DNA AMPLIFICATION FROM MICRODISSECTED CHROMOSOMES

在这个项目中，我们将开发试剂盒，服务和试剂， 产生和使用扩增的DNA微型文库的过程 (i.e.,扩增产物）从显微切割的人类染色体 片段 第一阶段研究的重点将是制定标准 用于产生这样的微型库的方案。 两个扩增 基于识别位点的使用，提出了策略 在整个人类基因组中以约250 bp的间隔分布 （确保最小值中基因组序列的代表性覆盖率） 图书馆）。这些策略是基于Mbo I对DNA序列的识别 切割并通过寡核苷酸杂交至最常见的6 bp 人类基因组中的序列 DNA产物从两个 策略是用单个衔接子寡核苷酸进行PCR扩增。 的 将分析扩增反应产物的量 和DNA的大小，基因组覆盖度，杂交特异性， 在再扩增时的可恢复性、易用性和再现性。 根据第一阶段开发的扩增策略，我们将 最终提供给人类基因组研究团体 标准化的试剂可以用于：a）获得区域特异性的 克隆（即，用于重叠群组装/图谱闭合的基因组克隆，和/或cDNA 克隆以鉴定新基因），B）作为分离的起始材料 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.