CBSE Class 12 Biology Chapter 11: Biotechnology Principles and Processes NCERT Solutions

Here are 10 recombinant proteins used in medical practice, along with their therapeutic applications:

1. Human insulin – Used to treat diabetes mellitus by regulating blood glucose levels.
2. Human growth hormone – Used to treat dwarfism caused by growth hormone deficiency.
3. Blood clotting factor VIII/IX – Used to treat haemophilia, a genetic disorder causing improper blood clotting.
4. TPA (tissue plasminogen activator) – Used to dissolve blood clots, treating heart attacks and strokes.
5. PDGF (platelet derived growth factor) – Stimulates wound healing and tissue repair.
6. Interferon – Used in the treatment of viral infections and certain types of cancer.
7. Interleukin – Enhances immune reactions, used in cancer therapy and treating immune deficiencies.
8. Hepatitis B vaccine – A recombinant vaccine used for the prevention of Hepatitis B infection.
9. Herpes Vaccine – Used for the prevention of infectious diseases caused by the herpes simplex virus.
10. DNase I – Used in the treatment of cystic fibrosis to break down DNA in mucus, making it easier to clear.

Here is a representation of a restriction enzyme's action:

Name of the Restriction Enzyme: Bam HI

Substrate DNA: A DNA molecule containing the recognition sequence for Bam HI.

Recognition Site (Site at which it cuts DNA): The specific DNA sequence that the restriction enzyme recognizes and binds to. For Bam HI, this is 5'—GGATCC—3'.

Recognition Site:

5'— G G A T C C —3'

3'— C C T A G G —5'

Action of the Enzyme: Bam HI cuts the DNA within its recognition site, typically between the G and G bases.

Cutting Pattern:

5'— G | G A T C C —3'

3'— C C T A G | G —5'

Product it Produces: The enzyme produces DNA fragments with 'sticky ends' – short, single-stranded overhangs that can readily base-pair with complementary sticky ends.

Sticky Ends:

5'— G

3'— C C T A G G —5' G A T C C —3'

5'— G G A T C C —3' G 3'— These sticky ends are essential for the subsequent ligation of DNA fragments in recombinant DNA technology.

Enzymes are generally bigger in molecular size than DNA. This is because enzymes are proteins, and proteins are macromolecules composed of amino acids. DNA, on the other hand, is a nucleic acid made up of nucleotides. Amino acids are larger molecules than nucleotides. Therefore, a protein molecule (enzyme) typically has a much larger molecular weight and size compared to a DNA molecule, especially considering that a single gene (a segment of DNA) is usually much smaller than a functional enzyme.

This difference in size can be observed using techniques like gel electrophoresis. When denatured proteins and DNA fragments are run on a gel, proteins (enzymes) would generally migrate slower through the gel matrix due to their larger size, indicating they are bigger molecules.

The molar concentration of human DNA in a typical human cell is not a fixed, easily stated value like a simple molarity (moles per liter) because it depends on the cell's volume and the amount of DNA present. However, the source material indicates that the concentration of DNA in a human cell extract is approximately 2 mg/ml. To determine the molar concentration, one would need to know the average molecular weight of the human genome and the volume of the cell. This calculation is complex and often requires consultation with specialized resources or teachers.

No, eukaryotic cells do not naturally possess restriction endonucleases. The justification is that the DNA molecules in eukaryotes are typically heavily methylated, which interferes with the action of restriction enzymes. Restriction endonucleases have been exclusively isolated from various strains of bacteria, where they function as a defense mechanism against invading viral DNA by cutting it at specific sites.

While shake flasks are suitable for small-scale laboratory cultivation and mixing of desired materials, large-scale production of biotechnological products is carried out in bioreactors. Besides improved aeration and mixing, stirred tank bioreactors offer several significant advantages over shake flasks:

• Sampling: Small volumes of cultures can be periodically withdrawn from the bioreactor for sampling and analysis without disrupting the entire process.
• Control Systems: Bioreactors are equipped with sophisticated control systems for monitoring and maintaining optimal conditions, including foam control, pH control, and temperature control.
• Uniform Conditions: They facilitate even mixing and ensure consistent oxygen availability throughout the entire volume of the bioreactor, promoting uniform cell growth and product formation.
• Sterility Maintenance: Bioreactors are designed to maintain sterile conditions, preventing contamination by unwanted microorganisms.
• Process Optimization: The controlled environment allows for precise optimization of growth parameters, leading to higher yields and product quality.

Palindromic nucleotide sequences in DNA are segments where the sequence of bases reads the same forwards and backwards on the complementary strand. This means the sequence on the 5' to 3' direction of one strand is identical to the sequence on the 5' to 3' direction of the complementary strand.

