High Fidelity Genomic Cloning and Amplification

高保真基因组克隆和扩增

• 批准号: 7110694
• 负责人: Ronald Godiska
• 金额: $ 41.7万
• 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 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7110694
• 关键词: DNA; DNA directed DNA polymerase; genes; genome; human; library

DESCRIPTION (provided by applicant): Unbiased and accurate cloning is essential for analysis of the human genome and other genomes important to our well-being. Complete genomic sequencing and comparative genomics reveal new information about metabolic pathways in normal tissue as well as diseased states, 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. However, current methods of constructing libraries and cloning individual genes are often extremely challenging, time consuming, and costly. Importantly, they also leave many uncloned gaps. These obstacles obscure important information about specific genes and genomic regions (e.g., centromeres) and limit the scope of genomes studied. The objectives of this proposal are to provide new vectors and streamlined methods to create clone 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 genes and 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 new methods of PCR amplification and rapid production of sequencing template from single bacterial colonies. These methods will be optimized for bench-scale and high-throughput amplification and sequencing. The advances proposed by this research will increase the accuracy and lower the costs of cloning and sequencing single genes or entire genomic or cDNA libraries. These advances will allow researchers to readily analyze DNA regions that are currently difficult or impossible to study. Improved sequence information will accelerate the advances in biotechnology and increase the potential of individual and comparative genomic analyses.

描述（由申请人提供）：无偏倚和准确的克隆对于分析人类基因组和其他对我们的福祉至关重要的基因组至关重要。完整的基因组测序和比较基因组学揭示了正常组织以及疾病状态（如癌症、糖尿病、衰老、艾滋病等）代谢途径的新信息。基因组测序还提供了关于水平基因转移、蛋白质家族进化、遗传库和物种进化的关键信息。然而，目前构建文库和克隆单个基因的方法通常极具挑战性，耗时且昂贵。重要的是，它们也留下了许多未克隆的缺口。这些障碍模糊了关于特定基因和基因组区域（如着丝粒）的重要信息，并限制了基因组研究的范围。本提案的目标是提供新的载体和简化的方法来创建克隆库，超越当前的保真度、复杂性、缺乏克隆偏见和易于分析的标准。这些技术将提供获取人类基因组中现有克隆空白的途径，并允许克隆其他顽固性基因和基因组。具体目标包括开发一种新的线性克隆载体，并证明其稳定维持“不可克隆”序列的能力，如倒置重复序列、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大克隆（bbb8 kb）。目标还包括开发新的PCR扩增方法和从单个细菌菌落快速生产测序模板。这些方法将被优化用于实验规模和高通量扩增和测序。本研究的进展将提高单个基因或整个基因组或cDNA文库的克隆和测序的准确性和降低成本。这些进步将使研究人员能够轻松地分析目前难以或不可能研究的DNA区域。改进的序列信息将加速生物技术的进步，增加个体和比较基因组分析的潜力。