Renewable Microcolumns for Automated DNA Purification and Flow-through Amplification: From Sediment Samples through Polymerase Chain Reaction
- BATTELLE (PACIFIC NW LAB)
- Pacific Northwest National Laboratory
- LOS ALAMOS TECH ASSOC
- OFFICE OF FELLOWSHIP PROGRAMS
There is an increasing need for field-portable systems for the detection and characterization of microorganisms in the environment. Nucleic acids analysis is frequently the method of choice for discriminating between bacteria in complex systems, but standard protocols are difficult to automate and current microfluidic devices are not configured specifically for environmental sample analysis. In this report, we describe the development of an integrated DNA purification and PCR amplification system and demonstrate its use for the automated purification and amplification of Geobacter chapelli DNA (genomic DNA or plasmid targets) from sediments. The system includes renewable separation columns for the automated capture and release of microparticle purification matrices, and can be easily reprogrammed for new separation chemistries and sample types. The DNA extraction efficiency for the automated system ranged from 3 to 25 percent, depending on the length and concentration of the DNA target . The system was more efficient than batch capture methods for the recovery of dilute genomic DNA even though the reagen volumes were smaller than required for the batch procedure. The automated DNA concentration and purification module was coupled to a flow-through, Peltier-controlled DNA amplification chamber, and used to successfully purify and amplify genomic and plasmid DNA from sediment extracts. Cleaning protocols were also developed to allow reuse of the integrated sample preparation system, including the flow-through PCR tube.
- Research Organization:
- Pacific Northwest National Lab., Richland, WA (US)
- Sponsoring Organization:
- US Department of Energy (US)
- DOE Contract Number:
- AC06-76RL01830
- OSTI ID:
- 15001079
- Report Number(s):
- PNNL-SA-34985; KJ0200000; TRN: US200401%%454
- Journal Information:
- Analytica Chimica Acta, Vol. 469, Issue 1; Other Information: PBD: 1 Dec 2001
- Country of Publication:
- United States
- Language:
- English
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