Natural microbial communities are complex assemblages of organisms composed of a variety of different physiological groups of bacteria, archaea, and microeucaryotes, including fungi, algae, and protozoa. Recent molecular biological studies have shown that the assemblage of organisms present in an environmental sample can contain thousands of distinct biotypes of bacteria and that many of these organisms represent undescribed microbial lineages, some of which lack even a single representative species available in pure culture. This complexity is further compounded by the high spatial and temporal variability of species represented in many natural communities. The characterization of natural microbial communities is clearly a difficult problem.

The goal of this project is to develop a tool for characterizing the vast diversity of microbes in environmental samples using a hierarchical phylogenetic framework. The phylochip provides a format for parallel hybridization reactions using a high density microarray of hundreds of oligonucleotides on a small surface area. Oligonucleotide are immobilized within individual polyacrylamide gel elements (100x100x20 µm) affixed to a glass slide.

Target nucleic acids are fragmented and fluorescently labeled before hybridization with immobilized probes. Our current chip design uses oligonucleotide probes of 15 to 25 nucleotides in length to identify microorganisms at different taxonomic ranks using 16S rRNA sequence as the target. After hybridization, the microchips are analyzed with a wide-field-high aperture fluorescence microscope equipped with a cooled CCD camera.

Specific objectives of this project are (1) to establish the rules governing non-specific and specific probe-target duplex hybridizations, (2) to determine the relationship between signal intensity and specificity of probe-target duplexes, (3) to develop directed strategies aimed at improving probe specificity, (4) to determine the effects of single-bp mismatches on dissociation temperatures and signal intensities of probe-target duplexes, and (5) to determine what factors (and/or conditions) contributing to signal intensity in order to optimize hybridization and washing protocols.

A fully developed DNA microchip might be able to characterize virtually any environment, using hybridization patterns to define microbial community structure and to monitor gene expression.

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