Chapter 9: Biotechnology : Principles and Processes Long Questions | biology Class 12 CBSE

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Chapter 9: Biotechnology : Principles and Processes Long Questions | biology Class 12 CBSE | ByteNotes.in

Long Answer

Hard difficulty • Structured explanation

Hard

Question 1

Long Form

Describe the features of a cloning vector that make it suitable for recombinant DNA technology, with reference to pBR322.

A cloning vector must have an origin of replication (ori) to replicate autonomously in the host cell and maintain linked foreign DNA; the ori also determines copy number.
It must carry a selectable marker, such as antibiotic resistance genes (ampR and tetR in pBR322), to distinguish transformed cells from non-transformed ones on selective media.
pBR322 contains multiple unique restriction sites (Hind III, EcoRI, BamHI, SalI, PvuII, PstI, ClaI), allowing insertion of foreign DNA at specific locations; rop gene codes for proteins involved in plasmid replication control.
Insertion of foreign DNA at the BamHI site within the tetR gene inactivates tetracycline resistance (insertional inactivation), enabling recombinants (AmpR, TetS) to be distinguished from non-recombinants (AmpR, TetR) by replica plating.
The vector must be small enough to be easily manipulated in vitro and to enter host cells efficiently; pBR322 fulfils this requirement with a compact, well-characterised structure.

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Long Answer

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Question 2

Long Form

Compare the roles of restriction endonucleases and DNA ligase in the construction of recombinant DNA. How do sticky ends facilitate this process?

Restriction endonucleases act as 'molecular scissors'; they recognise specific palindromic sequences in DNA and cleave both strands at or near the recognition site, generating defined DNA fragments.
They cut the DNA strands at staggered positions within the palindrome, producing short single-stranded overhangs called sticky ends on each fragment, which are sequence-specific.
Sticky ends from the same restriction enzyme are complementary to each other; they can base-pair (form hydrogen bonds) with matching ends from other DNA fragments cut by the same enzyme.
DNA ligase acts as 'molecular glue'; it seals the nicks in the sugar-phosphate backbone by forming covalent phosphodiester bonds between the 3'-OH and 5'-phosphate ends of base-paired sticky end fragments.
Together, both enzymes allow precise, reversible joining of DNA from different sources: restriction enzymes create compatible ends and ligase permanently seals them, forming a stable circular recombinant DNA molecule.

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Long Answer

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Hard

Question 3

Long Form

Outline the complete sequence of steps involved in recombinant DNA technology from isolation of DNA to obtaining the final product.

DNA isolation: Cells are broken open using lytic enzymes (lysozyme/cellulase/chitinase); RNA is removed with ribonuclease and proteins with protease; purified DNA precipitates in chilled ethanol.
Fragmentation and vector preparation: Source DNA and vector are treated with the same restriction endonuclease to generate compatible sticky ends; gel electrophoresis checks the digestion.
Ligation and amplification: The desired DNA fragment is ligated into the cut vector using DNA ligase, forming recombinant DNA; PCR can amplify the gene of interest prior to ligation if needed.
Transformation and selection: Recombinant DNA is introduced into competent host cells (heat shock, biolistics, or micro-injection); transformed cells are selected on antibiotic media using selectable markers.
Expression, scale-up, and downstream processing: The foreign gene is expressed in host cells; bioreactors are used for large-scale culture; the product is separated, purified, formulated, clinically tested, and quality-checked before marketing.

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Long Answer

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Hard

Question 4

Long Form

Analyse the different methods used to introduce recombinant DNA into host cells, highlighting the principle behind each method.

