Se ha denunciado esta presentación.
Presented by: Aleena Ahmad Khan
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
• 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
• 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.
• 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.
• 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
• 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.
• This technique can be used for the analysis of
genetically transmitted diseases, to look for
• Chromosome Walking is used in the discovery of
single-nucleotide polymorphism of different
• 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
• If the markers were too far away, it simply was not a
• 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.