Prokaryotic suppression subtractive hybridization PCR cDNA subtraction, a targeted method to identify differentially expressed genes.
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.
De Long
Susan K
SK
The University of Texas at Austin, Department of Civil, Architectural, and Environmental Engineering, 1 University Station C1786, Austin, TX 78712, USA.
Kinney
Kerry A
KA
Kirisits
Mary Jo
MJ
D019342:Acetic Acid / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D005827:Genetics, Microbial / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism; D014050:Toluene / Q000378:metabolism
D014050:Toluene; D019342:Acetic Acid