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. 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. One difficulty of chromosome walking is recognizing where the gene is located between the two markers.
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. The strategy of using a genetic map to hone in on the physical gene is a general one for isolating disease-associated genes.