Tools of recombinant DNA technology Questions in English

(C) molecular probe is a short,single-stranded segment of $DNA$ or $RNA$ that is radioactively labeled (e.g.,with $P^{32}$).
It typically consists of $20-40$ nucleotides.
The probe is designed to be complementary to a specific sequence within the desired gene,allowing it to hybridize with the target sequence for identification.
Therefore,statements $III$ and $IV$ are correct.

(D) In recombinant $DNA$ technology,a vector contains an antibiotic resistance gene (selectable marker) that allows for the identification and elimination of non-transformants.
When a foreign $DNA$ fragment is inserted into the vector,it often disrupts the antibiotic resistance gene (insertional inactivation).
This allows researchers to distinguish between transformed cells (those that have taken up the plasmid) and non-transformed cells,and further distinguish between recombinant cells (those containing the foreign $DNA$) and non-recombinant cells (those containing the original vector).

(C) The Western blotting technique,also known as protein immunoblotting,was developed by $Harry \ Towbin$,$Theophil \ Staehelin$,and $Julian \ Gordon$ in $1979$.
This technique is used to detect specific proteins in a sample of tissue homogenate or extract.
It involves the transfer of proteins from a gel to a membrane,followed by detection using antibodies specific to the target protein.
Therefore,the correct answer is $Towbin$.

(B) The ability of a cell to take up foreign $DNA$ from its environment is known as $Competence$.
$Competence$ is a physiological state that allows a bacterial cell to undergo transformation,which is the process of horizontal gene transfer where exogenous genetic material is taken up by the cell.
$Sexduction$ refers to the transfer of chromosomal genes by an $F'$ factor.
$Hfr$ stands for High frequency of recombination,which is a bacterium with a conjugative plasmid integrated into its main chromosomal $DNA$.
$Transduction$ is the process by which foreign $DNA$ is introduced into a cell by a virus or viral vector.

(A) Recombinant $DNA$ is formed by inserting a foreign $DNA$ fragment into a vector. In many cloning vectors,the foreign $DNA$ is inserted at specific restriction sites located within the antibiotic resistance genes. This insertion results in the insertional inactivation of the antibiotic resistance gene,which is a crucial step in identifying recombinant colonies.

(A) $DNA$ polymerase,which is stable at high temperatures $(>90^{\circ}C)$,is required to carry out the synthesis of new $DNA$ strands during the extension phase of $PCR$.
$DNA$ polymerases like $Taq$ polymerase are generally used in $PCR$ reactions.
$Taq$ polymerase is isolated from a thermophilic bacterium called $Thermus$ $aquaticus$.

(B) Genetic engineering,also known as recombinant $DNA$ technology,involves the manipulation of genetic material to produce desired traits.
Plasmids are small,circular,double-stranded $DNA$ molecules that are distinct from a cell's chromosomal $DNA$.
In genetic engineering,plasmids are extensively used as vectors to carry foreign genes into host cells (such as $E. coli$) for cloning or expression.
Therefore,plasmids are essential tools in genetic engineering.

(D) $Agrobacterium$ $tumefaciens$ is a soil bacterium that acts as a natural genetic engineer because it can transfer a piece of $DNA$ known as $T-DNA$ into the host plant cells,causing them to produce compounds that the bacterium can use. This ability to naturally transform plant cells is widely exploited in biotechnology to introduce foreign genes into plants.

(B) In recombinant $DNA$ technology,specific enzymes are used to break down cell walls to release $DNA$.
$1$. Plant cells have cell walls made of cellulose,which is degraded by the enzyme $Cellulase$.
$2$. Fungi have cell walls made of chitin,which is degraded by the enzyme $Chitinase$.
$3$. Bacteria have cell walls made of peptidoglycan,which is degraded by the enzyme $Lysozyme$.
$4$. Algae cell walls are primarily composed of cellulose,galactans,and mannans. $Methylase$ is an enzyme involved in $DNA$ methylation,not in cell wall degradation. Therefore,the pair $Algae-Methylase$ is incorrectly matched.

