31 Years NEET Previous Year Questions: Biotechnology: Principles & Processes

White color colonies will be selected:

• In blue-white screening, the lacZ gene in the plasmid is disrupted when a foreign DNA fragment is inserted at the EcoRI site.
• The disruption prevents the production of β-galactosidase, an enzyme responsible for converting X-gal (an artificial substrate) into a blue-colored product.
• As a result, colonies with recombinant plasmids appear white, while non-recombinant colonies (where lacZ is intact) appear blue. Selecting white colonies ensures that recombinant plasmids containing the alien DNA are identified.

Why Other Options Are Incorrect:

• Using ampicillin & tetracycline containing medium plate: While antibiotic selection (e.g., ampicillin resistance) is often used to ensure the presence of plasmids in transformed bacteria, this method does not distinguish between recombinant and non-recombinant plasmids. It only selects bacteria that contain the plasmid.
• Blue color colonies will be selected: Blue colonies indicate non-recombinant plasmids where the lacZ gene is intact and functional. These colonies do not contain the inserted alien DNA and are not the desired recombinants.
• Blue color colonies grown on ampicillin plates can be selected: Blue colonies represent non-recombinant plasmids. Additionally, while ampicillin selects for plasmid-containing bacteria, it does not distinguish between recombinant and non-recombinant colonies.

• The polymerase chain reaction (PCR) is a widely used molecular biology technique for amplifying specific DNA sequences exponentially.
• It involves repeated cycles of denaturation, annealing, and extension to double the amount of DNA with each cycle.
• The amplification follows the equation 2ⁿ, where n is the number of cycles performed. Each cycle doubles the amount of DNA present, leading to exponential growth.

Key Points about PCR:

• Denaturation: This is the first step of PCR. The reaction mixture is heated to around 94-98°C for 20-30 seconds, causing the double-stranded DNA to melt and separate into two single strands.
• Primer Annealing: The temperature is lowered to 50-65°C for 20-40 seconds to allow the primers to bind (anneal) to their complementary sequences on the single-stranded DNA template.
• Extension: The temperature is raised to around 72°C, the optimal temperature for Taq polymerase. This enzyme synthesizes a new DNA strand by adding nucleotides to the primer in a sequence-specific manner.
• Each cycle doubles the amount of DNA, leading to exponential amplification according to the formula 2ⁿ.
• PCR is used in various applications, including genetic testing, forensic analysis, and detecting pathogens

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• A bioreactor is a vessel or device in which biological reactions and processes are carried out under controlled conditions to produce biological products. 
• A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen).
• Foaming is a common issue in bioreactors, particularly in processes where gas sparging is employed. Foam formation can hinder the efficient operation of the bioreactor and may affect the quality of the product.
• Foam breaker: The foam breaker (identified as 'C' in the figure) is designed to disrupt and dissipate foam generated during the bioreactor operation. It ensures smooth processing by preventing foam overflow and maintaining the integrity of the biological processes.
• Foam breakers can be mechanical devices, such as rotating blades, or chemical agents that reduce surface tension and eliminate foam.

Statement I: "The blue-colored colonies have DNA insert in the plasmid and they are identified as recombinant colonies."

• This statement is incorrect because the blue-colored colonies represent non-recombinant colonies.
• These colonies have an intact lacZ gene, which produces β-galactosidase, resulting in the cleavage of X-gal and the formation of blue color.
• No DNA insert is present in these colonies, and thus they are not recombinant.

Statement II: "The colonies without blue color have DNA insert in the plasmid and are identified as recombinant colonies."

• This statement is correct because the absence of blue color (white colonies) indicates the disruption of the lacZ gene by the insertion of foreign DNA.
• The lack of β-galactosidase activity results in no cleavage of X-gal, and thus no blue color is produced.
• These colonies are recombinant as they contain the inserted DNA fragment in the plasmid.

