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Proceedings Paper

Strategies for automated sample preparation, nucleic acid purification, and concentration of low-target-number nucleic acids in environmental and food processing samples
Author(s): Cynthia J. Bruckner-Lea; David A. Holman; Beatrice L. Schuck; Fred J. Brockman; Darrell P. Chandler
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Paper Abstract

The purpose of this work is to develop a rapid, automated system for nucleic acid purification and concentration from environmental and food processing samples. Our current approach involves off-line filtration and cell lysis (ballistic disintegration) functions in appropriate buffers followed by automated nucleic acid capture and purification on renewable affinity matrix microcolumns. Physical cell lysis and renewable affinity microcolumns eliminate the need for toxic organic solvents, enzyme digestions or other time- consuming sample manipulations. Within the renewable affinity microcolumn, we have examined nucleic acid capture and purification efficiency with various microbead matrices (glass, polymer, paramagnetic), surface derivitization (sequence-specific capture oligonucleotides or peptide nucleic acids), and DNA target size and concentration under variable solution conditions and temperatures. Results will be presented comparing automated system performance relative to benchtop procedures for both clean (pure DNA from a laboratory culture) and environmental (soil extract) samples, including results which demonstrate 8 minute purification and elution of low-copy nucleic acid targets from a crude soil extract in a form suitable for PCR or microarray-based detectors. Future research will involve the development of improved affinity reagents and complete system integration, including upstream cell concentration and cell lysis functions and downstream, gene-based detectors. Results of this research will ultimately lead to improved processes and instrumentation for on-line, automated monitors for pathogenic micro-organisms in food, water, air, and soil samples.

Paper Details

Date Published: 12 January 1999
PDF: 9 pages
Proc. SPIE 3544, Pathogen Detection and Remediation for Safe Eating, (12 January 1999); doi: 10.1117/12.335782
Show Author Affiliations
Cynthia J. Bruckner-Lea, Pacific Northwest National Lab. (United States)
David A. Holman, Pacific Northwest National Lab. (United States)
Beatrice L. Schuck, Pacific Northwest National Lab. (United States)
Fred J. Brockman, Pacific Northwest National Lab. (United States)
Darrell P. Chandler, Pacific Northwest National Lab. (United States)

Published in SPIE Proceedings Vol. 3544:
Pathogen Detection and Remediation for Safe Eating
Yud-Ren Chen, Editor(s)

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