Monday, 21 June 2021

Steps of rDNA Technology

  Recombinant DNA or r-DNA technology is a technique that is used to refer, identification, isolation and insertion of gene of interest into a vector, Such as plasmid or bacteriophage to form a recombinant DNA molecule and production of large quantities of that gene fragment or product encoded by gene of interest.

Steps in rDNA technology

Step 1 : Identification and isolation of gene of interest or dna fragment to be cloned.

Step 2 : Insertion of this isolated gene into a suitable vector.

Step 3: Introduction of this vector into a suitable host the process is called as transformation.

Step 4 : Selection of the transformed host cell.

Step 5 : Multiplication or expression of the introduced gene of interest inside the host.

Let's discuss this step in detail.

Step 1 : Identification and isolation of gene of interest

  In recombinant DNA technology this gene of interest is isolate from different resources. These include
- Genomic library,
- cDNA library,
- Chemical synthesis of a gene if you know the sequence,
- If the number of copies of a particular gene is very less or not sufficient for gene cloning we can amplify that using polymerase chain reaction.

  For this step we have taken human insulin gene from genomic library. Genomic library is a source from where we get our gene of interest.

Step 2 : Insertion of Gene of interest in a suitable vector

Vector is any DNA molecule that has the ability to replicate inside the host to which the gene of interest has integrated for cloning.

  Vectors include Plasmids, Bacteriophages, Cosmids, BAC, Yeast vectors, Shuttle vectors etc.
most commonly used vector in rDNA technology is plasmid. Plasmid is extra chromosomal DNA that is present in bacteria.

 For the insertion of gene of interest we need to make a cut in the vector using a restriction enzyme. Restriction enzymes are enzymes that are capable of making internal cuts at specific sites in a DNA molecule.

  Now we need to incorporate our insulin gene into this vector. Our gene of interest is settled in between cut and ligated by ligase enzyme. Now we have the recombinant DNA molecule. Recombinant DNA molecule is a DNA molecule from which DNA are from different sources.

Step 3 : Introduction of this vector into a suitable host

  There are different gene transfer methods by which we can introduce our vector into a host. these methods are :
- Electroporation
- Microinjection
- Liposome mediated gene transfer
- Silicon carbide fiber mediated gene transfer
- Gene gun method.
- Poly Ethylene Glycol mediated gene transfer
- Calcium Chloride mediated gene transfer
- DEAD dextran mediated gene transfer.
- Natural transformation by micro-organism

So now our gene of interest is incorporated in the vector, we need to introduce this recombinant DNA into the host(bacterial cell) to manipulate. So now bacterial cell is genetically modified with our gene of interest.

Step 4 : Selection of the transformed host cell

After the transformation we will be getting three types of colonies
- Non-transformed bacterial cell, without any change
- Transform bacterial cell, with unaltered vector (vector is in the cell has transformed but without our gene of interest)
- Transformed bacterial cell with our recombinant vector

 We need Transformed bacterial cell with our gene of interest. Then we need to select this particular colony from the rest majority of the colonies.

There are different ways to select perticular colony which are
- Antibiotic resistance in selective medium
- Visible characters
- Assay for biological activity
- Colony hybridization
- Blotting test.

 Commonly used method for selection is antibiotic resistance selection in recombinant selective medium. In this method our gene of interest contains some antibiotics resistance gene. So in the medium which contain antibiotics only thos bacteria will survive which has our gene of interest.

Step 5 : Multiplication or expression of the introduced gene in the host

 Our intention is to make as many copies of that particular gene or we need to make that gene product to be synthesized inside the bacterium. We have genetically modified bacteria (host) with our recombinant DNA (plasmid). This plasmid will replicate inside the host as well as these bacteria will also replicate making millions of copies of our gene of interest.

 Our first intention is achieved, we have our gene of interest produced in millions of numbers. Our actual intention is to synthesize desired product inside bacteria. This is happening inside the host, gene that will be transcribed and translated and produce our desires protein or product.

 The forst and most common product which is produced by rDNA technology is human insulin. This insulin is called as Humulin.


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