Chromosome Walking

Chromosome walking is a method used to clone in an orderly fashion the DNA segments along the chromosome starting at any point for which we have a probe. To build up a series of overlapping cloned DNA fragments, it begins with one clone from a library, and then identifies a second clone whose insert overlaps with the insert in the first clone. This was the first method, called chromosome walking, devised for assembly of clone contigs. But there is a limitation to the speed of chromosome walking and that is 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. 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. The insert from one of the selected clones is then used as a hybridization probe with all other clones in the library.

A probe may have a genome wide repetition of sequences and this is a problem that is faced during the walk as then it will not only hybridize to overlapping clones but will also to non overlapping clone whose inserts also contain copies of repeat. This unwanted hybridization can be reduced by blocking the repeat sequence with pre-hybridization with unlabelled 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 (a short end-fragment) which has a lesser chance of repetition. The walk can also be sped up by using the PCR instead of hybridization to identify the clones with overlapping inserts. Primers, used in attempting PCRs with other clones in the library, are designed from the sequences of the DNA end fragments. Further higher speeds can be achieved by using groups of clones mixed together such that an unambiguous overlapping clone can be made. But care must be taken as ambiguities might arise in this case too.

This procedure is well suited for positional cloning as the objective here is to walk from a mapped mark to an inserting gene. But for the process of assembling clone contigs across entire genome, the procedure proves to be a bad choice. So as an alternative procedure clone finger printing technique is used. The method provides information regarding the physical structure of a cloned DNA fragment and this information (called a fingerprint) can be compared with data of other clones, thus allows identifying the overlaps which might have similarities.