Biotechnology: Principles and Processes

The correct answer is that white-colored colonies will be selected.

Since an alien piece of DNA is being inserted at EcoRI site, the gene $\beta$-galactosidase present here will undergo insertional inactivation.

This gene is responsible for producing blue-colored colonies, but since it has been insertionally inactivated, white colored colonies will be produced.

Ampicillin and tetracycline resistance genes present in the given DNA will remain intact. Thus, the given DNA will show amp ${ }^R$ and tet ${ }^R$.

Polymerase Chain Reaction (PCR) is a technique used to amplify DNA, and its amplification can be described by the equation $2^n$. In this equation, $n$ represents the number of cycles. For example, if PCR is run for 3 cycles, the calculation would be $2^3$, which equals $2 \times 2 \times 2 = 8$. Therefore, 8 DNA fragments would be produced after 3 cycles.

$\therefore \quad$ Part labelled as C is foam breaker.

In the method using blue and white selectable markers, recombinant colonies are differentiated from non-recombinant ones based on their ability to produce color in the presence of a chromogenic substrate.

Statement I is incorrect. The blue-colored colonies do not contain the DNA insert in the plasmid. They are non-recombinant because the enzyme β-galactosidase is still active and able to cleave the chromogenic substrate, producing a blue color.

Statement II is correct. Colonies that do not exhibit a blue color indicate that a DNA insert is present in the plasmid. This insert disrupts the coding sequence of the enzyme β-galactosidase, inactivating it. Thus, these colonies do not produce color and are identified as recombinant.

In summary, recombinant colonies lose their ability to produce blue color due to the insertional inactivation of the β-galactosidase gene, while non-recombinant colonies appear blue.

RNAi (RNA interference) takes place in all eukaryotic organisms as a method of cellular defense. This method involves silencing of a specific mRNA due to a complementary dsRNA molecule that binds to and prevents translation of the mRNA.

Gene cloning is a process where a specific gene or DNA sequence is isolated and replicated, creating multiple identical copies.

In gene cloning, restriction enzymes, DNA ligase and DNA polymerase are primarily used.

The cutting of DNA by restriction endonucleases results in the fragments of DNA. These fragments can be separated by a technique known as gel electrophoresis.

The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as elution. The DNA fragments purified in this way are used in constructing rDNA by joining them with cloning vectors.

$\bullet$ In gel electrophoresis, the DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel. Hence, the smaller the fragment size, the farther it moves from cathode towards anode.

Let's analyze each statement separately to determine its validity:

Statement I: The Indian Government has set up GEAC, which will make decisions regarding the validity of GM research.

The Genetic Engineering Appraisal Committee (GEAC) is indeed an authority established by the Indian Government under the Ministry of Environment, Forest and Climate Change. Its role is to evaluate and make decisions regarding the research, development, and commercialization of genetically modified (GM) organisms and products. Therefore, Statement I is true.

Statement II: Biopiracy is the term used to refer to the use of bio-resources by native people.

Biopiracy refers to the unethical or unauthorized exploitation of biological resources, typically involving indigenous knowledge, by entities (often corporations or researchers from developed countries) without fair compensation to the native people or countries from which these resources originate. Therefore, this statement is incorrect as it should mention the exploitation by external entities rather than the use by native people themselves. Therefore, Statement II is false.

Based on the analysis, the correct answer is:

Option A: Statement I is true but Statement II is false

To match List-I with List-II, we need to find the correct association for each item provided:

(A) Genetically engineered Human Insulin - This is produced using recombinant DNA technology and commonly involves the bacterium E. coli.

(B) GM Cotton - This is genetically modified cotton, which typically involves the incorporation of genes from Bacillus thuringiensis (Bt) to produce pest-resistant cotton.

(C) ADA Deficiency - Ada deficiency is often treated using gene therapy.

(D) ELISA - ELISA (Enzyme-Linked Immunosorbent Assay) is a test that uses antigen-antibody interactions to detect the presence of a substance, usually an antigen or antibody, in a liquid sample.

Now let's match them with List-II:

So, the correct answer is:

Option D: A-II, B-IV, C-I, D-III

The incorrect statement related to gel electrophoresis is Option A: Separated DNA fragments can be directly seen under UV radiation.

Here's why:

DNA itself is not fluorescent. To visualize DNA fragments after separation, a dye that binds to DNA and fluoresces under UV light is used. This dye, often ethidium bromide or SYBR Safe, intercalates between the DNA bases, making the DNA visible under UV light.

