Process of Recombinant DNA technology Questions in English

(D) bioreactor is a vessel in which raw materials are biologically converted into specific products,individual enzymes,etc.,using microbial,plant,animal,or human cells.
Bioreactors provide optimal conditions for achieving the desired product by providing optimum growth conditions such as temperature,$pH$,substrate,salts,vitamins,and oxygen.
They are essentially large-scale fermentation tanks used for the production of biotechnological products.

(B) $PCR$ (Polymerase Chain Reaction) is a powerful molecular diagnostic technique.
It is used to detect $HIV$ in suspected $AIDS$ patients by amplifying the viral $DNA$,even when the viral load is very low.
Additionally,$PCR$ is used to detect mutations in genes in suspected cancer patients,which helps in early diagnosis and personalized treatment.
$ELISA$ is primarily used for detecting antigens or antibodies,while $Widal$ test is for typhoid and $CT\, SCAN$ is an imaging technique.

(D) Gel electrophoresis is a technique used to separate charged molecules like $DNA$,$RNA$,or proteins based on their size and charge.
In the context of $DNA$ technology,it is specifically used to separate $DNA$ fragments based on their size.
Under an electric field,$DNA$ fragments (which are negatively charged) move towards the anode through a matrix (usually agarose gel).
Smaller fragments move faster and travel further through the gel pores compared to larger fragments,allowing for effective separation.

(D) The $Polyethylene \ Glycol$ $(PEG)$ method is a chemical-mediated transformation technique used in biotechnology.
It is primarily used for the direct transfer of genetic material $(DNA)$ into plant protoplasts without the need for a biological vector like $Agrobacterium$.
$PEG$ acts as a fusogenic agent that facilitates the uptake of $DNA$ by the protoplasts by altering the permeability of the cell membrane.
Therefore,it is a well-known method for vectorless gene transfer.

(D) Stirred-tank bioreactors are designed to provide optimal conditions for achieving the desired product by providing growth parameters like temperature,pH,substrate,salts,vitamins,and oxygen. The primary function of the stirring mechanism is to ensure proper mixing and oxygen availability throughout the bioreactor for the growing microbes.

(B) The blue-white screening method is based on the insertional inactivation of the $lacZ$ gene.
$1$. The $lacZ$ gene encodes the enzyme $\beta$-galactosidase.
$2$. In non-recombinant bacteria,the $lacZ$ gene is intact and functional,so the bacteria produce $\beta$-galactosidase. This enzyme reacts with the chromogenic substrate $X-gal$ to produce a blue color.
$3$. In recombinant bacteria,the foreign $DNA$ is inserted into the $lacZ$ gene,causing insertional inactivation. Consequently,no functional $\beta$-galactosidase is produced,and the colonies remain white.

(D) Restriction endonucleases cut $DNA$ at specific recognition sequences,resulting in fragments of varying lengths.
These $DNA$ fragments are negatively charged due to the phosphate backbone.
Gel electrophoresis is the technique used to separate these $DNA$ fragments based on their size (molecular weight).
Under an electric field,the fragments move through a matrix (usually agarose gel) towards the anode (positive electrode).
Smaller fragments move faster and travel further through the gel pores compared to larger fragments,allowing for their separation.

(D) Southern hybridization is a technique used for the detection of specific $DNA$ sequences in $DNA$ samples.
The steps involved in Southern hybridization are:
$1$. Digestion of $DNA$ with restriction endonucleases.
$2$. Separation of $DNA$ fragments by gel electrophoresis.
$3$. Transfer of separated $DNA$ fragments onto a nitrocellulose or nylon membrane (Blotting).
$4$. Hybridization with a labeled probe.
$5$. Detection of the hybridized probe using autoradiography.
$PCR$ (Polymerase Chain Reaction) is a separate technique used for the amplification of specific $DNA$ sequences and is not a step in the standard Southern hybridization procedure.

