DNA AMPLIFICATION FROM MICRODISSECTED CHROMOSOMES

在这个项目中，我们将开发有用的工具包、服务和试剂 扩增DNA微型文库的构建和应用 (即扩增产物)来自显微切割的人类染色体 碎片。第一阶段研究的重点将是制定标准 用于生成这种迷你库的协议。两次放大 根据他们对识别站点的使用情况，提出了策略 以大约250个碱基的间隔分布在整个人类基因组中 (确保最低限度基因组序列的代表性覆盖- 库)。这些策略是基于MBO I的DNA序列识别 切割和寡核苷酸杂交到最常见的6个碱基对 人类基因组中的序列。从两种方法获得的DNA产物 策略是用单个接头寡核苷酸进行聚合酶链式反应扩增的。这个 将对扩增反应的产物进行数量分析 DNA的大小，基因组覆盖的程度，杂交的特异性， 再扩增后的可检索性、易用性和重复性。 根据第一阶段制定的放大战略，我们将 最终提供给人类基因组研究界 可用于：a)用于获得特定地区的标准化试剂 克隆(即用于重叠群组装/图谱闭合的基因组克隆，和/或cDNA 克隆以识别新基因)，b)作为分离的起始材料 以及c)作为杂交/扩增靶标 用于亚染色体定位。

In this project we will develop kits, services, and reagents useful in the processes of generating and employing amplified DNA mini-libraries (i.e., amplified products) from microdissected human chromosome fragments. The focus of Phase I studies will be development of standard protocols for generating such mini-libraries. Two amplification strategies are proposed based on their use of recognition sites distributed throughout the human genome at about 250 bp intervals (insuring representative coverage of genomic sequences in the min- library). The strategies are based on DNA sequence recognition by Mbo I cleavage and by oligonucleotide hybridization to the most common 6 bp sequences in the human genome. DNA products obtained from both strategies are PCR amplified with a single adaptor oligonucleotide. The products of the amplification reactions will be analyzed for the amount and size of DNA, degree of genome coverage, hybridization specificity, retrievability upon reamplification, ease of use and reproducibility. Based on amplification strategies developed in Phase I, we will ultimately make available to the human genom research community standardized reagents that can be used: a) for obtaining region-specific clones (i.e., genomic clones for contig assembly/map closure, and/or cDNA clones to identify novel genes), b) as starting material for isolation of region-specific STSs, and c) as hybridization/amplification targets for subchromosome localization.