1 - Methods in Molecular Immunology: Chromosomal Walking in the Major Histocompatibility Complex

Abstract

This chapter focuses on the methods involved in molecular immunology. A chromosomal walk is the technique used to isolate DNA fragments adjacent to an existing region of cloned DNA. In most cases, the walk is accomplished by screening genomic libraries, constructed from partially digested DNA, with a labeled, unique restriction fragment isolated from the end of a cloned region. Clones containing overlapping DNA fragments are thus obtained, and after restriction mapping and alignment, they can be used to continue the walk. As cosmid vectors can accommodate inserts about twice as big as bacteriophage λ vectors, chromosomal walking experiments are more efficiently performed in cosmid cloning systems. It is advisable to verify chromosomal walks by comparing isolated cosmid clones with genomic DNA through Southern blot analysis. This can be done for clones isolated from the major histocompatibility complex of the mouse by using the congenic and recombinant congenic mouse stains available. Chromosomal jumping experiments and microdissection of defined regions of the chromosome can be used to isolate a large number of probes from the region of interest to speed up the walking process.