Primer design for RT-PCR.

Primer design is a crucial initial step in any experiment utilizing PCR to target and amplify a known nucleotide sequence of interest. Properly designed primers will increase PCR amplification efficiency as well as isolate the targeted sequence of interest with higher specificity. Many factors that may limit the success of a primer pair can be detected a priori with computational methods. For example, primer dimer detection, amplification of alternative products, stem loop interference, extreme melting temperatures, and genotype-specific variations in the target sequence can all be considered computationally to minimize subsequent PCR failures. The use of computational sequence analysis tools to select the best primer pair from the available candidates will not only reduce experimental rates of failure but also avoid the generation of misleading results arising from the amplification of alternative products.