Chromosome walking is a method to move systematically along a chromosome from a known location. • It is the only to search for a gene its position on a chromosome is only approximately known. The walking starts at the closest gene that has already been identified, known as a marker gene. Once the markers on either side of an unmapped sequence are found, the chromosome walk can begin from one of the markers. • the probe is taken from the end of an insert which has a lesser chance of repetition. STEPS Steps: 1- the gene is fragmented into overlapped fragments by the restriction enzymes. 2-the overlapped fragments are then inserted (cloned) into plasmids, phages or cosmids. 3-the first insert (clone)is then isolated and sequenced. Diagram 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. 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. Disadvantage There is a limitation to the speed of chromosome walking because 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. There is a limitation to the speed of chromosome walking because 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 unclone able sections of DNA. A solution to this problem was achieved with the advent of chromosome jumping (Marx, 1989), which allows the skipping of unclone able sections of DNA.

1. Chromosome
Walking
MADE BY ALLAH NAWAZ
FROM UOG

2. Chromosome Walking
Chromosome walking is a method to move systematically
along a chromosome from a known location.
• It is the only to search for a gene its position on a
chromosome is only approximately known.
The walking starts at the closest gene that has already been
identified, known as a marker gene.
Once the markers on either side of an unmapped sequence
are found, the chromosome walk can begin from one of the
markers.
• the probe is taken from the end of an insert which has a
lesser chance of repetition.

3. Chromosome Walking
• It 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.
• The walking starts at the closest gene that has already been
identified, known as a marker gene.

4. • 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.

5. Steps
Steps: 1- the gene is fragmented into
overlapped fragments by the restriction
enzymes.
2-the overlapped fragments are then
inserted (cloned) into plasmids, phages or
cosmids.
3-the first insert (clone)is then isolated and
sequenced.

6. steps
4- a small segment of DNA from one end of the
first recombinant (insert) is obtained and subcloned
(cloned from a clone )and is then used as a probe to
rescreen for the next insert of the genome
containing this overlapping piece of DNA.
5-the second insert is in turn sequenced and a
terminal piece of it is subcloned and then used to
rescreen for the next third DNA insert and so on the
process is repeated so as to walk down the
chromosome.

9. 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.

10. • This can be reduced by blocking the repeat sequence
with pre-hybridization with unlabeled genomic DNA..
• 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.

12. 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.

13. 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.
• when a gene is too large (>100 kb) to be
sequenced in one piece

14. Disadvantages
• There is a limitation to the speed of chromosome walking
because 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.

15. • If the markers were too far away, it simply was not a
viable option.
• Additionally, chromosome walking could easily be
stopped by unclone able sections of DNA.
• A solution to this problem was achieved with the advent
of chromosome jumping (Marx, 1989), which allows the
skipping of unclone able sections of DNA.
Disadvantages