insertion of GoI in vectors

To introduce gene of interest into a vector, three-step process s done:

Cutting out the gene

Scientists first remove their gene of interest from the DNA sequences on either side of it. They can use restriction enzymes to do the cutting. These enzymes, which came originally from bacteria, cut DNA at specific sites in the sequence. If there’s not enough DNA for successful cutting or no suitable restriction enzyme recognition sites around the gene, scientists first use polymerase chain reaction (PCR) to make many more copies. By designing their PCR primers carefully, they can introduce new restriction sites on either side of the copied DNA sequence.

Opening up the vector

Next, scientists make a cut in the DNA sequence of the vector. They use the same restriction enzymes as they used to cut out the gene in step 1. This turns the vector into a linear molecule and makes it ready to accept the new piece of DNA.

Sticking the vector and the gene together

The final step in cloning is to incorporate the DNA of interest into the vector. Scientists mix the gene and the opened vector together with a bacterial enzyme called DNA ligase. The ligase sticks DNA ends together to form a single circular molecule that includes both the vector and the gene.

Regards