第三节克隆新基因.ppt

一、图位克隆法 Positional Cloning Before the genomics era, geneticists seeking the genes responsible for human genetic disorders frequently faced a problem: They did not know the identity of the defective protein, so they were looking for a gene without knowing its function. Thus, they had to identify the gene by finding its position on the human genetic map. Positional cloning ——The process that commences with searching for markers linked to a particular inherited trait, then using those markers to identify the approximate location of the gene responsible for the trait, and then using various cloning strategies to identify, isolate and characterize the gene. The strategy of positional cloning begins with the study of a family or families afflicted with the disorder, with the goal of finding markers that are tightly linked to the disease gene，to be pinned down to a relatively small region of the genome. The next step is to search through the million or so base pairs to find a gene that is the likely culprit. 1. Searching for Markers 人基因组内存在(CA)n 形式的碱基重复。若父亲某个片段内n=12, 母亲n=15, 且要寻找的突变基因处在(CA) 12的近旁，那么获得遗传疾病的后代，在这一区段内,两种重复形式总是出现n=12，而在健康的子女中总是出现n=15。 在研究这个问题时，总是寻找尽可能大的有遗传疾病的家庭，以利追踪基因，在他们的基因组内寻找碱基重复的特征，只在患病成员身上出现而不在健康成员身上出现的DNA片段。 20世纪80年代中期，詹姆斯古西拉幸运地找到了只存在于患者身上的片段。该片段中一定含有“亨廷顿舞蹈病”基因。但那个片段太大。10年后才在那个片段上找到了亨廷顿基因。 RFLP 全基因组扫描 (1) Chromosome walking染色体步查 The strategy of map-based cloning is to find molecular markers very closely linked to the gene of interest. Those molecular markers can serve as the starting point for chromosome walking or jumping to the gene. Contig（重叠群）： A set of overlapping clones that provide a physical map of a portion of a chromosome. It refers to contiguous map. In the diagram, the walk begins with a clone containing mkrB. The ends of the clone (boxed) are used to probe a library. Clones from adjacent genome segments are thus identified and isolated. The distal ends of those clones are used to reprobe the library. These steps ar