We highly recommend using one of the many computer software programs available to design primers. This will help to accurately calculate the melting temperature (Tm) and help to identify potential secondary hybridization sites and secondary structure issues. The following must be taken into consideration when designing sequencing primers:
- Primers should be a minimum of 18 bases long, which will reduce the probability of second site hybridization on the DNA template.
- Keep the primer Tm around 50°C. Better results are obtained from primers with Tm > 45°C compared to primers with lower Tm.
- Runs of identical nucleotides should be avoided, particularly runs of four or more G's.
- The primer should not be designed over a SNP location.
- The G-C content should be in the range of 30% to 80%, with 50% to 55% being ideal. If the primers G-C content is less than 50%, the length of the primer may need to be increased to maintain the proper Tm.
- Ensure the primer is as pure as possible. HPLC purification is preferred.
- Primers that can hybridize to form primer dimers should be avoided.
- Designing primers that contain palindromes should be avoided for they can form secondary structures.
- If bisulfate-converted DNA is to be sequenced, then design primers using ABI's Methyl Primer Express Software, which is a free download found on ABI's web site.
If you have any questions regarding DNA sequencing or would like to schedule an appointment please contact the 51³Ô¹ÏºÚÁÏ Genomics Core manager Casey Hall-Wheeler.