A Novel Method to Specifically Cleave Chromosome-size Plant DNA

一种特异性切割染色体大小植物 DNA 的新方法

• 批准号: 9307162
• 负责人: Ray Wu
• 金额: --
• 依托单位: Cornell Univ - State: AWDS MADE PRIOR MAY 2010
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9307162&HistoricalAwards=false
• 关键词: Novel; Method; Specifically; Cleave; Chromosome

9307162 Wu The goal of this proposed research is to develop two new methods for cleaving chromosome-size DNA specifically into large fragments in order to construct a contiguous physical map of each chromosome. These fragments will be used as starting materials in producing a low-density physical map of each chromosome without gaps. The major advantage of this method is that it will allow for the cleavage and recovery of much larger DNA molecules than can be achieved by the three previously published methods. Four novel cleavage-specific plasmids have been designed and two have been constructed for this research. These plasmids will be used to transform Oryza sativa (rice), followed by regeneration of the resulting transgenic plants. Quantitative slot-blot hybridization and genomic blot analysis will be carried out to determine the copy number of the integrated copy of the cleavage-specific plasmid in each transgenic plant. A collection of over 100 transgenic plants will be obtained and characterized, each with several copies of the cleavage-specific plasmids integrated in a different chromosome. Next, the chromosomal DNA from different transgenic plants will be specifically cleaved using a novel enzyme-based strategy, followed by fractionation of the DNA using pulsed field gel electrophoresis and hybridization analysis. The final result of this proposed research is to produce a contiguous, low-density physical map, with between 2 to 7 megabases (mb) in between two markers, of at least 8 out of the 12 chromosomes in rice. ***

9307162 Wu 这项研究的目标是开发两种新方法，将染色体大小的 DNA 特异性切割成大片段，从而构建每条染色体的连续物理图谱。这些片段将用作起始材料，生成每条染色体的无间隙低密度物理图谱。该方法的主要优点是，与之前发布的三种方法相比，它可以切割和回收更大的 DNA 分子。本研究设计了四种新型切割特异性质粒，并构建了两种。这些质粒将用于转化稻（Oryza sativa），然后再生所得转基因植物。将进行定量槽印迹杂交和基因组印迹分析以确定每个转基因植物中切割特异性质粒的整合拷贝的拷贝数。将获得并鉴定 100 多种转基因植物的集合，每种植物都有多个拷贝的切割特异性质粒整合在不同的染色体中。接下来，来自不同转基因植物的染色体 DNA 将使用新型酶策略进行特异性切割，然后使用脉冲场凝胶电泳和杂交分析对 DNA 进行分级分离。这项拟议研究的最终结果是生成一个连续的、低密度的物理图谱，其中两个标记之间有 2 到 7 兆碱基 (mb) 之间的距离，涉及水稻 12 条染色体中的至少 8 条。 ***