• Gene cloning is a method used to create identical copies of a specific gene or DNA segment. It involves isolating a desired gene, inserting it into a vector, and introducing it into a host organism to amplify and express the gene.
• Enzymes play a crucial role in the various steps of gene cloning. However, not all enzymes are essential for the process.
• The most commonly used enzymes in gene cloning are restriction enzymes, DNA ligase, and DNA polymerase.
  • Restriction enzymes: These are essential enzymes for gene cloning. They recognize specific DNA sequences and cut the DNA at or near these sites, producing fragments that can be inserted into vectors.
  • DNA ligase: This enzyme is critical for gene cloning. It joins the DNA fragments (e.g., the insert DNA and the vector) by forming phosphodiester bonds, making a stable recombinant DNA molecule.
  • DNA polymerase: DNA polymerase is sometimes used in gene cloning for amplifying DNA through techniques like PCR (Polymerase Chain Reaction) or for filling in DNA overhangs after restriction digestion.
• DNA mutase: This enzyme is not essential for gene cloning. DNA mutase is involved in introducing mutations into DNA, which is not a requirement for cloning
• DNA recombinase: While recombinases facilitate site-specific recombination and have applications in advanced genetic engineering (e.g., CRISPR or recombineering), they are not essential for basic gene cloning processes.

• Statement I: The DNA fragments extracted from gel electrophoresis can be used in construction of recombinant DNA: This statement is correct. DNA fragments separated by gel electrophoresis can be extracted and purified for use in creating recombinant DNA, as they provide the necessary fragments for ligation into vectors.
• Statement II: Smaller size DNA fragments are observed near anode while larger fragments are found near the wells in an agarose gel: This statement is correct. In gel electrophoresis, smaller DNA fragments migrate further away from the wells towards the anode, whereas larger fragments travel a shorter distance and thus remain closer to the wells. DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field through a medium/matrix.

The correct answer is option (b).
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 (a), (c) and (d) are incorrect because they have either more than 6 or less than 6bp.

When a piece of DNA carrying a gene of interest is transferred into an alien organism, its fate depends on several factors, including the mechanisms it has for replication, integration, and inheritance. Here is an analysis of each statement provided:

A. The piece of DNA would be able to multiply itself independently in the progeny cells of the organism.
This is incorrect unless the DNA contains an origin of replication that is compatible with the host cell's replication machinery.

B. It may get integrated into the genome of the recipient.
This is a likely scenario if the DNA has sequences that facilitate recombination with the host genome or if the host has natural mechanisms of DNA integration such as those observed in bacteria with transformation capabilities, or other eukaryotic organisms with similar processes.

C. It may multiply and be inherited along with the host DNA.
This statement can be true if the introduced DNA is replicated along with the host DNA, possibly through integration into the host genome or existence in the form of an episome that replicates independently yet synchronously with the host DNA.

D. The alien piece of DNA is not an integral part of chromosome.
This statement is generally accurate before integration. The introduced DNA, before integration, exists extrachromosomally and thus is not part of any chromosome, unless mechanisms are present for its integration into the host genome.

E. It shows ability to replicate.
This statement depends on specific sequences in the DNA, such as the origin of replication compatible with the host cell's machinery, mentioned in statement A. Without these, it will not replicate independently.

Given these explanations, the most accurate answer that describes the fate of the DNA in the recipient organism is: Option C: B and C only
'B' is correct as the DNA may integrate into the host genome, and 'C' is correct as the DNA might also multiply if replicated in sync with the host organism's DNA, particularly if it integrates or exists as an episome compatible with the host's cellular machinery.

Correct answer is option (2), because
'X' in the given diagram is ori while 'Y' is rop.
'X' which is ori is responsible for controlling the copy number of the linked DNA and 'Y' which is rop codes for protein involved in the replication of plasmid.
Options (1), (3) and (4) are incorrect as 'X' and 'Y' are not related to these functions.

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.

Bio-reactors are typically used for large-scale production, not small-scale bacterial cultures. They provide a controlled environment to cultivate microorganisms, plant cells, or animal cells for producing various products like antibiotics, enzymes, biofuels, and more.
The other statements are correct:

• Statement 1: Most commonly used bio-reactors are of the stirring type, known as stirred-tank bio-reactors.
• Statement 3: Bio-reactors are equipped with systems like an agitator system, oxygen delivery system, and foam control system to maintain optimal conditions for microbial growth.
• Statement 4: A bio-reactor indeed provides optimal growth conditions to achieve the desired product, such as maintaining temperature, pH, and nutrient levels.

Selectable markers are genes introduced into cloning vectors to enable the identification of successfully transformed cells. These markers usually provide resistance to specific antibiotics, allowing only the transformed cells to grow in the presence of the antibiotic.