(A) Stirred-tank bioreactors are cylindrical vessels designed to facilitate the mixing of the reactor contents.
They are equipped with an agitator system,an oxygen delivery system,a foam control system,a temperature control system,a pH control system,and sampling ports.
The primary purpose of the stirring mechanism is to ensure even mixing and,crucially,to provide optimal oxygen availability throughout the entire culture medium for the aerobic growth of microorganisms.

(B) Downstream processing refers to the stages of processing that occur after the biosynthesis of the product in a bioreactor.
It includes the following steps:
$1$. Separation of the product from the culture medium.
$2$. Purification of the product.
$3$. Formulation with suitable preservatives.
$4$. Quality control and clinical trials.
'Expression' is a part of the upstream processing,where the gene of interest is expressed in the host organism to produce the desired product. Therefore,it is not a part of downstream processing.

(D) The process of separating $DNA$ fragments based on their size is known as gel electrophoresis.
After the $DNA$ fragments are separated on an agarose gel,they cannot be seen directly under visible light.
To visualize the $DNA$ fragments,the gel is stained with a fluorescent dye called Ethidium bromide $(EtBr)$.
When the stained gel is exposed to $UV$ radiation,the $DNA$ fragments appear as bright orange-colored bands.

(B) Gel electrophoresis is a technique used to separate $DNA$ fragments based on their size.
$DNA$ molecules are negatively charged due to the phosphate groups in their backbone.
When an electric field is applied,the $DNA$ fragments move towards the anode (positive electrode).
The agarose gel acts as a molecular sieve.
Smaller $DNA$ fragments can navigate through the pores of the gel matrix more easily and quickly than larger fragments.
Therefore,smaller fragments travel a greater distance from the well compared to larger fragments.

(B) In recombinant $DNA$ technology,the production of a desired protein involves two main stages:
$1$. Upstream processing: This involves the preparation of the desired gene,selection of a suitable vector,and the cultivation of the host cells in a bioreactor to produce the protein.
$2$. Downstream processing: This stage involves the separation and purification of the expressed protein from the culture medium before it is formulated for marketing.
Therefore,the process of separation and purification is known as downstream processing.

(B) The $t-DNA$ (transfer $DNA$) is a segment of $DNA$ found in the $Ti$ (tumor-inducing) plasmid of the bacterium $Agrobacterium$ $tumefaciens$.
When this bacterium infects a plant,it naturally transfers this $t-DNA$ into the host plant's genome.
This process is widely used in biotechnology to introduce foreign genes into plants for genetic modification.

(A) The Polymerase Chain Reaction $(PCR)$ is a technique used to amplify a specific $DNA$ segment.
It consists of three main steps performed in a cyclic manner:
$1$. Denaturation: The double-stranded $DNA$ is heated to a high temperature (usually around $94-98^{\circ}C$) to separate the two strands.
$2$. Annealing: The temperature is lowered (usually $50-65^{\circ}C$) to allow the primers to bind to the complementary sequences on the single-stranded $DNA$ templates.
$3$. Extension: The temperature is adjusted (usually $72^{\circ}C$) for the $DNA$ polymerase enzyme to synthesize the new $DNA$ strand by adding nucleotides to the primers.
Therefore,the correct order is Denaturation,Annealing,and Extension.

(D) For the industrial production of enzymes or other products on a large scale,microbes are grown in large vessels known as bioreactors.
Bioreactors provide optimal growth conditions such as temperature,$pH$,substrate,salts,vitamins,and oxygen to achieve the desired product.
$BOD$ incubators are used for laboratory-scale incubation,while sludge digesters are used in sewage treatment plants.
Therefore,the correct equipment for large-scale industrial production is the bioreactor.

(B) In the process of recombinant $DNA$ technology,the isolation of genetic material $(DNA)$ involves several steps.
After the removal of other biomolecules like $RNA$,proteins,and lipids using specific enzymes,the purified $DNA$ remains in the aqueous phase.
To precipitate the $DNA$ out of this mixture,chilled ethanol is added.
This causes the $DNA$ to form a fine thread-like precipitate,which can be collected by spooling.

