Primer3: Improvements for the design of qPCR primers

Andreas Untergasser1, Ioana Cutcutache2, Steve Rozen2
1University of Heidelberg, Germany; 2Duke-NUS Graduate Medical School, Singapore

For over one decade Primer3 assisted scientists in the process of primer selection. The popularity of the Primer3 software in the scientific community is indicated by over 5000 citations of the initial publication. Several software tools employ Primer3 for the primary selection of primers and reevaluate the output according to their special needs, like the tool primer-blast at NCBI. Primer-blast uses Primer3 for the selection of primers and evaluates their specificity by blasting them against one of the NCBI nucleotide databases. We think the success of Primer3 is based on the accuracy of primer selection, the fast and stable implementation, the flexibility of the primer selection process and the open source license which allows the free use, modification and distribution of Primer3. Users of Primer3 fall into three groups: bioinformaticians, who embed Primer3 within their web servers, bioinformatics scripts or pipelines, expert users who select large numbers of primers and need tight control over the selection process, and occasional users who simply want a convenient way to design a few primer pairs. Bioinformaticians usually rely on the Primer3 command-line interface and run Primer3 on a local machine (download at Expert or occasional users usually rely on the Primer3 web interface, Primer3plus ( The functionality of Primer3 has expanded in the last decade considerably since it was first released. Primer3 now offers a cleaner interface on the command line, improved primer selection algorithms and up to date melting temperature calculations as well as thermodynamic secondary structure calculations. The primary function of Primer3 is the selection of primers for the amplification a given sequence. The latest version can also pick lists of primers which are not matched into primer pairs, primers required for Sanger-sequencing, cloning primers, and can reevaluate user-specified primer pairs. The old algorithm had the tendency to over represent very good primers in the result list and to favor certain regions. To overcome this limitation, a region around a selected primer can be defined, in which no second primer is allowed to bind and thereby creating a list of truly unique primer pairs which are well distributed over the sequence. In qPCR applications it is desired to discriminate DNA derived from mRNA from genomic DNA by using primers spanning an exon-exon junction. Primer3 can be provided with a list of junctions and will ensure that either the forward or the reverse primer span one of these junctions. Primer3 is a complex tool, that allows control of the selection process with many parameters. Primer3 now has the option to save settings for these parameters in a file, enabling expert users to easily provide optimal parameter settings to less experienced users. We are confident that the new modifications position Primer3 to be a valuable for the coming decade.

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