CBSE Class 12 Biology Biotechnology Principles And Processes Notes Set 02

• Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans.
• According to EFB, biotechnology is defined as ‘the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services’.

Principles Of Biotechnology

There are two core techniques that enabled birth of modernbiotechnology. 
• Genetic engineering
• Maintenance of sterile (microbial contaminationfree)ambience 
• In a chromosome, there is a specific DNA sequence called the origin of replication, which is responsible for initiating replication.
• Molecular scissors: Restriction enzymes are called molecular scissors as they cut the DNA at specific locations.

There are three basic steps in genetically modifying an organism
• Identification of DNA with desirable genes
• Introduction of the identified DNA into the host
• Maintenance of introduced DNA in the host and transfer of the DNA to its progeny

Tools Of Recombinant DNA Technology

Restriction Enzymes
• The first restriction endonuclease-Hind II, always cut DNA molecules at a particular point by recognizing a specific sequence of six base pairs. This specific base sequence is known as the recognition sequence.
• In EcoRI, the letter ‘R’ is derived from the name of strain.
• Roman numbers following the names indicate the order in which the enzymes were isolated from that strain of bacteria.

Restriction enzymes are of two kinds:
• Exonucleases: Remove nucleotides from the ends of the DNA
• Endonucleases: Make cuts at specific positions within the DNA
• Palindromic nucleotide sequences: It is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same.
• Sticky ends: These are the over hanging stretches created by restriction enzymes. These are named so because they form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends facilitates the action of the enzyme DNA ligase.

Separation And Isolation of DNA Fragments
• DNA fragments can be separated, according to their size through sieving effect provided by the agarose gel, by a technique known as gel electrophoresis.
• The smaller the fragment size, the farther it moves.
• The separated DNA fragments can be visualized only after staining with ethidium bromide, followed by exposure to UV radiation.
• The orange coloured separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as elution.

Cloning Vectors
• Cloning vector is a DNA segment that replicates independent of the chromosome and can be transferred between hosts. E.g., plasmid and bacteriophage.

The following are the features that are required to facilitate cloning into a vector.
• Origin of replication (ori)
• Selectable marker: helps in identifying and eliminating non-transformants and selectively permitting the growth of the transformants.
• Cloning sites: In order to have the alien DNA, the vector needs to preferably have single, recognition sites for the commonly used restriction enzymes.
• Insertional inactivation: The insertion of an enzyme, β-galactosidase within the coding sequence results into inactivation of the enzyme, which is referred to as insertional inactivation. This method is used differentiate recombinants from non-recombinants.
• Colour selection: Presence of insert results into insertional inactivation of the α-galactosidase and the colonies do not produce any colour, these are identified as recombinant colonies.
• Vectors for cloning genes in plants and animals Competent Host (For Transformation With Recombinant DNA)
• The bacterial cells are made ‘competent’ to take up DNA by treating them with calcium, through heat shock, microinjection and biolistic or gene gun.
• Micro-injection: A recombinant DNA is directly injected into the nucleus of an animal cell.
• Biolistic or gene gun: This method is suitable for plants; cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA.
• Disarmed pathogen vectors when allowed to infect the cell, transfer the recombinant DNA into the host

Process Of Recombinant DNA Technology

Isolation of the Genetic Material (DNA)
Bacterial membrane – Lysozyme
Plant cells – Cellulase
Fungus – Chitinase
• RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease.

Cutting of DNA at Specific Locations
• This is done by restriction enzymes.

Amplification of Gene of Interest using PCR:
• The repeated amplification is achieved by the use of a thermostable DNA polymerase (isolated from a bacterium, Thermus aquaticus), which remain active during the high temperature induced denaturation of double stranded DNA.

Insertion of Recombinant DNA into the Host Cell/Organism
• The cells harbouring cloned genes of interest may be grown on a small scale in the laboratory.
• The cultures may be used for extracting the desired protein and then purifying it by using different separation techniques.

Downstream Processing
• After completion of the biosynthetic stage, the product has to be subjected through a series of processes before it is ready for marketing as a finished product.
• The processes include separation and purification, which are collectively referred to as downstream processing.

Bioreactor
• To produce in large quantities, the development of bioreactors, where large volume of culture can be processed, was required.
• A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen).

Stirred-tank reactor
• It is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents.
• The stirrer facilitates even mixing and oxygen availability throughout the bioreactor.
• The bioreactor has an agitator system, an oxygen delivery system and a foam control system, a temperature control system, pH control system and sampling ports so that small volumes of the culture can be withdrawn periodically.