Improvements in BAC fingerprinting and end sequencing

BAC 指纹识别和末端测序的改进

• 批准号: 6733577
• 负责人: Marco A. Marra
• 金额: $ 140.48万
• 依托单位: BRITISH COLUMBIA CANCER AGENCY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-09 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6733577
• 关键词: artificial chromosomes; biomedical automation; biotechnology; capillary electrophoresis; computer program /software; computer system design /evaluation; gel electrophoresis; genetic mapping; genetic techniques; informatics; method development; molecular cloning; nucleic acid purification; nucleic acid sequence; restriction fragment length polymorphism; technology /technique development

DESCRIPTION (provided by applicant): The work described in the Research Plan will have two important consequences. First, it will result in more efficient, less expensive methods for the production of accurate BAC fingerprint maps and end sequences. Second, it will result in the generation of at least 2,800,000 fingerprinted clones, assembled into fingerprint maps for 14 large (e.g. mammalian) genomes. We will undertake our proposed software and "wet lab" technology development exercises in the context of a production fingerprint mapping effort, using methods we established to generate the maps over the three years of the proposal. Software development will include automated lane tracking of fingerprinting gels, improving our automated restriction fragment identification software (BandLeader), and modification of it so that it produces probability-based measures of restriction fragment quality. These "quality values", produced for each restriction fragment, will be exploited by new software we are developing to correctly and automatically order clones within contigs. Automatic selection of clone tiling sets will also be developed. Fingerprint maps will be made available for download on our website in FPC format and for viewing using our Internet Contig Explorer (ICE) software. We will provide lists of tiling set clones to the sequencing centers to support their sequencing goals. BAC-end sequencing technology development will emphasize experiments aimed at reduction in volume of expensive sequencing reagents, less expensive DNA purification and reduced use of expensive plasticware. We will place special emphasis on improving capillary array electrophoresis and low volume thermocycling methods as these may impact both costs of sequencing reagents and DNA purification. Finally, we will evaluate commercially available BAC DNA purification reagents for performance in both end sequencing and fingerprinting. If the performance and cost of the reagents are favorable, we will undertake experiments to automate one or more of the commercially available kits. Should this be successful, we will integrate the new protocols and processes into our production fingerprinting group.

描述（由申请人提供）：研究计划中描述的工作将产生两个重要后果。首先，它将带来更高效、更便宜的方法来生成准确的 BAC 指纹图谱和末端序列。其次，它将产生至少 2,800,000 个带指纹的克隆，并组装成 14 个大型（例如哺乳动物）基因组的指纹图谱。我们将在生产指纹图谱工作的背景下进行我们提议的软件和“湿实验室”技术开发练习，使用我们在提案的三年内建立的生成图谱的方法。软件开发将包括指纹凝胶的自动泳道跟踪、改进我们的自动限制性片段识别软件 (BandLeader) 以及对其进行修改，以便生成基于概率的限制性片段质量测量。这些为每个限制性片段产生的“质量值”将被我们正在开发的新软件利用，以正确、自动地对重叠群内的克隆进行排序。还将开发克隆平铺集的自动选择。指纹图谱将以 FPC 格式在我们的网站上下载，并使用我们的 Internet Contig Explorer (ICE) 软件进行查看。我们将向测序中心提供平铺集克隆列表，以支持他们的测序目标。 BAC 末端测序技术的开发将强调旨在减少昂贵测序试剂体积、降低 DNA 纯化成本并减少昂贵塑料器具使用的实验。我们将特别重视改进毛细管阵列电泳和低容量热循环方法，因为这些可能会影响测序试剂和 DNA 纯化的成本。最后，我们将评估市售 BAC DNA 纯化试剂在末端测序和指纹识别方面的性能。如果试剂的性能和成本有利，我们将进行实验以自动化一种或多种市售试剂盒。如果成功，我们将把新的协议和流程集成到我们的生产指纹识别组中。