Multi-User Cyanobacterial Resource (MCR)

Oklahoma State University, Stillwater OK

Investigators

Abstract

The Synechocystis sp. PCC6803 genomic sequence will be used to develop DNA microarrays, thus enabling efficient techniques to monitor global patterns of differential transcription in this important model system. The project will produce and utilize a PCR-generated gene set representing the entire 3168 genes of the Synechocystis sp. PCC6803 genome, a unicellular, transformable cyanobacterium that is an important model organism for the study of photosynthesis and environmental gene regulation. The amplified gene set will be arrayed on microscope slides to be probed with fluorescently-labeled cDNA produced from mRNA for the purpose of analyzing global patterns of differential transcriptional activity. The PCR-generated gene set will have utility beyond the primary application of differential transcriptional profiling, because the PCR amplification oligonucleotides incorporate a pair of common adaptamer sequences connected to the gene-specific sequences. The ordered library of PCR-generated gene fragments will thus be flanked by adaptamer sequences. The presence of these adaptamers will have two important functions. First, it will allow re-amplification of 'master-sets' of the primary gene fragment sets from common PCR primers, thereby maximizing yields of printable product and minimizing the depletion of the original stock of gene-specific PCR primers. This will provide ample DNA for these arrays and allow experimentation, for example, with different coupling chemistries for the DNA hybridization microarrays. Second, this approach allows in-frame directional cloning of the amplified genes, thus permitting the future connection of these nucleic acid-based procedures with high-throughput protein analysis. The resulting availability of DNA microarrays for global transcriptional profiling will provide researchers with a powerful new tool to address fundamental problems in many different areas of biological research.