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- 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.
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- 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.
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