PRIMEGENSw3: A Web-Based Tool for High-Throughput Primer and Probe Design

  • Garima Kushwaha
  • Gyan Prakash Srivastava
  • Dong XuEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1275)


Highly specific and efficient primer and probe design has been a major hurdle in many high-throughput techniques. Successful implementation of any PCR or probe hybridization technique depends on the quality of primers and probes used in terms of their specificity and cross-hybridization. Here we describe PRIMEGENSw3, a set of web-based utilities for high-throughput primer and probe design. These utilities allow users to select genomic regions and to design primer/probe for selected regions in an interactive, user-friendly, and automatic fashion. The system runs the PRIMEGENS algorithm in the back-end on the high-performance server with the stored genomic database or user-provided custom database for cross-hybridization check. Cross-hybridization is checked not only using BLAST but also by checking mismatch positions and energy calculation of potential hybridization hits. The results can be visualized online and also can be downloaded. The average success rate of primer design using PRIMEGENSw3 is ~90 %. The web server also supports primer design for methylated sequences, which is used in epigenetic studies. Stand-alone version of the software is also available for download at the website.

Key words

Primer Probe PCR Primer-design High-throughout design DNA methylation PRIMEGENS 



This work has been supported by National Institute of Health (Grant 1R01-DA025779) and the Congressionally Directed Medical Research Programs, U.S. Army Medical Research and Materiel Command (Grant BC062135). This research is also supported in part by the Paul K. and Diane Shumaker Endowment in Bioinformatics.


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Garima Kushwaha
    • 1
  • Gyan Prakash Srivastava
    • 2
  • Dong Xu
    • 3
    Email author
  1. 1.Informatics Institute and Christopher S. Bond Life Sciences CenterUniversity of MissouriColumbiaUSA
  2. 2.Department of NeurologyBrigham and Women’s Hospital, Harvard Medical SchoolBostonUSA
  3. 3.Computer Science Department, Informatics Institute, and Christopher S. Bond Life Sciences CenterUniversity of MissouriColumbiaUSA

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