• Ampicillin, Chloramphenicol, and Tetracycline are all common selectable markers used in cloning vectors because they confer resistance to these antibiotics.
• Metformin, however, is a drug used primarily for the treatment of type 2 diabetes and is not used as a selectable marker in cloning vectors.

Statement I: Restriction Endonuclease recognizes specific sequences of nucleotides in the DNA, known as the recognition sequence, and binds to them. This statement is true.
Statement II: After binding to the recognition sequence, Restriction Endonuclease cuts both strands of the DNA double helix at specific points in their sugar-phosphate backbone. This is also true, as these enzymes typically create staggered or blunt ends by cleaving the DNA at these specific sites.
Thus, both statements are correct.

The ori site (origin of replication) is the specific DNA sequence responsible for initiating DNA replication in a chromosome. It is the site where the process of DNA replication begins, allowing the DNA to be copied for cell division. The ori site is crucial for the proper replication of the chromosome in both prokaryotes and eukaryotes.
The other options:

• Recognition sequence: This refers to a specific sequence recognized by enzymes like restriction endonucleases but is not involved in replication initiation.
• Cloning site: This refers to a specific site in vectors where foreign DNA can be inserted, not related to DNA replication initiation.
• Restriction site: This refers to a specific sequence recognized and cut by restriction enzymes but is not involved in replication initiation.

Incorrect Statement: Separated DNA fragments cannot be directly seen under UV radiation. After electrophoresis, DNA fragments are typically stained with a dye such as ethidium bromide, which intercalates with the DNA and fluoresces under UV light. Without staining, DNA fragments cannot be directly observed under UV light.
The other statements are correct:

• Statement 2: Separated DNA can indeed be extracted from the gel after electrophoresis using a process called gel extraction.
• Statement 3: DNA is negatively charged, and during electrophoresis, it moves toward the positive electrode (anode).
• Statement 4: The sieving effect of agarose gel helps separate DNA fragments based on their size, with smaller fragments moving faster through the gel than larger ones.

The p^BR322 cloning vector contains restriction sites for several restriction endonucleases. The tetracycline resistance (tet^R) gene is part of the pBR322 vector and has specific restriction enzyme sites.
The Bam HI and Sal I restriction enzymes have their restriction sites within or near the tet^R gene in p^BR322. These enzymes can be used to cut the plasmid at specific sites, making it possible to insert foreign DNA or manipulate the plasmid.
The other options do not match the specific restriction sites within the tetracycline resistance gene in p^BR322.

To create a recombinant DNA molecule, the vector DNA and source DNA must be cut with the same restriction enzyme to ensure compatible sticky ends that can be ligated together. In this case, Hind II is used to cut both the vector DNA and the source DNA, producing compatible ends for ligation.
The other options involve different restriction enzymes that would produce incompatible ends or not the correct cutting pattern for creating recombinant DNA with compatible ends.

B. Cut the DNA with sticky end: EcoRI is a restriction enzyme that produces sticky (cohesive) ends by cutting between the G and A in the sequence GAATTC.
C. Recognises a specific palindromic sequence: EcoRI recognizes the palindromic sequence GAATTC, where the sequence reads the same in both directions.
D. Cut the DNA between the base G and A where it encounters the DNA sequence 'GAATTC': This is the specific action of EcoRI, cutting the DNA between the G and A of the GAATTC sequence.
A. Cut the DNA with blunt end: EcoRI cuts the DNA with sticky ends, not blunt ends.
E. Exonuclease: EcoRI is not an exonuclease. It is a restriction endonuclease, which cuts DNA at specific sequences, rather than progressively trimming nucleotides from the ends.

The correct sequence of steps in the Polymerase Chain Reaction (PCR) process is as follows:
C. Denaturation: The DNA sample is heated to around 94-98°C to separate the two strands of the DNA template.
A. Annealing: The temperature is lowered to 50-65°C to allow the primers to bind to the complementary sequences on the single-stranded DNA templates.
D. Treatment with Taq polymerase and deoxynucleotides: The Taq polymerase enzyme, along with free nucleotides (dNTPs), is added for the extension process.
E. Extension: The Taq polymerase synthesizes a new strand of DNA starting from the primers at around 75-80°C.
B. Amplification (~1 billion times): The process is repeated through many cycles (usually 20-40), resulting in exponential amplification of the target DNA sequence.
Thus, the correct sequence is C → A → D → E → B.

Option (d) 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 (b), (c) and (a) are not correct because YAC, BAC, pBR322 are vectors.

