class 12 notes

Class 12 Biology Notes Chapter 11 (Biotechnology: Principles and Processes) – Examplar Problems Book

Philoid

Philoid

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Examplar Problems
Alright class, let's begin our detailed study of Chapter 11: Biotechnology: Principles and Processes. This is a fundamentally important chapter, laying the groundwork for understanding how we manipulate life at the molecular level for human benefit. Pay close attention to the tools and techniques involved, as they are frequently tested in competitive exams.

Chapter 11: Biotechnology: Principles and Processes - Detailed Notes

1. Definition and Principles:

  • Biotechnology: Defined by the European Federation of Biotechnology (EFB) as 'the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services'. Essentially, it uses living organisms or their components to create useful products or processes.

  • Two Core Techniques:

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

  • Conceptual Development leading to Genetic Engineering:

    • Traditional hybridisation often leads to the inclusion and multiplication of undesirable genes along with desired genes.
    • Genetic engineering overcomes this limitation by allowing the isolation and introduction of only one or a set of desirable genes without introducing undesirable genes into the target organism.
    • Key milestones: Understanding DNA structure, restriction enzymes, DNA ligase, plasmid vectors, and transformation processes.

  • 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.

2. Tools of Recombinant DNA Technology:

These are the essential components required to perform genetic engineering.

  • (a) Restriction Enzymes ('Molecular Scissors'):

    • Enzymes that cut DNA at specific recognition nucleotide sequences. They belong to a larger class called nucleases.
    • Types:
      • Exonucleases: Remove nucleotides from the ends of the DNA.
      • Endonucleases: Make cuts at specific positions within the DNA. Restriction endonucleases are the key type used in rDNA technology.
    • Discovery: The first restriction endonuclease characterised was Hind II. Its recognition sequence is specific (6 base pairs).
    • Recognition Sequence: Each restriction endonuclease recognizes a specific palindromic nucleotide sequence in the DNA. A palindrome is a sequence of base pairs that reads the same on the two strands when the orientation of reading is kept the same (e.g., 5'-GAATTC-3' on one strand, and 3'-CTTAAG-5' on the complementary strand).
    • Mechanism: They inspect the DNA sequence, bind to the specific recognition site, and cut each of the two strands of the double helix at specific points in their sugar-phosphate backbones.
    • Types of Cuts:
      • Sticky Ends (Cohesive Ends): Cut the strands slightly away from the center of the palindrome site, leaving single-stranded overhanging stretches. Example: EcoRI. These ends can base-pair (hydrogen bond) with their complementary counterparts, facilitating the action of DNA ligase.
      • Blunt Ends: Cut both strands at the same point, usually in the center of the recognition sequence.
    • Nomenclature: Named based on the prokaryotic cell from which they were isolated.
      • First letter: Genus (e.g., E from Escherichia).
      • Second two letters: Species (e.g., co from coli).
      • Next letter/number: Strain (e.g., R from RY13).
      • Roman numeral: Order of discovery from that strain (e.g., I for the first). Example: EcoRI comes from Escherichia coli RY13, first identified.

  • (b) Cloning Vectors ('Vehicle DNA'):

    • DNA molecules that can carry a foreign DNA segment and replicate inside the host cell. Plasmids and bacteriophages are commonly used vectors.
    • Essential Features of a Cloning Vector:
      • Origin of Replication (ori): A specific sequence where replication starts. Any piece of DNA linked to this sequence can be made to replicate within the host cells. This site also controls the copy number of the linked DNA. High copy number vectors are preferred for obtaining many copies of the target DNA.
      • Selectable Marker: Helps in identifying and eliminating non-transformants and selectively permitting the growth of the transformants. Genes encoding resistance to antibiotics (e.g., ampicillin resistance - ampR, tetracycline resistance - tetR, chloramphenicol, kanamycin) are useful selectable markers for E. coli. Normal E. coli cells do not carry resistance against these antibiotics.
      • Cloning Sites (Recognition Sites): Specific recognition sites for commonly used restriction enzymes, where the foreign DNA is inserted. Preferably, the vector should have single recognition sites for several restriction enzymes within the selectable marker gene or at a separate location. Multiple sites within the vector will generate several fragments, complicating gene cloning.
      • Insertional Inactivation: Ligation of foreign DNA at a restriction site present within a selectable marker gene leads to the inactivation of that gene. This allows for the selection of recombinants (cells with vector containing the insert) from non-recombinants (cells with vector but no insert).
        • Example: In pBR322, if foreign DNA is ligated at the BamHI site within the tetR gene, the tetracycline resistance is lost, but ampicillin resistance (ampR) remains. Recombinants will grow on ampicillin medium but not on tetracycline medium. Non-recombinants will grow on both.
        • Alternative: Using insertional inactivation of a gene coding for a chromogenic substrate (e.g., lacZ gene coding for β-galactosidase). If foreign DNA is inserted within the lacZ gene, the enzyme is not produced (insertional inactivation). Recombinant colonies do not produce colour (appear white), whereas non-recombinant colonies produce blue colour in the presence of the chromogenic substrate (X-gal).
    • Examples of Vectors:
      • Plasmids: Extrachromosomal, self-replicating, circular DNA in bacteria (e.g., pBR322). Suitable for cloning small DNA fragments.
      • Bacteriophages: Viruses that infect bacteria (e.g., Lambda phage, M13 phage). Can carry larger DNA fragments than plasmids.
      • BACs (Bacterial Artificial Chromosomes) & YACs (Yeast Artificial Chromosomes): Used for cloning very large DNA fragments (e.g., in genome sequencing projects).
      • Vectors for Cloning Genes in Plants and Animals:
        • Agrobacterium tumefaciens (plant pathogen): Its Ti plasmid (Tumour inducing) can be modified into a cloning vector by removing the tumour-causing genes and inserting the gene of interest. It naturally delivers a piece of DNA ('T-DNA') to transform normal plant cells into a tumor.
        • Retroviruses: Can be disarmed (disease-causing genes removed) and used to deliver genes into animal cells.

