Chromosome walking-short and easy summary

1. Chromosome Walking
Ms Saajida Sultaana Mahusook

3.  Chromosome walking is a method of positional cloning used to
find, isolate, and clone a particular allele in a gene library.
 An allele is a gene for a particular genetic trait passed on from
adults to their offspring, such as the allele for brown eyes in a
gene for eye color.
 Sometimes, the approximate location of a single allele in a string
of deoxyribonucleic acid (DNA) may be known.
 To isolate a particular allele for a genetically transmitted disease,
chromosome walking needs to explore for the desired specimen in
an unmapped DNA sequence outside of previously mapped
sequences.

4.  To locate a particular disease gene, the walking starts at the closest
gene that has been identified, known as a marker gene.
 Each successive gene in the sequence is tested repeatedly for
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.
 Once the gene is cloned, its function can be fully identified.
Throughout this process, tests are done to fully identify the properties
of each successive clone, to map their locations.
 There are positional cloning tests that are done prior to a
chromosome walk, to narrow down possible particular genetic
sequences that may contain the desired mutant gene for a disease.

5.  Once the markers on either side of an unmapped probable
sequence are found, the chromosome walk can begin from one of
the markers.
 The testing on each successive clone is complex, time-
consuming, and varied by species. There are different tests for
genes related to plant diseases than for genes involved in a
human gene library.
 Positional cloning uses genetic markers that are known to inhabit
the chromosomes of individuals who have specific diseases.
 These databases of known common traits allow testing that can
be used to identify individuals who may or may not have certain
recessive genes for a disease that has not presented as yet.

6.  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.
 By obtaining 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 can 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 by chromosome walking.

7. Steps involved in chromosome walking

8.  Select a clone of interest (identified by a probe) from the genomic library.
 The gene is fragmented into overlapping fragments with the help of
restriction enzymes.
 Subclone a small fragment from one end of the clone.
 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.
 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.
 The third clone identified is also subcloned and hybridized with clones in
the same manner and the procedure may be continued
 Restriction map of each selected clone may be prepared and compared
to know the regions of overlapping so that identification of new
overlapping restriction sites will amount to walking along the
chromosome or along a long chromosome segment.

9.  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.
 Chromosome walking, along with all of the other tests done prior
to chromosome walking, requires exceptionally well equipped
laboratories within which to perform all of the cloning phases,
examinations, and analysis of mutations found.

10. Applications
 Used to find mutation and analysis of genetically transmitted
diseases.
 Used in the detection of single nucleotide polymorphism in
different organism.

11. Disadvantages
 Limitation in the speed of chromosome walking due to small size
of the fragments.
 Difficulty of walking through the repeated sequence which is
scattered throughout the gene.
 Not a viable option if the markers are too far from each other.
 The process can be stopped easily by the presence of unclonable
sections of DNA. A solution to this problem is Chromosome
jumping that skips unclonable sections of DNA.

12. Reference
 http://www.eplantscience.com/index/genetics/genetic_engineering_a
nd_biotechnology_restriction_maps_and_molecular_genetic_maps/c
hromosome_walking_and_characterization_of_chromosome_segme
nts.php
 https://www.slideshare.net/zohaibkhan404/chromosome-walking-
49440731
 http://www.wisegeek.com/what-is-chromosome-walking.htm#
 http://www.slideshare.net/ereenerezk/chromosomewalking

13. THANK YOU