101. The Ti plasmid of Agrobacterium tumefaciens can be adapted as a cloning vector for plants after it is disarmed. In this context, disarming means that
ⓐ. all DNA-transfer functions are removed together with pathogenicity
ⓑ. the plasmid is converted into a non-pathogenic bacterial chromosome
ⓒ. pathogenic functions are removed but DNA-delivery ability is retained
ⓓ. the plasmid is destroyed after each plant transformation event
Correct Answer: pathogenic functions are removed but DNA-delivery ability is retained
Explanation: A disarmed Ti plasmid is engineered so that the harmful functions associated with the natural pathogenic interaction are eliminated, while the biological machinery that allows DNA delivery into plant cells is retained. This is an important biotechnology principle: useful properties of a natural pathogen can be separated from its disease-causing effects. The modified system can then be used to carry a gene of interest into plant cells without preserving the original harmful role. Disarming therefore does not mean eliminating the entire plasmid or abolishing transfer. Instead, it means retaining the useful delivery function while removing the undesirable pathogenic activity.
102. A proposed modification of the Ti plasmid removes both its pathogenic effect and its ability to transfer DNA into plant cells. Why would this modification make the plasmid unsuitable as a plant cloning vector?
ⓐ. DNA-delivery ability must be retained even after pathogenicity is removed
ⓑ. successful plant transformation requires the plasmid to remain pathogenic
ⓒ. DNA-delivery ability is required only for animal-cell transformation
ⓓ. loss of pathogenicity necessarily blocks plasmid replication in bacteria
Correct Answer: DNA-delivery ability must be retained even after pathogenicity is removed
Explanation: The purpose of disarming a natural pathogenic vector is selective: the harmful functions should be removed, but the useful gene-transfer capability must remain. If both pathogenicity and DNA delivery are eliminated, the modified plasmid loses the very property that makes it useful for plant transformation. The biotechnology strategy is therefore based on functional separation. Natural Agrobacterium biology provides a mechanism for moving DNA into plant cells, and vector engineering redirects that mechanism toward transfer of a desired gene rather than disease production. Removing the transfer mechanism would defeat the cloning objective.
103. Consider the following statements about Agrobacterium-derived vectors:
I. Natural Agrobacterium can transfer T-DNA into susceptible plant cells.
II. A disarmed Ti plasmid retains useful DNA-delivery capacity.
III. Disarming is intended to preserve the original pathogenic effect.
IV. The engineered system can be used as a plant gene-delivery vector.
Which option is correct?
ⓐ. I and III only
ⓑ. II and III only
ⓒ. I, II and IV only
ⓓ. I, II, III and IV
Correct Answer: I, II and IV only
Explanation: Statements I, II and IV correctly describe the logic of Agrobacterium-based plant transformation. The natural bacterium transfers T-DNA into plant cells, demonstrating an efficient biological gene-delivery mechanism. Biotechnology modifies the Ti plasmid so that harmful pathogenic functions are removed while useful DNA-transfer capacity is preserved. Statement III is therefore incorrect because disarming does not aim to preserve pathogenicity; it aims to eliminate the harmful effect while retaining the transfer mechanism. The resulting system converts a natural pathogen-derived process into a controlled biotechnology tool for introducing desired genes into plant cells.
104. Retroviruses are relevant to recombinant DNA technology because their natural biology demonstrates an ability to
ⓐ. separate DNA fragments according to size
ⓑ. digest plant cell walls using cellulase
ⓒ. deliver genetic information into animal cells
ⓓ. precipitate purified DNA with chilled ethanol
Correct Answer: deliver genetic information into animal cells
Explanation: Retroviruses naturally possess mechanisms that allow them to introduce genetic information into animal cells. This biological property provides a conceptual model for the development of engineered gene-delivery vectors. Biotechnology can modify such systems so that harmful or pathogenic functions are disabled while useful delivery capability is retained. The important principle is the relationship between a natural biological mechanism and its engineered use as a vector. Retroviral systems are associated primarily with animal-cell gene delivery, whereas Agrobacterium-derived Ti plasmids are the corresponding major natural model discussed for plant cells.
105. A retroviral vector is described as disarmed. Which modification best fits this description?
ⓐ. harmful functions are disabled while gene-delivery ability is retained
ⓑ. all viral genetic material is removed, preventing any cell interaction
ⓒ. gene-delivery ability is disabled while pathogenic functions are retained
ⓓ. viral DNA is converted into a Ti plasmid before delivery
Correct Answer: harmful functions are disabled while gene-delivery ability is retained
Explanation: Disarming a retroviral vector follows the same general biotechnology logic seen with the Ti plasmid: preserve the useful DNA-delivery property but remove the original harmful functions. A natural retrovirus can introduce genetic information into animal cells, and this ability can be exploited after suitable modification. The vector is therefore not made biologically inactive in every respect. Instead, its harmful role is separated from its capacity to deliver a desired gene. This allows a naturally evolved transfer mechanism to be redirected toward controlled biotechnology applications without retaining the original pathogenic effect.