Ethidium bromide is a fluorescent dye commonly used in molecular biology to visualize nucleic acids, especially DNA, under UV light. When exposed to UV light, ethidium bromide intercalates between the base pairs of DNA, emitting a bright orange fluorescence. This property makes it extremely useful for detecting DNA bands in agarose gel electrophoresis. The fluorescence is specific to DNA and is not emitted by other molecules, which is why it is widely used in DNA analysis.

In recombinant DNA technology, ethanol precipitation is used to purify DNA from a solution. DNA is soluble in water but is precipitated out when chilled ethanol is added. This is because ethanol reduces the solubility of DNA, causing the DNA molecules to clump together and form a visible precipitate. This process is essential for isolating DNA after various stages such as restriction digestion or PCR amplification. The addition of salt, such as sodium acetate, helps in neutralizing the charge of the DNA, making it more likely to precipitate out when ethanol is added.

The gene gun, also known as the biolistic method, is a technique used to deliver foreign DNA into cells. In this method, tungsten or gold microparticles are coated with the DNA of interest and then fired into plant or animal cells under high pressure. The heavy metal particles penetrate the cell membrane, carrying the DNA with them. This method is particularly useful in plant biotechnology, as it does not require the use of plant-specific transformation methods like Agrobacterium-mediated transformation. The use of tungsten or gold ensures that the particles are dense and can effectively carry the DNA into cells.

The correct sequence of steps in recombinant DNA technology is as follows:

• B (Cutting of DNA): First, the DNA of interest and the vector are cut using restriction enzymes to generate sticky ends.
• C (Isolation of desired DNA fragment): The fragment of interest is then isolated from the rest of the DNA.
• D (Amplification of gene of interest using PCR): The desired DNA fragment is often amplified using PCR to produce enough copies for cloning.
• A (Insertion of recombinant DNA into the host cell): Finally, the recombinant DNA is inserted into the host cell for expression or further manipulation. This process is crucial in genetic engineering and cloning.

Taq polymerase, a thermostable DNA polymerase, was originally isolated from the bacterium Thermus aquaticus, which thrives in the high-temperature environment of hot springs. The enzyme is capable of withstanding the high temperatures used in the Polymerase Chain Reaction (PCR), which involves repeated cycles of heating and cooling. This property makes Taq polymerase ideal for PCR, as it does not denature at high temperatures, unlike most other enzymes. It efficiently synthesizes new DNA strands during the extension step of PCR, enabling rapid amplification of DNA sequences.

In the pBR322 plasmid, the tetracycline resistance (tetR) gene is located in a region that contains a BamH I restriction site. If foreign DNA is inserted into the BamH I site of the tetracycline resistance gene, it disrupts the gene's function, leading to the loss of tetracycline resistance. This is a key feature in recombinant DNA technology because plasmids are often designed so that when foreign DNA is inserted into a specific site, it can deactivate a reporter gene like tetR. This allows researchers to easily identify transformed cells by their ability to resist or not resist tetracycline.

Agrobacterium tumefaciens is primarily used to transfer genes into eukaryotic plant cells, not prokaryotic cells. It uses a plasmid known as the Ti plasmid to insert a segment of DNA (T-DNA) into the plant genome. This process is a natural form of horizontal gene transfer that has been harnessed in genetic engineering for plant transformation. Agrobacterium is not used for gene transfer in prokaryotic cells, as they do not possess the machinery to process the T-DNA in the same way eukaryotic plant cells do. The other statements about Agrobacterium tumefaciens are correct, such as its ability to deliver T-DNA into plant cells and its role in transforming plant cells into tumor cells.

Restriction enzymes, also known as restriction endonucleases, act as "molecular scissors" because they can cut DNA at specific sequences. These enzymes recognize particular sequences of nucleotides in the DNA and make cuts, typically resulting in sticky or blunt ends, depending on the enzyme. This property is widely used in molecular biology for DNA manipulation, such as in cloning, genetic engineering, and DNA analysis.

• DNA ligase is an enzyme that joins two DNA fragments together by forming phosphodiester bonds, not cutting DNA.
• RNA polymerase is responsible for synthesizing RNA from a DNA template in transcription, not for cutting DNA.
• DNA polymerase synthesizes DNA from a template strand during replication or PCR, but it does not act as molecular scissors.

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.