Improvements in BAC fingerprinting and end sequencing

BAC 指纹识别和末端测序的改进

• 批准号: 6594660
• 负责人: Marco A. Marra
• 金额: $ 174.69万
• 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/6594660
• 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; 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的后果。首先，它将导致更有效，更便宜的方法来生产准确的BAC指纹图和最终序列。其次，这将导致至少生成2,800,000个指纹克隆，并将14个大（例如哺乳动物）基因组的指纹图组装成指纹图。我们将在生产指纹映射工作的背景下，使用我们建立的方法来生成图中的三年中，我们将在生产指纹映射工作的背景下进行提出的软件和“湿实验室”技术开发练习。软件开发将包括对指纹凝胶的自动车道跟踪，改善我们的自动限制片段识别软件（BandLeader），以及对其进行修改，从而产生基于概率的限制片段质量的测量。这些针对每个限制片段产生的“质量值”将被我们开发的新软件利用，以正确，自动在重叠群中订购克隆。也将开发自动选择克隆瓷砖集。指纹图将以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设定的克隆列表，以支持其测序目标。 BAC-END测序技术开发将强调旨在减少昂贵的测序试剂量，较便宜的DNA纯化和减少昂贵塑料软件的使用的实验。我们将特别强调改善毛细管阵列电泳和低体积热环节方法，因为这些方法可能会影响测序试剂的成本和DNA纯化。最后，我们将评估在末端测序和指纹识别中性能的市售BAC DNA纯化试剂。如果试剂的性能和成本是有利的，我们将进行实验，以使一个或多个市售套件自动化。如果这是成功的，我们将将新的协议和流程整合到我们的生产指纹组中。