Chromosome walking


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Chromosome walking

  1. 1. Chromosome Walking Presented by: Aleena Ahmad Khan
  2. 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. 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. 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. 5. • 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.
  6. 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. 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. 8. References • • • • eb&cd=1&cad=rja&ved=0CCoQFjAA&url=http%3A%2F%2Flectures sig2=KTMTKSwexvcC8PTx4ux3NA&bvm=bv.53899372,d.Yms • • 2AA6wM9U • _and_jumping#ref1 • •