameter. It refers to 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 dimers or hairpin structures can cause PCR failure. These secondary structures should be minimized during the design phase to ensure successful amplification.