Exercise 4: Designing degenerate primers

A degenerate primer contains a mix of bases at one or more sites. They are useful when you only have the protein sequence of your gene of interest so want to allow for the degeneracy in the genetic code, or when you want to isolate similar genes from a variety of species where the primer binding sites may not be identical.

The degeneracy value of a primer is the number of different primers that the primer sequence represents. For example, a primer which contains the nucleotide character N once (and no other ambiguities) has a degeneracy of 4 because N represents the four bases A,C,G and T. A primer that contains an N and an R has degeneracy 4 x 2 = 8 because R represents the two bases A and G. Because a degenerate primer is really a mix of different primers, only a fraction of the primer mix will work in an actual PCR. Thus it is best to limit degeneracy to under 100, as with higher values any one primer will become too diluted to work effectively, and non-specific target fragments may also be amplified. Degenerate bases at the 3` end of a primer should also be avoided.

In Geneious, you can design degenerate primers by using an alignment as the template. In this example, we will design primers to amplify an MHC class II gene, a highly polymorphic immune gene found in vertebrates.

Click on the MHC class II alignment. In the Display window next to the sequence viewer, check Highlighting and select Disagreements to Consensus.


From the alignment you can see that this gene contains polymorphic regions interspersed with conserved regions. We wish to design primers to sit in the relatively conserved regions at ends of the sequence to amplify across the polymorphic regions. We can do this by specifying the Target Region to amplify.

Select bases 41 to 264 in the alignment then click Primers → Design New Primers. These base numbers should show up in both the Target Region and Included Region options. As this is the region we wish to sequence, we don't want our PCR primers within this region, so uncheck the Included Region box and check the Target Region box. Uncheck the product size range and optimal product size boxes if these are checked, and set Number of pairs to generate to 1. As the region from bases 1-40 where the forward primer will sit is still somewhat polymorphic, we will need a degenerate primer to bind to this region. Expand the Characteristics panel, check the Allow Degeneracy box and set the number to 300.

At the bottom of the window are options that allow you to control how primers are designed on alignments. For degenerate primers, choose to design them on the Consensus. Then click the Consensus options to set how variable a position should be to be made degenerate. In this window set the Threshold to 75%. This means that at any given site in the primer, the primer will match at least 75% of the sequences in the alignment. Thus if more than 75% of the sequences in an alignment have the same base at any given position, that position will not be degenerate in the primer. If you want a primer where every single variant base in every sequence is included in the primer, set the consensus threshold on 100%.

Your settings should now look as in the picture below. Click OK


You should find that one primer pair has been added to the consensus sequence. Mouse over the forward primer to bring up the tool tip. You'll see that this primer has 4 degenerate sites specified by the letters Y, M, B, and R. These letters are IUPAC degeneracy codes which specify the mix of bases at that position. For a list of these codes see here. The degeneracy score for this primer is 24, which is a reasonable value for successful PCR. The second position in the primer is not degenerate as only 1 sequence in the alignment has a variant base (T) in this position, thus 5/6 (83%) of sequences match the primer, which is greater than the 75% threshold.

Click on each primer annotation and Extract them to your Document Table so they can be used in other functions.

This concludes the Primer Design tutorial

Back to Start