High Fidelity Genomic Cloning And Amplification

描述（由申请人提供）：鸟枪克隆文库是人类基因组和其他对我们的福祉很重要的基因组的分子分析的基本资源。全基因组测序和比较基因组学揭示了关于正常和患病组织中代谢途径的新信息，如癌症，糖尿病，衰老，艾滋病等。基因组测序还提供了关于水平基因转移、蛋白质家族进化以及物种遗传库和进化的关键信息。基因组学的潜在好处增加了对更多具有更高保真度和更完整覆盖范围的文库的需求。 然而，目前的图书馆建设方法极具挑战性，耗时且成本高昂。对于较大的基因组，它们也留下了许多未克隆的缺口。这些障碍模糊了重要的序列信息，限制了基因组研究的范围。 该提案的目标是提供新的载体和简化的方法，以创建超越当前保真度，复杂性，缺乏克隆偏见和易于分析的标准的文库。这些技术将为人类基因组中现有的克隆缺口提供途径，并允许克隆其他野生动物的基因组。具体的目标包括开发一种新的线性克隆载体，并证明其稳定维持其他“不可克隆”序列的能力，如反向重复序列、微卫星重复序列和来自富含AT的病原体（如卡氏肺孢子虫）的基因组DNA的大克隆（>8 kb）。目标还包括开发从单个细菌菌落快速制备模板的方法，用于实验室规模和高通量测序。长期目标包括克隆人类基因组序列中现有的缺口，从富含AT和其他困难的基因组中创建完整的文库，以及体外扩增100 kb的DNA区域。这项研究提出的进展将允许以更高的准确性和更低的成本克隆和测序额外的基因组和cDNA文库。来自这些文库的改进的序列信息将加速进步并增加个体和比较基因组分析的潜力。

DESCRIPTION (provided by applicant): Shotgun clone libraries are essential resources for molecular analysis of the human genome and of other genomes important to our well-being. Complete genomic sequencing and comparative genomics reveal new information about metabolic pathways in normal and diseased tissue, such as cancer, diabetes, aging, AIDS, and others. Genomic sequencing also provides critical information about horizontal gene transfer, evolution of protein families, and the genetic repertoire and evolution of species. The potential benefits of genomics have increased the demand for additional libraries, with higher fidelity and more complete coverage. However, current methods of library construction are extremely challenging, time consuming, and costly. For larger genomes, they also leave many uncloned gaps. These obstacles obscure important sequence information and limit the scope of genomes studied. The objectives of this proposal are to provide new vectors and streamlined methods to create libraries that surpass current standards of fidelity, complexity, lack of cloning bias, and ease of analysis. These techniques will provide access to existing clone gaps in the human genome and allow cloning of other recalcitrant genomes. Specific aims include development of a novel linear cloning vector and demonstration of its capacity for stable maintenance of otherwise "unclonable" sequences, such as inverted repeats, microsatellite repeats, and large clones of genomic DNA (>8 kb) from AT-rich pathogens, such as Pneumocystis carinii. The aims also include developing methods of rapid template preparation from single bacterial colonies for bench-scale and high-throughput sequencing. Long-term goals include cloning existing gaps in the human genomic sequence, creating complete libraries from AT-rich and other difficult genomes, and in vitro amplification of 100-kb regions of DNA. The advances proposed by this research will allow cloning and sequencing of additional genomic and cDNA libraries with greater accuracy and lower costs. Improved sequence information from these libraries will accelerate the advances and increase the potential of individual and comparative genomic analyses.