Figure 13.4:
The Design for Sequencing dialog
This option will design primers for Sanger sequencing, placing primers at a specified interval across a whole sequence or selected region. Choose Unidirectional if you wish to sequence with forward primers only. Choose Bidirectional to design forward and reverse primers that will enable recovery of double stranded sequence.
The specified interval is the distance from the 3′ end of one primer to the 3′ end of the next primer on the same strand. Primer3 will attempt to place primers within 20 bp of the specified distance, although this may not always be possible.
If a selected region is chosen for sequencing primer design, the first primer will be placed approximately 50 bp 5′ to the selection if there is enough flanking sequence to do so. If there is no sequence upstream of the selection the first primer is placed as close to the beginning of the selection as possible.
If designing bidirectional primers, the minimum distance from the 3′ end of the fwd primer to the 3′ end of the next primer on the reverse strand is 20 bp.