Tag: rDNA technology

Recombinant DNA Technology OR rDNA Technology – An Overview

First things first, what is recombinant DNA technology? rDNA technology is widely used technique in the field of genetic engineering. What basically happens in rDNA technique is, we take our gene of interest and insert into a vector. A vector is a vehicle which transports our gene of interest to the host cell. E.g. Plasmid – it is extrachromosomal DNA which has the ability replicate independently.

Therefore, rDNA is combination of two different types of DNA. Let’s say I take a gene of interest e.g. human insulin gene and insert it in a vector e.g. plasmid from E.coli. So what we get is a recombined DNA and this construct is called recombinant DNA (Figure 1).

Figure 1. rDNA

Steps Involved in rDNA Technology :

• Isolation of Gene of Interest –

  First thing would be to isolate our gene of interest. Say for example, we were talking about human insulin gene or it can be any other gene you are interested in. So from where can we isolate the gene of interest? It can be obtain from,
  Genomic library – which contains many different genes.
  cDNA library – which is a complementary DNA library or
  Chemically synthesize it – if we know the sequence of our gene of interest.
  Now that we have got our gene of interest, all we need is a vector to make rDNA. So the next step would be to insert our gene of interest into a vector.

• Construction of rDNA by joining the Gene of Interest into a Suitable Vector –

  In order to insert the gene of interest into a vector we need to cut open the vector first and after insertion, seal it back. So cutting of a vector would be done by molecular scissors called restriction enzymes. And sealing or ligation of gene of interest with the vector would be done by ligase enzyme.So at the end of this step we would get rDNA (Figure 1).
  Our rDNA is ready now but we need this rDNA in multiple copies to get good amount of product. Also we need a living system where the gene of interest can be expressed to give us our product. So the next step is,

• Introduction of rDNA into a Suitable Organism –

  How can we transfer rDNA in a host (suitable organism)? For this purpose we have specific gene transfer methods such as,

  Physical gene transfer – electroporation, liposome mediated gene transfer, microinjection
  Chemical gene transfer – PEG method, calcium chloride method
  Virus mediated gene transfer – virus is used to transfer the rDNA
  

Figure 2. Introduction of rDNA into a Host Cell

Once we perform the gene transfer method (Figure 2), there can be three possible outcomes (Figure 3).

Figure 3. Three Possible Outcomes following the Gene Transfer Methods

Non-transformed cells – not all the host cells would take up the rDNA. Chances are there that some cells would be non-transformed cells.
Transformed cells with non-recombinant vector – here we have got transformed cells but the vector is non-recombinant and this can happen when in step 2, while preparing rDNA some of the vectors do not take up the gene of interest.
Transformed cells with recombinant vector – cells that have got rDNA and this is the type we are looking for.

So the next step would be to isolate transformed cells with rDNA right!

• Isolation of Transformed Cells with rDNA –

  We need some kind of screening method to differentiate between the transformed cells with rDNA from other cells. For this we can use antibiotics in the media and while constructing the rDNA it is made sure that it contains some antibiotic resistant gene. So when we grow all three types of cells only the cells with rDNA can survive and form colonies. We can also use visible pigmentation assay such as blue-white screening where non-transformed or without gene of interest cell would produce blue colonies whereas transformed cells with rDNA would produce white colonies (Figure 4).

Figure 4. Blue-White Screening Showing Transformed Cells with rDNA

• Multiplication and Expression of Gene of Interest –

  Once we isolate the cells with rDNA we need to multiply them to get multiple copies of gene of interest and express it to get desired protein product.

Figure 5. Multiplication of rDNA within a Host Cell

The plasmid in rDNA has the ability to replicate itself. So it will multiply in the host producing multiple copies (Figure 5) and the cell will express the gene of interest to give desired protein.

Examples of rDNA Products –

Production of recombinant human insulin, recombinant human growth hormones, recombinant blood clotting factor VIII, recombinant hepatitis B vaccine etc.

I hope this post was helpful 🙂

Understand this topic in more details in this video.