Advertisement

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

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

Abstract

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 

Notes

Acknowledgements

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.

References

  1. 1.
    Xu D, Li G, Wu L, Zhou J, Xu Y (2002) PRIMEGENS: robust and efficient design of gene-specific probes for microarray analysis. Bioinformatics 18:1432–1437CrossRefPubMedGoogle Scholar
  2. 2.
    Srivastava GP, Xu D (2007) Genome-scale probe and primer design with PRIMEGENS. Methods Mol Biol 402:159–176CrossRefPubMedGoogle Scholar
  3. 3.
    Srivastava GP, Guo J, Shi H, Xu D (2008) PRIMEGENS-v2: genome-wide primer design for analyzing DNA methylation patterns of CpG islands. Bioinformatics 24:1837–1842CrossRefPubMedGoogle Scholar
  4. 4.
    Srivastava GP, Kushwaha G, Shi H, Xu D (2010) High-throughput primer and probe design for genome-wide DNA methylation study using PRIMEGENS. In: A practical guide to bioinformatics analysis. iConcept Press Ltd, G. Fung ed. Hong Kong, pp 151–171Google Scholar
  5. 5.
    Lee EJ, Pei L, Srivastava GP, Joshi T, Kushwaha G, Choi JH, Wang X, Mockaitis K, Colbourne J, Zhang L, Schroth GP, Xu D, Zhang K, Shi H (2011) Targeted bisulfate sequencing by solution hybrid selection and massively parallel sequencing. Nucleic Acids Res 39(19):e127CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Srivastava GP, Hanumappa M, Kushwaha G, Nguyen HT, Xu D (2011) PRIMEGENS-v2: homolog-specific PCR primer design for profiling splice variants. Nucleic Acids Res:39(10):e69Google Scholar
  7. 7.
    Kushwaha G, Srivastava GP, Xu D (2011) PRIMEGENSw3: a web-based tool for high-throughput primer and probe design. In: Bioinformatics and Biomedicine (BIBM), 2011 IEEE international conference, 2011, 345, 351. doi:  10.1109/BIBM.2011.43
  8. 8.
    Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386PubMedGoogle Scholar
  9. 9.
    Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410CrossRefPubMedGoogle Scholar
  10. 10.
    Zhang Z, Schwartz S, Wagner L, Miller W (2000) A greedy algorithm for aligning DNA sequences. J Comput Biol 7:203–214CrossRefPubMedGoogle Scholar
  11. 11.
    Crothers DM, Zimm BH (1964) Theory of the melting transition of synthetic polynucleotides: evaluation of the stacking free energy. J Mol Biol 9:1–9CrossRefPubMedGoogle Scholar
  12. 12.
    DeVoe H, Tinoco I Jr (1962) The stability of helical polynucleotides: base contributions. J Mol Biol 4:500–517CrossRefPubMedGoogle Scholar
  13. 13.
    Gray DM, Tinoco I Jr (1970) A new approach to the study of sequence-dependent properties of polynucleotides. Biopolymers 9:223–244CrossRefGoogle Scholar
  14. 14.
    Borer PN, Dengler B, Tinoco I Jr, Uhlenbeck OC (1974) Stability of ribonucleic acid double-stranded helices. J Mol Biol 86:843–853CrossRefPubMedGoogle Scholar
  15. 15.
    Tinoco I Jr, Borer PN, Dengler B, Levine MD, Uhlenbeck OC, Crothers DM, Gralla J (1973) Improved estimation of secondary structure in ribonucleic acids. Nat New Biol 246:40–41CrossRefPubMedGoogle Scholar
  16. 16.
    Uhlenbeck OC, Borer PN, Dengler B, Tinoco I Jr (1973) Stability of RNA hairpin loops. J Mol Biol 73:483–496CrossRefPubMedGoogle Scholar
  17. 17.
    Breslauer KJ et al (1986) Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A 83(11):3746–3750CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Rychlik W, Spencer WJ, Rhoads RE (1990) Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res 18(21):6409–6412CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Owczarzy R, Vallone PM, Gallo FJ, Paner TM, Lane MJ, Benight AS (1997) Predicting sequence-dependent melting stability of short duplex DNA oligomers. Biopolymers 44(3):217–239CrossRefPubMedGoogle Scholar
  20. 20.
    Ke S-H, Wartell RM (1993) Influence of nearest neighbor sequence on the stability of base pair mismatches in long DNA: determination by temperature-gradient gel electrophoresis. Nucleic Acids Res 21(22):5137–5143CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    SantaLucia J Jr, Allawi HT, Seneviratne PA (1996) Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry 35:3555–3562CrossRefPubMedGoogle Scholar
  22. 22.
    Santalucia JA (1998) Unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA 95:1460–1465CrossRefPubMedCentralPubMedGoogle Scholar
  23. 23.
    Peyret N, Seneviratne PA, Allawi HT, SantaLucia J (1999) Nearest-neighbor thermodynamics and NMR of DNA sequences with internal AA, CC, GG, and TT mismatches. Biochemistry 38:3468–3477CrossRefPubMedGoogle Scholar
  24. 24.
    Hyndman D, Cooper A, Pruzinsky S, Coad D, Mitsuhashi M (1996) Software to determine optimal oligonucleotide sequences based on hybridization simulation data. Biotechniques 20:1090–1094, 1096–1097PubMedGoogle Scholar
  25. 25.
    Allawi HT, SantaLucia J Jr (1997) Thermodynamics and NMR of Internal G∙T Mismatches in DNA. Biochemistry 36:10581–10594CrossRefPubMedGoogle Scholar
  26. 26.
    Allawi HT, SantaLucia J Jr (1998) Nearest-neighbor thermodynamics of internal A∙C mismatches in DNA: sequence dependence and pH effects. Biochemistry 37:9435–9444CrossRefPubMedGoogle Scholar
  27. 27.
    Allawi HT, SantaLucia J Jr (1998) Thermodynamics of internal C·T mismatches in DNA. Nucleic Acids Res 26:2694–2701CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Allawi HT, SantaLucia J Jr (1998) Nearest neighbor thermodynamic parameters for internal G∙A mismatches in DNA. Biochemistry 37:2170–2179CrossRefPubMedGoogle Scholar
  29. 29.
    Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811CrossRefPubMedGoogle Scholar
  30. 30.
    Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, Li B, Cavet G, Linsley PS (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 21:635–637CrossRefPubMedGoogle Scholar
  31. 31.
    Baulcombe DC (2007) Molecular biology. Amplified silencing. Science 315:199–200CrossRefPubMedGoogle Scholar
  32. 32.
    Allen E, Howell MD (2010) MiRNAs in the biogenesis of trans-acting siRNAs in higher plants. Semin Cell Dev Biol 8:798–804CrossRefGoogle Scholar

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

Personalised recommendations