基因,蛋白,免疫 课程3.doc

/silver/10.3.shtml Previous Next 10.3 PHYSICAL MAPS AND POSITIONAL CLONING There are two stages in the process of positional cloning. The first stage is the focus of a major portion of this book: to use formal linkage analysis and other genetic approaches — as tools — to find flanking DNA markers that lie very close to the locus of interest. With these markers in hand, one can move to the second stage of this pathway: obtaining clones that cover the critical region, then identifying the gene of interest apart from all other genes and non-genic sequences within this region. This second stage will be the focus of the remaining portion of this chapter. In what follows, I will move away from the realm of the formal geneticist to that of the molecular biologist. However, for several reasons, my intention is only to provide an overview of the conceptual framework that underlies the various approaches being used at the current time. First, the topics of physical mapping and positional cloning have filled entire books and many excellent review articles. Second, these linked topics are driven by technology, and new improved protocols are constantly moving old ones onto the shelves. Consequently, any detailed discussion of actual molecular techniques will quickly become outdated. 10.3.1 Prerequisites to positional cloning The absolute first step in the process of positional cloning is the high resolution mapping of the locus of interest relative to closely linked DNA markers. This process (described at length in Chapter 9) provides an investigator with two sets of complementary tools that are essential prerequisites to the actual generation of a physical map around the locus of interest. The first set of tools will be represented by the small number of animal samples with crossover sites in the vicinity of the locus. The second set of tools is the small group of closely linked DNA markers. Once the phenotypically defined gene has been closely linked to one or more DNA mark