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 isnt 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. 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. 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. 8. 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