In addition of screening the entire iGEM registry, we have developed a tool to help a biology iGEM team (Download). This team is the Virginia United team. A part of this team was indeed working at Virginia Bioinformatics Institute, so we had the opportunity to meet each other. It consisted in finding the DNA sequence of the primer, given three input sequences: the two sequences that will undergo the PCR, and the complete resulting sequence.

The software will find 4 primers: the forward primer overlapping with the first input, the reverse user primer that overlaps with the first sequence and the gap sequence added on the PCR, the forward user sequence, overlapping with the second sequence and the gap sequence, and endly the reverse primer, which overlaps with the second sequence. If some suffix, prefix or terminator are added on the full sequence, they will be added in the forward and reverse primers. To detect the length of the overlapping part of the primers, we search for the correct length that will have the desired melting temperature. This melting temperature is calculated with the equation:

deltaH

Tm = ----------------------------------------- - 273.15 + 16.6log[Na+]

-10.8 + deltaS + Rln([oligo]/4)

with R = 1.987 cal/(mol K) Gas constant, [oligo] = 5E-07 M, [Na+] = 0.05 M. The find the position of the cutting enzyme (where the nucleotide U was placed), we search for an A in the gap sequence, and a matching T 8~13bp further. The A will be replaced by the U in the reverse user primer, and the T will be replace by the U in the forward user primer.

This tool will be useful also for biologist working on the VBI lab, as they had to do it manually before, and it was pretty slow and subject to human errors.

Example: here is a detailled example of an extraction of PCR primers.

Download the User Primer Calculator

An help file to use this software is given here 1. This software can be used with the interface shown previously, or with command line only (to automatisized it easily). The input can be the 3 sequences or a file containing these 3 sequences.

An executable working on both windows or on linux is available here 2. As it is too big for the iGEM wiki, it has been compressed with pack200 (then zip). In linux, you can uncompress it by typing unpack200 name.zip name.jar. On windows, you could use Unpack200.exe to uncompress it. Also, the executable is easy to reconstruct from the source code given. You just have to create a java project with this code with eclipse and then go to export, create a runnable jar file. The jar file is the executable.

The source code of this software (in Java) is also available 3.