(A) Transformation is a process where a bacterium takes up naked $DNA$ from its environment. This process is not a passive diffusion of $DNA$ molecules through the cell wall and membrane.
It is an active,energy-requiring process that occurs only in specific bacteria that possess the necessary enzymatic machinery for active uptake and subsequent recombination of the $DNA$ into the host genome.
Even within a population of such bacteria,only 'competent' cells,which express specific competence factors,are capable of performing this uptake.
Therefore,both the Assertion and the Reason are correct,and the Reason provides the correct explanation for why the process is active.

(C) The Southern blot technique is a method used to detect specific $DNA$ sequences in a sample.
The process begins with the isolation of $DNA$ from the sample,followed by the digestion of the $DNA$ using restriction enzymes.
Restriction enzymes act as molecular scissors that cut the $DNA$ at specific recognition sites,resulting in $DNA$ fragments of varying lengths.
After digestion,these fragments are separated based on their size using gel electrophoresis.

(C) In the polymerase chain reaction $(PCR)$,the number of $DNA$ molecules produced after $n$ cycles is given by the formula $2^n$,where $n$ is the number of cycles.
Given that the number of cycles $n = 4$,the number of $DNA$ molecules formed will be $2^4 = 2 \times 2 \times 2 \times 2 = 16$.

(A) Alternative selectable markers have been developed to differentiate recombinants from non-recombinants based on their ability to produce color in the presence of a chromogenic substrate.
When a recombinant $DNA$ is inserted into the coding sequence of the enzyme $\beta -$ galactosidase,the gene becomes non-functional.
This process is known as insertional inactivation.
Consequently,the bacteria containing the recombinant plasmid cannot produce the functional $\beta -$ galactosidase enzyme.
As a result,they fail to produce color in the presence of a chromogenic substrate,leading to the formation of colorless colonies.
Non-recombinant bacteria,which do not have the insert,produce functional $\beta -$ galactosidase and form blue-colored colonies.

(D) Let's analyze each statement:
$(a)$ $DNA$ is negatively charged due to the phosphate backbone. In gel electrophoresis,it moves towards the positive electrode (Anode). Therefore,it is loaded at the cathode end,not the anode end. This statement is incorrect.
$(b)$ $DNA$ fragments move through the matrix of the gel,not just the surface. The concentration of the gel (sieve effect) significantly affects the movement of $DNA$ fragments. This statement is incorrect.
$(c)$ Smaller $DNA$ fragments move faster and travel a greater distance through the gel matrix compared to larger fragments. This statement is correct.
$(d)$ $DNA$ is colorless and cannot be seen under visible light. It must be stained with a dye like Ethidium Bromide and then exposed to $UV$ radiation to be visualized. Pure $DNA$ cannot be visualized directly. This statement is incorrect.
Since statements $(a), (b),$ and $(d)$ are incorrect,the correct option is $(d)$.

(N/A) Bioreactors are large vessels in which raw materials are biologically converted into specific products,individual enzymes,etc.,using microbial,plant,animal,or human cells.
They provide optimal growth conditions for achieving the desired product by providing optimum temperature,$pH$,substrate,salts,vitamins,and oxygen.
The most commonly used bioreactors are of the stirring type,known as stirred-tank bioreactors.

(N/A) Shake flasks are used for growing and mixing the desired materials on a small scale in the laboratory. $A$ large-scale production of desired biotechnological products is done by using $bioreactors$. Besides better aeration and mixing properties,the bioreactors have the following advantages:
$(i)$ Small volumes of cultures can be periodically withdrawn from the bioreactor for sampling.
$(ii)$ They have a foam control system,a pH control system,and a temperature control system to regulate the environment during the process.
$(iii)$ They facilitate even mixing and oxygen availability throughout the bioreactor with the help of baffles.

