Quick Revision Notes

Class 12 Biology: Biotechnology - Principles and Processes

MP Board Examination

1. Introduction to Biotechnology

• Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans.
• European Federation of Biotechnology (EFB) defines biotechnology as: "The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services."

2. Main Principles of Biotechnology

Modern biotechnology mainly uses two core techniques:

1. Genetic Engineering: Techniques to alter the chemistry of genetic material (DNA and RNA), to introduce these into host organisms and thus change the phenotype of the host organism.
2. Bioprocess Engineering: Maintenance of sterile (contamination-free) ambiance in chemical engineering processes to enable growth of only the desired microbe/hainan cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes, etc.

3. Tools of Recombinant DNA Technology (rDNA Technology)

The key tools required for genetic engineering are:

A. Restriction Enzymes (Molecular Scissors)

• Belong to a class of enzymes called Nucleases.
• Exonucleases: Remove nucleotides from the ends of the DNA.
• Endonucleases: Cut DNA at specific points within the DNA strand.
• Restriction Endonucleases: Inspect the DNA sequence, find a specific recognition sequence, and bind to DNA to cut each of the two strands at specific points.
• Palindromic Nucleotide Sequences: The sequence of base pairs reads the same on the two strands when orientation of reading is kept the same.
  • Example: 5' - GAATTC - 3' 3' - CTTAAG - 5'

B. Cloning Vectors (Vehicles for Gene Transfer)

• Plasmids and bacteriophages have the ability to replicate within bacterial cells independent of chromosomal DNA control.
• Features required to facilitate cloning into a vector:
  1. Origin of replication (ori): Sequence responsible for initiating replication.
  2. Selectable marker: Helps in identifying and eliminating non-transformants and selectively permitting the growth of the transformants (e.g., resistance genes for Ampicillin, Tetracycline).
  3. Cloning sites: Recognition sites for the restriction enzymes where the foreign DNA can be inserted.

C. Competent Host (For Transformation with rDNA)

• DNA is a hydrophilic molecule, so it cannot pass through cell membranes.
• Bacterial cells are made 'competent' to take up DNA by treating them with specific concentrations of a divalent cation (such as calcium), followed by Heat Shock method (incubating at 42°C and then placing on ice).
• Other methods: Microinjection, Biolistics (gene gun), and Disarmed pathogen vectors.

4. Processes of Recombinant DNA Technology

Steps involved in recombinant DNA technology are executed in a specific sequence:

1. Isolation of Genetic Material (DNA):

   • Treating bacterial cells/plant/animal tissue with enzymes like Lysozyme (bacteria), Cellulase (plants), Chitinase (fungus).
   • Chilled ethanol is added to precipitate out purified DNA.

2. Fragmentation of DNA by Restriction Endonucleases:

   • DNA is cut using restriction enzyme restriction sites, monitored via Agarose Gel Electrophoresis.
   • DNA fragments move towards the anode (+) because they are negatively charged. Smaller fragments move faster and further.
   • Stained with Ethidium Bromide and visualized under UV light (orange bands).
   • Elution: Cutting out separated bands of DNA from the agarose gel.

3. Amplification of Gene of Interest using PCR (Polymerase Chain Reaction):

   • Multiple copies of desired DNA are synthesized in vitro.
   • Three main steps in each PCR cycle:
     1. Denaturation: Heating target DNA at 94°C to separate strands.
     2. Annealing: Cooling down to allow primers to bind to complementary sequences.
     3. Extension: DNA polymerase (Taq polymerase, isolated from Thermus aquaticus) extends the primers using nucleotides at ~72°C.

4. Insertion of Recombinant DNA into the Host Cell/Organism:

   • Direct introduction of foreign DNA into host cell.

5. Culturing the Host Cells on a Large Scale:

   • Using Bioreactors (vessels where raw materials are biologically converted into specific products).
   • Types of Bioreactors:
     • Simple stirred-tank bioreactor
     • Sparged stirred-tank bioreactor (air is bubbled through)

6. Downstream Processing:

   • Final stage before a product is marketed.
   • Includes Separation and Purification.
   • Product is formulated with suitable preservatives and undergoes strict quality control testing.

Important Terms for MP Board Exam (Glossary)

• Recombinant DNA: DNA formed by combining DNA from different organisms.
• Plasmid: Extra-chromosomal, self-replicating circular DNA molecule found in bacteria.
• Palindromic Sequence: Base sequence that reads the same forward and backward on complementary strands.
• Taq Polymerase: Heat-stable DNA polymerase enzyme used in PCR, obtained from Thermus aquaticus.
• Bioreactor: Large vessel used to grow microbes or cells to produce biotechnology products in large quantities.