  • (c) Competent Host (For Transformation with Recombinant DNA):

    • DNA is hydrophilic and cannot easily pass through cell membranes.
    • Host cells (e.g., bacteria, yeast, plant or animal cells) must be made 'competent' to take up foreign DNA (like a plasmid).
    • Methods to make cells competent:
      • Chemical Treatment: Treating cells with a specific concentration of a divalent cation (e.g., calcium chloride, CaCl2) increases the efficiency with which DNA enters the bacterium through pores in its cell wall.
      • Heat Shock: Cells treated with CaCl2 are incubated with rDNA on ice, then briefly placed at 42°C (heat shock), and then put back on ice. This facilitates DNA uptake.
      • Micro-injection: Recombinant DNA is directly injected into the nucleus of an animal cell using a micro-needle.
      • Biolistics or Gene Gun: Cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA. Suitable for plant cells.
      • Electroporation: Brief electrical pulses create transient pores in the cell membrane, allowing DNA entry.
      • Disarmed Pathogen Vectors: Using modified viruses or bacteria (like Agrobacterium) that infect the cell and transfer the rDNA.

  • (d) DNA Ligase ('Molecular Glue'):

    • Enzyme that joins DNA fragments by forming phosphodiester bonds between them.
    • It seals the nicks in the sugar-phosphate backbone after complementary sticky ends have base-paired, creating a stable recombinant DNA molecule.

3. Processes of Recombinant DNA Technology:

  • (a) Isolation of the Genetic Material (DNA):

    • The cell containing the DNA of interest must be broken open (lysis).
    • Enzymes used for lysis depend on the cell type: Lysozyme (bacteria), Cellulase (plant cells), Chitinase (fungus).
    • Other macromolecules like RNA (treated with ribonuclease), proteins (treated with protease), etc., are removed.
    • Purified DNA finally precipitates out after adding chilled ethanol. This appears as a collection of fine threads in the suspension.

  • (b) Cutting of DNA at Specific Locations:

    • Purified DNA is incubated with the chosen restriction enzyme under optimal conditions (temperature, pH, buffer).
    • The vector DNA is also cut with the same restriction enzyme to generate compatible sticky ends.
    • Agarose Gel Electrophoresis: Used to check the progression of restriction enzyme digestion and to purify DNA fragments. DNA fragments separate (resolve) according to their size through the sieving effect of the agarose gel matrix. Smaller fragments move farther. The separated DNA fragments can be visualised after staining with Ethidium Bromide (EtBr) followed by exposure to UV radiation (bright orange bands). The desired band is cut out from the gel (elution).

  • (c) Amplification of Gene of Interest using PCR (Polymerase Chain Reaction):

    • Principle: In vitro synthesis of multiple copies (billions) of a specific DNA sequence using a thermostable DNA polymerase.
    • Requirements:
      • DNA Template: The DNA containing the sequence to be amplified.
      • Primers: Two sets of chemically synthesised oligonucleotides (short DNA sequences) that are complementary to the regions flanking the target DNA sequence (one for each strand).
      • Enzyme: DNA polymerase, usually Taq polymerase (isolated from thermophilic bacterium Thermus aquaticus), which remains active at high temperatures.
      • Deoxynucleotides (dNTPs): dATP, dGTP, dCTP, dTTP.
      • Buffer and Mg2+ ions.
    • Steps in each cycle:
      1. Denaturation: Heating the reaction mixture to a high temperature (e.g., 94-96°C) to separate the two strands of the template DNA.
      2. Annealing: Lowering the temperature (e.g., 50-65°C) to allow the primers to bind (anneal) to their complementary sequences on the separated strands.
      3. Extension: Raising the temperature (e.g., 72°C, optimal for Taq polymerase) to allow the DNA polymerase to synthesize new DNA strands starting from the primers, using the template strands and dNTPs.
    • These three steps constitute one cycle. The process is repeated many times (e.g., 30 cycles), leading to exponential amplification of the target DNA segment.

