Perhaps,the first thing that comes to mind on hearing the word cloning, is Dolly the sheep. She was the first mammal to be ‘cloned’–in other words, she was an exact copy of a sheep.
There is another type of cloning, one that is done on a smaller scale. It is called molecular cloning and is the omnipresent technique in molecular biology labs.
A bit of background first –our genetic information is coded in long, long stretches of DNA molecules. In some sense, this is a primary information repository of a cell/organism. It is the blueprint for producing proteins. Proteins, on the other hand, are the guys who carry out various functions to keep the cell up and running. They are versatile creatures–they help in making other proteins, act as a catalyst (enzymes) in biological reactions, regulate what goes in and out of the cell and much more.
Reverting back to cloning.Let’s say,you want to produce one of these proteins in large amounts.One of the classic examples for this is insulin –it is required for diabetic patients on a regular basis. Culturing human cells to produce insulin in a mass scale is not viable, ‘cause they are very finicky and costly to maintain. Instead, if we could have an easygoing, happily multiplying E. coli to produce the human protein, it would be ideal.
This is where molecular cloning comes in.It gives a bunch of steps to help the piece of DNA (or the gene -blueprint for insulin) to incorporate into the E. coli’s own junk. E. coli can now produce insulin that humans can extract by breaking open the E. coli cells and purifying the required protein.
The entire process of cloning involves multiple steps. Since you can’t just force a random piece of DNA inside the E. coli, it needs to be attached to a mediator of sorts–this would help the DNA to produce insulin without any hassle. In a lot of cases, the mediator used is something called a plasmid. A lot of bacteria can take in these plasmids without any fuss.
The first step in the process is to have multiple copies of the DNA that produces your required protein. This is obtained through a process of polymerase chain reaction. Next,the DNA and plasmid need to be cut at precise locations to glue them together. This is done by a class of proteins called restriction enzymes. The plasmid and DNA of interest are pasted together by another enzyme called DNA ligase.
Now that your mediator is all set, the E. coli needs to be convinced to allow it inside its kingdom. The E. coli needs a little prodding to do this –it could be a heat shock or an electric shock. In case of other mediators,like a virus, there are other methods to do the same. These processes are not 100% efficient–to choose only those cells which have accepted the mediator, there are two ways –using markersin the mediatorsor a crude DNA extract method.
In the first, the mediator codes for some marker which signals that it is present in that particular cell. The most famous example is the puC19 plasmid that imparts a blue color to the colonies in a galactose medium. There are certain subtleties in differentiating the cells that do not have the insert DNA from thosewhich do –these depend on the kind of markers the plasmid has.
In the second method, a large number of the E.coli colonies are picked at random and a crude DNA extract of each of these colonies are made. From this extract, we can identify the colonies that have the plasmid containing the insert DNA.
Bingo, you have got your insulin producing gene in E. coli,all set for mass scale production.
.svg)
.png)