Here are five examples of palindromic DNA sequences:

1. 5'—GGATCC—3' 3'—CCTAGG—5'
2. 5'—AAGCTT—3' 3'—TTCGAA—5'
3. 5'—ACGCGT—3' 3'—TGCGC A—5'
4. 5'—ACTAGT—3' 3'—TGATCA—5'
5. 5'—AGGCCT—3' 3'—TCCGGA—5'

To create a palindromic sequence, you can start with a sequence on one strand (e.g., 5'—GATC—3') and then write its complementary sequence in the opposite direction (3'—CTAG—5'). Reading the top strand 5' to 3' gives GAATTC. Reading the bottom strand 5' to 3' (which is written 3' to 5' in the standard representation) also gives GAATTC.

Recombinant DNA, in the context of natural genetic recombination, is made during Meiosis I, specifically at the Pachytene stage. During this stage, homologous chromosomes pair up to form synaptonemal complexes. Following the formation of these complexes, crossing over occurs between non-sister chromatids at points called chiasmata. This exchange of genetic material between homologous chromosomes results in the formation of recombinant DNA molecules.

A reporter enzyme can be used to monitor the transformation of host cells by foreign DNA, providing a visual or easily detectable signal of successful gene insertion, complementing the function of a selectable marker. For example, the $\backslash beta$-galactosidase (Lac Z) gene is often used as a reporter gene. When a foreign DNA fragment is successfully inserted into the cloning vector within the Lac Z gene's coding sequence (disrupting it), the enzyme $\backslash beta$-galactosidase is not produced. Host cells transformed with such a vector will not be able to metabolize a specific substrate (like X-gal), and thus will appear white when grown on a medium containing X-gal. In contrast, cells that were not successfully transformed or contain a vector without the inserted foreign DNA will produce functional $\backslash beta$-galactosidase, metabolize X-gal, and appear blue. This allows for easy identification and selection of recombinant cells based on their color phenotype, in addition to the selectable marker (e.g., antibiotic resistance) which ensures only transformed cells survive.

(a) Origin of Replication (ori): This is a specific DNA sequence from where the process of DNA replication begins. Any DNA fragment, when linked to this sequence, can be replicated within the host cell. The 'ori' also controls the copy number of the linked DNA. If one desires to recover many copies of the target DNA, it should be cloned in a vector that supports a high copy number originating from a strong 'ori'.

(b) Bioreactors: Bioreactors are large vessels designed for the biological conversion of raw materials into specific products using microorganisms, plant cells, or animal cells and their enzymes. They provide optimal conditions for microbial growth and product formation. The most common type is the stirred tank bioreactor, which is typically a cylindrical vessel with a curved base. It includes systems for agitation, oxygen delivery, foam control, pH and temperature control, and sampling ports. Sterile air is often sparged into the reactor to ensure adequate oxygen supply.

(c) Downstream Processing: This refers to the series of processes required to isolate and purify the desired product obtained from a biotechnological process. After the biological conversion in the bioreactor, the product is separated from the host cells and other impurities. This involves steps like cell lysis (if the product is intracellular), filtration, centrifugation, chromatography, and crystallization. The purified product is then formulated with suitable preservatives and may undergo clinical trials before being made into a finished product ready for marketing.

(a) PCR (Polymerase Chain Reaction): PCR is a molecular biology technique used to amplify a specific segment of DNA in vitro, meaning outside a living organism. It involves repeated cycles of three main steps:

1. Denaturation: The double-stranded DNA template is heated to a high temperature (around 94-98°C) to separate it into single strands.
2. Annealing: The temperature is lowered (around 50-65°C) to allow short synthetic DNA sequences called primers to bind (anneal) to complementary regions on the single-stranded DNA templates.
3. Extension: The temperature is raised to an optimal point for a thermostable DNA polymerase (like Taq polymerase, isolated from Thermus aquaticus) to synthesize new DNA strands, starting from the primers.

This process is repeated for many cycles, exponentially increasing the quantity of the target DNA sequence. PCR is widely used for gene cloning, genetic fingerprinting, and diagnostics.

(b) Restriction enzymes and DNA: Restriction enzymes are a class of enzymes, primarily endonucleases, that recognize specific short DNA sequences, known as recognition sites, and cleave the DNA strands at or near these sites. These enzymes are a natural defense mechanism in bacteria to restrict the growth of foreign DNA, such as that from viruses, by degrading it. The recognition sites are typically palindromic. Restriction enzymes are essential tools in genetic engineering for cutting DNA molecules precisely, allowing for the isolation of genes and the construction of recombinant DNA molecules. They can produce either blunt ends or sticky ends (overhanging single-stranded regions) depending on the enzyme and the cleavage site.

(c) Chitinase: Chitinase is an enzyme that catalyzes the hydrolysis of chitin, a long-chain polymer of N-acetylglucosamine. Chitin is a major structural component of the cell walls of fungi and the exoskeletons of arthropods (like insects and crustaceans). In biotechnology, chitinase can be used as part of the process to break down the cell walls of fungi or to extract cellular components from arthropods. For instance, if a gene of interest is located within a fungal cell, chitinase could be used to degrade the fungal cell wall, facilitating access to the cell's contents.