Chemical method (heat shock): Bacterial cells are treated with divalent cations (Ca2+) to make them competent; they are incubated with DNA on ice, heat-shocked at 42°C, and returned to ice, allowing DNA uptake through cell wall pores.
Micro-injection: A fine glass needle is used to directly inject recombinant DNA into the nucleus of animal cells; it physically delivers DNA without chemical treatment, suitable for embryonic or oocyte cells.
Biolistics (gene gun): DNA-coated gold or tungsten microparticles are fired at high velocity into plant cells, physically penetrating cell walls and membranes to deliver DNA; preferred for plant cells with rigid walls.
Disarmed pathogen vectors: Modified (disarmed) pathogens like Agrobacterium tumifaciens (Ti plasmid) or retroviruses naturally infect host cells and transfer recombinant DNA into the host genome, exploiting natural infection machinery.
Each method is chosen based on the cell type: chemical/heat shock for bacteria, micro-injection for animal cells, biolistics for plants, and vector-mediated infection for a wide range of eukaryotic hosts.

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Long Answer

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Medium

Question 5

Long Form

Explain the principle and steps of PCR. How does this technique overcome the limitations of conventional DNA cloning?

PCR (Polymerase Chain Reaction) amplifies a specific DNA sequence in vitro using primers, thermostable Taq polymerase, and repeated thermal cycles; one billion copies can be generated from a single template.
Step 1 — Denaturation: The reaction mixture is heated (~94°C) to separate the double-stranded DNA into single strands that serve as templates.
Step 2 — Primer annealing: The temperature is lowered (~50–60°C) so that short oligonucleotide primers complementary to the flanking regions of the target sequence bind to the template strands.
Step 3 — Extension: Temperature is raised (~72°C); Taq polymerase extends the primers using supplied dNTPs, synthesising new complementary strands and doubling the copies of the target sequence each cycle.
Advantage over conventional cloning: PCR amplifies DNA without the need for host cells or vectors; Taq polymerase's thermostability enables repeated cycles without adding fresh enzyme, allowing rapid and massive amplification of even trace amounts of target DNA.

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Long Answer

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Hard

Question 6

Long Form

Discuss how traditional hybridisation in plant and animal breeding differs from genetic engineering and why genetic engineering is considered superior for targeted trait introduction.

Traditional hybridisation involves crossing two organisms with different traits; because genes are inherited in groups on chromosomes, desirable genes are often co-inherited with many undesirable genes.
Conventional breeding requires multiple backcrossing cycles over several generations to eliminate undesired traits, which is time-consuming and often imprecise.
Genetic engineering allows isolation of a single gene or a specific set of genes encoding a desired trait, using restriction enzymes and gene cloning, without disturbing other genes in the organism's genome.
The identified gene can be transferred into any target organism (even across species barriers) using vectors or physical methods, offering a precision impossible in classical breeding.
Genetic engineering also overcomes reproductive barriers between distantly related species, enabling introduction of novel traits (e.g., pest resistance, drought tolerance) that do not exist in the species' gene pool.

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Long Answer

Medium difficulty • Structured explanation

Medium

Question 7

Long Form

Explain the role of bioreactors in large-scale production of biotechnological products. Describe the features of a stirred-tank bioreactor.

Bioreactors are large vessels (100–1000 litre capacity) that provide the optimal environment for biological conversion of raw materials into desired products using microbial, plant, animal, or human cells expressing recombinant genes.
They allow continuous culture where spent medium is continuously replaced with fresh medium, maintaining cells in the exponential growth phase and maximising biomass and product yield.
A stirred-tank bioreactor is cylindrical (or with a curved base) containing an agitator system that ensures uniform mixing of nutrients and even oxygen distribution throughout the vessel.
It incorporates an oxygen delivery system (sterile air sparging), foam control system, temperature and pH control systems, and sampling ports for periodic culture monitoring and quality assessment.
Bioreactors are essential because small-volume laboratory cultures cannot yield commercially significant quantities of recombinant proteins; scale-up in bioreactors bridges the gap between laboratory-scale production and marketable quantities.

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Long Answer

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Medium

Question 8

Long Form

Describe the isolation of genetic material (DNA) from cells. What impurities must be removed and how?