106. Match each natural biological system with the host context in which its engineered gene-delivery ability is most appropriately used.
| System | Host context |
|---|
| P. Disarmed Ti plasmid | I. Animal-cell gene delivery |
| Q. Disarmed retroviral vector | II. Plant-cell gene delivery |
ⓐ. P-I, Q-II
ⓑ. P-II, Q-I
ⓒ. P-I, Q-I
ⓓ. P-II, Q-II
Correct Answer: P-II, Q-I
Explanation: Agrobacterium tumefaciens naturally transfers T-DNA into plant cells, so its disarmed Ti plasmid is adapted for plant gene delivery. Retroviruses naturally introduce genetic information into animal cells, so disarmed retroviral systems provide a corresponding strategy for animal-cell gene transfer. The comparison is useful because both systems illustrate the same general engineering principle: remove the undesirable pathogenic function while retaining the naturally evolved DNA-delivery mechanism. The host context differs, but the conceptual biotechnology strategy is parallel. The host context follows directly from the natural transfer mechanism retained in each engineered vector.
107. A researcher needs to introduce a desired gene into plant cells using a biological vector derived from an organism with a natural gene-transfer mechanism. Which choice is most appropriate?
ⓐ. A disarmed retroviral vector retaining animal-cell delivery functions
ⓑ. An intact pathogenic retrovirus
ⓒ. A disarmed Ti plasmid derived from Agrobacterium tumefaciens
ⓓ. An exonuclease lacking any vector DNA
Correct Answer: A disarmed Ti plasmid derived from Agrobacterium tumefaciens
Explanation: For plant cells, the natural gene-transfer ability of Agrobacterium tumefaciens provides the relevant biological model. Its Ti plasmid can be modified so that pathogenic functions are removed while the capacity to deliver a gene of interest into plant cells is retained. A retroviral system is associated instead with gene delivery into animal cells. An exonuclease is an enzyme that removes nucleotides from DNA ends and does not function as a gene-delivery vector. The correct choice therefore depends on connecting the vector's natural biological mechanism with the host type into which DNA must be delivered.
108. DNA does not normally enter bacterial cells efficiently simply because it is present in the surrounding medium. One important reason is that DNA is
ⓐ. hydrophobic and readily soluble in the lipid bilayer
ⓑ. hydrophilic and unable to cross cell membranes readily
ⓒ. converted into protein before it reaches the cell surface
ⓓ. electrically neutral and unaffected by membrane barriers
Correct Answer: hydrophilic and unable to cross cell membranes readily
Explanation: DNA is a large, highly polar and negatively charged molecule, making it hydrophilic rather than lipid-soluble. The cell membrane contains a hydrophobic lipid-bilayer core, which creates an effective barrier to spontaneous passage of DNA. Therefore bacterial cells generally require treatment that makes them competent for DNA uptake before transformation can occur efficiently. The problem is not a lack of DNA outside the cell but a mismatch between the chemical nature of DNA and the permeability properties of the membrane. Competence-inducing procedures are designed to increase the probability that recombinant DNA can cross this barrier and enter recipient cells.
109. Bacterial cells are treated with an appropriate concentration of a divalent cation such as \(\mathrm{Ca^{2+}}\) before recombinant DNA is added. The main purpose of this treatment is to
ⓐ. increase the ability of bacterial cells to take up plasmid DNA
ⓑ. digest the bacterial chromosome into cloning-size fragments
ⓒ. replace DNA ligase during recombinant-vector construction
ⓓ. convert plasmid DNA into protein before it enters the bacterial cell
Correct Answer: increase the ability of bacterial cells to take up plasmid DNA
Explanation: Treatment with a divalent cation such as \(\mathrm{Ca^{2+}}\) is used to induce bacterial competence, making cells more capable of taking up recombinant DNA from their surroundings. DNA does not naturally cross the bacterial membrane efficiently because it is hydrophilic and negatively charged. Competence treatment helps overcome this barrier and increases transformation efficiency. The cation does not function as a restriction enzyme, ligase or protein-synthesis system. Its role belongs specifically to the host-preparation stage of recombinant DNA technology. This distinction is important because successful cloning requires not only construction of recombinant DNA but also an appropriate method for delivering that DNA into a recipient cell.
