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Home » Biology Homework Help » Biotechnology » Chromosome Jumping and Walking
Chromosome Jumping and Walking
The techniques of chromosome jumping and chromosome walking are used for the construction of restriction maps of genomes. Chromosome jumping is used for rather large DNA fragments, usually of several hundred kb in size, while chromosome walking is applicable to much smaller DNA fragments. A simple approach to chromosome jumping uses ‘jumping’ and ‘linking’ libraries generated by a rare cutting enzyme, e.g. Not I for humans. Each clone in a jumping library contains the DNA sequences on one side each, e.g. sequences 2 and 3, of two neighbouring cutting sites of enzyme Not I, while a clone in linking library has the DNA sequence located on either side of a single Not I site, e.g. regions 1 and 2 in clone 1 and regions 3 and 4 in clone 2.

The procedure for construction of a Not I jumping library is, in simple terms, as follows. Genomic DNA is completely digested with Not I, and the resulting fragments are circularized in the presence of supF marker. The circularized fragments are digested with a frequent cutting enzyme like BamHI to delete bulk of the DNA leaving only a small DNA sequence on either side of the supF marker. The resulting fragments are ligated in a suitable λ vector and cloned.

Similarly, the linking library clones are prepared. The genomic DNA is partially digested with a frequent cutting enzyme like Sau 3A and the fragments are circularized in the presence of supF marker. The circularized fragments are cut open with the enzyme Not I, and the linear fragments are integrated in a suitable λ vector. Only those circles that have a Not I site will become linear and hence, integrate in the λ vector. It is important to note that the regions surrounding a single Not I site are present in one clone.

Physical map of chromosome is constructed by digesting the genomic DNA with Not I and separating the fragments by pulse field gradient electrophoresis. Each clone of the jumping and the linking libraries is hybridized with all the DNA fragments so obtained. Each clone should hybridize with two fragments which contain the sequences represented in the clone. The data from the jumping library and linking library are pooled together to determine the correct order of the various fragments in the organisation of different chromosomes in the genome.

Chromosome walking

This technique is a more conventional approach and is used to prepare restriction maps of small regions of chromosomes. It also helps in identifying DNA fragments having overlapping regions, and as a result to locate two or more fragments that may contain a large sequence of interest, e.g. parts of a single large gene (some eukaryotic genes may be over 100 kb long). The technique uses conventional cloning techniques to obtain DNA fragments. First, a single DNA fragment is identified to serve as the reference point. The restriction map of this fragment is prepared and one end of it is used as a probe to locate a neighbouring overlapping fragment. Restriction map of this new fragment is prepared and its other end is used as a probe to locate the next overlapping neighbour fragment.

The techniques of chromosome jumping and chromosome walking enable the identification of contigs. Contigs (contiguous fragments) are clones containing neighbouring DNA fragments having an overlapping region. Contigs greatly facilitate mapping and, ultimately, the correct alignment of base sequence data.

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