HUMAN RESTRICT MAP DERIVED FROM GENOMIC INSERTION DATA

这个项目的目标是创建一张限制图 人类基因组的平均限制位点间距为50 千个碱基对(0.05厘米器官)。次要目标是 评估提议的方法论是否可以 用于快速创建基因组DNA的限制图 个人在两到三个月内，只需要血液或组织 以样品为起始材料。方法论，基因组插入 测绘，涉及到引入一种极其罕见的限制 在人类基因组中使用一个 复制缺陷逆转录病毒结构。限制点 位置信息从克隆细胞系中获得，该克隆细胞系包含 逆转录病毒的单个完整拷贝，在最初的 感染了构建物。来自给定克隆系的DNA是 分离，整合的逆转录病毒在罕见的 限制位置，并且DNA被部分地用 显示所需切割频率的限制性内切酶(S) 人类DNA(如测绘酶)。然后对DNA样本进行脉冲处理 与大小标准(波长噬菌体)相邻的现场电泳法 梯形图)和映射酶部分切割的位置 通过印迹，然后用逆转录病毒探测印迹来可视化 DNA来创建间接末端标记。项目可能会设置 为产生高分辨率基因型的基础工作， 任何个人的预后和/或诊断医学目的。

The objective of this project is to create a restriction map of the human genome at an average restriction site spacing of fifty thousand base-pairs (0.05 centimorgans). A secondary objective is to evaluate the possibility that the proposed methodology can be used to rapidly create restriction maps of genomic DNA from any individual in two or three months, requiring only a blood or tissue sample as starting material. The methodology, Genomic Insertion Mapping, involves introduction of an extremely rare restriction site into random positions within the human genome using a replication defective retrovirus construct. Restriction site position information is obtained from clonal cell lines containing a single integrated copy of the retrovirus, cloned after initial infection with the construct. DNA from a given clonal line is isolated, the integrated retrovirus is cleaved at the rare restriction position, and the DNA is partially digested with a restriction enzyme(s) showing the desired frequency of cleavage in human DNA (e.g. mapping enzyme). The DNA sample is then pulsed field electrophoresed adjacent to size standards (lambda phage ladder) and the positions of partial cleavage by the mapping enzyme visualized by blotting and then probing the blot with retroviral DNA to create an indirect end label. The project may set the ground work for the generation of high resolution genotypes, for prognostic and/or diagnostic medical purposes, of any individual.