  • (d) Ligation of DNA Fragment into a Vector:

    • The cut gene of interest (source DNA fragment) and the cut vector DNA are mixed.
    • DNA ligase enzyme is added. It joins the source DNA fragment to the vector DNA by forming phosphodiester bonds, creating recombinant DNA (rDNA).

  • (e) Insertion of Recombinant DNA into the Host Cell/Organism:

    • The rDNA (e.g., plasmid) is introduced into a competent host cell (e.g., E. coli) using methods like heat shock, electroporation, etc. This process is called transformation (for bacteria).

  • (f) Selection and Screening of Transformed Cells:

    • Using selectable markers (e.g., antibiotic resistance, chromogenic substrate) to identify cells that have taken up the plasmid (transformants) and specifically those containing the recombinant plasmid (recombinants). (Refer back to 'Insertional Inactivation').

  • (g) Obtaining the Foreign Gene Product (Recombinant Protein):

    • The ultimate aim is often to produce a desired protein encoded by the gene of interest.
    • The foreign gene gets expressed under appropriate conditions provided in the host cell.
    • Bioreactors: For large-scale production, cells containing the rDNA are cultured in large vessels called bioreactors (100-1000 litres).
      • Provide optimal growth conditions: temperature, pH, substrate, salts, vitamins, oxygen.
      • Common type: Stirred-tank reactor (usually cylindrical, curved base for mixing). Has an agitator system, oxygen delivery system, foam control, temperature control, pH control, sampling ports.
      • Sparged stirred-tank reactor: Air is bubbled through the reactor for increased oxygen transfer.
    • The culture leads to the production of the target protein (or other biochemical).

  • (h) Downstream Processing (DSP):

    • A series of processes after the biosynthetic stage (bioreactor).
    • Includes:
      • Separation: Isolating the product from the reactor medium.
      • Purification: Removing impurities to obtain a pure product.
      • Formulation: Adding preservatives and preparing the product suitable for use (e.g., as a drug).
      • Quality Control Testing: Ensuring the product meets required standards of safety and efficacy.
    • DSP varies depending on the product and is crucial for the final marketable product.

4. Significance:

Recombinant DNA technology has revolutionized biology and medicine, enabling the production of therapeutic drugs (e.g., insulin, growth hormone), vaccines, diagnostic tools, genetically modified crops with improved traits, and advancements in basic research like gene function studies.

Multiple Choice Questions (MCQs):

  1. Which enzyme is responsible for cutting DNA at specific recognition nucleotide sequences?
    a) DNA Ligase
    b) DNA Polymerase
    c) Restriction Endonuclease
    d) Helicase

  2. The sequence 5'-GAATTC-3' is a recognition site for which restriction enzyme?
    a) Hind III
    b) BamHI
    c) EcoRI
    d) Sal I

  3. Which of the following is NOT an essential feature of a cloning vector?
    a) Origin of replication (ori)
    b) Selectable marker
    c) Restriction sites
    d) Interferon gene

  4. The thermostable DNA polymerase used in PCR is isolated from:
    a) Escherichia coli
    b) Agrobacterium tumefaciens
    c) Thermus aquaticus
    d) Saccharomyces cerevisiae

  5. The process of introducing foreign DNA into a bacterial host cell by treating it with calcium chloride followed by a brief heat exposure is called:
    a) Transduction
    b) Transformation (Heat Shock method)
    c) Conjugation
    d) Microinjection

  6. In the pBR322 vector, if a foreign gene is inserted at the BamHI site, the recombinant plasmid will confer resistance to:
    a) Tetracycline only
    b) Ampicillin only
    c) Both Ampicillin and Tetracycline
    d) Neither Ampicillin nor Tetracycline

  7. Agarose gel electrophoresis separates DNA fragments primarily based on their:
    a) Charge
    b) Sequence
    c) Size
    d) A-T content

  8. The technique used to introduce foreign DNA into plant cells by bombarding them with high-velocity micro-particles coated with DNA is:
    a) Electroporation
    b) Microinjection
    c) Biolistics (Gene Gun)
    d) Lipofection

  9. The final series of processes involving separation, purification, and formulation of the product obtained from bioreactors is known as:
    a) Upstream processing
    b) Bioprocessing
    c) Downstream processing
    d) Fermentation

  10. The role of DNA ligase in recombinant DNA technology is to:
    a) Cut DNA at specific sites
    b) Synthesize DNA from an RNA template
    c) Join DNA fragments by forming phosphodiester bonds
    d) Separate DNA strands

Answer Key for MCQs:

  1. c) Restriction Endonuclease
  2. c) EcoRI
  3. d) Interferon gene
  4. c) Thermus aquaticus
  5. b) Transformation (Heat Shock method)
  6. b) Ampicillin only (Tetracycline resistance is lost due to insertional inactivation)
  7. c) Size
  8. c) Biolistics (Gene Gun)
  9. c) Downstream processing
  10. c) Join DNA fragments by forming phosphodiester bonds

Make sure you understand the 'why' behind each step and tool, not just the 'what'. This understanding is key for tackling application-based questions in your exams. Good luck with your preparation!

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