This Article Full Text Full Text (PDF) Supplemental material Other Versions of this Article: AEM.01647-07v1 74/1/225    most recent 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 Google Scholar Google Scholar Articles by De Long, S. K. Articles by Kirisits, M. J. Search for Related Content PubMed PubMed Citation Articles by De Long, S. K. Articles by Kirisits, M. J. Agricola Articles by De Long, S. K. Articles by Kirisits, M. J.

Prokaryotic Suppression Subtractive Hybridization PCR cDNA Subtraction, a Targeted Method To Identify Differentially Expressed Genes ^,

The University of Texas at Austin, Department of Civil, Architectural, and Environmental Engineering, 1 University Station C1786, Austin, Texas 78712

Received 18 July 2007/ Accepted 29 October 2007

Molecular biology tools can be used to monitor and optimize biological treatment systems, but the application of nucleic acid-based tools has been hindered by the lack of available sequences for environmentally relevant biodegradation genes. The objective of our work was to extend an existing molecular method for eukaryotes to prokaryotes, allowing us to rapidly identify differentially expressed genes for subsequent sequencing. Suppression subtractive hybridization (SSH) PCR cDNA subtraction is a technique that can be used to identify genes that are expressed under specific conditions (e.g., growth on a given pollutant). While excellent methods for eukaryotic SSH PCR cDNA subtraction are available, to our knowledge, no methods previously existed for prokaryotes. This work describes our methodology for prokaryotic SSH PCR cDNA subtraction, which we validated using a model system: Pseudomonas putida mt-2 degrading toluene. cDNA from P. putida mt-2 grown on toluene (model pollutant) or acetate (control substrate) was subjected to our prokaryotic SSH PCR cDNA subtraction protocol to generate subtraction clone libraries. Over 90% of the sequenced clones contained gene fragments encoding toluene-related enzymes, and 20 distinct toluene-related genes from three key operons were sequenced. Based on these results, prokaryotic SSH PCR cDNA subtraction shows promise as a targeted method for gene identification.

Published ahead of print on 9 November 2007.

Supplemental material for this article may be found at http://aem.asm.org/.