High Fidelity Genomic Cloning And Amplification

高保真基因组克隆和扩增

• 批准号: 6736574
• 负责人: Ronald Godiska
• 金额: $ 9.95万
• 依托单位: LUCIGEN CORPORATION
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2004-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6736574
• 关键词: biotechnology; comparative genomic hybridization; functional /structural genomics; genetic library; genome; high throughput technology; molecular biology information system; nucleic acid amplification techniques; nucleic acid sequence; technology /technique development; transfection /expression vector

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.

描述（由申请人提供）：shot弹枪克隆文库是对人类基因组和其他对我们福祉重要的基因组分子分析的重要资源。完整的基因组测序和比较基因组学揭示了有关正常组织和患病组织中代谢途径的新信息，例如癌症，糖尿病，衰老，艾滋病等。基因组测序还提供了有关水平基因转移，蛋白质家族进化以及物种的遗传库和进化的关键信息。基因组学的潜在好处增加了对额外图书馆的需求，并具有更高的保真度和更完整的覆盖范围。 但是，当前的图书馆构建方法极具挑战性，耗时且昂贵。对于较大的基因组，它们还留下了许多无关的差距。这些障碍掩盖了重要的序列信息，并限制了研究的基因组范围。 该提案的目标是提供新的向量和简化的方法，以创建超过忠诚度，复杂性，缺乏克隆偏见和易于分析的当前标准的库。这些技术将为人类基因组中现有的克隆间隙提供访问，并允许克隆其他顽固基因组。具体目的包括开发新型的线性克隆载体以及其稳定维持原本“不吻合”序列的能力，例如倒置重复序列，微卫星重复序列以及来自富裕病原体的基因组DNA（> 8 kb）的大克隆，例如肺炎脑性carinii。目的还包括开发从单个细菌菌落进行快速模板制备的方法，用于台式和高通量测序。长期目标包括克隆人类基因组序列中的现有差距，从富裕和其他困难基因组创建完整的库，以及对DNA的100-kb区域的体外扩增。这项研究提出的进步将允许以更高的精度和较低的成本进行克隆和测序。这些文库的改进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。

项目成果

ExCyto PCR amplification.

ExCyto PCR 扩增。

• DOI: 10.1371/journal.pone.0012629
• 发表时间: 2010
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Dhodda,Vinay;Godiska,Ronald;Vanwye,JeffreyD;Mead,David;Hochstein,Rebecca;Sheets,Lynne;VandeZande,Sarah;Niebauer,Chris;Crawford,DouglasL;Oleksiak,MarjorieF
• 通讯作者: Oleksiak,MarjorieF

Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli.

• DOI: 10.1093/nar/gkp1181
• 发表时间: 2010-04
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Godiska R;Mead D;Dhodda V;Wu C;Hochstein R;Karsi A;Usdin K;Entezam A;Ravin N
• 通讯作者: Ravin N