Primer Specificity Testing enables you to design target specific primers and test their specificity against an off-target sequence(s) in a single step. The Specificity Testing tool uses Geneious BLAST to detect and exclude areas of high similarity between your target and the selected off-target database. Primers are then designed (using Primer3) on areas of low similarity according to the specified criteria.
How to use the Specificity Testing tool:
1. Select the sequence(s) you want to off-target test against
To design your target-specific primers, select your target sequence then choose Primers > Design New Primers. Under Specificity Testing check the checkbox and Choose the off-target sequence(s) to test against. Primers can be tested against a single sequence (including Alignments), list of sequences, or a folder containing multiple sequences.
2. Configure your off-target binding settings
The next section determines what you consider to be off-target binding. Any primers that are considered to have off-target binding are not returned as a primer annotation. If you are not sure what options to use, we suggest using the default options and tweaking from there.
Primers with less mismatches than those entered in the first dropdown are considered to be likely to bind to off-target sequence(s) and excluded as primer candidates for the target sequence.
The value in the second dropdown is a subset of the first dropdown and gives more weight to the location of the mismatches, due to annealing chemistry.
The third dropdown indicates the location of the mismatches. Binding at the 3' end is required for initial annealing to the template.
Primer candidates with at least as many mismatches as indicated in the fourth dropdown are considered unlikely to bind to the off-target sequence(s) and will be accepted as primer candidates for the target sequence.
Off-target primer pairs that result in amplicon sizes larger than that indicated in the fifth dropdown are considered as target primer candidates due to the decreasing efficiency of PCR as the amplicon size increases.
How to increase your chances of returning primer results
Due to the way Geneious Prime and Primer3 communicate, it is worth simply increasing the number of pairs to generate if you are not returning any primers. Failing that, in addition to increasing the number of pairs to generate, we recommend decreasing your stringency settings, if that is appropriate.
How to use Whole Genome sequences as your off-target database
To off-target test against a whole genome, download (or import) the genome in Geneious Prime and use this as your off-target database. You can either choose to Use Folder containing the genome sequences or individually choose chromosome sequences present in that folder. Please see tip below about performance if using large off-target databases.
How to get unique primers for Alignments
You can now design primers specific to a sequence in the alignment while making sure they do not bind to other sequences in the alignment. The simplest way to do this is to select the alignment document you are designing your primers on as the off-target sequence(s). Ignore the expected warning you will get about identical sequences detected.
Ensure you have selected Design primers on: Every Sequence if you want to design unique primer pairs specific to every sequence in the alignment.
⚠️ We recommend in silico testing your unique primers with tools such as Test with Saved Primers. Primer Specificity Testing works with primer pairs so depending on your design settings, you may get an unpaired primer binding on multiple alignment sequences. This lone primer is unlikely to yield an efficient product though it is worth testing and being aware of in case you wish to use more stringent design criteria.
A note of factors that impact performance
In order of impact, expect the tool to run slower as you:
- Increase the size of the off-target database
- Increase the size/length of the sequence to design primers on