(D) The popularity and success of $PCR$ (polymerase chain reaction) are primarily due to the availability of thermostable $DNA$ polymerase.
During the $PCR$ process,the reaction mixture is heated to high temperatures $(>90^{\circ} C)$ to denature the double-stranded $DNA$.
Standard $DNA$ polymerases would denature and become inactive at these temperatures.
Therefore,a thermostable $DNA$ polymerase,such as $Taq$ polymerase (isolated from the bacterium $Thermus$ $aquaticus$),is essential as it remains active even after repeated heating cycles.

(C) $pBR322$ was the first artificial cloning vector constructed in $1977$ by Boliver and Rodriquez.
It is widely used in gene cloning experiments.
In the name $pBR322$:
$p$ denotes that it is a plasmid.
$BR$ stands for Boliver and Rodriquez,the scientists who constructed this plasmid.
$322$ is a number assigned to distinguish this specific plasmid from others developed in the same laboratory.

(C) In genetic engineering,$Restriction$ $endonucleases$ are used to cut $DNA$ at specific recognition sequences,while $DNA$ $ligase$ is used to join the $DNA$ fragments together. Therefore,these two enzymes are fundamental tools in recombinant $DNA$ technology.

(B) The key tools required for recombinant $DNA$ technology include:
$1$. Enzymes (such as restriction endonucleases,$DNA$ ligases,etc.)
$2$. Cloning vectors (to carry the foreign $DNA$ into the host)
$3$. Competent host organisms (to receive the recombinant $DNA$)
$RNA$ primers are specifically used in the Polymerase Chain Reaction $(PCR)$ technique,and $DNA$ probes are used for the detection of specific $DNA$ sequences,but they are not considered the fundamental 'key tools' for the basic construction of recombinant $DNA$ molecules in the same category as enzymes,vectors,and host organisms. Therefore,the correct combination is $I, IV$ and $V$.

(C) $RNA$ polymerase is an enzyme involved in the process of transcription,where $RNA$ is synthesized from a $DNA$ template. It is not directly involved in the synthesis of $RNA$ primers (which is done by primase).
Respiration occurs in the mitochondria,not the lysosome.
Restriction enzymes are essential tools in genetic engineering used to cut $DNA$ at specific recognition sequences.
The Central Dogma describes the flow of genetic information from $DNA$ to $RNA$ to protein,not just the structure of $DNA$.

(B) Plasmids are extrachromosomal,self-replicating,circular $DNA$ molecules found in bacteria.
They are suitable as cloning vectors because they possess an origin of replication ($ori$ site),which allows them to replicate independently of the host chromosomal $DNA$.
This independent replication ensures that the inserted gene of interest is copied multiple times within the host cell.

(D) Restriction enzymes are known as molecular knives or molecular scissors and are used to cut $DNA$ at specific sites. These enzymes were first discovered by $Smith$, $Nathan$, and $Arber$ in the year $1970$.

(B) During gene cloning,a plasmid is referred to as a gene taxi or vector.
Molecular biologists insert the desired gene into the plasmid $DNA$.
This recombinant plasmid is then introduced into a host cell,such as a living bacterium,to replicate the gene.

(C) $Agrobacterium \text{ } tumefaciens$ contains a tumor-inducing $(Ti)$ plasmid, which causes the formation of crown gall tumors in infected plants.
This interaction is the most thoroughly studied because scientists have successfully modified the $Ti$ plasmid to act as a cloning vector.
By removing the tumor-causing genes and replacing them with genes of interest, it has become a fundamental tool in plant genetic engineering to transfer foreign $DNA$ into host plants.

(D) cloning vector is a small piece of $DNA$ taken from any organism into which a foreign $DNA$ fragment can be inserted for cloning purposes.
These vectors act as carriers to transport foreign genetic material into a host cell.
They are also commonly referred to as vehicle $DNA$ or gene carriers because they facilitate the transfer and replication of the inserted $DNA$ within the host organism.
Therefore,all the given terms are used to describe the same biological tool.

(B) vector is a $DNA$ molecule used as a vehicle to artificially carry foreign genetic material into another cell,where it can be replicated and/or expressed.
Plasmids and bacteriophages are the most commonly used vectors in recombinant $DNA$ technology because they have the ability to replicate within bacterial cells independent of the control of chromosomal $DNA$.

