SATURATING REGIONS WITH PCR DETECTABLE MARKERS

用 PCR 可检测标记饱和区域

• 批准号: 2444958
• 负责人: Don Straus
• 金额: $ 15.46万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 1997-11-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444958
• 关键词: genetic mapping; genetic markers; genetic techniques; genome; genotype; laboratory mouse; nucleic acid sequence; polymerase chain reaction; restriction fragment length polymorphism; subtraction hybridization; technology /technique development

DESCRIPTION (Adapted from the Investigator's Abstract): The investigator will develop new technology to saturate specifically targeted regions of the genome with a novel type of PCR detectable marker. The significance of the technology lies in its promise to accelerate the assembly of physical maps which in turn would expedite genome sequencing and gene identification. The ease of making a physical map depends on the local density of genetically mapped markers. Even for the mouse genome, with almost 6,000 markers mapped, laborious chromosome "walks" must often be undertaken. The goal of saturation mapping is to expedite physical mapping by isolating markers at intervals substantially smaller than the size of a Yeast Artificial Chromosome clone. Mapping tightly linked markers requires an efficient system for high throughput genotyping because a large number of strains must be screened to uncover infrequent recombination events. The investigator proposes to saturate regions with Restriction Site Polymorphism (RSP) markers, which have attributes that make them well suited for this type of analysis. RSPs are codominant PCR detectable makers, are detectable by hybridization, are amenable to detection by agarose gel electrophoresis or non-electrophoretic methods, are well suited for automated high throughput genotyping, and can be disseminated by distributing primer sequences. The technology the investigator will develop is based on Targeted RFLP Subtraction, a method that the investigator recently used to efficiently isolate region specific markers in a model organism. Specifically, the investigator will (1) modify RFLP Subtraction for isolating the versatile RSP markers and (2) optimize and demonstrate the use of Targeted RFLP Subtraction for saturating a region of the mouse genome with the PCR detectable RSPs. The Targeted RFLP Subtraction method will be useful in any organism in which controlled matings are possible. Saturating regions of the human genome with the aid of rodent-human hybrid cell lines or in the context of linkage disequilibrium studies may enhance identification of human disease genes.

描述（改编自研究者摘要）：研究者 将开发新技术，以饱和特定目标区域的 基因组与一种新型的PCR检测标记。 的意义 技术在于它有希望加速物理地图的组装 这反过来将加快基因组测序和基因鉴定。 的 绘制物理地图的难易程度取决于遗传物质的局部密度。 映射标记。 即使是老鼠的基因组，有近6,000个标记， 必须经常进行绘制的、费力的染色体“行走”。 的目标 饱和作图是通过分离标记来加速物理作图， 间隔大大小于酵母人工的大小 染色体克隆。 定位紧密连锁的标记需要高效的 高通量基因分型系统，因为大量菌株必须 进行筛选以揭示罕见的重组事件。 研究者 限制性位点多态性（RSP） 标记，这些标记具有使它们非常适合这种类型的 分析. RSP是共显性PCR可检测标记，可通过 杂交，可通过琼脂糖凝胶电泳检测，或 非电泳方法非常适合自动化高通量 基因分型，并且可以通过分配引物序列传播。 的 研究人员将开发的技术基于靶向RFLP 减法，研究人员最近使用的一种方法， 分离模式生物中区域特异性标记。 具体而言是 研究者将（1）修改RFLP减法，以分离多功能 RSP标记和（2）优化和演示靶向RFLP的使用 用PCR饱和小鼠基因组区域的减法 可检测的RSPs。 靶向RFLP减法方法将在任何 可以控制交配的有机体。 饱和区域 人类基因组的帮助下，啮齿动物-人类杂交细胞系或在 连锁不平衡研究的背景可能会提高识别 人类疾病基因