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SNP Genotyping by the 5′-Nuclease Reaction: Advances in High-Throughput Genotyping with Nonmodel Organisms

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Single Nucleotide Polymorphisms

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 578))

Abstract

Population genetics studies play an increasingly important role in the management and conservation of nonmodel organisms. Unlike studies with model organisms, a typical population genetics study of a nonmodel organism may be conducted by analyzing thousands or hundreds of thousands of individuals for several dozen single nucleotide polymorphisms (SNPs). The use of robust, robotically mediated TaqMan reactions provides substantial advantages in these types of studies. We describe the methods and laboratory setup for analyzing a sustained high throughput of SNP assays in routine university or natural resource agency laboratories with a handful of thermal cyclers. Agencies sustain rates of nearly 150,000 assays per week using uniplex reactions with the Applied Biosystems 7900HT Fast Real-Time PCR System (AB 7900HT). We further describe the medium-density array run on the BioMark from Fluidigm, which increases this rate to over 500,000 assays per week by multiplexing 96 samples for 96 SNPs.

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References

  1. Holland, P. M., Abramson, R. D., Watson, R. and Gelfand, D. H. (1991) Detection of specific polymerase chain reaction product by utilizing the 5′ 3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 88, 7276–7280.

    Article  PubMed  CAS  Google Scholar 

  2. Holland, P. M., Abramson, R. D., Watson, R., Will, S., Saiki, R. K., and Gelfand, D. H. (1992) Detection of specific polymerase chain reaction product by utilizing the 5′ 3′ exonuclease activity of Thermus aquaticus DNA polymerase. Clin. Chem. 38, 462–463.

    Google Scholar 

  3. Hauser, L. and Seeb, J. E. (2008) Advances in molecular technology and their impact on fisheries genetics. Fish Fish. 9, 473–486.

    Article  Google Scholar 

  4. Moen, T., Hayes, B., Nilsen, F., Delghandi, M., Fjalestad, K. T., Fevolden, S. E. et al. (2008) Identification and characterisation of novel SNP markers in Atlantic cod: Evidence for directional selection. BMC Genetics 9, 18.

    Article  PubMed  Google Scholar 

  5. Narum, S. R., Banks, M., Beacham, T. D., Bellinger, M. R., Campbell, M. R., Dekoning, J. et al. (2008) Differentiating salmon populations at broad and fine geographical scales with microsatellites and single nucleotide polymorphisms. Mol. Ecol. 17, 3464–3477.

    PubMed  CAS  Google Scholar 

  6. Schlotterer, C. (2004) The evolution of molecular markers – just a matter of fashion? Nat. Rev. Genet. 5, 63–69.

    Article  PubMed  Google Scholar 

  7. Seddon, J. M., Parker, H. G., Ostrander, E. A. and Ellegren, H. (2005) SNPs in ecological and conservation studies: a test in the Scandinavian wolf population. Mol. Ecol. 14, 503–511.

    Article  PubMed  CAS  Google Scholar 

  8. Livak, K. J. (2003) SNP genotyping by the 5′-nuclease reaction. In Kwok, P.-Y. (Ed.) Single Nucleotide Polymorphisms: Methods and Protocols. Methods in Molecular Biology, Totowa, Humana Press, pp. 129–147.

    Google Scholar 

  9. Heid, C. A., Stevens, J., Livak, K. J. and Williams, P. M. (1996) Real time quantitative PCR. Genome Res. 6, 986–994.

    Article  PubMed  CAS  Google Scholar 

  10. Ranade, K., Chang, M. S., Ting, C. T., Pei, D., Hsiao, C. F., Olivier, M. et al. (2001) High-throughput genotyping with single nucleotide polymorphisms. Genome Res. 11, 1262–1268.

    PubMed  CAS  Google Scholar 

  11. Smith, C. T., Elfstrom, C. M., Seeb, L. W. and Seeb, J. E. (2005) Use of sequence data from rainbow trout and Atlantic salmon for SNP detection in Pacific salmon. Mol. Ecol. 14, 4193–4203.

    Article  PubMed  CAS  Google Scholar 

  12. The International HapMap Consortium. (2007) A second generation human haplotype map of over 3.1 million SNPs. Nature 449, 851-U3.

    Google Scholar 

  13. Kwok, P.-Y. (2003) Single nucleotide polymorphisms; Methods and Protocols, Methods in Molecular Biology, vol. 212, Totowa, Humana Press.

    Google Scholar 

  14. Smith, C. T. and Seeb, L. W. (2008) Number of alleles as a predictor of the relative assignment accuracy of short tandem repeat (STR) and single-nucleotide-polymorphism (SNP) baselines for chum salmon. Trans. Am. Fish. Soc. 137, 751–762.

    Article  CAS  Google Scholar 

  15. Fox, C. J., Taylor, M. I., Pereyra, R., Villasana, M. I. and Rico, C. (2005) TaqMan DNA technology confirms likely overestimation of cod (Gadus morhua L.) egg abundance in the Irish Sea: implications for the assessment of the cod stock and mapping of spawning areas using egg-based methods. Mol. Ecol. 14, 879–884.