Let's look at the other options:

Option B: Separated DNA can be extracted from gel piece is correct. DNA fragments can be extracted from the gel by cutting out the desired band and using a method called gel extraction. This allows for further analysis or cloning of the extracted DNA.

Option C: Fragment of DNA moves toward anode is also correct. DNA molecules carry a negative charge due to the phosphate groups in their backbone. In gel electrophoresis, the DNA is loaded into wells at the negative end of the gel. When an electric current is applied, the negatively charged DNA fragments migrate towards the positive electrode (anode).

Option D: Sieving effect of agarose gel helps in separation of DNA fragments is correct. Agarose gel acts as a sieve, with pores that allow smaller fragments to travel through more easily than larger fragments. This difference in mobility, based on size, allows for separation of DNA fragments.

Recombinant DNA technology involves the insertion of a specific gene from one organism into the DNA of another organism. This process requires the use of restriction enzymes to cut both the vector DNA and the source DNA at specific sites, creating compatible ends that can then be joined together.

Here's a breakdown of the options provided:

Option A: Hind II, Hind II

Both the vector DNA and the source DNA are cut with Hind II. However, since Hind II would cut both DNAs at the same sequence, it may not necessarily create sticky ends that are compatible for recombination.

Option B: Hind II, Alu I

Using Hind II for the vector DNA and Alu I for the source DNA means they are cut with different restriction enzymes. This typically wouldn’t create compatible ends for recombination.

Option C: Hind II, EcoR I

Similar to Option B, Hind II for the vector DNA and EcoR I for the source DNA won’t create compatible ends, as different enzymes recognize and cut at different sequences.

Option D: Hind II, BamHI

In this option, Hind II cuts the vector DNA and BamHI cuts the source DNA. Like Options B and C, using different restriction enzymes typically won’t create compatible ends for recombinant DNA formation.

Therefore, the correct answer is:

Option A: Hind II, Hind II

Because using the same restriction enzyme for both the vector and the source DNA increases the likelihood of generating compatible "sticky" ends that can be joined together to form a recombinant DNA molecule.

The correct answer is Option A: Creating pores in the midgut. Here's why:

Bt toxin, short for Bacillus thuringiensis toxin, is a naturally occurring insecticide produced by the bacterium Bacillus thuringiensis. When ingested by certain insect pests, it acts specifically on their digestive system.

Here's how it works:

• Ingestion: Insect pests consume Bt toxin along with plant material.
• Activation: The alkaline environment of the insect's midgut (the middle part of its digestive tract) activates the Bt toxin.
• Binding: The activated toxin binds to specific receptor proteins on the epithelial cells lining the insect's midgut.
• Pore Formation: This binding causes the toxin to insert itself into the cell membrane of the midgut cells, forming pores or holes.
• Cell Disruption: The formation of these pores disrupts the cell's ability to maintain its structure and function. The cell leaks nutrients and fluids, ultimately leading to cell death.
• Death: The widespread damage to the midgut cells results in the insect's death due to starvation and/or internal organ failure.

Let's look at why the other options are incorrect:

• Option B: Damaging the respiratory system: Bt toxin primarily targets the digestive system of insects, not their respiratory system.
• Option C: Degenerating the nervous system: While some insecticides do affect the nervous system, Bt toxin's primary mode of action is through the digestive system.
• Option D: Altering the pH of body fluids: While Bt toxin's activation is influenced by pH, its primary effect is not on altering the overall pH of the insect's body fluids.

In summary, Bt toxin works by creating pores in the midgut of susceptible insect pests, leading to their death.

The pBR322 cloning vector is a commonly used plasmid in molecular biology that contains two antibiotic resistance genes: the tetracycline resistance (tet^R) gene and the ampicillin resistance (amp^R) gene. Various restriction endonuclease enzymes can cut specific sequences within this plasmid, and each enzyme has specific restriction sites.

For the tetracycline resistance (tet^R) gene, the restriction sites of interest are those that either flank or are within the gene sequence. The enzymes listed in the options are BamHI, SalI, PstI, and PvuI. We need to determine which of these enzymes have sites located within the tet^R gene region in pBR322.

Upon analysis of the restriction map of the pBR322 vector, it is noted that:

• BamHI: Has a recognition site within the tet^R gene.
• SalI: Cuts within the tet^R gene.
• PstI: Cuts outside the tet^R gene.
• PvuI: Cuts outside the tet^R gene.

Therefore, the correct choice of restriction endonuclease enzymes whose restriction sites are present within the tetracycline resistance (tet^R) gene in the pBR322 cloning vector is:

Option A: BamHI and SalI

The correct answer is option (D) as

EcoRI does not cut the DNA with blunt ends. Instead, it cuts the DNA with sticky / cohesive / staggered ends on each strand.

