Nanoliter High-Throughput PCR for DNA and RNA Profiling

  • Colin J. H. Brenan
  • Douglas Roberts
  • James Hurley
Part of the METHODS IN MOLECULAR BIOLOGY™ book series (MIMB, volume 496)


The increasing emphasis in life science research on utilization of genetic and genomic information underlies the need for high-throughput technologies capable of analyzing the expression of multiple genes or the presence of informative single nucleotide polymorphisms (SNPs) in large-scale, population-based applications. Human disease research, disease diagnosis, personalized therapeutics, environmental monitoring, blood testing, and identification of genetic traits impacting agricultural practices, both in terms of food quality and production efficiency, are a few areas where such systems are in demand. This has stimulated the need for PCR technologies that preserves the intrinsic analytical benefits of PCR yet enables higher throughputs without increasing the time to answer, labor and reagent expenses and workflow complexity. An example of such a system based on a high-density array of nanoliter PCR assays is described here. Functionally equivalent to a microtiter plate, the nanoplate system makes possible up to 3,072 simultaneous end-point or real-time PCR measurements in a device, the size of a standard microscope slide. Methods for SNP genotyping with end-point TaqMan PCR assays and quantitative measurement of gene expression with SYBR Green I real-time PCR are outlined and illustrative data showing system performance is provided.

Key Words

5′-exonuclease assay TaqMan PCR SNP genotyping nanofluidic high-throughput genotyping SYBR Green I real-time PCR nanoliter PCR quantitative PCR 


  1. 1.
    Murray, S., Oliphant, A., Shen, R., McBride, C., Steeke, R., Shannon, S., Rubano, T., Kermani, B., Fan, J-B., Chee, M., Hansen, M. (2004) A highly informative SNP linkage panel for human genetic studies. Nature Meth 1, 113–117.CrossRefGoogle Scholar
  2. 2.
    Saito, K., Nakayama T., Sato N., Morita A., Takahashi T., Soma M., Usami, R. (2006) Haplotypes of the plasminogen activator gene associated with ischemic stroke. Thromb Haemost 96, 331–336.PubMedGoogle Scholar
  3. 3.
    Shi, M. (2001) Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies. Clin Chem 47, 164–172.PubMedGoogle Scholar
  4. 4.
    Beuselinck K., van Ranst M., van Eldere, J. (2005) Automated extraction of viral-pathogen RNA and DNA for high-throughput quantitative real-time PCR. J Clin Microbiol 43, 5541–5546.Google Scholar
  5. 5.
    Balashov S. V., Gardiner R., Park S., Perlin D. S. (2005) Rapid, high-throughput, multiplex, real-time PCR for identification of mutations in the cyp51A gene of Aspergillus fumigatus that confer resistance to itraconazaole. J Clin Microbiol 43, 214–222.CrossRefPubMedGoogle Scholar
  6. 6.
    Stephens, A. J., Huygens F., Inman-Bamber J., Price E. P., Nimmo G. R., Schooneveldt J., Munckhof W., Giffard P. M. (2006) Methicillin-resistant Staphylococcus aureus gentoytping using a small set of polymorphisms. J Med Microbiol 55, 43–51.CrossRefPubMedGoogle Scholar
  7. 7.
    Denomme G. A., Van Oene, M. (2005) High-throughput multiplex single-nucleotide polymorphism analysis for red cell and platelet antigen genotypes. Transfusion 45, 660–666.Google Scholar
  8. 8.
    Higgins, M., Hughes, A., Buzzacott, N., Lown J. (2004) High-throughput genotyping of human platelet antigens using the 5’-nuclease assay and minor groove binder probe technology. Vox Sang 87, 114–117.CrossRefPubMedGoogle Scholar
  9. 9.
    Smith, C. T., Baker, J., Park, L., Seeb, W., Elfstrom, C., Abe, S., Seeb, J. E. (2005) Characterization of 13 single nucleotide polymorphism markers for chum salmon. Molecular Ecology Notes 5, 259–262.CrossRefGoogle Scholar
  10. 10.
    Smith, C. T., Templin, W. D., Seeb, J. E., Seeb, L. W. (2005) Single nucleotide polymorphisms provide rapid and accurate estimates of the proportions of U.S. and Canadian chinook salmon caught in Yukon river fisheries. North Am J Fisheries Management 25, 944–953.CrossRefGoogle Scholar
  11. 11.
    Giancola, S., McKhann, H., Berard, A., Camilleri, C., Durand, S., Libeau, P., Roux, F., Reboud, X., Gut, I., Brunel, D. (2006) Utilization of the three high-throughput SNP genotyping methods, the GOOD assay, Amplifluor and TaqMan, in diploid and polyploidy plants. Theoretical Appl Genetics 112, 1115–1124.CrossRefGoogle Scholar
  12. 12.
    Liu, R. H. et al. (2004) Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection. Anal Chem 76, 1824–1831.CrossRefPubMedGoogle Scholar
  13. 13.
    Lee, D. -S. et al. (2004) Bulk-micromachined submicroliter-volume PCR chip with very rapid thermal response and low power consumption. Lab Chip 4, 401–407.CrossRefPubMedGoogle Scholar
  14. 14.
    Zhang, C., Xing, D. (2007) Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends Nucleic Acids Research 35, 4223–4237.Google Scholar
  15. 15.
    Dahl, A. et al. (2007) Quantitative PCR based expression analysis on a nanoliter scale using polymer nano-well chips Biomed Microdevices 9, 307–314.CrossRefPubMedGoogle Scholar
  16. 16.
    Kanigan T. K., Brenan C. J. H., Lafontaine S., et al. (2000) Living chips for drug discovery. SPIE Proceedings 3929, 172–180.Google Scholar
  17. 17.
    Brenan C. J, H., Morrison T., Stone K., et al. (2002) A massively parallel microfluidics platform for storage and ultra high throughput screening. SPIE Proceedings 4626, 560–569.Google Scholar
  18. 18.
    Morrison T., Hurley J., Garcia J., et al. (2006) Nanoliter high throughput quantitative PCR. Nucleic Acids Res 34, e123.CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Colin J. H. Brenan
    • 1
  • Douglas Roberts
    • 1
  • James Hurley
    • 1
  1. 1.BioTrove Inc.WoburnUSA

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