Can I get a copy of your software? How about the source code?

Sure! This software is released under the GPLv3 license, and is available here.

What's the difference between the standalone and server version? How do I install it? What are the system requirements?

Please refer to the Download page for this information.

What version of Primer3/BlastN are you using?

This server uses Primer3 version 1.1.4 (Apr 2008) and BlastN version 2.2.22 (Sept 2009).

What is the difference between the two tiling types?

The different tiling types have an influence solely on how the target sequence is split into regions to tile. It has no effect whatsoever on the subsequent steps (Primer3 and BlastN). It is meant to simplify mental math for users according to their requirements: do they want a fixed number of amplicons (regardless of the distance between them) or have one amplicon at each X base pairs (and let the program figure out how many amplicons will be required).

For example, given an input sequence 10,000 nucleotides long, specifying that you want 50 primer pairs designed, or that you want them to be 200 base pairs appart will give precisely the same result.

How is the "amplicon count" determined?

The amplicon count is the number of plausible DNA fragments that will be amplified using the suggested primer pair, according the the specificity parameters provided (total mismatches, 3' mismatches and amplification distance threshold). To compute the amplicon count, all potential hybridization sites, for each oligonucleotide of the primer pair, are enumerated. Any pair of hybridization sites found within the specified amplification distance threshold, with appropriate strandedness, counts for an amplicon. Properly designed primer pairs should lead to a single specific amplicon. Please note that this method will also detect amplicons resulting from mispriming of an individual oligonucleotide, if any exist.

How is the primer pair score computed?

Briefly, the score of a primer pair is inversely proportional to the number of possible amplicons and inversely proportional to the number of hybridization sites for each primer of the pair. The scoring function gives a lot more weight to the number of possible amplicons, since a primer pair with multiple amplicons should never be used. Also, a small bonus proportional to the total number of mismatches to the most similar unintended target is used to discriminate primer pairs with an equal number of amplicons and cross-hybridization sites.

The exact formula used is:

score=   1 / ((1000 * (amplicon count-1)) + (hybridiztion count for forward primer -1) + (hybridization count for reverse primer - 1))
+ ( 0.5 * (mismatch count to next-best site for forward primer + mismatch count to next-best site for reverse primer) / ( forward primer length + reverse primer length ) )