Slide 1: Map-based Cloning: Chromosome
Walking 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.
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 are continued until a clone contains either mkrA or mkrC sequences.
Clones between mkr B and mkrC must then be evaluated for the presence of yfg (see right).
Chromosome walking has been used in the isolation of centromere sequences, among others. If the target species is a species whose genome has been completely molecularly mapped, an ordered set of YACs, PACs, BACs or cosmid clones will be available. Knowing which molecular markers are adjacent to the target gene automatically identifies the YACs and/or cosmids that need to be tested
One difficulty of chromosome walking is recognizing where the gene is located between the two markers. Zoo (or garden) blots where DNAs of a variety of species have been restricted, electrophoresed and Southern blotted can be useful. Gene sequences are more likely to be conserved during evolution than intergenic sequences. The identification of GC islands or the use of exon trapping can also be useful. There are other problems with walking. Copyright of original figures used in this presentation from other sources is duly acknowledged and the copyright remains with its original creator. These have not been used for commercial purpose but for educational use only. Slide 2: Map-based Cloning: Jumping-Linking Chromosome walks can be interrupted if a segment of the region to be walked through is non-clonable (for example if it is toxic to the host cell).
Chromosome walks can be detoured in many directions if one of the clone end probes (filled box) is a repeated sequence.
The jumping-linking strategy was invented to overcome the problems of unclonable segments and repeated sequences and to enable larger regions of the genome to be scanned. Use is made of a restriction enzyme that recognizes few sites in the DNA (large thick bars) and an enzyme that cuts frequently (small thin bars).
The jumping library contains clones in which the two double digestion fragments at either end of a large fragment are cloned in the same plasmid in the library (segments connected by dotted lines).
The linking library consists of plasmids containing the fragments generated by the frequent cutter that also contain a recognition site for the rare cutter.
The two libraries are used in alternation to hop down the molecular chromosome. © A.K. Chhabra