What is Gene Cloning?
What do you mean by gene cloning? Gene means a segment of DNA that expresses for a particular protein. And cloning means making multiple copies. So, gene cloning means a technique used for making multiple copies of a gene. If we use PCR (Polymerase Chain Reaction) method for cloning genes then such type of method is called cell free gene cloning method or molecular cloning using PCR. If we use a host cell for cloning genes then such method is called cell based gene cloning method. In this article, we’ll be discussing cell based gene cloning method in detail.
Why Gene Cloning?
Before learning the process of gene cloning method, it is important to first understand why do we do gene cloning? To understand why gene cloning is required, we take an example of insulin protein. Here gene cloning method is used to make multiple copies of insulin protein genes. Why insulin gene is important? Insulin gene is responsible for making insulin proteins and this insulin protein is used as a drug therapy for patients suffering from diabetes.
Diabetic patients fail to produce sufficient amount of insulin inside the body. And we know that insulin allows cells to take up glucose. In the absence of insulin, cells fail to get glucose inside the cell and high amount of glucose circulates in the blood. Insulin injection is given to diabetic patients so that their body cell could absorb glucose from the blood.
There are millions of people suffering from diabetes. They need insulin on a regular basis. How so much amount of insulin is produced? Well, the answer to this question is gene cloning. First of all, insulin gene is cloned using a host cell and as gene expresses, it forms insulin protein. This insulin is isolated from the host cell and used in drug therapy for diabetic patients.
Gene Cloning Steps
- Isolation of double stranded DNA containing the gene of interest: we know that beta cell of pancreas is responsible for the synthesis of insulin. We also know that insulin gene is located on the short arm of chromosome 11 in humans. So, the first step would be to isolate DNA from chromosome 11 of beta cell. Now, DNA contains many genes, but we’re only interest in insulin gene right? So, we need to cut that part of double stranded DNA that contains insulin gene. For this purpose, the double stranded DNA is incubated with restriction endonuclease enzyme. The restriction endonuclease will cut double stranded DNA into fragments – one fragment would contain our gene of interest.
- Vector molecule: Based on the size and length of the gene of interest, vector molecule is selected, which is nothing but a double stranded self-replicating DNA molecule. Vector molecules are DNA molecules that are responsible for carrying the gene of interest. In other words, we can say that vector molecules act as vehicle for carrying the gene of interest. Some of the examples of vector molecules include plasmid, bacteriophage lambda, BAC, and YAC etc. Vector molecules possess two characteristics – the first is that they are self-replicating and the second is they possess selectable marker, such as antibiotic resistant sites. The vector molecule is cleaved with the same restriction enzyme that we used to cleave the double stranded DNA isolated from the beta cell of pancreas. After cutting vector DNA – it is mixed with the gene of interest and using DNA ligase, both the DNAs are joined together. Now, this modified vector molecule is called recombinant DNA molecule.
- Transfer of recombinant DNA molecule into the host cell: The recombinant DNA molecule is transferred into the host cell (for example bacterial cell). In order for bacteria to take up recombinant DNA, the bacterial cell is first made competent. This is achieved by treating bacterial cell with a specific concentration of divalent cation such as Calcium ions. Calcium ions increase the efficiency with which DNA enters the bacterium. After that heat and cold treatment is given alternatively to enable bacteria to take up recombinant DNA. In addition to bacterial cell, we can also use plant cells or yeast cells for the transfer of recombinant DNA. As the host cell divides so as the recombinant DNA molecules and thus make multiple copies of the gene of interest.
- Growing bacterial cells in agar plate: Agar is a nutrient medium where bacterial cells are grown. Bacterial cells take up nutrients from agar to multiply and grow. Agar plate is added with antibiotics such as ampicillin or tetracycline. It is important to mention that we expect only those cells to grow on agar medium who have received recombinant DNA. There might be other bacterial cells that didn’t receive recombinant DNA. And to filter transformant bacteria from non transformant bacteria – agar media is added with antibiotics. Now, in this example, say, the recombinant DNA is having ampicillin resistant gene – it means, those bacteria having recombinant DNA would grow on agar medium and vice versa. And those bacteria without recombinant DNA would not survive on agar medium.
- Isolation of bacterial cells: Once bacterial cells grow on agar medium – they are picked up from the medium. Well, again vector molecule could be of two types: cloning vector and expression vector. If we just want to make a gene library – we need multiple copies of that particular gene. For this purpose, we use only cloning vector. On the other hand, if we want the product of a gene (insulin in this case), then we would need to use an expression vector. This kind of vector possess all the machinery required for the expression of a gene. So, if we take an expression vector then along with the host cell genes, our gene of interest present in the recombinant vector would also express and form products of our interest.