(N/A) reporter enzyme is used to monitor the transformation of host cells by tracking the activity of its corresponding gene.
For example,the enzyme $\beta$-galactosidase $(LacZ)$ is used in blue-white screening.
When a foreign $DNA$ fragment is inserted into the $LacZ$ gene,the gene becomes inactivated (insertional inactivation).
Consequently,the transformed cells do not produce $\beta$-galactosidase and appear white in the presence of a chromogenic substrate.
In contrast,non-transformed cells or cells with non-recombinant plasmids produce the enzyme and appear blue.

(N/A) Origin of Replication: This is a specific sequence in the genome from where replication starts,and any piece of $DNA$ when linked to this sequence can be made to replicate within the host cells. This sequence is also responsible for controlling the copy number of the linked $DNA$. Thus,if one wants to recover many copies of the target $DNA$,it should be cloned in a vector whose origin supports a high copy number.
$(b)$ Bioreactor: Bioreactors are vessels in which raw materials are biologically converted into specific products by microbes,plant cells,animal cells,and/or their enzymes. The bioreactor provides optimum growth conditions (temperature,$pH$,substrate,salts,vitamins,oxygen) and facilitates the production of desired products. The most commonly used bioreactor is the stirred-tank type. $A$ stirred-tank bioreactor is usually a cylindrical vessel with a curved base to facilitate the mixing of contents. In a sparged stirred-tank bioreactor,sterile air bubbles are sparged. The stirrer facilitates mixing and oxygen availability throughout the bioreactor. It includes an agitator system,oxygen delivery system,foam control system,temperature control system,$pH$ control system,and sampling ports.
$(c)$ Downstream Processing: After the biosynthesis of the product,it is subjected to a series of processes collectively called downstream processing before it is marketed as a finished product. The two main processes are separation and purification. The product is then formulated with suitable preservatives. Such formulations must undergo clinical trials in the case of drugs.

(N/A) The main phases included in recombinant $DNA$ technology are as follows:
$(i)$ Identification and isolation of $DNA$ with desirable genes.
$(ii)$ Insertion of the isolated $DNA$ into a suitable vector to form recombinant $DNA$.
$(iii)$ Introduction of recombinant $DNA$ into the host cell.
$(iv)$ Selection of transformed host cells and maintenance of recombinant $DNA$ in the host.
$(v)$ Expression of the foreign gene to obtain the desired product.

(N/A) The cutting of $DNA$ by restriction endonucleases results in the fragments of $DNA$. These fragments can be separated by a technique known as $gel$ $electrophoresis$. Since $DNA$ 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.
Nowadays,the most commonly used matrix is agarose,which is a natural polymer extracted from sea weeds. The $DNA$ fragments separate according to their size through the sieving effect provided by the agarose gel. Hence,the smaller the fragment size,the farther it moves. Look at the figure and guess at which end of the gel the sample was loaded.
Separated $DNA$ fragments can be visualized only after staining the $DNA$ with a compound known as ethidium bromide followed by exposure to $UV$ radiation (you cannot see pure $DNA$ fragments in the visible light and without staining).
Bright orange-colored bands of $DNA$ can be observed in an ethidium bromide-stained gel exposed to $UV$ light (see figure). 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 recombinant $DNA$ by joining them with cloning vectors.

(N/A) $DNA$ is a hydrophilic molecule,so it cannot pass through cell membranes. To force bacteria to take up plasmids,the bacterial cells must be made competent. This is achieved through the following methods:
$1$. Chemical Treatment: Bacterial cells are treated with a specific concentration of a divalent cation,such as calcium. This increases the efficiency of $DNA$ entry through pores in the cell wall.
$2$. Heat Shock Method: $r-DNA$ is forced into these cells by incubating them with recombinant $DNA$ on ice,followed by a brief heat shock at $42^{\circ} C$,and then placing them back on ice. This enables the bacteria to take up the recombinant $DNA$.
$3$. Micro-injection: Recombinant $DNA$ is directly injected into the nucleus of an animal cell.
$4$. Biolistics (Gene Gun): Cells are bombarded with high-velocity micro-particles of gold or tungsten coated with $DNA$.
$5$. Disarmed Pathogen Vectors: These vectors are allowed to infect cells,thereby transferring the recombinant $DNA$ into the host.
$6$. Electroporation: Cells are subjected to high-voltage electric pulses to make the cell membrane permeable for $DNA$ entry.
$7$. Lipofection: Recombinant $DNA$ is encapsulated in a lipid layer,allowing it to pass through the cell membrane.