110. Two bacterial cultures receive identical recombinant plasmid DNA. Culture P is first treated with an appropriate concentration of \(\mathrm{Ca^{2+}}\), while culture Q receives no competence-inducing treatment. Under otherwise comparable conditions, which outcome is most reasonable?
ⓐ. Culture Q, because untreated membranes always allow greater plasmid uptake.
ⓑ. Culture Q, because \(\mathrm{Ca^{2+}}\) treatment blocks plasmid entry into bacterial cells.
ⓒ. Both cultures, because competence treatment does not affect transformation efficiency.
ⓓ. Culture P, because \(\mathrm{Ca^{2+}}\) treatment increases competence for plasmid uptake.
Correct Answer: Culture P, because \(\mathrm{Ca^{2+}}\) treatment increases competence for plasmid uptake.
Explanation: The purpose of \(\mathrm{Ca^{2+}}\)-mediated treatment is to make bacterial cells competent for DNA uptake. Because untreated bacterial membranes normally present a substantial barrier to entry of hydrophilic DNA, culture Q would generally be expected to transform less efficiently. Culture P has undergone the treatment specifically designed to increase the probability of DNA entry and should therefore produce more transformed cells under otherwise comparable conditions. The comparison demonstrates why recipient-cell preparation is a distinct and necessary step. Even a perfectly constructed recombinant plasmid cannot be useful if the host cells are not capable of receiving it efficiently.
111. During standard bacterial heat-shock transformation, the correct sequence is
ⓐ. \(42^\circ\text{C}\) \(\rightarrow\) ice \(\rightarrow\) prolonged boiling
ⓑ. ice \(\rightarrow\) approximately \(42^\circ\text{C}\) briefly \(\rightarrow\) ice
ⓒ. room temperature \(\rightarrow\) freezing \(\rightarrow\) \(100^\circ\text{C}\)
ⓓ. approximately \(42^\circ\text{C}\) \(\rightarrow\) ultraviolet exposure \(\rightarrow\) ice
Correct Answer: ice \(\rightarrow\) approximately \(42^\circ\text{C}\) briefly \(\rightarrow\) ice
Explanation: Competent bacterial cells mixed with recombinant DNA are first kept on ice, then briefly exposed to approximately \(42^\circ\text{C}\), and finally returned to ice. This ordered temperature treatment is part of the standard transformation procedure used to promote entry of DNA into competent bacterial cells. The sequence matters because heat shock is not simply a high-temperature incubation; it is a brief temperature transition applied to cells that have already undergone competence-inducing treatment. Excessive heating would damage cells rather than improve transformation. The key learning point is therefore both the temperature and the order: cold treatment, brief heat shock, and return to cold conditions together form the transformation sequence.
112. A laboratory follows every bacterial transformation step correctly except that the recombinant DNA and competent cells are never exposed to the brief \(42^\circ\text{C}\) heat-shock step. What is the most reasonable prediction?
ⓐ. The recombinant plasmid will be converted into a bacteriophage when heat shock is omitted.
ⓑ. Cells will become recombinant automatically even without plasmid entry.
ⓒ. Plasmids will replicate outside cells and fully compensate for the omitted heat shock.
ⓓ. Transformation efficiency will fall because the heat-shock uptake step was omitted.
Correct Answer: Transformation efficiency will fall because the heat-shock uptake step was omitted.
Explanation: The heat-shock stage is part of the procedure used to facilitate uptake of recombinant DNA by previously prepared competent bacterial cells. If this step is omitted, the cells may remain less efficient at taking up the plasmid, so the number of successful transformants is expected to fall. The effect should be interpreted at the DNA-delivery stage, not at the earlier recombinant-construction stage: the plasmid may have been assembled correctly but still fail to enter enough recipient cells. This distinction illustrates an important troubleshooting principle in biotechnology. A failed experiment can result from problems in vector construction, host preparation or DNA delivery, and identifying the affected stage helps explain the observed outcome.
113. A researcher wants to introduce recombinant DNA directly into the nucleus of an animal cell using a physical delivery method. Which technique is most appropriate?
