Arbitrarily-Primed Polymerase Chain Reaction (AP-PCR) for the Identification of Degradative Bacteria

Effective in situ bioremediation relies on an assessment of the degradative potential of naturally occurring organisms at contaminated sites, and/or our ability to monitor the performance of added microorganisms. Arbitrarily-primed polymerase chain reaction (AP-PCR) involves the use of a short piece of single-stranded DNA, of arbitrary mucleic acid sequences, to facilitate the amplification of genomic DNA. The pattern of amplified DNA fragments produced provides a unique "finger print" that allows the identification of bacterial isolates. The procedure is rapid and specific, and it may be adapted to identify large numbers of isolates. The proposed project will employ AP-PCR to identify bacteria which may be used for bioremediation, and to identify bacteria from contaminated environments. Phase I will involve an initial assessment and optimization of the technique for identifying degradative bacteria. Variations in PCR primers and reaction conditions, as well as, methods for sample preparation, cell lysis, DNA isolation, and pattern recognition and comparison will be evaluated to insure reproducible results. The goal of the project is to develop a rapid, cost effective, commercially attractrive method for preliminarily characterization of potential bioremediation sites, and to rapidly identify added organisms to assess their survival of proliferation.