Question

18. Describe the process of DNA cloning. What do we need? Describe what a vector is. Describe what the vector needs to have to be a good vector. How do you know when it works? Make sure to describe how we make recombinant DNA. I

Answer 1

DNA cloning is the process in which a desired DNA sequence is inserted into a vector. This vector can the be inserted into bacteria for them to use the desired DNA sequence.

The vector is a DNA molecule that will carry on the task of transporting the desired DNA clone. A vector must have an origin of replication, a cloning site, and a sequence that will help us to confirm success of cloning (selectable marker).

The process of cloning needs a vector with a multicloning site, that means regions where there are sequences that can be recognized by restriction enzymes to cut. The vector with its respective multicloning site is mixed with restriction enzymes, ligases and the desired DNA sequence. The restriction enzymes will cut the cloning site, then the desired DNA will approach and ligases will correctly link the nucleotides, including the DNA sequence into the vector. Now we have a recombinant DNA molecule.

Once we have the recombinant DNA we may confirm the succes by inserting the vector into a cell. We are going to take a bacteria strain and we will transform it. Transformation is the process in which the vector is inserted. The bacteria is exposed to electrical currents that will cause loosen the membrane and will allow the entrance of the vector, this process is called electroporation. Then we have to nurish the bacteria, so they survive, and then we can test for the correct cloning and transformation. The vector may have a selectable marker that is the gene for an antibiotic resistance, in this case we grow the bacteria in such antibiotic and the positive growth it in will confirm the proper DNA cloning. Some other vectors have different markers, changing the dynamics of the confirmation test.