ⓐ. \(\mathrm{Ca^{2+}}\)-mediated transformation of competent bacterial cells
ⓑ. Micro-injection directly into the animal-cell nucleus
ⓒ. Agarose electrophoresis of DNA fragments before delivery
ⓓ. Biolistic delivery using DNA-coated metal particles
Correct Answer: Micro-injection directly into the animal-cell nucleus
Explanation: Micro-injection is a direct physical method in which recombinant DNA is introduced into the nucleus of an animal cell using a very fine injection apparatus. The defining clues are therefore the animal-cell context and direct delivery into the nucleus. This method is fundamentally different from bacterial transformation, where cells are made competent and recombinant DNA is encouraged to cross the bacterial membrane. It is also different from biolistics, which uses high-velocity DNA-coated metal particles and is particularly useful for introducing DNA into plant cells. Micro-injection bypasses the need for a biological vector to cross the membrane because the DNA is physically placed at the required intracellular location.
114. Match each DNA-delivery method with the most appropriate description.
| Method | Description |
|---|
| P. Micro-injection | I. DNA-coated metal particles are accelerated into cells |
| Q. Biolistics | II. Recombinant DNA is directly introduced into the nucleus of an animal cell |
| R. \(\mathrm{Ca^{2+}}\)-mediated bacterial transformation | III. Recipient bacterial cells are made competent for DNA uptake |
| S. Disarmed-pathogen delivery | IV. A modified biological vector retains gene-transfer ability but lacks the original harmful role |
ⓐ. P-I, Q-II, R-IV, S-III
ⓑ. P-III, Q-I, R-II, S-IV
ⓒ. P-IV, Q-III, R-I, S-II
ⓓ. P-II, Q-I, R-III, S-IV
Correct Answer: P-II, Q-I, R-III, S-IV
Explanation: Micro-injection physically places recombinant DNA into the nucleus of an animal cell, whereas biolistics delivers DNA by accelerating DNA-coated metal particles into target cells. In bacterial transformation, treatment with an appropriate divalent cation such as \(\mathrm{Ca^{2+}}\) helps produce competent cells capable of taking up DNA more efficiently. Disarmed-pathogen systems use the naturally evolved ability of certain biological agents to enter cells and deliver genetic material, but their harmful functions are removed before biotechnology use. These methods therefore solve the same broad problem, getting recombinant DNA into a host, by very different mechanisms. Correct method selection depends on the type of host and the route by which DNA is to be delivered.
115. In the biolistic or gene-gun method, recombinant DNA is commonly delivered into plant cells by
ⓐ. precipitating DNA with chilled ethanol before exposing plant tissue
ⓑ. injecting DNA through a fine needle directly into an animal-cell nucleus
ⓒ. treating bacterial cells with restriction enzymes before DNA uptake
ⓓ. accelerating DNA-coated gold or tungsten particles into plant cells
Correct Answer: accelerating DNA-coated gold or tungsten particles into plant cells
Explanation: Biolistics is a physical DNA-delivery technique particularly associated with plant cells. Recombinant DNA is coated onto microscopic particles made of materials such as gold or tungsten, and these particles are propelled at high velocity into the target cells. The particles act as carriers that physically cross cellular barriers and bring the attached DNA into the cells. This approach is conceptually different from micro-injection, in which DNA is directly injected into an animal-cell nucleus, and from bacterial competence procedures involving \(\mathrm{Ca^{2+}}\) and heat shock. The essential features of biolistics are therefore DNA-coated metal particles, high-velocity delivery and its usefulness for introducing DNA into plant cells.
116. A laboratory plans three experiments:
| Experiment | Target |
|---|
| P | Direct delivery of recombinant DNA into an animal-cell nucleus |
| Q | Physical delivery of DNA into plant cells using metal particles |
| R | Uptake of plasmid DNA by specially treated bacterial cells |
Which assignment is correct?
ⓐ. P-biolistics, Q-micro-injection, R-disarmed Ti-vector transfer
ⓑ. P-\(\mathrm{Ca^{2+}}\)-mediated uptake, Q-heat-shock treatment, R-micro-injection
ⓒ. P-micro-injection, Q-biolistics, R-competence-based transformation
ⓓ. P-biolistic delivery, Q-replica-plating screening, R-gel electrophoresis
Correct Answer: P-micro-injection, Q-biolistics, R-competence-based transformation
Explanation: The appropriate delivery method depends strongly on the type of recipient cell. Micro-injection is suited to direct introduction of recombinant DNA into the nucleus of an animal cell. Biolistics is used to bombard plant cells with DNA-coated gold or tungsten particles, allowing DNA to cross physical cellular barriers. Bacterial plasmid uptake is handled differently: bacterial cells are first made competent, for example using \(\mathrm{Ca^{2+}}\), and then subjected to the transformation procedure. The three experiments therefore represent distinct host-specific delivery strategies rather than interchangeable methods.
