Objective

Application of Prokaryotic cDNA Subtraction from the molecular scale to the field scale.

Molecular biological tools (MBT) have the potential to improve the design and operation of both in-situ and ex-situ biological treatment processes. However, the application of many MBTs is predicated on a priori knowledge of target nucleic acid sequences (i.e., biomarkers), and the current dearth of such biomarkers is a barrier to the routine application of MBTs at Department of Defense field sites. A critical need, therefore, exists for tools that can rapidly identify the functional target genes necessary for biomarker development. To address this need, this project will deploy Prokaryotic cDNA Subtraction to rapidly identify the key genes relevant to the biodegradation process of interest; in this case, the process of interest is perchlorate reduction in unsequenced environmental isolates. The development of a biomarker that accurately represents the diversity of functional gene sequences for perchlorate reduction is challenging. A wide range of taxonomically diverse perchlorate-reducing bacteria has been detected at contaminated sites, but their perchlorate-reduction genes remain largely unsequenced.

The primary objective of this project is to demonstrate that the combination of Prokaryotic cDNA Subtraction and reverse transcription real-time polymerase chain reaction (rtRT-PCR) can be used to rapidly identify functional gene biomarkers from unsequenced environmental microorganisms. More specifically, the project seeks to identify functional gene biomarkers for perchlorate reduction in two environmental isolates.