Chromosome Walking and Characterization of Chromosome Segments | Genetic Engineering and Biotechnology Restriction Maps and Molecular Genetic Maps | Genetics, Biotechnology, Molecular Biology, Botany

⇒	Genetic Engineering and Biotechnology 2. Restriction Maps and Molecular Genetic Maps
⇒	Restriction mapping
	⇒	Restriction cleavage and gel electrophoresis
	⇒	Construction of a restriction map
	⇒	Use of partial digests, end labeling and hybridization in restriction mapping
⇒	Restriction fragment length polymorphisms (RFLPs) as markers for genetic maps
⇒	Linkage and recombination between molecular and phenotypic markers
⇒	Random amplified polymorphic DNA (RAPDs) using PCR
	⇒	Minisatellites (VNTRs) and Microsatellites (SSRs)
⇒	Chromosome Walking and Characterization of Chromosome Segments
⇒	Reverse Genetics and Chromosome Jumping (or Hopping) Libraries

Chromosome Walking and Characterization of Chromosome Segments
When a probe is used for the identification of a gene sequence in a genomic library, the probe may hybridize with a number of clones, each carrying a part of a large gene fragmented during preparation of genomic library. If we obtain partial digests (by digesting the DNA only partially) from the genome, different genomes (from large number of cells) may give fragments which have overlapping sequences, because sites cleaved in different genomes of the same organism, will differ being random. Since none of these fragments may have its entire sequence represented in the probe, overlapping sequences may be used to construct the original genomic sequence. Identification of fragments with an overlapping sequence may be a key to the reconstruction or characterization of large chromosome regions. This is achieved by the technique popularly called chromosome walking.

The technique of chromosome walking involves the following steps : (i) from the genomic library select a clone of interest (identified by a probe) and subclone a small fragment from one end of the clone (there is a technique available to subclone a fragment from the end); (ii) the subcloned fragment of the selected clone may be hybridized with other clones in the library and a second clone hybridizing with the subclone of the first clone is identified due to presence of overlapping region; (iii) the end of the second clone is then subcloned and used for hybridization with other clones to identify a third clone having overlapping region with the subcloned end of the second clone; (iv) third clone identified as above is also subcloned and hybridized with clones in the same manner and the procedure may be continued; (v) restriction map of each selected clo.ne may be prepared and compared to know the regions of overlapping as shown in Figure 40.11, so that identification of new overlapping restriction sites will amount to walking along the chromosome or along a long chromosome segment.

Fig. 40.11. The technique of walking through successive hybridization between chromosome overlapping genomic clones.

Regions of chromosome approaching 1000kb have been mapped following the above technique. Restriction maps of entire chromosomes can be prepared in this manner following the technique of chromosome walking.





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