EcoRI is a restriction endonuclease that recognises a specific palindromic sequence and cuts at a specific site within the DNA, known as the restriction site. It is not an exonuclease as exonucleases remove nucleotides from the free ends of the DNA.

The recognition sequence for EcoRI is $ \begin{aligned} & 5^{\prime}-\mathrm{G}^{\downarrow}-\mathrm{A}-\mathrm{A}-\mathrm{T}-\mathrm{T}-\mathrm{C}-3^{\prime} \\ & 3^{\prime}-\mathrm{C}-\mathrm{T}-\mathrm{T}-\mathrm{A}-\mathrm{A}_{\uparrow}-\mathrm{G}-5^{\prime} \end{aligned}$ and it cuts the DNA between bases G and A only when the sequence GAATTC is present in the DNA.

Therefore, ‘A’ and ‘E’ represent incorrect features about EcoRI, whereas ‘B’, ‘C’ and ‘D’ are correct features of EcoRI.

The other options, i.e., (A), (B) and (C) are incorrect as they represent incorrect combinations of features w.r.t. EcoRI.

The Polymerase Chain Reaction (PCR) is a method used to amplify DNA. The correct sequence of steps involved in the process of PCR is:

1. Denaturation: The double-stranded DNA is heated to separate it into two single strands.
2. Annealing: The temperature is lowered to allow primers to attach to the single-stranded DNA.
3. Extension: Taq polymerase extends the primers to form a new DNA strand.
4. Amplification: This cycle of heating and cooling is repeated multiple times (~1 billion times) to amplify the DNA.

Therefore, the correct sequence of steps of PCR from the options given is:

Option A:

$\mathrm{C} \rightarrow \mathrm{A} \rightarrow \mathrm{D} \rightarrow \mathrm{E} \rightarrow \mathrm{B}$

This option correctly represents the sequence of steps in PCR where:

1. $\mathrm{C}$ represents Denaturation.
2. $\mathrm{A}$ represents Annealing.
3. $\mathrm{D}$ represents Treatment with Taq polymerase and deoxynucleotides (which occurs during the Extension step).
4. $\mathrm{E}$ represents Extension.
5. $\mathrm{B}$ represents Amplification (~1 billion times).

Hence, Option A is the correct sequence.

The correct answer is option (2).

The first restriction endonuclease - Hind II, whose functioning depends on a specific DNA nucleotide sequence was isolated. It was found that Hind II always cut DNA molecules at a particular point by recognising sequence of six base pairs.

Option (1), (3) and (4) are incorrect because they have either more than 6 or less than $6 \mathrm{~bp}$.

The correct answer is Option C: Tumor inducing plasmid.

The "Ti plasmid" is a type of plasmid (a small DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently) found in the bacterium Agrobacterium tumefaciens. This bacterium is known for its ability to transfer a segment of its DNA to a plant, integrating this DNA into the plant's genome, causing the plant to develop tumors, known as crown gall disease.

The term "Ti plasmid" stands for "Tumor inducing plasmid" because of its role in this process. When Agrobacterium tumefaciens infects a plant, the Ti plasmid transfers a portion of its DNA-known as T-DNA (transfer DNA)-into a random site in the plant's genome. The expression of genes in the T-DNA leads to the overproduction of certain growth hormones (auxins and cytokinins), which results in the formation of tumors on the plant.

Thus, Option C "Tumor inducing plasmid" most accurately describes the Ti plasmid's function of inducing tumor formation in infected plants. Options A, B, and D do not correctly describe the properties or functions of the Ti plasmid.

In pBR322, a commonly used plasmid in genetic engineering, certain restriction sites are present within the antibiotic resistance genes, which provide resistance to tetracycline and ampicillin. Here's what would happen if foreign DNA is inserted at each of the following restriction sites:

Option A : Pst I - The PstI site is present in the ampR gene (ampicillin resistance gene). Insertion of DNA here would disrupt the ampicillin resistance gene, causing a loss of ampicillin resistance, but it would not affect tetracycline resistance.

Option B : Pvu I - Similar to PstI, the PvuI site is also present in the ampR gene. Therefore, insertion of DNA here would cause a loss of ampicillin resistance, but it wouldn't affect the plasmid's tetracycline resistance.

Option C : EcoR I - The EcoRI site is not present within either the ampR or tetR genes in pBR322. Inserting DNA at this site would not disrupt either the ampicillin or tetracycline resistance genes.

