Can you recall meiosis and indicate at what stage of prophase I, recombinant DNA is made? (Leptotene/ Zygotene/ Pachytene)

1. Principles of Biotechnology:

(i) Biotechnology is defined as 'The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.'

(ii) The processes such as in vitro fertilisation leading to a 'test-tube' baby, synthesising a gene and using it, developing a vaccine, or correcting a defective gene, are all parts of biotechnology.

(iii) Conventional biotechnology deals with large-scale production and marketing of products and processes using natural strains of microorganisms and cell lines.

(iv) Modern biotechnology is associated with recombinant DNA (rDNA) technology or genetic engineering involving the manipulation of desired genes. It enhances their productivity and also gives rise to new products.

2. Tools of Recombinant DNA Technology:

(i)The key tools of rDNA technology are restriction endonucleases and cloning vectors.

(ii) Restriction enzymes are called molecular scissors because these enzymes cut at specific sites.

(iii) Cloning vector: Plasmids and bacteriophages can replicate within bacterial cells independent of the control of chromosomal.

3. Restriction Endonucleases:

(i) Restriction endonucleases cut the desired DNA at specific sites to produce sticky ends.

(ii) These endonucleases are also called scalpels or molecular scissors.

(iii) Restriction endonucleases are of three types as Type $\mathrm{I}$, Type $\mathrm{II}$, and Type $\mathrm{III}$.

(iv) Type $\mathrm{II}$ is used in gene cloning and is the most stable.

(v) Restriction endonucleases are present in bacteria only, providing a defence mechanism called the restriction-modification system.

4. Cloning Vectors:

(i) Cloning vectors are also called vehicle DNA or gene carriers.

(ii) The vectors are also the DNA molecules that can carry the alien DNA and replicate inside the host cell.

(iii) Plasmids are the most commonly used vectors.

(iv) Other types of the vector include bacteriophages, cosmids, phagemids, Yeast Artificial Chromosome (YAC), Bacterial Artificial Chromosome (BAC), and also some plant and animal viruses.

(v) The first artificial cloning vector is pBR $322$ from E. coli plasmid and is $4,362$ base pairs long.

(vi) Passenger DNA or foreign DNA is the desired DNA, also called a DNA insert. It is to be ligated with vector DNA to construct rDNA.

5. Processes of Recombinant DNA Technology:

(i) Electrophoresis is a technique used in rDNA technology for separation of DNA fragments by the use of the electric field.

(ii) Passenger DNA can also be obtained through the genomic library, cDNA library or Polymerase Chain Reaction (PCR).

(iii) A plasmid's other antibiotic resistance gene remains active and acts as a selective marker in selecting the transformant (cloned plasmid).

(iv) Alternatively, selectable markers have been developed which differentiate recombinants from non-recombinants based on their ability to produce color in the form of a chromogenic substrate.

6. Transformation:

(i) The introduction of the rDNA molecule into the host cell is carried out for further propagation.

(ii) The host cells are poorly accessible to DNA molecules, so the host cells are made 'competent' to take up rDNA easily.

(iii) The process of entering the plasmid containing a foreign DNA fragment into the host cell (a bacterium) is called transformation.

(iv) When rDNA is allowed to enter through a gene carrier bacteriophage, it is called transfection.

7. DNA Delivery Systems:

(i) Direct delivery of DNA into the target cells at a higher speed is carried out by microprojectile bombardment, microinjection, electroporation, liposome-mediated gene transfer, etc.

(ii) Expression of a foreign gene takes place in the form of a protein called rec protein within the host organism.

8. Bioreactors and Downstream Processing:

(i) Downstream processing involves separating and purifying the final product.

(ii) The product is formulated along with suitable preservatives, and the formulation must undergo thorough clinical trials to ensure the end user's safety.

(iii) The rec protein is subjected to downstream processing involving two steps—separation and purification before it is marketed.

(iv) Large-scale production of functional protein or rec protein is carried out with the help of bioreactors.