DESIGN TASKS We discriminate ﬁve distinct primer design user scenarios, or tasks, that have been integrated in the new interface. These tasks can be selected using a drop down list at the top of the Primer3Plus page. Currently the tasks include Detection, Cloning, Sequencing, Primer Check and Primer List. More tasks might be added in future versions of the Primer3Plus web interface. Detection The Detection task can be used for designing standard PCR primers or hybridization oligos to detect a given sequence. The user provides the template sequence and optionally indicates target regions within the sequence or changes the parameters for primer picking. By pressing the Pick Primers button a list of suggested primer pairs is presented, ordered with the best pair at the top. The Detection task is most reminiscent to the original Primer3 web interface. Cloning For cloning a PCR product in a speciﬁc reading frame in an expression vector, it is important to control precisely the start and end of the PCR product. With the Cloning task either the 50 or the 30 ends of the primers can be ﬁxed to the boundary of the included region, marked with curly braces. The output lists the predicted best ﬁtting primer pairs that start or end at the given position. Subsequently, the user can modify the primer sequence, e.g. by adding a restriction enzyme recognition site that facilitates cloning of the PCR product. Fixing primer ends signiﬁcantly limits the number of possible candidates that fulﬁl the stringent default settings. If this is the case, alternative primers are given that are accompanied by a warning concerning the relaxed stringencies that have been applied (Figure 2). Sequencing The Sequencing task is developed to design a series of primers on both the forward and reverse strands that can be used for custom primer-based (re-)sequencing of clones. Under default settings, the primers are spaced by a 500 nt interval on both strands. The spacing of primers is conﬁgurable within the Advanced settings tab, as is the overlap between the forward and reverse primers. A conﬁgurable number of nucleotides immediately follow- ing the primer that usually cannot be read reliably, are taken into account when selecting the forward and reverse primers. Primer Check The Primer Check task can be used to obtain information on a speciﬁed primer, like its melting temperature or self complementarity. This can, for instance, be useful when certain primers are to be reused under diﬀerent experi- mental conditions or if the original primer characteristics were lost. The Primer Check option is also available on the Primer3Manager page, enabling a re-check of primers that have been modiﬁed by the user, e.g. for cloning Nucleic Acids Research, 2007, Vol. 35, Web Server issue W73 purposes. When this task is selected, no template sequence is required and only the primer sequence has to be provided. Primer List With the Primer List task, all possible primers that can be designed on the target sequence and meet the current settings will be returned with their corresponding char- acteristics. This task basically allows manual selection of primers, which could be of interest in case of speciﬁc tasks that are not (yet) implemented in Primer3Plus (like picking overlapping primers). The run time of the Primer List task can be relatively long when compared to the other Task options, and can take up to a minute, especially when lengthy target sequences are submitted. TECHNOLOGY The Primer3Plus web pages consist of HTML with JavaScript for interactivity. The JavaScript is not essential for proper function of Primer3Plus and can be disabled. Custom settings are stored locally using the Boulder IO format (a simple data format: tag ¼ value). The Primer3Plus web interface has been tested to run properly on the most recent versions of Firefox, Internet Explorer, Safari and Konqueror. The server side software has been written in Perl, using the CGI standard for communicat- ing with the Apache web server. It runs on both SuSE Enterprise Linux 9 and Windows XP, and should run unaltered on any modern Unix/Linux platform. Primer3Plus is designed with interoperability in mind. It is relatively easy to integrate with other (web) applications. Settings, like the template sequence, can be passed in via HTML POST or GET operations, so applications can be linked directly to an already ﬁlled Primer3Plus form. FUTURE WORK An annotated view of the template DNA sequence showing, for instance, the open reading frames or intron/exon boundaries would facilitate ﬁnding the regions of interest for ampliﬁcation or detection. For this, Primer3Plus will need to be extended with functionality to extract and display features from uploaded sequence ﬁles. Primer3Plus was recently included in the Primer3 SourceForge project (http://sourceforge.net/projects/ primer3/). This allows for tight integration of the web interface with the core program and creates a platform for both developers and end users to discuss further modiﬁca- tions and additions to Primer3Plus. ACKNOWLEDGEMENTS The authors would like to thank Steve Rozen for helpful suggestions and ongoing support of Primer3, and Catarina Cardoso and Gerben Bijl for extensive beta testing. R.G. was funded by NWO-VIDI (864.06.007) and NWO-HORIZON (050-71-052). H.N. was sup- ported by the BioAssist program of the Netherlands