(C) Herbert Boyer discovered that restriction enzymes have the capability of cutting $DNA$ strands in a particular fashion,which left what has become known as 'sticky ends' on the strands. This discovery was fundamental to the development of recombinant $DNA$ technology.

(A) Agarose is a natural polysaccharide extracted from sea weeds (specifically red algae like Gelidium and Gracilaria).
In the process of gel electrophoresis,$DNA$ fragments are separated based on their size as they move through the pores of the agarose gel matrix under an electric field.

(A) In gel electrophoresis,the process of separating $DNA$ fragments requires the $DNA$ to be cut into smaller pieces first.
This cutting process is performed by specific enzymes known as restriction endonucleases.
These enzymes recognize specific palindromic nucleotide sequences in the $DNA$ molecule and cleave the phosphodiester bonds at or near these sites,resulting in fragments of varying lengths that can then be separated by gel electrophoresis.

(B) In recombinant $DNA$ technology,a selectable marker is a gene introduced into a cell that confers a trait suitable for artificial selection.
It allows the identification and elimination of non-transformants and selectively permits the growth of the transformants.
Since the chloramphenicol resistance gene allows for the selection of transformed cells in the presence of the antibiotic chloramphenicol,it functions as a selectable marker.

(B) In recombinant $DNA$ technology,vectors are $DNA$ molecules used as vehicles to carry a particular foreign $DNA$ segment into the host cell.
$A$ refers to $Vector$ (e.g.,plasmids or bacteriophages).
$B$ refers to $DNA$ (the gene of interest).
Therefore,the correct option is $(B)$.

(D) Restriction enzymes,also known as molecular scissors,are essential tools in biotechnology.
$1$. They are used to cut $DNA$ at specific recognition sequences,which is the first step in creating recombinant $DNA$ molecules.
$2$. They are used in gene mapping by creating specific $DNA$ fragments (Restriction Fragment Length Polymorphism - $RFLP$) that help in locating genes on chromosomes.
$3$. They are used in the diagnosis of genetic diseases by identifying mutations that alter restriction sites,allowing for the detection of specific genetic disorders.
Therefore,all the given options are correct applications of restriction enzymes.

(C) Restriction enzymes are a class of enzymes that recognize specific nucleotide sequences (palindromic sequences) in $DNA$ and cut the $DNA$ at specific sites.
These enzymes are derived from bacteria,where they serve as a defense mechanism against bacteriophages.
However,in genetic engineering,they are used as 'molecular scissors' to cut $DNA$ molecules from any source,whether it is bacterial,viral,or eukaryotic,provided the specific recognition site is present.
Therefore,restriction enzymes can be used to cut any $DNA$ fragment.

(D) Restriction endonucleases are enzymes that cut $DNA$ at specific sites.
These specific sites are known as recognition sequences.
These sequences are typically palindromic in nature,meaning they read the same in both directions $(5' \rightarrow 3')$ on the two strands.
Since all the terms provided describe the nature and function of these sites,the correct answer is $D$.

(C) Apart from $Taq$ polymerase,which is isolated from the bacterium $Thermus$ $aquaticus$,other thermostable $DNA$ polymerases such as $Vent$ polymerase (isolated from $Thermococcus$ $litoralis$) and $Pfu$ polymerase (isolated from $Pyrococcus$ $furiosus$) are also used in $PCR$. These enzymes are preferred because they possess high fidelity and remain stable at the high temperatures required for $DNA$ denaturation.

(C) The given $DNA$ sequence is $5^{\prime}-GTCGAC-3^{\prime}$ / $3^{\prime}-CAGCTG-5^{\prime}$.
This sequence represents the recognition site for the restriction enzyme $Hind\;II$.
$Hind\;II$ was the first restriction endonuclease to be isolated and it always cuts $DNA$ molecules at a particular point by recognizing a specific recognition sequence of $6$ base pairs.
The recognition sequence for $Hind\;II$ is $GTCGAC$.

(D) $1$. Bacteriophages are viruses that infect bacteria. Therefore,$A$ is $Virus$ and $B$ is $Bacteria$.
$2$. Cosmids are hybrid vectors constructed by combining features of plasmids and the $cos$ site of the $\lambda$ (lambda) phage. Therefore,$C$ is $Cosmid$.
$3$. Matching these with the options,we get $A-$Virus,$B-$Bacteria,$C-$Cosmid.

