This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chandler, D. P. Articles by Jarrell, A. E. Search for Related Content PubMed PubMed Citation Articles by Chandler, D. P. Articles by Jarrell, A. E. Agricola Articles by Chandler, D. P. Articles by Jarrell, A. E.

Automated Purification and Suspension Array Detection of 16S rRNA from Soil and Sediment Extracts by Using Tunable Surface Microparticles

Biochip Technology Center, Argonne National Laboratory, Argonne, Illinois 60439,¹ Environmental Microbiology Group, Pacific Northwest National Laboratory, Richland, Washington 99352²

Received 15 October 2003/ Accepted 22 January 2004

Autonomous, field-deployable molecular detection systems require seamless integration of complex biochemical solutions and physical or mechanical processing steps. In an attempt to simplify the fluidic requirements for integrated biodetection systems, we used tunable surface microparticles both as an rRNA affinity purification resin in a renewable microcolumn sample preparation system and as the sensor surface in a flow cytometer detector. The tunable surface detection limits in both low- and high-salt buffers were 1 ng of total RNA (10⁴ cell equivalents) in 15-min test tube hybridizations and 10 ng of total RNA (10⁵ cell equivalents) in hybridizations with the automated system (30-s contact time). RNA fragmentation was essential for achieving tunable surface suspension array specificity. Chaperone probes reduced but did not completely eliminate cross-hybridization, even with probes sharing <50% identity to target sequences. Nonpurified environmental extracts did not irreparably affect our ability to classify color-coded microparticles, but residual environmental constituents significantly quenched the Alexa-532 reporter fluor. Modulating surface charge did not influence the interaction of soluble environmental contaminants with conjugated beads. The automated system greatly reduced the effects of fluorescence quenching, especially in the soil background. The automated system was as efficacious as manual methods for simultaneous sample purification, hybridization, and washing prior to flow cytometry detection. The implications of unexpected target cross-hybridization and fluorescence quenching are discussed relative to the design and implementation of an integrated microbial monitoring system.

This article has been cited by other articles:

• Chandler, D. P., Jarrell, A. E., Roden, E. R., Golova, J., Chernov, B., Schipma, M. J., Peacock, A. D., Long, P. E. (2006). Suspension Array Analysis of 16S rRNA from Fe- and SO42-Reducing Bacteria in Uranium-Contaminated Sediments Undergoing Bioremediation.. Appl. Environ. Microbiol. 72: 4672-4687 [Abstract] [Full Text]  
• Wick, L. M., Rouillard, J. M., Whittam, T. S., Gulari, E., Tiedje, J. M., Hashsham, S. A. (2006). On-chip non-equilibrium dissociation curves and dissociation rate constants as methods to assess specificity of oligonucleotide probes. Nucleic Acids Res 34: e26-e26 [Abstract] [Full Text]  
• Lim, D. V., Simpson, J. M., Kearns, E. A., Kramer, M. F. (2005). Current and Developing Technologies for Monitoring Agents of Bioterrorism and Biowarfare. Clin. Microbiol. Rev. 18: 583-607 [Abstract] [Full Text]