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Multiplexed Identification and Quantification of Analyte DNAs in Environmental Samples Using Microspheres and Flow Cytometry

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Environmental Microbiology

Part of the book series: Methods in Biotechnology ((MIBT,volume 16))

Abstract

Complex mixtures of nucleic acids occur in numerous systems, including environmental samples (e.g., groundwater, sediment), skin, feces, and blood. Often, it is desirable to be able to identify and measure the amount of a particular analyte DNA in a mixture. When there are multiple analyte DNAs of interest, multiplexing techniques can speed up the analysis.

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References

  1. Spiro, A., Lowe, M., and Brown, D. (2000) A bead-based method for multiplexed identification and quantitation of DNA sequences using flow cytometry. Appl. Environ. Microbiol. 66, 4258–4265.

    Article  PubMed  CAS  Google Scholar 

  2. Spiro, A. and Lowe, M. (2002) Quantitation of DNA sequences in environmental PCR products by a multiplexed, bead-based method. Appl. Environ. Microbiol. 68, 1010–1013.

    Article  PubMed  CAS  Google Scholar 

  3. Fulton, R. J., McDade, R. L., Smith, P. L., Kienker, L. J., and Kettman, J. R. (1997) Advanced multiplexed analysis with the FlowMetrix system. Clin. Chem. 43, 1749–1756.

    PubMed  CAS  Google Scholar 

  4. Smith, P. L., WalkerPeach, C. R., Fulton, R. J., and DuBois, D. B. (1998) A rapid, sensitive, multiplexed assay for detection of viral nucleic acids using the FlowMetrix system. Clin. Chem. 44, 2054–2056.

    PubMed  CAS  Google Scholar 

  5. Dunbar, S. A. and Jacobson, J. W. (2000) Application of the Luminex LabMAP in rapid screening for mutations in the cystic fibrosis transmembrane conductance regulator gene. Clin. Chem. 46, 1498–1500.

    PubMed  CAS  Google Scholar 

  6. Iannone, M. A., Taylor, J. D., Chen, J., Li, M.-S., Rivers, P., Slentz-Kesler, K. A., et al. (2000) Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry. Cytometry 39, 131–140.

    Article  PubMed  CAS  Google Scholar 

  7. Taylor, J. D., Briley, D., Nguyen, Q., Long, K., Iannone, M. A., Li, M. S., et al. (2001) Flow cytometric platform for high-throughput single nucleotide polymorphism analysis. BioTechniques 30, 661–669.

    PubMed  CAS  Google Scholar 

  8. Ye, F., Li, M. S., Taylor, J. D., Nguyen, Q., Colton, H. M., Casey, W. M., et al. (2001) Fluorescent microsphere-based readout technology for multiplexed human single nucleotide polymorphism analysis and bacterial identification. Hum. Mutat. 17, 305–316.

    Article  PubMed  CAS  Google Scholar 

  9. Armstrong, B., Stewart, M., and Mazumder, A. (2000) Suspension arrays for high throughput, multiplexed single nucleotide polymorphism genotyping. Cytometry 40, 102–108.

    Article  PubMed  CAS  Google Scholar 

  10. Colinas, R. J., Bellisario, R., and Pass, K. A. (2000) Multiplexed genotyping of beta-globin variants from PCR-amplified newborn blood spot DNA by hybridization with allele-specific oligodeoxynucleotides coupled to an array of fluorescent microspheres. Clin. Chem. 46, 996–998.

    PubMed  CAS  Google Scholar 

  11. Cai, H., White, P. S., Torney, D., Deshpande, A., Wang, Z., Marrone, B., et al. (2000) Flow cytometry-based minisequencing: a new platform for high-throughput single-nucleotide polymorphism scoring. Genomics 66, 135–143.

    Article  PubMed  CAS  Google Scholar 

  12. Nolan, J. P. and Mandy, F. F. (2001) Suspension array technology: new tools for gene and protein analysis. Cellular and Molecular Biology 47, 1241–1256.

