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

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

Application Question

Medium difficulty • Concept in a practical situation

Medium

Question 1

Applied Concept

A scientist wants to clone a human insulin gene into E. coli. She cuts both the human DNA and the plasmid vector with EcoRI. Explain why it is necessary to use the same restriction enzyme for both the source DNA and the vector, and what would happen if different enzymes were used.

EcoRI recognises the palindromic sequence 5'-GAATTC-3' and cuts both strands, leaving identical 4-nucleotide 5'-AATT-3' sticky ends on all fragments; using the same enzyme on both human DNA and the plasmid ensures that the insulin gene fragment and the linearised vector have complementary sticky ends that can base-pair with each other.
If different restriction enzymes were used (e.g., EcoRI for the insert and BamHI for the vector), the sticky ends generated would be non-complementary—the single-stranded overhangs would not be able to form hydrogen bonds with each other, preventing ligation by DNA ligase.
The consequence would be failure to form a recombinant plasmid; even if blunt-end ligation were attempted, it would be far less efficient and would not be sequence-specific, potentially allowing the vector to re-ligate to itself without the insulin gene insert, reducing recombinant clone frequency.

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Application Question

Medium difficulty • Concept in a practical situation

Medium

Question 2

Applied Concept

A student performing gel electrophoresis of a restriction-digested DNA sample loads the sample at one end of the agarose gel. After running the gel, she observes that some bands are near the loading end and others have migrated far. With reference to the principles of gel electrophoresis, explain the basis for the observed pattern.

DNA fragments are negatively charged due to the phosphate groups in their backbone; when an electric field is applied, all fragments migrate from the negatively charged end (cathode, where the sample is loaded) toward the positively charged end (anode).
Agarose gel acts as a molecular sieve; smaller DNA fragments encounter less resistance as they thread through the pores of the gel matrix and therefore migrate faster, travelling farther from the loading end in a given time period.
Larger DNA fragments are retarded more by the gel matrix and remain closer to the loading end; thus bands near the loading end represent large fragments and bands farther away represent small fragments, allowing size determination by comparison with a DNA ladder of known fragment sizes.

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Application Question

Hard difficulty • Concept in a practical situation

Hard

Question 3

Applied Concept

A biotechnology company wants to produce a therapeutic enzyme in large quantities for clinical use. They have successfully expressed the gene in E. coli at laboratory scale. What challenges would they face in scaling up production, and how would a bioreactor help overcome them?

At laboratory scale (shake flasks), volumes are limited to a few hundred millilitres, yielding insufficient quantities of the enzyme; aeration and mixing are inconsistent, leading to uneven growth and variable product quality, neither of which is acceptable for clinical-grade production.
A stirred-tank bioreactor with capacities of 100–1000 litres allows massively increased culture volume; its agitator system, oxygen sparging, and foam control ensure uniform mixing and oxygenation, supporting consistent exponential-phase growth and high, reproducible enzyme yields.
Bioreactors also allow continuous culture mode where spent medium is continuously replaced with fresh medium, maintaining cells in optimal physiological state for prolonged production; precise automated control of temperature and pH minimises batch-to-batch variability essential for therapeutic product consistency.

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Application Question

Hard difficulty • Concept in a practical situation

Hard

Question 4

Applied Concept

With reference to the vector pBR322, a researcher inserts a foreign gene at the BamHI restriction site. She then plates the transformed bacteria on ampicillin medium and on tetracycline medium. Interpret the results she would observe for recombinant and non-recombinant colonies.

The BamHI site lies within the tetracycline resistance gene (tetR) of pBR322; inserting foreign DNA at this site disrupts the tetR gene (insertional inactivation) while leaving the ampicillin resistance gene (ampR) intact, so recombinant bacteria carry a functional ampR but a non-functional tetR.
On ampicillin medium, both recombinant and non-recombinant transformants (i.e., all bacteria that took up the plasmid) will grow, since both carry functional ampR; non-transformed bacteria (no plasmid) will die on this medium.
On tetracycline medium, only non-recombinant bacteria (intact tetR) will grow; recombinant bacteria (disrupted tetR) will not grow on this medium—so colonies that grow on ampicillin but fail to grow when replica-plated on tetracycline are identified as recombinants containing the foreign insert.