(D) Genetic engineering,or recombinant $DNA$ technology,relies on the ability to manipulate $DNA$ molecules precisely.
Restriction endonucleases are enzymes that act as 'molecular scissors' by cutting $DNA$ at specific recognition sequences.
These enzymes were originally discovered in bacteria as a defense mechanism against bacteriophages.
Once purified,these restriction endonucleases can be used in vitro (outside the living organism) to cut $DNA$ molecules at specific sites,which is a fundamental step in creating recombinant $DNA$ constructs.

(D) The gene gun method,also known as the biolistic technique,is a direct gene transfer method used primarily for plants.
Statement $I$ is correct: It is indeed called the biolistic technique.
Statement $II$ is correct: In this process,cells are bombarded with high-velocity micro-particles of gold or tungsten coated with the target $DNA$.
Statement $III$ is correct: This technique has been successfully used to transform important crop plants such as maize,rice,and wheat.
Therefore,all three statements are correct.

(D) In the $pBR322$ vector,$amp^R$ stands for ampicillin resistance gene and $tet^R$ stands for tetracycline resistance gene. These are antibiotic resistance genes that serve as selectable markers. $Ori$ is the origin of replication,and $Rop$ codes for proteins involved in the replication of the plasmid.

(A) The primary role of $DNA$ ligase in the construction of a recombinant $DNA$ molecule is the formation of a phosphodiester bond between two $DNA$ fragments.
$DNA$ ligase acts as a molecular glue by sealing the nicks or gaps in the sugar-phosphate backbone of $DNA$ strands.
It catalyzes the joining of the $3'$-hydroxyl end of one nucleotide to the $5'$-phosphate end of another.
This enzyme is essential for joining the vector $DNA$ and the insert $DNA$ to create a functional recombinant molecule.

(B) Ligases are enzymes that catalyze the joining of two large molecules by forming a new chemical bond. In the context of $DNA$ technology, $DNA$ ligase catalyzes the formation of a phosphodiester bond between the $3'-OH$ end of one nucleotide and the $5'-phosphate$ end of another nucleotide. This bond involves the linkage of $C-O-P-O-C$ atoms, specifically forming the $P-O$ bond within the sugar-phosphate backbone.

(D) Recombinant $DNA$ technology involves the insertion of a specific gene into a vector to create a recombinant molecule.
$Plasmids$ and $Phages$ are commonly used as vectors to carry the foreign $DNA$ into the host cell.
$Restriction$ enzymes are essential for cutting the $DNA$ at specific recognition sequences to create sticky or blunt ends.
$DNA$ polymerase $III$ is the primary enzyme responsible for $DNA$ replication in prokaryotes (like $E. coli$). It is not typically used in the construction of recombinant $DNA$ molecules in vitro; instead,$DNA$ ligase is used to join the $DNA$ fragments,and sometimes $DNA$ polymerase $I$ or $Taq$ polymerase is used for specific applications like filling in ends or $PCR$ amplification.

(C) In recombinant $DNA$ technology,a desired segment of $DNA$ or a gene is combined with the $DNA$ of a vector to multiply and produce copies of the gene.
Viruses (such as bacteriophages) and plasmids are the most commonly used cloning vectors in recombinant $DNA$ technology because they can replicate within host cells.

(A) Chemical synthesis of a gene is possible when the exact sequence of nucleotides in the gene is known.
This process involves the synthesis of short oligonucleotides that are subsequently joined together to form the complete gene sequence.
This technique is particularly useful for synthesizing genes that are small or for creating synthetic genes with specific modifications.
Other options like restriction enzymes,Sanger method (used for sequencing),or cDNA synthesis (reverse transcription) are different approaches to obtaining genes,not chemical synthesis.

(D) In recombinant $DNA$ technology,the donor cell provides the gene of interest. Since the genetic code is universal,genes from any living organism (prokaryotic or eukaryotic) can be isolated and inserted into a host bacterium for replication and expression. Therefore,any kind of cell can serve as a donor.

