To do hairpin or dimer analysis, click on the appropriate option to the right of your sequence. For hairpin analysis, you can change the default concentrations provided to match your reaction conditions.

The most valuable piece of information on this screen is the T_m for each of your structures. If the T_m of the structure is lower than your reaction conditions, then this structure will not cause any problems. If it is higher, this oligo may be problematic and should be redesigned. For self-dimer analysis, click on 'Self-Dimer' to bring up a new window with each possible self-dimer your oligo can form.

For each diagram you will be able to see the calculated delta G value for this secondary structure. If you have a strong delta G (–9 kcal/mol or more negative) this oligo could be problematic. Heterodimer analysis works the same way as self-dimer analysis. Use the 'Hetero-Dimer' button in the OligoAnalyzer program to check for primer dimers.

Enter the sequence of your forward primer into the sequence box, and then click 'Hetero-Dimer.' This will open a second box below the original sequence box, in which you enter the sequence of your reverse primer.

Then click the 'Calculate' button below the second box. In general, a primer pair with a delta G of –9 kcal/mol or more negative will be problematic.

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