Background Information on Recombinant DNA
As you know, DNA is the material within a cell that determines what and organism looks like and how it functions. DNA does all f this via the proteins for which it codes.
Today, scientists are able to insert pieces of “foreign” DNA into an organism’s DNA so that the organism will express a desired characteristic, produce a certain substance, or even not express an undesirable characteristic. This moving of DNA pieces between
unrelated organisms is called recombinant DNA technology.
There are many ways to insert a piece of DNA from one organism into the cells of another organism. In recombinant DNA technology, one of the most widely used mechanisms for DNA insertion is the plasmid from Agrobacterium tumefaciens.
A plasmid is a circular ring of DNA which is found in some bacteria. The agrobacterium’s plasmid is unique because it has a DNA transforming region. When the bacterium bumps up against another cell, the DNA within the transfer region (T-DNA) “jumps out” of the plasmid and is inserted into the other cell’s chromosome.
As a biotechnologist, you could use your knowledge about this special capability of the T-DNA region of this plasmid in order to transfer desirable genes into plant cells of your choice.
So how do you “recombine” DNA using this technology? Once you have found the DNA that contains the characteristics you want, you must isolate or remove this specific DNA section.
Restriction enzymes are special molecules that cut the DNA in specific places so that the section you are looking for can be removed. Once the DNA fragment is cut, it needs to be inserted into the vector DNA (the agrobacterium’s plasmid). You must first isolate the plasmid from the Agrobacterium and then expose the plasmid to the restriction enzyme so that a gap in this circular DNA opens to combine with the new piece of DNA.
The restriction enzymes must be selected carefully so that 1) it cuts the DNA fragment (the new piece of DNA) that contains the specific characteristics you want, and 2) it splices the T-DNA out of the plasmid but leaves the genes responsible for the transfer intact!
Now you must “recombine” the plasmid with the DNA fragment coding for the specific characteristic you want. Once the plasmid and the new DNA piece are mixed together they must be joined. Ligase is a molecule that helps to join the exposed ends of the plasmid with the new DNA piece. Ligase acts like tape, binding the pieces together.
The new plasmid is put back into the agrobacterium and when the bacterium replicates, the new DNA will too. Very often this agrobacterium’s plamid is then inserted