Specific options for alignment-based primer and probe design
Compared to the primer view of a single sequence the most notable difference is that the alignment primer view has no available graphical information. Furthermore, the selection boxes found to the left of the names in the alignment play an important role in specifying the oligo design process. This is elaborated below. The Primer Parameters group in the Side Panel has the same options for specifying primer requirements, but differs by the following (see figure 30.12):- In the Mode submenu which specifies the reaction types the following options are found:
- Standard PCR. Used when the objective is to design primers, or primer pairs, for PCR amplification of a single DNA fragment.
- TaqMan. Used when the objective is to design a primer pair and a probe set for TaqMan quantitative PCR.
The work flow when designing alignment based primers and probes is as follows:
- Use selection boxes to specify groups of included and excluded sequences. To select all the sequences in the alignment, right-click one of the selection boxes and choose Mark All.
- Mark either a single forward primer region, a single reverse primer region or both on the sequence (and perhaps also a TaqMan region). Selections must cover all sequences in the included group. You can also specify that there should be no primers in a region (No Primers Here) or that a whole region should be amplified (Region to Amplify).
- Adjust parameters regarding single primers in the preference panel.
- Click the Calculate button.