    PubMed  CAS  Google Scholar 

  13. Nolan, J. P. and Sklar, L. A. (2002) Suspension array technology: evolution of the flat-array paradigm. Trends in Biotechnology 20, 9–12.

    Article  PubMed  CAS  Google Scholar 

  14. Jacobsen, C. S. (1995) Microscale detection of specific bacterial DNA in soil with a magnetic capture-hybridization and PCR amplification assay. Appl. Environ. Microbiol. 61, 3347–3352.

    PubMed  CAS  Google Scholar 

  15. Yang, L., Tran, D. K., and Wang, X. (2001) BADGE, Beads Array for the Detection of Gene Expression, a high-throughput diagnostic bioassay. Genome Research 11, 1888–1898.

    PubMed  CAS  Google Scholar 

  16. Chandler, D. P., Stults, J. R., Anderson, K. K., Cebula, S., Schuck, B. L., and Brockman, F. J. (2000) Affinity capture and recovery of DNA at femtomolar concentrations with peptide nucleic acid probes. Analytical Biochem. 283, 241–249.

    Article  CAS  Google Scholar 

  17. Bruckner-Lea, C. J., Stottlemyre, M. S., Holman, D. A., Grate, J. W., Brockman, F. J., and Chandler, D. P. (2000) Rotating rod renewable microcolumns for automated, solid-phase DNA hybridization studies. Anal. Chem. 72, 4135–4141.

    Article  PubMed  CAS  Google Scholar 

  18. Dojka, M. A., Hugenholtz, P., Haack, S. K., and Pace, N. R. (1998) Microbial diversity in a hydrocarbon-and chlorinated-solvent-contaminated aquifer undergoing intrinsic bioremediation. Appl. Environ. Microbiol. 64, 3869–3877.

    PubMed  CAS  Google Scholar 

  19. Ravenschlag, K., Sahm, K., Knoblauch, C., Jørgensen, B. B., and Amann, R. (2000) Community structure, cellular rRNA content, and activity of sulfate-reducing bacteria in marine arctic sediments. Appl. Environ. Microbiol. 66, 3592–3602.

    Article  PubMed  CAS  Google Scholar 

  20. Lin, C. and Stahl, D. A. (1995) Taxon-specific probes for the cellulolytic genus Fibrobacter reveal abundant and novel equine-associated populations. Appl. Envir. Microbiol. 61, 1348–1351.

    CAS  Google Scholar 

  21. Fuchs, B. M., Glockner, F. O., Wulf, J., and Amann, R. (2000) Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes. Appl. Environ. Microbiol. 66, 3603–3607.

    Article  PubMed  CAS  Google Scholar 

  22. Nikiforov, T. T., Rendle, R. B., Kotewicz, M. L., and Rogers, Y.-H. (1994) The use of phosphorothioate primers and exonuclease hydrolysis for the preparation of single-stranded PCR products and their detection by solid-phase hybridization. PCR Methods Appl. 3, 285–291.

    PubMed  CAS  Google Scholar 

  23. Ramette, R. W. (1981) Chemical Equilibrium and Analysis. Addison-Wesley, Reading, MA. Also, a useful website is http://oncampus.richmond.edu/~rdominey/301/SAM_General.html.

    Google Scholar 

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© 2004 Humana Press Inc.

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Lowe, M., Spiro, A., Summers, A.O., Wireman, J. (2004). Multiplexed Identification and Quantification of Analyte DNAs in Environmental Samples Using Microspheres and Flow Cytometry. In: Walker, J.M., Spencer, J.F.T., Ragout de Spencer, A.L. (eds) Environmental Microbiology. Methods in Biotechnology, vol 16. Humana Press. https://doi.org/10.1385/1-59259-765-3:051

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  • DOI: https://doi.org/10.1385/1-59259-765-3:051

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-116-5

  • Online ISBN: 978-1-59259-765-9

  • eBook Packages: Springer Protocols

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