How to use primique

Given a set of sequences in the Fasta format, primique suggests primer pairs for each sequence in the set such that each pair only amplifies its target sequence.

For example, primique might be used to design a specific pair of primers for each sequence in a family of closely related and very similar transcription factors. Each primer pair suggested by the program will, theoretically, only amplify its target sequence and none of the other sequences. Also, primique might be used to design specific primers for a group of very similar pathogen strains.

You can upload a local database of other sequences and make sure that the primers do not amplify any of these database sequences either. Further, you may choose to blast the primers against the non-redundant nucleotide database of NCBI to check whether they amplify anything there.

You can set various parameters regarding desired primer and amplicon length, melting temperature, GC content, etc.

The suggested primer sets are scored and ranked. The score depends on whether the individual primers have 1- or 2-mismatch alignments to any non-target sequences. Such "close match" alignments may be considered problematic since they represent potential mis-primings. If any are found, close match alignments where the (right-most) mismatch position is located closer to the 5'-end than the 3'-end are considered worse than the other way around. A score of 3000 indicates the the primer pair has no 1- or 2-mismatch alignments to any non-target sequences.

Further, variation among the locations of the suggested primers is promoted.

If you do not succeed in finding primers for all of your sequences, you can do a re-run only on these sequences with relaxed parameter settings. If you do so, you could first store the primers already found under the more restrictive settings - or choose the option of doing the re-run in a new browser window. You can also try again with all sequences.

Note that primers are not allowed to contain characters other than A, C, G, T, a, c, g or t.

Before uploading homologous sequences, it may be a good idea to truncate 'singlet tails' outside homologous regions to prevent primers from being suggested here.

Go back to the front page of primique.