The following points highlight the seven main steps involved in gene cloning. Some of the steps are: 1. Isolation of DNA (gene of interest) fragments to be cloned 2. Insertion of Isolated DNA into the a suitable vector to form the recombinant DNA 3. Introduction of the recombinant DNA into a suitable organism known as host and other steps too.
Gene Cloning Step # 1.
Isolation of DNA (Gene of Interest) Fragments to be Cloned:
Before we carry out the operation of gene cloning we need two basic things in their purified state – the gene of our interest (GI) and the vector. A GI is a fragment of gene whose product (a protein, enzyme or a hormone) interests us. For example, gene encoding for the hormone insulin.
Similarly, the vector is a carrier molecule which can carry our GI into a host, replicate there along with the GI making its multiple copies. In this state the GI can also be expressed in the host cell producing the product of the gene which is needed by us.
Gene Cloning Step # 2.
Insertion of Isolated DNA into the a Suitable Vector to Form the Recombinant DNA:
Once the ingredients are ready we can start the operation. Our next step will be to cut both the vectors as well as the GI by using a special type of enzyme, called restriction endonuclease. A restriction endonuclease is an enzyme that cuts double-stranded or single-stranded DNA at specific recognition nucleotide sequences known as restriction sites towards the inner region (hence endonuclease).
They are also regarded as molecular scissors as they cut open the DNA strands. After this cutting step we move to pasting. Here the GI is taken and pasted to the cut vector. This procedure also needs an enzyme, called DNA ligase. They are also considered as molecular glue.
The resulting DNA molecule is a hybrid of two DNA molecules – our GI and the vector. In the terminology of genetics this intermixing of different DNA strands is called recombination (which naturally takes place in the prophase 1 of meiosis 1). Hence, this new hybrid DNA molecule is also called a recombinant DNA molecule and this technology is called recombinant DNA technology (RDT).
Gene Cloning Step # 3.
Introduction of the Recombinant DNA into a Suitable Organism known as Host:
When our recombinant DNA molecule is ready we need to introduce it into a living system known as host.
This is done either for one or both of the following reasons:
(a) To replicate the recombinant DNA molecule in order to get the multiple copies of our GI.
(b) To let our GI get express and produce the protein which is needed by us.
Introduction of the recombinant DNA into the host cell is done by various ways and strictly depends upon the size of the DNA molecule and the nature of GI. Some of the methods followed to carry out this step includes electroporation, micro-injection, lipofection, etc.
When we carry out this process some of the host cells will take up the recombinant DNA and some will not. The host cells which have taken up the recombinant DNA are called transformed cells and the process is called transformation.
Gene Cloning Step # 4.
Selection of the Transformed Host Cells and Identification of the Clone Containing the Gene of Interest:
The transformation process generates a mixed population of transformed and non-trans- formed host cells. As we are interested only in transformed host cells it becomes necessary to filter them out. This is exactly what is done in the selection process. There are many existing selection strategies some of which include taking the help of reporter genes, colony hybridization technique, etc.
Gene Cloning Step # 5.
Multiplication/Expression of the Introduced Gene in the Host:
Once we have purified our transformed host cells by the screening process; it is now our job to provide them optimum parameters to grow and multiply. In this step the transformed host cells are introduced into fresh culture media which provide them rich nourishment followed by an incubation in the oven at right temperature.
At this stage the host cells divide and re-divide along with the replication of the recombinant DNA carried by them. Now at this point we have two choices.
When the aim of the cloning process is to generate a gene library, then our target will be obtaining numerous copies of GI. So with this plan in our mind we will simply go for the replication of the recombinant DNA and not beyond that.
If the aim of the cloning experiment is to obtain the product of GI, then we will go for a step ahead where we will provide favourable conditions to the host cells in which the GI sitting in the vector can express our product of interest (PI).
Gene Cloning Step # 6.
Isolation of the Multiplied Gene Copies/Protein Expressed by the Introduced Gene:
In this step we isolate our multiplied GI which is present attached with the vector or the protein encoded by it. This can be rightly compared with the process of harvesting where we collect the crop from the field. There are many processes of isolation, the selection of which varies from case to case.
Gene Cloning Step # 7.
Purification of the Isolated Gene Copy/Protein:
After the harvesting of the isolated gene copy or the protein it is now our job to purify them.