(N/A) Recombinant $DNA$ technology involves several steps in a specific sequence:
$1$. Isolation of the genetic material $(DNA)$.
$2$. Fragmentation of $DNA$ by restriction endonucleases.
$3$. Isolation of a desired $DNA$ fragment.
$4$. Ligation of the $DNA$ fragment into a vector.
$5$. Transferring the recombinant $DNA$ into the host.
$6$. Culturing the host cells in a medium at a large scale.
$7$. Extraction of the desired product.

(N/A) Nucleic acid is the genetic material of all organisms without exception. In the majority of organisms,it is $DNA$.
To cut the $DNA$ with restriction enzymes,it needs to be in a pure form,free from other macromolecules.
Since $DNA$ is enclosed within membranes,we must break the cell open to release $DNA$ along with other macromolecules such as $RNA$,proteins,polysaccharides,and lipids.
This is achieved by treating the bacterial cells,plant,or animal tissue with specific enzymes: lysozyme for bacteria,cellulase for plant cells,and chitinase for fungus.
$RNA$ can be removed by treatment with ribonuclease,whereas proteins can be removed by treatment with protease.
Other molecules are removed by various treatments,and the purified $DNA$ ultimately precipitates out after the addition of chilled ethanol. This can be observed as a collection of fine threads in the suspension,which can be removed by spooling.

(N/A) $PCR$ stands for $\text{Polymerase Chain Reaction}$. In this reaction,multiple copies of the gene (or $DNA$) of interest are synthesized in vitro using two sets of primers (small chemically synthesized oligonucleotides that are complementary to the regions of $DNA$) and the enzyme $DNA$ polymerase.
The enzyme extends the primers using the nucleotides provided in the reaction and the genomic $DNA$ as a template. If the process of replication of $DNA$ is repeated many times,the segment of $DNA$ can be amplified to approximately $1$ billion times.
Such repeated amplification is achieved by the use of a thermostable $DNA$ polymerase (isolated from a bacterium,$\text{Thermus aquaticus}$),which remains active during the high-temperature-induced denaturation of double-stranded $DNA$. The amplified fragment,if desired,can now be used to ligate with a vector for further cloning.

(N/A) $PCR$ stands for Polymerase Chain Reaction. In this reaction,multiple copies of the gene (or $DNA$) of interest are synthesized in vitro using two sets of primers (small chemically synthesized oligonucleotides that are complementary to the regions of $DNA$) and the enzyme $DNA$ polymerase.
The enzyme extends the primers using the nucleotides provided in the reaction and the genomic $DNA$ as a template. If the process of replication of $DNA$ is repeated many times,the segment of $DNA$ can be amplified to approximately $1$ billion times. Such repeated amplification is achieved by the use of a thermostable $DNA$ polymerase (isolated from a bacterium,Thermus aquaticus),which remains active during the high-temperature-induced denaturation of double-stranded $DNA$. The amplified fragment,if desired,can now be used to ligate with a vector for further cloning.

(N/A) There are several methods for introducing ligated $DNA$ into recipient cells. Recipient cells,after being made competent,can take up $DNA$ present in their surroundings.
If a recombinant $DNA$ molecule carrying a gene for antibiotic resistance (e.g.,ampicillin resistance) is transferred into $E. coli$ cells,the host cells become transformed into ampicillin-resistant cells.
When these transformed cells are spread on agar plates containing ampicillin,only the transformants will grow,while untransformed recipient cells will die.
Because of the ampicillin resistance gene,it is possible to select the transformed cells in the presence of ampicillin. In this context,the ampicillin resistance gene is referred to as a selectable marker.

