What is rDNA Technology? Steps involved in rDNA technology

Recombinant DNA Technology

Recombinant DNA (rDNA) technology is also known as DNA cloning, that is, it allows us to insert a DNA fragment of interest such as a new gene into a new organism (which doesn't contain that fragment of DNA).

It is one of the practical applications of microbial genetics and thus forms the major part of genetic engineering, where an organism's genetic information is modified by directly changing its nucleic acid genome through a collection of methods known as recombinant DNA technology or rDNA technology.

  

Though we can have various strategies to achieve this, but the simplest of it is to introduce a new gene into bacteria through plasmid vectors.

Plasmids are small circular DNA molecules present in bacteria and can be used to carry a foreign gene into the bacteria, therefore said to be a vector.

Steps involved in this simple strategy, i.e., Bacterial Gene Cloning

1. A bacterial plasmid designed with an antibiotic resistant marker gene is selected as a vector.
2. The circular plasmid vector is cleaved at specific sites using special enzymes called restriction endonuclease which are enzymes that cleave DNA at specific sequence patterns. For instance, EcoRI is a restriction enzyme which cleaves at the sequence GAATC.
3. The gene to be cloned is inserted in the gap produced by the endonuclease activity and then an enzyme DNA ligase is used to join the DNA pieces. This results in a circular plasmid vector having the gene of interest or the recombinant plasmid.
4. This recombinant plasmid vector is now transferred into the cell by means of a process called transformation.
5. Now these bacteria can be grown in a media containing specific antibiotics as per the resistant gene present in the plasmid vector. The bacterial cells which have taken up the recombinant plasmid vector can only grow in the presence of antibiotics (positive clones).
6. Then these positive clones can be subjected to further applications such as production of recombinant proteins, studying expression etc., This method has been successfully used for commercial production of many proteins like:

• Human insulin
• Antibodies
• Enzymes of medical & industrial interests etc.,

Basic Principles of Recombinant DNA Technology

Recombinant DNA technology unveils up totally new areas of research and applied biology, and therefore is an essential part of biotechnology, which is nowadays experiencing a stage of rapid growth and development.

Where in general sense, recombination is said to be the process in which one or more nucleic acids molecules are combined or rearranged to produce a new nucleotide sequence, for instance, usually the genetic material from two parents is combined to form a recombinant chromosome with a new or different genotype resulting in a new arrangement of genes or parts of genes and is accompanied by a phenotypic change.

Such manipulation of genes can be followed by several techniques, thus there are many diverse and complex techniques.

The basic principles of recombinant DNA technology is one such simple technique usually suggested.

rDNA Technology (bacterial gene cloning)

Steps involved in this short of technology

1. Isolation of the DNA from the source, that is, the donor
2. Generation of DNA fragments and selection of the desired piece of DNA, that is, the gene of interest
3. Insertion of the selected DNA into a cloning vector (Example: a plasmid) to create a recombinant DNA.
4. Introduction of the recombinant vectors into host cells (Example: bacteria)
5. Multiplication and selection of clones containing the recombinant molecules
6. Expression of the gene to produce the desired product.

Reference

• Recombinant DNA Technology By Keya Chaudhuri · 2013
  
• State Council of Educational Research and Training (SCERT 2018)