Multi-User Cyanobacterial Resource (MCR)

多用户蓝藻资源 (MCR)

• 批准号: 0084457
• 负责人: Robert Burnap
• 金额: $ 24.99万
• 依托单位: Oklahoma State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0084457&HistoricalAwards=false
• 关键词: Multi; User; Cyanobacterial; Resource; MCR

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.

Synechocystis sp。 PCC6803基因组序列将用于开发DNA微阵列，从而实现有效的技术来监视此重要模型系统中差分转录的全局模式。该项目将产生并利用代表Synechocystis sp的整个3168基因的PCR生成的基因集。 PCC6803基因组是一种单细胞，可转化的蓝细菌，是研究光合作用和环境基因调节的重要模型生物。放大的基因集将在显微镜载玻片上排列，以用mRNA产生的荧光标记的cDNA探测，以分析差异转录活性的全局模式。 PCR生成的基因集将具有差异转录分析的主要应用，因为PCR扩增寡核苷酸均包含了连接到基因特异性序列的一对公共适应性序列。因此，PCR生成的基因片段的有序文库将由适应器序列侧面。这些适应器的存在将具有两个重要的功能。首先，它将允许从普通PCR引物中重新扩大主要基因片段集的“主集”，从而最大化可打印产品的产量，并最大程度地减少基因特异性PCR引物的原始库存的消耗。这将为这些阵列提供足够的DNA，并允许实验，例如，与DNA杂交微阵列的化学偶联化学不同。其次，这种方法允许对放大基因的框架定向克隆，从而通过高通量蛋白分析允许这些基于核酸的程序的未来连接。 DNA微阵列用于全球转录分析的可用性将为研究人员提供有力的新工具，以解决许多不同生物学研究领域的基本问题。