117. An engineered pathogen-derived vector can be useful in recombinant DNA technology when
ⓐ. pathogenic functions are enhanced while host-cell damage is increased
ⓑ. harmful functions are removed while DNA-delivery capacity is retained
ⓒ. host-entry mechanisms are removed while pathogenicity is preserved
ⓓ. its genetic material is replaced with agarose before delivery
Correct Answer: harmful functions are removed while DNA-delivery capacity is retained
Explanation: Certain pathogens naturally possess efficient mechanisms for entering host cells and transferring genetic material. Biotechnology can exploit this delivery ability after the system has been disarmed, meaning that the original disease-causing or harmful functions are removed. The useful transfer machinery is retained so that a gene of interest can still be introduced into the appropriate host cell. This is the same broad principle illustrated by disarmed Ti plasmids and engineered retroviral vectors. The goal is not to preserve pathogenicity but to separate the valuable gene-delivery property from the unwanted harmful biological effect.
118. Several DNA-delivery strategies must be chosen for different target cells. The intended procedures are:
P. Direct nuclear delivery into an animal cell
Q. Particle-mediated DNA delivery into a plant cell
R. Biological transfer into a host using a modified pathogen-derived vector
Which combination is most appropriate?
ⓐ. P-biolistics, Q-micro-injection, R-agarose electrophoresis
ⓑ. P-heat-shock treatment, Q-micro-injection, R-DNA precipitation
ⓒ. P-restriction digestion, Q-biolistics, R-PCR amplification
ⓓ. P-micro-injection, Q-biolistics, R-disarmed-pathogen delivery
Correct Answer: P-micro-injection, Q-biolistics, R-disarmed-pathogen delivery
Explanation: Each description points to a distinct mechanism. Direct placement of recombinant DNA into an animal-cell nucleus is characteristic of micro-injection. Bombardment of plant cells using DNA-coated metal particles defines biolistics or the gene-gun method. A modified pathogen-derived vector, in contrast, takes advantage of a biological system that can enter host cells and transfer DNA while no longer retaining its original harmful function. These methods should be distinguished from laboratory procedures such as PCR, electrophoresis and restriction digestion, which manipulate or analyse DNA but are not themselves equivalent host-delivery mechanisms.
119. The major steps of recombinant DNA technology are listed below:
P. Isolation of genetic material
Q. Cutting DNA with a restriction endonuclease and obtaining the desired fragment
R. Ligation of the desired fragment into a vector
S. Transfer of recombinant DNA into a suitable host
T. Growth of the transformed host and recovery of the desired product
Which sequence is correct?
ⓐ. \(P\rightarrow Q\rightarrow R\rightarrow S\rightarrow T\)
ⓑ. \(Q\rightarrow P\rightarrow S\rightarrow R\rightarrow T\)
ⓒ. \(P\rightarrow R\rightarrow Q\rightarrow T\rightarrow S\)
ⓓ. \(S\rightarrow P\rightarrow Q\rightarrow R\rightarrow T\)
Correct Answer: \(P\rightarrow Q\rightarrow R\rightarrow S\rightarrow T\)
Explanation: Recombinant DNA technology follows an ordered biological and experimental sequence. The genetic material must first be isolated from the source and made suitable for manipulation. Restriction digestion then generates DNA fragments, after which the fragment containing the desired gene is obtained and joined to an appropriate vector using DNA ligase. The newly constructed recombinant DNA is then introduced into a suitable host, where it can be maintained, multiplied and, when appropriate, expressed. Finally, the host is cultured and the desired biological product is recovered. Later steps depend on the successful completion of earlier ones, so the order is biologically functional rather than arbitrary.
120. During recombinant DNA construction, a scientist digests source DNA but does not isolate the particular fragment containing the gene of interest before attempting ligation with the vector. What is the principal problem with this procedure?
ⓐ. Restriction digestion must always follow ligation of the vector and insert.
ⓑ. The vector can selectively capture the correct source fragment without purification.
ⓒ. The desired gene fragment was not specifically recovered before vector ligation.
ⓓ. DNA ligase will convert unwanted source fragments into RNA during ligation.
Correct Answer: The desired gene fragment was not specifically recovered before vector ligation.
Explanation: Restriction digestion can produce several DNA fragments from the source genetic material, not all of which contain the desired gene. The fragment containing the required sequence must therefore be identified and recovered before controlled recombinant construction. If the entire mixture is taken directly into ligation, many irrelevant DNA fragments may compete with the desired fragment for interaction with the opened vector. The problem is therefore one of specificity and purification, not a failure of restriction digestion itself. Recombinant DNA technology relies on selecting the correct DNA fragment before it is joined to the vector, so that the resulting construct contains the intended genetic information.
