Question

When a cDNA carrying a protein coding region is inserted into a prokaryotic expression vector in order to produce the protein in bacteria, the orientation of the insert relative to the prokaryotic promoter must be known. Describe the use of a restriction endonuclease to determine the insert’s orientation in the vector (support your explanation with a drawing).

Answer 1

We explain how to use restriction endonuclease to determine the insert's orientation with an example.

Suppose we have a gene insert of size 1200 bp which is inserted in a plasmid whose original size (before insertion) is 500bp. The size of the recombinant plasmid after inserting the gene will become 5000 bp + 1200 bp = 6200 bp.

Now, to determine the orientation of the insert (relative to the prokaryotic promoter), we choose a restriction enzyme (or two restriction enzymes) such that two cuts are made: one cut within the insert, but significantly off center; the other cut in the backbone on one side of the insert. This will be followed by running on agarose gel by electrophoresis. The inserts with correct orientation and the inserts with incorrect orientation will show band with different sizes. Theoretically, we know the size of the band for the correct orientation. The lane with the band that matches this theoretical value will be the one with correct orientation.

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