W72 Nucleic Acids Research, 2007, Vol. 35, Web Server issue Figure 1. Screenshot of the Primer3Plus start page. In contrast to the original Primer3 web interface, detailed settings that are not frequently used are hidden behind tabbed panels. A pull-down task menu is added (top left) that provides ﬁve scenarios for which Primer3Plus can be used: gene detection, primers for cloning purposes, sequencing, list of all possible primers and check option for already available primers. Here an example sequence (GFP) was loaded, the Cloning task selected and the open reading frame marked as included region. Primer3 web interface presents all options and settings in one large web page, Primer3Plus starts with a more simple screen containing only the input boxes for the sequence and the option to select the target region for ampliﬁcation (Figure 1). The target sequence can be pasted or uploaded in any common format, such as FASTA and EMBL. Furthermore, the ‘Pick Primers’ button is moved to the top of the page to make sure it is always available. Also a new task selection box is included (top left corner), that provides the possibility to select between ﬁve diﬀerent scenarios for which Primer3Plus can be used (see subsequently). For most purposes the default parameter settings are adequate. Adjusting these parameters is possible by using the tabbed panels. The tabs are ordered in the most likely frequency of use, with the left most panel being the start page, followed by the general settings, advanced settings, internal oligo options, penalty weights and sequence quality. The parameter names were kept the same as in the original Primer3 interface to minimize time needed to get familiar with using Primer3Plus. The labels of the parameters are web links to a context-sensitive help text, just like in the original Primer3 web interface. Primer3Plus adds to this a ﬂoating tool tip with a brief description for some of the most prominent parameters. Next to the default settings, the user can choose speciﬁc settings for special tasks like primer design for qPCR applications. It is also possible to store custom settings locally to save the user from having to go through the conﬁgura- tion each time primers are designed for speciﬁc non-standard conditions, like AT-rich organisms or high-salt environments. The Primer3Plus results page shows the suggested primers with their characteristics, ordered from best to worst. The best scoring primer pair is marked on the template sequence. The selected primers can be submitted to the Primer3Manager where the user can manage a primer collection. Designed primers are stored on the web server for a limited period, allowing users to design multiple primer pairs and combine them in a single order. Users, or actually web clients, are identiﬁed using cookies that expire after a set period (currently one week). Primers can also be saved locally as a FASTA ﬁle and previously saved primers can be uploaded allowing the addition of new primers to an existing primer collection. Within the Primer3Manager selected primers can be re-analyzed, submitted to the NCBI BLAST service (7) or combined on an order form. In case Primer3Plus is run on a local server, the order form can be customized allowing tight integration into a laboratory primer order management system.