can someone explain in a easy way chromosome walking I know

can someone explain in a easy way chromosome walking? I know it\'s with overlapping fragments and a gen nearby the gen you want to find, but I don\'t clearly understand it, I have images in my book, but an image with steps and explanation would be nice,

Solution

Chromosome Walking:

Chromosome walking is positional cloning of gene sequences or upstream promoter sequences.

Eukaryotic DNA contains a lot of junk DNA between the genes. The technique relies upon screening of gDNA library for overlapping fragments in a sequential manner to tap the upstream or downstream region.

Procedure:

1. Isolation of Genomic DNA: First of all isolate genomic DNA (gDNA) of the test organism and then partially digest it with some restriction enzyme, say Hind III that would yield unequal random fragments.

2. Cloning of the Digested into E.coli: Clone the digested gDNA into a cloning vector (between the Hind III sites in this case) to prepare a gDNA library of the organism. Each colony of the library would have only one clone.

3. Preparation of marker probe: Prepare a radioactively labelled probe from the neighboring gene (say A), from where you want to start walking.

4. Colony Hybridization: Hybridize the probe with your gDNA library. The probe would show the signal in the colonies carrying DNA in and around your gene.

5. Sequencing: Pick the colonies showing the signal and isolate the plasmid DNA and sequence each using the plasmid-specific primers. You would get only one sequence per bacterial colony of your library.

The sequence, thus obtained must be analyzed. Select longer sequences towards the direction of your walk.

6. Preparation of the probe: Prepare new probes from the region towards your target gene.

7. Repeat the steps 4-6 till you get the clone and sequence of your target gene.