(C) $DNA$ probe is a single-stranded sequence of $DNA$ or $RNA$ that has a known sequence.
It is labeled with a radioactive isotope or a fluorescent dye.
It is used to detect the presence of complementary nucleotide sequences in a sample of $DNA$ or $RNA$ through the process of hybridization.
Therefore,the correct option is $C$.

(A) Thermostable enzymes 'Taq' and 'Vent' isolated from thermophilic bacteria are $DNA$ polymerase.
$Taq$ polymerase is isolated from the thermophilic bacterium $Thermus \; aquaticus$.
$Vent$ polymerase is isolated from the thermophilic bacterium $Thermococcus \; litoralis$.
These enzymes are widely used in the Polymerase Chain Reaction $(PCR)$ technique because they remain stable at high temperatures required for the denaturation of $DNA$.

(A) The naming convention for restriction enzymes follows a specific pattern:
$1$. The first letter comes from the Genus name of the organism.
$2$. The next two letters come from the species name.
$3$. The fourth letter represents the strain of the organism.
$4$. These enzymes are typically extracted from prokaryotic organisms (bacteria).
Therefore, $A = \text{Genus}$, $B = \text{species}$, $C = \text{strain}$, and $D = \text{bacteria}$.

(D) Statement $I$ is correct: $Eco RI$ is indeed isolated from the bacterium $Escherichia \; coli \; RY13$.
Statement $II$ is correct: The recognition sequence for $Eco RI$ is $5^{\prime}-GAATTC-3^{\prime}$ and $3^{\prime}-CTTAAG-5^{\prime}$.
Statement $III$ is correct: The enzyme cleaves the $DNA$ between the bases $G$ and $A$ on both strands,resulting in sticky ends.
Since all the given statements are correct,the correct answer is 'None of the above'.

(A) The most important feature in a plasmid to be used as a vector is the origin of replication $(Ori)$.
Origin of replication is a specific sequence of $DNA$ bases that is responsible for initiating the replication process.
Without an $Ori$ site,the plasmid cannot replicate within the host cell,making it impossible to maintain the recombinant $DNA$ molecule.
While selectable markers and restriction sites are also essential for a vector,the origin of replication is the fundamental requirement for the autonomous replication of the plasmid.

(D) Plasmids are small,circular,double-stranded $DNA$ molecules that are distinct from a cell's chromosomal $DNA$.
They are widely used as vectors in genetic engineering because they possess an origin of replication $(ori)$,which allows them to replicate independently within the host cell.
Because they are circular,they lack free ends,which protects them from degradation by exonucleases.
Furthermore,their ability to integrate or carry foreign $DNA$ makes them ideal for transferring genes into host organisms,including eukaryotic cells.

(A) Primers are small,chemically synthesized oligonucleotides,typically $10-18$ nucleotides in length.
They are designed to be complementary to the specific sequences present at the $3'$ ends of the target $DNA$ segment.
These primers provide a free $3'$-$OH$ group,which is essential for the $DNA$ polymerase enzyme to initiate the process of $DNA$ replication during $PCR$.

(A) In bacteria,the restriction-modification system serves as a defense mechanism against bacteriophages. The restriction endonuclease recognizes specific $DNA$ sequences and cleaves them. To prevent the host's own $DNA$ from being degraded by its own restriction enzymes,the bacterium produces a methylase enzyme. This enzyme adds a methyl group to specific bases within the recognition sequence of the host $DNA$. This modification prevents the restriction endonuclease from binding to or cutting the host $DNA$,thereby protecting it.

(D) $Agrobacterium \; tumefaciens$ is a soil-inhabiting bacterium that acts as a natural pathogen for many dicot plants.
It contains a $Ti-plasmid$ (Tumour-inducing plasmid).
During infection,it transfers a specific segment of $DNA$ known as $T-DNA$ into the host plant cell genome.
This $T-DNA$ integrates into the plant genome and causes the formation of tumours (galls) by inducing uncontrolled cell division.
Therefore,all three statements ($I, II,$ and $III$) are correct.

(C) The first restriction endonuclease discovered was Hind $II$.
It was isolated by Smith,Wilcox,and Kelley in $1970$ from the bacterium Haemophilus influenzae.
Hind $II$ always cuts $DNA$ molecules at a particular point by recognizing a specific sequence of six base pairs,which is known as the recognition sequence.