    Article  PubMed  CAS  Google Scholar 

  16. Sprowles, A. E., Stephens, M. R., Clipperton, N. W. and May, B. P. (2006) Fishing for SNPs: A targeted locus approach for single nucleotide polymorphism discovery in rainbow trout. Trans. Am. Fish. Soc. 135, 1698–1721.

    Article  CAS  Google Scholar 

  17. Weidmann, M., Armbruster, K. and Hufert, F. T. (2008) Challenges in designing a Taqman-based multiplex assay for the simultaneous detection of herpes simplex virus types 1 and 2 and Varicella-zoster virus. J. Clin. Virol. 42, 326–334.

    Article  PubMed  CAS  Google Scholar 

  18. Dahl, A., Sultan, M., Jung, A., Schwartz, R., Lange, M., Steinwand, M. et al. (2007) Quantitative PCR based expression analysis on a nanoliter scale using polymer nano-well chips. Biomed. Microdevices 9, 307–314.

    Article  PubMed  CAS  Google Scholar 

  19. Spurgeon, S. L., Jones, R. C. and Ramakrishnan, R. (2008) High throughput gene expression measurement with real time PCR in a microfluidic dynamic array. PLoS ONE 3, e1662.

    Article  PubMed  Google Scholar 

  20. Osman, F., Leutenegger, C., Golino, D. and Rowhani, A. (2008) Comparison of low-density arrays, RT-PCR and real-time TaqMan® RT-PCR in detection of grapevine viruses. J. Virol. Methods 149, 292–299.

    Article  PubMed  CAS  Google Scholar 

  21. Tadros, S. F., D'souza, M., Zhu, X. X. and Frisina, R. D. (2008) Apoptosis-related genes change their expression with age and hearing loss in the mouse cochlea. Apoptosis 13, 1303–1321.

    Article  PubMed  CAS  Google Scholar 

  22. Perkel, J. (2008) SNP genotyping: six technologies that keyed a revolution. Nat. Methods 5, 447–453.

    Article  CAS  Google Scholar 

  23. Qin, J., Jones, R. C. and Ramakrishnan, R. (2008) Studying copy number variations using a nanofluidic platform. Nucleic Acids Res. 36, e16.

    Article  Google Scholar 

  24. Tranah, G. J., Lescault, P. J., Hunter, D. J. and De Vivo, I. (2003) Multiple displacement amplification prior to single nucleotide polymorphism genotyping in epidemiologic studies. Biotechnol. Lett. 25, 1031–1036.

    Article  PubMed  CAS  Google Scholar 

  25. Walsh, P. S., Erlich, H. A. and Higuchi, R. (1992) Preferential PCR amplification of alleles: mechanisms and solutions. Genome Res. 1, 241–250.

    Article  CAS  Google Scholar 

  26. Dube, S., Qin, J. and Ramakrishnan, R. (2008) Mathematical analysis of copy number variation in a DNA sample using digital PCR on a nanofluidic device. PLoS ONE 3, e2876.

    Article  PubMed  Google Scholar 

  27. Morin, P. A. and McCarthy, M. (2007) Highly accurate SNP genotyping from historical and low-quality samples. Mol. Ecol. Notes 7, 937–946.

    Article  CAS  Google Scholar 

  28. Morin, P. A., Saiz, R. and Monjazeb, A. (1999) High-throughput single nucleotide polymorphism genotyping by fluorescent 5′ exonuclease assay. Biotechniques 27, 538–552.

    PubMed  CAS  Google Scholar 

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Acknowledgement

This work was supported by a grant from the Gordon and Betty Moore Foundation to J.E.S. and L.W.S.

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Authors and Affiliations

  1. School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA, USA

    James E. Seeb, Carita E. Pascal & Lisa W. Seeb

  2. Fluidigm Corporation, South San Francisco, CA, USA

    Ramesh Ramakrishnan

Authors
  1. James E. Seeb
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  2. Carita E. Pascal
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  3. Ramesh Ramakrishnan
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  4. Lisa W. Seeb
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Editor information

Editors and Affiliations

  1. Center for Gene Regulation in, Cleveland State University, Euclid Ave. 2121, Cleveland, 44115, U.S.A.

    Anton A. Komar

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC 2003

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Seeb, J.E., Pascal, C.E., Ramakrishnan, R., Seeb, L.W. (2009). SNP Genotyping by the 5′-Nuclease Reaction: Advances in High-Throughput Genotyping with Nonmodel Organisms. In: Komar, A. (eds) Single Nucleotide Polymorphisms. Methods in Molecular Biology™, vol 578. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-411-1_18

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  • DOI: https://doi.org/10.1007/978-1-60327-411-1_18

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-410-4

  • Online ISBN: 978-1-60327-411-1

  • eBook Packages: Springer Protocols

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