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Separation of 16S rRNA PCR Products From a Model Community Using Temporal Temperature Gradient Gel Electrophoresis, Rev A

Menu B. Leddy, Biotechnology Research Department, Orange County Water District, 10500 Ellis Ave., Fountain Valley, CA 92708


Introduction
Identification and characterization of bacterial species from membrane biofilms has been a microbiological challenge. A vast majority (>90%) of bacterial cells from membrane biofilms cannot be cultivated by standard microbiological techniques which have traditionally used culture-dependent methods. In addition, culture-based identification methods may be affected by the physiological state of the cells while growing on a specific medium. As a consequence of these limitations, microbial populations from membrane biofilms have not currently been well characterized.

Several molecular approaches now provide powerful culture-independent techniques that can be used for identification and characterization of a multi-species consortia (i.e. membrane biofilm community structure). One strategy is based on a combination of polymerase chain reaction (PCR*), restriction fragment length analysis and/or DNA sequencing to characterize multi-species consortia from a membrane biofilm.1 A limitation of this approach is that DNA sequencing is expensive and laborious when dealing with a multi-species consortia. Additionally, restriction fragment length analysis requires fragment pattern matches against a large database (Ribosomal Database Project, RDP),2 which contains more than 6,000 organisms, to identify a list of potential organisms in the mixture. This can result in spurious pattern matches because a combination of patterns from two or more bacteria may resemble unrelated restriction fragment patterns found in the RDP. In order to c
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