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Application Question

Medium difficulty • Concept in a practical situation

Medium

Question 5

Applied Concept

A forensic scientist needs to identify a suspect from a tiny blood sample found at a crime scene. The DNA available is in very small quantities. Which biotechnological technique would be most appropriate, and how would it work?

PCR (Polymerase Chain Reaction) is the most appropriate technique, as it can amplify even a trace amount of DNA (even from a few cells) to produce billions of copies, sufficient for analysis.
Specific primers complementary to the target DNA region (e.g., short tandem repeat loci used in DNA fingerprinting) are designed; repeated cycles of denaturation, primer annealing, and extension by thermostable Taq polymerase exponentially amplify the target sequence.
After approximately 30 cycles, the target sequence is amplified approximately one billion-fold; the amplified DNA can then be analysed by gel electrophoresis to generate a DNA profile unique to the individual, which can be compared with the suspect's reference profile for identification.

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Application Question

Medium difficulty • Concept in a practical situation

Medium

Question 6

Applied Concept

A plant genetic engineer wants to introduce a pest-resistance gene into a wheat plant. She cannot use chemical transformation as wheat is a monocot and Agrobacterium tumifaciens-based vectors are not efficient for monocots. What alternative method should she use and why?

The engineer should use biolistics (gene gun), which physically delivers DNA into plant cells by bombarding them with high-velocity gold or tungsten microparticles coated with the pest-resistance gene DNA.
The gene gun is effective for monocots and other plants where Agrobacterium-mediated transformation is inefficient, because the physical force of the particles penetrates the tough cell walls of cereal crop cells without requiring biological infection.
Once delivered into the cell, the coated DNA is released from the particles and can integrate into the plant genome; regeneration of whole transgenic plants from transformed cells is then achieved through tissue culture, allowing the pest-resistance gene to be stably inherited by all progeny.

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Application Question

Medium difficulty • Concept in a practical situation

Medium

Question 7

Applied Concept

With reference to the properties of DNA and cell membranes, explain why simply mixing recombinant plasmid DNA with bacterial cells in a test tube is insufficient to achieve transformation.

DNA is a hydrophilic (water-loving) molecule with a highly negatively charged phosphate backbone; because cell membranes are hydrophobic lipid bilayers, DNA cannot spontaneously diffuse through the membrane under normal conditions.
The electrostatic repulsion between the negatively charged DNA and the negatively charged lipopolysaccharide components of the bacterial outer membrane further prevents DNA from approaching and entering the cell.
Therefore, bacterial cells must first be made 'competent' by treating them with divalent cations such as Ca2+ ions, which neutralise charge repulsion and promote the formation of DNA–cell interactions; subsequent heat shock at 42°C is then required to induce the cells to actively take up the DNA.

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Application Question

Hard difficulty • Concept in a practical situation

Hard

Question 8

Applied Concept

Imagine you are working in a diagnostic laboratory and need to determine whether a patient's DNA sample carries a specific disease-associated mutation. Describe how PCR and gel electrophoresis together would help you diagnose the condition.

PCR would be used first to specifically amplify the region of the patient's DNA flanking the mutation site, using primers designed to flank the mutation; this generates millions of copies of the diagnostic region from even a small blood or tissue sample.
If the mutation creates or destroys a restriction enzyme recognition site, the PCR product can be digested with the appropriate restriction enzyme; the resulting fragments will differ in size between the normal and mutant allele.
Agarose gel electrophoresis of the digested PCR product will show distinct band patterns for the normal versus mutant genotype; a patient homozygous for the mutation shows only mutant bands, a carrier shows both, and a normal individual shows only normal bands, allowing unambiguous diagnosis.

