Bio::POD provides a graphical interface to the primer picking software, Primer3, developed by the Whitehead Institute for Biomedical Research.
Start by selecting the region of the gene which you would like to span with primers. Once you have selected a region, you may start the primer picking process one of two ways:
Click "Pick Primers..." from the "Primers" menu as shown here:
- OR -
Right click (or Ctrl+click) the selected region and click "Span with Primers..." as shown here:
Both of these methods will open the primer picking dialog shown here:
Click "Generate Primers" to span the selected region with primer pairs using the default settings.
Alternatively, you may alter the default settings such as the Minimum Overlap between primer pairs or any of the options in the drop-down list:
By selecting one of the options in the drop-down list, the option will automatically be displayed along with the default value as shown here:
You may alter the default value or uncheck the option which will make it disappear once again. A description of the options can be found here: http://frodo.wi.mit.edu/primer3/README.primer3_1.0.0
The Minimum Overlap option is the minimum overlap of the right primer of one primer pair and the left primer of the next primer pair (default: 0).
Once you have decided on the primer picking options and clicked "Generate Primers", the primer pairs will appear as dumbbell-shaped objects in the annotation area of each gene view. You can see the primer pairs in both the Transcript View and Genomic View as shown here (circled):
Here is an up-close view after zooming in to the 5th exon:
To get details on one or more primer pairs, select the primer pairs, right click (or Ctrl+click) the primer pairs, and click "Get Details...". An Annotation Details window will appear with all the details of the primer pair which can be used to order the primer pair(s) from an oligo synthesis company.