Chromosome walking A technique with which an unknown region of a chromosome can be explored. It is generally used to isolate a locus of interest for which no probe is available but that is known to be linked to a gene which has been identified and cloned. A fragment containing a known gene is selected and used as a probe to identify other overlapping fragments which contain the same gene. The nucleotide sequences of these fragments can then be characterized. This process continues for the length of the chromosome

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Chromosome walking
A technique with which an unknown region of a chromosome can be explored. It is generally
used to isolate a locus of interest for which no probe is available but that is known to be linked to
a gene which has been identified and cloned. A fragment containing a known gene is selected
and used as a probe to identify other overlapping fragments which contain the same gene. The
nucleotide sequences of these fragments can then be characterized. This process continues for the
length of the chromosome.
Method
The library comprises 96 clones, each containing a different insert. To begin the walk, the insert
from one of the clones is used as a hybridization probe against all the other clones in the library
The main problem that arises is that if the probe contains a genome-wide repeat sequence then it
will hybridize not only to overlapping clones but also to non-overlapping clones whose inserts
also contain copies of the repeat.
The extent of this non-specific hybridization can be reduced by blocking the repeat sequences by
prehybridization with unlabeled genomic DNA. But this does not completely solve the problem,
especially if the walk is being carried out with long inserts from high-capacity vectors such as
BACs or YACs. For this reason, intact inserts are rarely used for chromosome walks with human
DNA and similar DNAs which have a high frequency of genome-wide repeats.
Instead, a fragment from the end of an insert is used as the probe, there being less chance of a
genome-wide repeat occurring in a short end-fragment compared with the insert as a whole. If
complete confidence is required then the end-fragment can be sequenced before use to ensure
that no repetitive DNA is present.

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If the end-fragment has been sequenced then the walk can be speeded up by using PCR rather
than hybridization to identify clones with overlapping inserts. Primers are designed from the
sequence of the end-fragment and used in attempted PCRs with all the other clones in the
library. A clone that gives a PCR product of the correct size must contain an overlapping insert

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The use of overlapping DNA clones to find a new gene by "chromosome
walking"
To speed up the walk, genomic libraries containing very large cloned DNA molecules are
optimal. To probe for the next clone in the walk by DNA hybridization, a short DNA fragment
(labeled with a chemical or a radioisotope) from one end of the previously identified clone is
purified: If a "right-handed" end is used, for example, the walk will go in the "rightward"
direction, as shown in this example. Use of a small end fragment as a probe also reduces the
probability that the probe will contain a repeated DNA sequence that would hybridize with many
clones from different parts of the genome and thereby interrupt the walk.
as shown in the figure below

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Applications
 This technique can be used for the analysis of genetically transmitted diseases, to look for
mutations.
 Chromosome Walking is used in the discovery of single-nucleotide polymorphism of
different organisms
Disadvantages
 There is a limitation to the speed of chromosome walking because of the small size of the
fragments that are to be cloned.
 Another limitation is the difficulty of walking through the repeated sequence that are
scattered through the gene.
 If the markers were too far away, it simply was not a viable option
 Additionally, chromosome walking could easily be stopped by unclonable sections of
DNA.
 A solution to this problem was achieved with the advent of chromosome jumping (Marx,
1989), which allows the skipping of unclonable sections of DNA.
References:
1. http://pt.slideshare.net/linokhan/chromosome-walking
2. http://www.ncbi.nlm.nih.gov/books/NBK21116/figure/A6290/?report=objectonly
3. http://www.ncbi.nlm.nih.gov/books/NBK28329/figure/A1425/?report=objectonly
4. http://www.ncbi.nlm.nih.gov/books/NBK21117/
5. http://feedforbiotech.blogspot.com/2011/03/chromosome-walking.html
6. http://www.genevoguebiotech.com/2012/09/chromosomewalking.html
7. http://www.wisegeek.com/what-is-chromosome-walking.html