(A) When you insert a piece of alien $DNA$ into a cloning vector and transfer it into a bacterial,plant,or animal cell,the alien $DNA$ gets multiplied.
$\Rightarrow$ In almost all recombinant technologies,the ultimate aim is to produce a desirable protein. Hence,there is a need for the recombinant $DNA$ to be expressed.
The foreign gene gets expressed under appropriate conditions.
If any protein-encoding gene is expressed in a heterologous host,it is called a recombinant protein.
$\Rightarrow$ The cells harbouring cloned genes of interest may be grown on a small scale in the laboratory.
$\Rightarrow$ The cultures may be used for extracting the desired protein and then purifying it by using different separation techniques.
The cells can also be multiplied in a continuous culture system wherein the used medium is drained out from one side while fresh medium is added from the other to maintain the cells in their physiologically most active log/exponential phase. This type of culturing method produces a larger biomass,leading to higher yields of the desired protein.

(N/A) Small volume cultures cannot yield appreciable quantities of products. To produce in large quantities,the development of bioreactors,where large volumes ($100$-$1000$ litres) of culture can be processed,was required.
Bioreactors can be thought of as vessels in which raw materials are biologically converted into specific products,individual enzymes,etc.,using microbial,plant,animal,or human cells.
$A$ bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature,$pH$,substrate,salts,vitamins,oxygen).
The most commonly used bioreactors are of the stirring type.
$A$ stirred-tank reactor is usually cylindrical or has a curved base to facilitate the mixing of the reactor contents.
The stirrer facilitates even mixing and oxygen availability throughout the bioreactor. Alternatively,air can be bubbled through the reactor.
The bioreactor has an agitator system,an oxygen delivery system,a foam control system,a temperature control system,a $pH$ control system,and sampling ports so that small volumes of the culture can be withdrawn periodically.

(N/A) After the completion of the biosynthetic stage,the product must undergo a series of processes before it is ready for marketing as a finished product.
The processes include separation and purification,which are collectively referred to as downstream processing.
$\rightarrow$ The product must be formulated with suitable preservatives.
$\rightarrow$ Such formulations must undergo thorough clinical trials,as in the case of drugs.
Strict quality control testing for each product is also required.
The downstream processing and quality control testing vary from product to product.

(B) Downstream processing is a crucial stage in recombinant $DNA$ technology that involves the separation and purification of the synthesized product.
$1$. Purification: The product must be isolated from the complex mixture of the bioreactor to ensure it is free from contaminants.
$2$. Quality Control: The product undergoes rigorous testing to ensure it meets safety and efficacy standards.
$3$. Formulation: The product is formulated with suitable preservatives and additives to make it stable and ready for clinical use.
$4$. Safety Assessment: Before being released for human welfare,the product is tested for potential side effects or toxicity to ensure it is safe for consumption or medical application.

(N/A) $1$. Cloning: The process of creating multiple copies of a specific segment of foreign $DNA$ by inserting it into a suitable vector and allowing it to replicate within a host organism.
$2$. Elution: The process of extracting the separated $DNA$ fragments from the agarose gel after gel electrophoresis. The specific $DNA$ band is cut out from the gel and the $DNA$ is purified from the gel piece.
$3$. Insertional Inactivation: $A$ method used to identify recombinant colonies. When a foreign gene is inserted into a selectable marker gene (e.g.,an antibiotic resistance gene) present in the plasmid,the marker gene becomes inactivated. This loss of function allows for the differentiation between recombinant and non-recombinant cells.

(B) In transformation experiments,'competent' refers to the ability of bacterial cells to take up foreign $DNA$ from their surroundings.
Normally,$DNA$ is a hydrophilic molecule and cannot pass through cell membranes.
To facilitate this,bacterial cells are treated with a specific concentration of a divalent cation,such as $CaCl_2$,which increases the efficiency with which $DNA$ enters the bacterium through pores in its cell wall.

