Strategies for automated sample preparation, nucleic acid purification, and concentration of low-target-number nucleic acids in environmental and food processing samples

Cynthia J. Bruckner-Lea; David A. Holman; Beatrice L. Schuck; Fred J. Brockman; Darrell P. Chandler

doi:10.1117/12.335782

12 January 1999 Strategies for automated sample preparation, nucleic acid purification, and concentration of low-target-number nucleic acids in environmental and food processing samples

Cynthia J. Bruckner-Lea, David A. Holman, Beatrice L. Schuck, Fred J. Brockman, Darrell P. Chandler

Author Affiliations +

Proceedings Volume 3544, Pathogen Detection and Remediation for Safe Eating; (1999) https://doi.org/10.1117/12.335782
Event: Photonics East (ISAM, VVDC, IEMB), 1998, Boston, MA, United States

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.

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Cynthia J. Bruckner-Lea, David A. Holman, Beatrice L. Schuck, Fred J. Brockman, and Darrell P. Chandler "Strategies for automated sample preparation, nucleic acid purification, and concentration of low-target-number nucleic acids in environmental and food processing samples", Proc. SPIE 3544, Pathogen Detection and Remediation for Safe Eating, (12 January 1999); https://doi.org/10.1117/12.335782

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