Question

Betimar is studying resistance genes to a fungus that attacks wheat. She managed to find a resistance candidate gene and would like to study it in more detail. Vivian, who is his collaborator in this research, gave him the idea of cloning this gene in a vector. The vector they chose possesses 3 regions where restriction enzymes can cut, however they noted that this resistance candidate gene did not possess those restriction regions. What solution would you give Betimar and Vivian to use this vector and clone their resistance candidate gene?

Answer 1

1.

The fact that the restriction site for the three regions available in the vector is a good news. This ensures that while clonind the gene in the vector, the restriction enzyme will not cut the resistance gene itself.

Now our aim will be to introduce the restriction site of one of the three restriction enzymes on one side of our resistance gene and introduce the restriction site of another restriction enzyme (out of the remaining two) on the other site of our resistance gene.

This will be achieved by designing primers for the amplification of the resistance gene. However, the primers will have one additional feature that they will have the restriction site of the selected restriction enzymes upstream to that part of them which is complimentary to the resistance gene. The forward primer will have the restriction site for one of the two restriction enzymes while the reverse primer will have the same for the other.

After this, the gene will be PCR amplified by using the designed primers. At the end of the PCR, we will have our amplified gene with restriction sites flanking it.

This gene can now be inserted into the vector by digesting the gene and the vector with the selected restriction enzyme followed by ligation of the digested vector and digested gene.

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