(B) The role of proteases is to degrade the proteins present inside a cell from which $DNA$ is being isolated.
Proteins are often associated with $DNA$ (e.g.,histones) or present as cellular debris.
If these proteins are not removed,they can interfere with downstream processes such as the action of restriction endonucleases or $DNA$ ligases during recombinant $DNA$ technology.

(B) The $PCR$ process relies on the denaturation step to separate the double-stranded $DNA$ into single strands at high temperatures (around $94-98^{\circ}C$).
If the denaturation step is missed,the $DNA$ remains double-stranded.
Consequently,the primers cannot bind (anneal) to the complementary sequences on the template $DNA$.
As a result,the $DNA$ polymerase cannot initiate the extension process,and no amplification of the target $DNA$ sequence will occur.

(A) Gene cloning is a molecular biology technique used to produce multiple identical copies of a specific gene or $DNA$ fragment.
$1$. The process begins by isolating the gene of interest and ligating it into a suitable vector (such as a plasmid).
$2$. This creates a recombinant $DNA$ molecule.
$3$. The recombinant $DNA$ is then introduced into a host cell (usually a bacterium) through a process called transformation.
$4$. As the host cell divides,the recombinant $DNA$ is replicated along with the host's genome.
$5$. Eventually,a bacterial colony is formed where each cell contains multiple copies of the cloned gene.

(N/A) The process of cloning and expressing a human gene in $E. coli$ involves the following steps:
$1$. Isolation of the target gene: The human gene encoding for growth hormone is isolated using restriction enzymes.
$2$. Selection of a vector: $A$ suitable cloning vector,such as a plasmid,is chosen.
$3$. Construction of recombinant $DNA$: The human gene is inserted into the vector using $DNA$ ligase to form recombinant $DNA$.
$4$. Transformation: The recombinant $DNA$ is introduced into the host bacterium,$E. coli$.
$5$. Selection and Screening: Transformed bacteria are selected using antibiotic resistance markers.
$6$. Expression: The $E. coli$ cells are cultured under optimal conditions to express the human growth hormone protein.

(N/A) Yes,a disease can be detected before its symptoms appear.
Normally,the presence of a pathogen (bacteria,viruses,etc.) is suspected only when it has produced disease symptoms. By this time,the concentration of the pathogen is already very high in the body.
However,a very low concentration of a bacteria or virus can be detected even when symptoms are not yet visible.
The principle involved is the amplification of their nucleic acid ($DNA$ or $RNA$) using $PCR$ (Polymerase Chain Reaction).
$PCR$ creates millions of copies of the specific $DNA$ sequence of the pathogen,making it detectable even if the initial sample contains only a few copies.

(N/A) $PCR$ (Polymerase Chain Reaction) is a highly sensitive technique that allows for the specific amplification of a target $DNA$ sequence from a very small amount of $DNA$ template. Because of this high sensitivity,it can detect the presence of pathogenic organisms in an infected patient at a very early stage of infection,even before the pathogen has multiplied to a large number in the host's body.

(N/A) single-stranded $DNA$ or $RNA$,tagged with a radioactive molecule (probe),is allowed to hybridize to its complementary $DNA$ in a clone of cells,followed by detection using autoradiography.
The clone having the mutated gene will not appear on the photographic film because the probe will not have complementarity with the mutated gene.

(D) The experiment will not be affected because the recombinant $DNA$ molecule is a circular,closed structure with no free ends.
Exonucleases are enzymes that remove nucleotides specifically from the free ends of $DNA$ molecules.
Since the recombinant plasmid $DNA$ lacks free $3'$ or $5'$ ends,it will not act as a substrate for the exonuclease enzyme.
Therefore,the integrity of the recombinant $DNA$ remains intact,and the bacterial transformation process can proceed as planned.

(B) compound called Ethidium Bromide $(EtBr)$ is used to stain $DNA$ molecules.
When the agarose gel is treated with $EtBr$ and subsequently exposed to Ultraviolet $(UV)$ light,the $DNA$ fragments emit an orange fluorescence.
This allows the visualization of $DNA$ bands on the gel.