Cells are lysed using cell-wall-degrading enzymes: lysozyme for bacteria, cellulase for plant cells, and chitinase for fungal cells, releasing DNA along with other macromolecules such as proteins, RNA, polysaccharides, and lipids.
The mixture is treated with ribonuclease (RNase) to degrade contaminating RNA, since RNA may interfere with downstream applications requiring pure DNA.
Protease enzymes are added to remove proteins, including histones that are tightly associated with DNA in eukaryotic cells, ensuring the DNA is in a free, unbound form.
Other contaminating molecules (polysaccharides, lipids) are removed by appropriate chemical treatments and differential centrifugation or extraction methods.
Purified DNA finally precipitates out of solution upon addition of chilled ethanol and can be seen as fine threads or spools, which are collected (spooling) and used in downstream recombinant DNA procedures.

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Long Answer

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Hard

Question 9

Long Form

Explain why an alien piece of DNA must be linked to an origin of replication to multiply in a host organism. How does this relate to the concept of cloning?

When a foreign DNA fragment is introduced into a host cell, it will not survive or multiply unless it can initiate its own replication independently of the host chromosome.
A chromosome carries a specific sequence called the origin of replication (ori), which is the site where DNA replication begins; only DNA that contains or is linked to an ori can be replicated by the host's DNA polymerase.
By linking an alien DNA fragment to a vector that carries an ori, the alien DNA effectively becomes part of a replicating unit and is copied every time the vector replicates, multiplying along with it.
Cloning refers to making multiple identical copies of the alien DNA; the vector's ori ensures that every time a host cell divides, copies of the recombinant DNA (including the alien fragment) are passed to daughter cells.
Thus, the ori is the foundation for cloning, because without it the alien DNA is lost from the population; with it, the DNA is stably maintained and amplified in all progeny of the transformed host.

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Long Answer

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Hard

Question 10

Long Form

Compare the use of plasmids and bacteriophages as cloning vectors. What advantages do bacteriophages have in terms of copy number?

Plasmids are small, circular, extrachromosomal DNA molecules that replicate independently in bacterial cells and are present in limited copy numbers (1–2 or 15–100 copies per cell depending on the type).
Bacteriophages are viruses that infect bacteria and have a very high copy number per cell (many phage genomes replicate per infected bacterium), making them superior for producing many copies of a cloned gene.
Plasmids are easier to isolate and manipulate in vitro and are preferred for expressing foreign genes in bacteria because they can be introduced by simple transformation.
Bacteriophage vectors (such as lambda phage) can accommodate larger DNA inserts than typical plasmids, making them useful for constructing genomic libraries.
Both vectors must be engineered with appropriate selectable markers, restriction sites, and ori sequences; modern vectors are hybrids (cosmids, phagemids) combining advantages of both plasmids and phages.

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Chapter 9: Biotechnology : Principles and Processes | Long Questions

Describe the features of a cloning vector that make it suitable for recombinant DNA technology, with reference to pBR322.

Compare the roles of restriction endonucleases and DNA ligase in the construction of recombinant DNA. How do sticky ends facilitate this process?

Outline the complete sequence of steps involved in recombinant DNA technology from isolation of DNA to obtaining the final product.

Analyse the different methods used to introduce recombinant DNA into host cells, highlighting the principle behind each method.

Explain the principle and steps of PCR. How does this technique overcome the limitations of conventional DNA cloning?

Discuss how traditional hybridisation in plant and animal breeding differs from genetic engineering and why genetic engineering is considered superior for targeted trait introduction.

Explain the role of bioreactors in large-scale production of biotechnological products. Describe the features of a stirred-tank bioreactor.

Describe the isolation of genetic material (DNA) from cells. What impurities must be removed and how?

Explain why an alien piece of DNA must be linked to an origin of replication to multiply in a host organism. How does this relate to the concept of cloning?

Compare the use of plasmids and bacteriophages as cloning vectors. What advantages do bacteriophages have in terms of copy number?

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