How do I design degenerate primers based on an alignment?

 Using the Design New Primers operation

Geneious Prime can design degenerate primers based on an alignment using the Design New primers tool under the Primers menu. 

For example, if you want a degenerate primer pair with primer Tm's of around 54°C, a minimum PCR product length of 150 bp (alignment is 684 bp long), and a maximum allowed degeneracy of 32 then you would use settings similar to those shown below.

Check the Option to Allow Degeneracy and set an appropriate value (see the online manual, section 13.1.6 above for an explanation of degeneracy).

Ensure the option Design primers on: Consensus is set.

Click the Consensus Options button and set the threshold to 100% - identical if you want the primer degeneracy to incorporate every variant base.  If you are using a larger alignment, and want your degeneracy to cover bases found in 90% of the sequences, set the Consensus threshold to 90% rather than 100%.  With this setting, at sites where 9 out of 10 sequences are identical the most common base will be used, rather than adding a degenerate base for the single variant nucleotide.  



The Design New Primers tool may be suitable for your needs.  However, be aware that it will allow degeneracy very near to the 3' ends of the primers which may significantly reduce their ability to prime efficiently, even if the overall degeneracy is low.


Manually designing degenerate primers

 You can also do degenerate primer design manually using the following procedure.

1. Select the alignment, make sure the consensus is on and the Threshold is set appropriately for the level of degeneracy you require.  Make sure Highlighting is on to easily see differences.


2. Visually identify suitable regions for a degenerate primer location using the Identity graph as a guide, zoom in and identify ideal regions for degenerate primer locations.  Look for regions with minimal differences, and if possible a perfectly conserved "clamp" (as long as possible) at the 3' end of the primer.

3. On the Consensus line in the alignment, select the region you want to define as the primer, drag/select left to right for a forward primer, drag/select right to left for a reverse primer. 

If you are using Geneious Prime 2019 you will see a Floating Tooltip which details the calculated Tm for the selected region, and an Add Primer button (boxed red).



If you are using an earlier version of Geneious, select the region, view the Statistics side pane to see the estimated Rough Tm, then go Primers -> Characteristics for Selection to add a new primer.

4. Click the green Add Primer button, the Add Annotation window will open, you will see the calculated characteristics of the primer, including the Tm range based on the degeneracy, if you are happy with the primer characteristics, give the primer a Name:, go OK and the primer will be added to the consensus as an annotation.




5. If you manually create a forward and reverse primer and you want to pair them, select them both on the sequence (select the first, then CTRL or command click on the second), then right (or CTRL) click on one and choose to Pair Selected primers (available in Primer 2019.2.1 and later).



The primers will now have associated information calculated for product size, and potential primer dimer issues.  Hover over the primer annotation to see this information.




6. Select each primer annotation (select the first, then CTRL or command click on the second) and hit the Extract to extract the annotations to create new stand-alone Primer sequence files.


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