Here are a few guidelines to help in the design of oligos.
- Avoid primers with strings of a single base, especially G or C.
- Design oligos that are at least 18 bases long for good hybridization.
- Try to design primers with melting temperatures between 45 and 55C. The melting temperature can be roughly calculated by the following procedure:
- Add the number of G's and C's in the oligo. Multiply the total by 4.
- Add the number of A's and T's in the oligo. Multiply the total by 2.
- Add the results from a and b. This is your estimated melting temperature.
- If a primer has a GC content of less than 50%, you may need to design a longer oligo to keep the melting temperature above 45C.
- To decrease the chance of spurious priming, oligos shorter than 18 bases are not recommended.
- Avoid primers that form secondary structures, such as hairpins and primer dimers.
- Base order also influences the template/primer stability. Try to place a GC pair at the 5' and 3' ends of the oligo to stabilize binding under higher temperatures, such as those used with Taq DNA polymerase, but try to avoid strings of G's and C's, which can form internal, non-Watson-Crick base pairing (Sarocchi et al., 1970).