Chromosome walking is a method used to isolate and clone a particular gene or allele through positional cloning. It involves using overlapping clones that contain DNA fragments near the target gene to "walk" through the chromosome until reaching the gene. Each successive clone is tested to map its precise location until eventually reaching the target gene. Chromosome walking was developed in the early 1980s and can be used to analyze genetically transmitted diseases and find single nucleotide polymorphisms. However, it has limitations such as being a slow process and difficulty walking through repeated sequences.

1. Chromosome Walking
Presented by: Aleena Ahmad Khan

2. Chromosome Walking
• Chromosome
walking
is
a
method
of
positional cloning used to find, isolate, and clone a
particular allele in a gene library.
• Chromosome Walking was developed by Welcome
Bender, Pierre Spierer, and David S. Hogness in the
Early 1980's.
• There are nearly half a dozen positional cloning tests
that are done prior to a chromosome walk.
• Each clone in the cosmic library has a DNA insert of 50
KB.
• The walking starts at the closest gene that has already
been identified, known as a marker gene.

3. • Once the markers on either side of an unmapped
sequence are found, the chromosome walk can
begin from one of the markers.
• Each successive gene in the sequence is tested
repeatedly, known as overlap restrictions and
mapped for their precise location in the sequence.
• Eventually, walking through the genes reaches the
mutant gene in an unmapped sequence that binds
to a fragment of a gene of that particular disease.
• The testing on each successive clone is
complex, time-consuming, and varied by species.
• This series of overlapping clones could for example
consist of Bacterial Artificial Chromosomes.

4. Hybridization Probe
• A more straightforward approach thus is to use the
insert DNA from the starting clone as a hybridization
probe to screen all the other clones in the library.
• Positive hybridization signals that are given by
clones, whose inserts overlap with the probe, are
used as new probes to continue the walk.
• There are about 96 clones that a library consists of
and each clone contains a different insert.
• A probe may have a genome wide repetition of
sequences.

5. • This can be reduced by blocking the repeat sequence
with pre-hybridization with unlabeled genomic DNA.
• But this isn’t that affective solution especially in the
case when high capacity vectors such as BACs or
YACs are used in the walk.
• Therefore for chromosome walks with human DNA
which have a high rate of repetition, intact inserts are
not used in general.
• Instead the probe is taken from the end of an insert
which has a lesser chance of repetition.
• The walk can also be sped up by using
the PCR instead of hybridization.

7. Application
• 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.

8. 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.

9. References
• http://answers.google.com/answers/threadview/id/139820.html
• http://feedforbiotech.blogspot.com/2011/03/chromosomewalking.html
• http://dmohankumar.files.wordpress.com/2012/08/chromosomewalking.pdf
• https://www.google.com.pk/url?sa=t&rct=j&q=&esrc=s&source=w
eb&cd=1&cad=rja&ved=0CCoQFjAA&url=http%3A%2F%2Flectures
ug3.files.wordpress.com%2F2012%2F09%2Flecture15.ppt&ei=lNBbUpScEKv4QTUsYHIBw&usg=AFQjCNH25Coi17nNd4IwD7sUCgKIP479WQ&
sig2=KTMTKSwexvcC8PTx4ux3NA&bvm=bv.53899372,d.Yms
• http://nar.oxfordjournals.org/content/33/13/e122.full
• http://uk.answers.yahoo.com/question/index?qid=2007030302020
2AA6wM9U
• http://wiki.answers.com/Q/Applications_of_chromosome_walking
_and_jumping#ref1
• http://www.wisegeek.com/what-is-chromosome-walking.htm
• http://www.genevoguebiotech.com/2012/09/chromosomewalking.html