Designing an optimal set of primers for a difficult PCR target can sometimes feel like panning for gold. Lengthy or GC-rich sequences, or targets from complex sources can prove frustrating when the PCR does not work and exhilarating when it finally does. Oligo Primer Analysis software can be useful in optimizing primer design to amplify either the straightforward or difficult DNA sequence.
Oligo Primer Analysis Software is produced by Molecular Biology Insights. Molecular Biology Insights produces software for either Macintosh or Windows based operating systems. The program requires 8 Mb of disk space for both operating systems and 4 Mb of RAM for the Macintosh and 8 Mb of RAM for Windows. Oligo accepts sequence formats from GenBank and EMBL and files from other sequence analysis programs that are converted to a text format such as ASCII.
Oligo has a very simple user interface with very self-explanatory graphics and menu options. It is very versatile in choosing primers, whether you allow the program to perform an automated search or select a region manually. The automated search feature can be used by specifying the region to be amplified in base pairs and then the program goes through a series of subsearches based on preset parameters that can be customized to the user’s preferences. To select primers manually, an ideal area can be visualized by the sequence melting temperature and/or internal stability graph.
Oligo’s guidance on designing optimal PCR primers, sequencing primers, and hybridization probes lies in three essential criteria: specificity, primer does not form dimers or hairpins, and primer pairs do not form stable duplexes.
Once the primers are selected, they can be easily modified by adding restriction sites or edited for mutagenesis. Changes made to the primer sequence can be visualized to see changes in secondary structure of the primer and its influence upon PCR conditions. The primers can then be stored in a convenient database that Oligo provides or can be exported to Microsoft’s Excel if desired.
Some of the drawbacks to Oligo are that it’s not meant for high-throughput applications, primers can only be designed to one sequence at a time. There is also competition from oligo vendors such as Operon, Invitrogen, and IDT that provide free online primer design software.
Oligo provides free updates to its software, however there is not an automatic notification of available updates. Updates can be found by visiting the company’s website, proof of registration must be provided before you can download.
I have been reliably using Oligo Primer Design software for 6 years for routine and complex cloning projects.
Research Associate III
Molecular Biology
R&D Systems, Inc.