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Application Question

Hard difficulty • Concept in a practical situation

Hard

Question 9

Applied Concept

A microbiologist discovers a new bacterial strain that grows optimally at 65°C. Why would Taq polymerase from Thermus aquaticus be preferred over polymerases from this newly discovered strain for PCR applications?

PCR requires DNA denaturation at approximately 94–95°C to separate the double-stranded DNA template into single strands; a polymerase from a bacterium that grows optimally at 65°C would likely be inactivated at 94°C, as its optimal temperature is far below the denaturation step.
Taq polymerase from Thermus aquaticus (a hot-spring bacterium) is specifically adapted to function at high temperatures, with optimal activity around 72°C and stability at 94°C, allowing it to survive repeated denaturation cycles without losing activity.
Without a thermostable polymerase like Taq, fresh polymerase would need to be added after every denaturation step, making PCR technically impractical and prohibitively expensive for large-scale or routine use.

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Application Question

Hard difficulty • Concept in a practical situation

Hard

Question 10

Applied Concept

In a blue-white selection experiment using β-galactosidase, a student observes that all colonies on the plate are blue. What does this indicate about the transformation experiment, and what could have gone wrong?

Blue colonies indicate that the β-galactosidase gene in all transformants is intact and functional, meaning no foreign DNA insert has been incorporated into the gene; all colonies are therefore non-recombinant.
The most likely explanation is that the vector DNA was not properly cut by the restriction enzyme (incomplete digestion), allowing the circular vector to re-ligate to itself without incorporating the insert, regenerating intact β-galactosidase gene.
Another possible reason is that the foreign DNA fragment was not present in the ligation reaction, or the ligation conditions were suboptimal; to prevent this, one can treat the linearised vector with alkaline phosphatase to remove 5'-phosphate groups, preventing self-ligation and increasing the frequency of insert incorporation.

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Chapter 9: Biotechnology : Principles and Processes | Application Questions

A scientist wants to clone a human insulin gene into E. coli. She cuts both the human DNA and the plasmid vector with EcoRI. Explain why it is necessary to use the same restriction enzyme for both the source DNA and the vector, and what would happen if different enzymes were used.

A biotechnology company wants to produce a therapeutic enzyme in large quantities for clinical use. They have successfully expressed the gene in E. coli at laboratory scale. What challenges would they face in scaling up production, and how would a bioreactor help overcome them?

With reference to the vector pBR322, a researcher inserts a foreign gene at the BamHI restriction site. She then plates the transformed bacteria on ampicillin medium and on tetracycline medium. Interpret the results she would observe for recombinant and non-recombinant colonies.

A forensic scientist needs to identify a suspect from a tiny blood sample found at a crime scene. The DNA available is in very small quantities. Which biotechnological technique would be most appropriate, and how would it work?

A plant genetic engineer wants to introduce a pest-resistance gene into a wheat plant. She cannot use chemical transformation as wheat is a monocot and Agrobacterium tumifaciens-based vectors are not efficient for monocots. What alternative method should she use and why?

With reference to the properties of DNA and cell membranes, explain why simply mixing recombinant plasmid DNA with bacterial cells in a test tube is insufficient to achieve transformation.

Imagine you are working in a diagnostic laboratory and need to determine whether a patient's DNA sample carries a specific disease-associated mutation. Describe how PCR and gel electrophoresis together would help you diagnose the condition.

A microbiologist discovers a new bacterial strain that grows optimally at 65°C. Why would Taq polymerase from Thermus aquaticus be preferred over polymerases from this newly discovered strain for PCR applications?

In a blue-white selection experiment using β-galactosidase, a student observes that all colonies on the plate are blue. What does this indicate about the transformation experiment, and what could have gone wrong?

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