Question

Early gene cloning experiments involved insertion at one restriction site in the vector; for example, the insert would have and EcoRI site at each end, and he vector would be opened at an RI site prior to litigation. Under what circumstances would asymmetric cloning be desirable, with the insert having a different site at each end?

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Answer 1

If the gene is too big and hard to ligate into the plasmid. Meaning if the effeciency of the cloning is low for any reason and a single enzyme is used to digest the gene and vector, it is also possible that the gene that is ligated is reversed since the ligase enzyme do not recognise the initial part and final part of the gene. So the clones that would be obtained would contain both the correctly ligated recombinant clone as well as incorrectly ligated. And also there is no way to identify which clone is which unless the function of the protein of that gene is known and its activity can be observed.

If the gene is new and its exact function is not known, double restriction digetion is preferred because this way, all the observed clones after ligation would be correctly ligated and the correct protein would be formed.

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