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. 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.
4. 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.
5.
6. 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.
7. 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.
