5 Important Steps of Shotgun Method of Genome Sequencing

The following are the steps of the shotgun method:

1. Using specific restriction enzymes the isolated DNA from a particular organism is cleaved into many bits. As the restriction enzymes may bring about even or staggered cuts many sticky ends are left. The resultant DNA fragments are of different length.

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2. Host cells of E.coli are selected and the outer membrane is first dissolved by a detergent and its DNA is released.

The plasmids of E.coli are then separated from the main genome by ultracentrifugation. The plasmids are then kept in a solution containing the same restriction enzyme which will cut the plasmid circle into a linear form. Linear plasmid DNA also has overlapping sticky ends.

3. In this step isolated DNA and linear plasmid DNA are mixed. As a result random association occurs between the foreign DNA and the plasmid DNA.

Subsequently the foreign DNA gets integrated into plasmid DNA to form a long linear DNA molecule which then becomes a circle due to covalent joining. The resultant DNA is a recombinant or chimeric DNA.

4. The chimeric DNA molecules are now placed in a solution of calcium chloride which also has E.coli cells. On heating suddenly up to 42°C the membranes of bacteria become permeable to chimeric DNA molecules. Thus the E.coli cells incorporate the chimeric DNA plasmids.

5. The bacterial cells containing the chimeric plasmids are then cultured on a medium. The cells express not only their genes but the genes of the chimeric plasmids also thus producing a desired product.

All these cells are produced by the division of the original bacterial cell having a single chimeric plasmid hence they are called clones. Inserting a chimeric plasmid into a bacterial cell is of very rare occurrence.

It has been estimated that only one in 10s of 106 cells are capable of undergoing transformation.

In shotgun method several genes can be cloned at a time and it is an omnibus method because the genes to be cloned are not picked and identified before.