Nucleic Acids Research, 2007, Vol. 35, Web Server issue W71–W74 doi:10.1093/nar/gkm306 Primer3Plus, an enhanced web interface to Primer3 Andreas Untergasser1,*, Harm Nijveen 2 , Xiangyu Rao 2 , Ton Bisseling 1 , Rene´ Geurts1 and Jack A.M. Leunissen2 1Laboratory of Molecular Biology and 2Laboratory of Bioinformatics, Department of Plant Science, Wageningen University, Dreijenlaan 3, 6703HA, Wageningen, The Netherlands Received January 31, 2007; Revised April 2, 2007; Accepted April 4, 2007 ABSTRACT Here we present Primer3Plus, a new web interface to the popular Primer3 primer design program as an enhanced alternative for the CGI- scripts that come with Primer3. Primer3 consists of a command line program and a web interface. The web interface is one large form showing all of the possible options. This makes the interface powerful, but at the same time confusing for occasional users. Primer3Plus provides an intuitive user interface using present- day web technologies and has been developed in close collaboration with molecular biologists and technicians regularly designing primers. It focuses on the task at hand, and hides detailed settings from the user until these are needed. We also added functionality to automate specific tasks like design- ing primers for cloning or step-wise sequencing. Settings and designed primer sequences can be stored locally for later use. Primer3Plus supports a range of common sequence formats, such as FASTA. Finally, primers selected by Primer3Plus can be sent to an order form, allowing tight integration into laboratory ordering systems. Moreover, the open architecture of Primer3Plus allows easy expansion or integration of external software pack- ages. The Primer3Plus Perl source code is available under GPL license from SourceForge. Primer3Plus is available at http://www.bioinformatics.nl/ primer3plus. INTRODUCTION Oligonucleotide primers are widely used in various molecular biology techniques like DNA sequencing and the polymerase chain reaction (PCR). Since a primer serves as the starting point for DNA replication, speciﬁc binding of the oligonucleotide to the target sequence on the template strand is essential for a successful experiment. The binding speciﬁcity of a primer is determined by several of its properties, like the melting temperature (Tm), GC-content and self complementarity. Designing primers is usually done with the help of computer programs, among which Primer3 is most widely used judging from the hundreds of citations of the primary publication (1). Primer3 is popular since the program can be used online and is redistributed free of charge. Recently, a SourceForge project was started for Primer3 in which several improvements are being discussed and implemented. Subsequently Primer3 was updated to include an additional method for Tm calculation [SantaLucia model, (2)], since it was argued that primer design based on the Breslauer model shows variation in the predicted Tm (3,4). Primer3 consists of a command line C program, and an HTML web interface written in Perl. The web interface is one large form showing all possible options. This makes it powerful, but at the same time confusing for the occasional user; without in-depth knowledge it is hard to tell which of the settings are important for a speciﬁc design task. Some tasks, like designing PCR primers for position speciﬁc cloning, are not easy to perform with the current web interface, since this would require multiple runs of the program under diﬀerent settings. Because of these issues with the current web interface and facilitated by its liberal license, several academic and commercial parties have created their own command line and/or web interface to Primer3, tailored to their speciﬁc needs and audience; e.g. EMBOSS’ EPRIMER3 and the PCR Suite (5,6). We took this eﬀort one step further and developed a general purpose, easy to use and powerful new web interface for Primer3, called Primer3Plus that is available at http://www.bioinformatics.nl/primer3plus. WEB INTERFACE We aimed to design the Primer3Plus web interface in such a way that it is comprehensible for occasional users, and yet powerful for the experienced users that need to do more complex or laborious tasks. Where the original *To whom correspondente should be addressed. Tel: þ31 317 482037; Fax: þ31 317 483584; Email: andreas@untergasser.de The authors wish it to be known that, in their option, the first two authors should be regarded as joint First Authors 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.