Option D : BamH I - The BamH I site is within the tetR gene. If foreign DNA is inserted at this site, it would disrupt the tetracycline resistance gene, causing a loss of tetracycline resistance.

So, the correct answer to the question "Ligation of foreign DNA at which of the following site will result in loss of tetracycline resistance of pBR322?" is Option D : BamH I.

The correct answer is Option A :

(A) Kanamycin - (II) Selectable marker : Kanamycin is an antibiotic, and a resistance gene for kanamycin is often used as a selectable marker in genetic engineering. Cells that have been successfully transformed with the desired DNA will also carry the kanamycin resistance gene, and thus will survive in a culture medium containing kanamycin, while cells without the gene will not.

(B) ClaI - (III) Restriction site : ClaI is a restriction enzyme used in molecular biology to cut DNA at specific sites (restriction sites).

(C) Disarmed retroviruses - (I) Delivers genes into animal cells : Retroviruses are often used as vectors in gene therapy to deliver genes into animal cells. They can be "disarmed" to make them safe for use in humans or other animals.

(D) Kanamycin $^\mathrm{R}$ gene - (IV) Antibiotic resistance : The kanamycin resistance (Kan^R) gene is a type of antibiotic resistance gene. It produces a protein that inactivates kanamycin, allowing bacteria containing this gene to survive in the presence of this antibiotic.

Option (C) is correct answer because a single stranded DNA or RNA tagged with a radioactive molecule is called a probe and it helps in the detection of mutated gene.

Option (A), (B) and (D) are not correct because YAC, BAC, pBR322 are vectors.

Option (b) is the correct answer because when restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands, then single stranded portions are left at the ends. These overhanging stretches on each strand are called sticky ends.

Option (1) is the correct answer because separation of DNA fragments which is carried out after restriction enzyme digestion, is done by a technique known as gel electrophoresis.

Option (2) is not the correct answer because polymerase chain reaction (PCR) is a technique used for amplification of gene of interest.

Option (3) is not the correct answer because recombinant DNA technology comprises altering genetic material outside an organism to obtain enhanced and desired characteristics in living organisms or as their products.

Option (4) is not the answer because Southern blotting is a method used for detection of specific DNA sequences in samples.

Option (3) is the correct answer because cellulase is used to isolate genetic material from plant cells and chitinase is used to isolate genetic material from fungal cells.

Option (2) is incorrect because protease is used for digestion of proteins.

Option (1) and (4) are incorrect because lipase is used for the breakdown of lipids.

Option (2) is the correct answer because

$\bullet$ Gene gun or biolistics is a direct gene transfer method suitable for plant cell.

$\bullet$ Gene therapy involves replacement of a faulty gene by a normal healthy gene.

$\bullet$ Gene cloning is done to make identical copies of a particular DNA molecule.

$\bullet$ Genome is the total DNA in the cells of an organism.

Plasmid is small circular DNA outside the genomic DNA. The plasmid DNA confers certain unique phenotypic characters to the bacteria such as resistance to antibiotics.

Option (a) is the correct answer because bacteriophages act as a cloning vector for bacterial cells, not the plant cells.

Options (b), (c) and (d) are not the correct answers because Agrobacterium, gene gun and disarmed pathogens as vectors are used for introducing foreign DNA into the plant cells.

Option (c) is the correct answer as the most commonly used matrix in gel electrophoresis is agarose which is a natural polymer extracted from sea weeds.

In agarose gel electrophoresis, the DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel.

Since, DNA fragments are negatively charged molecules, they migrate towards the anode (positively-charged electrode) under an electric field through a medium/martix.

Both the statements are correct but the given reason is not the correct explanation. Polymerase chain reaction is used in DNA amplification.

Ampicillin resistance gene is a selectable marker that helps to check transformation by selection of transformants.

Option (c) is the incorrect statement, as bright coloured bands of DNA can be observed in the gel when EtBr (Ethidium bromide) treated DNA is exposed to UV light.

Option (a) is the correct answer because both the statements I and II are correct. Each restriction endonuclease recognises a specific palindromic nucleotide sequences in the DNA. It will bind to the DNA and cut each of the two strands of double helix at specific points. Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome site; but between the same two bases on the opposite strands. So both the statements I and II are correct.

Option (d) is the correct answer. Cloning vectors are the carriers of the desired gene in the host cell. The features desirable in a cloning vector are :-

$\bullet$ Presence of origin of replication

$\bullet$ Presence of marker genes

$\bullet$ Presence of very few, preferably single recognition site for the commonly used restriction enzymes