Tools of recombinant DNA technology Questions in English

(B) The gene gun method,also known as biolistics or microprojectile bombardment,is a technique used to introduce foreign $DNA$ into host cells. In this method,microparticles made of heavy metals like $Gold$ or $Tungsten$ are coated with the target $DNA$. These particles are then bombarded into the target cells at high velocity. This method is particularly effective for plant cells where the cell wall acts as a barrier.

(A) In genetic engineering,$Escherichia$ $coli$ $(E. coli)$ is widely used as a cloning host due to its well-understood genetics and rapid growth.
$Agrobacterium$ $tumefaciens$ is a soil bacterium that naturally transfers a segment of its $DNA$ ($T$-$DNA$) into plant cells,making it an essential tool for plant genetic engineering and the creation of transgenic plants.
Therefore,these two bacteria are considered the most useful in genetic engineering applications.

(D) Restriction enzymes are known as molecular scissors because they cut $DNA$ at specific recognition sequences.
$EcoRI$,$HindIII$,and $BamHI$ are all examples of restriction endonucleases.
Therefore,all the given options are correct.

(A) $Agrobacterium$ $tumefaciens$ is a soil bacterium that is widely used in genetic engineering as a natural vector for plant transformation. It possesses a $Ti$ (tumor-inducing) plasmid,which it uses to transfer a specific segment of its $DNA$,known as $T-DNA$,into the host plant genome. This ability makes it an essential tool for creating transgenic plants.

(D) In the nomenclature of restriction enzymes, the first letter of the name comes from the genus and the second two letters come from the species of the prokaryotic cell from which they were isolated.
For $EcoRI$, the name is derived from $Escherichia$ $coli$ $RY13$.
- $E$ stands for the genus $Escherichia$.
- $co$ stands for the species $coli$.
- $R$ stands for the strain $RY13$.
- $I$ indicates the order in which the enzyme was isolated from that strain.
Therefore, the part "co" represents $coli$.

(B) Gel electrophoresis is a laboratory technique used to separate mixtures of $DNA$,$RNA$,or proteins according to molecular size.
In this process,the molecules are pushed by an electrical field through a gel that contains small pores.
$DNA$ fragments are negatively charged,so they move towards the positive electrode (anode).
Smaller fragments move faster through the gel matrix than larger ones,allowing for their separation based on size.

(C) The first restriction endonuclease was discovered by $Werner \text{ } Arber$, $Hamilton \text{ } Smith$, and $Daniel \text{ } Nathans$.
$Hamilton \text{ } Smith$ and $Daniel \text{ } Nathans$ are specifically credited with the isolation and characterization of the first restriction enzyme, $HindII$, in $1970$.
Therefore, the correct option is $C$.

(B) Restriction enzymes,often called 'molecular scissors',are used in genetic engineering to cut $DNA$ at specific recognition sequences. By cutting the vector $DNA$ and the foreign $DNA$ with the same restriction enzyme,complementary sticky ends are produced. These ends can be joined using $DNA$ ligase to form recombinant $DNA$ molecules. Therefore,they are essential tools for creating recombinant $DNA$.

(B) In biotechnology,vectors are used to deliver foreign genes into host cells. For higher organisms (including animals),retroviruses are commonly used as vectors because they have the natural ability to infect host cells and integrate their genetic material into the host genome. By modifying these viruses to remove their pathogenic genes,they become safe and effective tools for gene transfer.

(B) In genetic engineering,$Plasmids$ are small,circular,double-stranded $DNA$ molecules that are distinct from a cell's chromosomal $DNA$. They are widely used as vectors to carry foreign genetic material into another cell,making them a fundamental tool in recombinant $DNA$ technology.

(B) The enzymes $Taq$ polymerase (isolated from $Thermus$ $aquaticus$) and $Pfu$ polymerase (isolated from $Pyrococcus$ $furiosus$) are thermostable $DNA$ polymerases.
These enzymes are essential in the Polymerase Chain Reaction $(PCR)$ technique because they can withstand the high temperatures required for the denaturation of $DNA$ strands without getting denatured themselves.

(D) $Agrobacterium$ $tumefaciens$ is a soil-borne pathogen that naturally infects a wide range of dicotyledonous plants by transferring a segment of its $DNA$ (known as $T-DNA$) into the host plant genome.
In biotechnology,this natural gene transfer mechanism is exploited to introduce foreign genes into plants.
While $Agrobacterium$ is highly effective for dicots,it is less efficient for monocots like cereal plants.
However,it remains the most common biological vector used in plant genetic engineering for transformation purposes.

(A) Plasmids are extrachromosomal,self-replicating,circular $DNA$ molecules found in bacteria.
They are not part of the main chromosome.
In genetic engineering,plasmids are widely used as 'vectors' to transfer desired genes into a host cell.
Therefore,option $A$ is correct.

(C) In recombinant $DNA$ technology,a 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 are the most commonly used vectors because they are small,circular,extrachromosomal $DNA$ molecules that can replicate independently within a host cell.
Therefore,a vector acts as a vehicle to transfer $DNA$ into a host cell.

(B) Restriction endonucleases are a class of enzymes that recognize specific nucleotide sequences in a $DNA$ molecule,known as recognition sequences or palindromic sequences.
They cut the $DNA$ at these specific sites,which is why they are often referred to as 'molecular scissors'.
This property is fundamental to recombinant $DNA$ technology,as it allows for the precise cutting of $DNA$ fragments to be inserted into vectors.

(D) In recombinant $DNA$ technology,the antibiotic resistance gene in a plasmid vector serves as a selectable marker.
When foreign $DNA$ is inserted into the plasmid,it often disrupts the antibiotic resistance gene (insertional inactivation).
$1$. Transformants are cells that have taken up the plasmid (either recombinant or non-recombinant).
$2$. Non-recombinants contain the original plasmid with a functional antibiotic resistance gene and can grow on antibiotic-containing media.
$3$. Recombinants contain the plasmid with the foreign $DNA$,which disrupts the resistance gene,making them sensitive to the antibiotic.
Therefore,the antibiotic resistance gene is used to distinguish between transformants (recombinants vs. non-recombinants) and to specifically identify recombinants.

(D) The sequence provided in such problems typically represents a palindromic sequence,which is a sequence of base pairs that reads the same forward and backward on the two strands of $DNA$ (e.g.,$5'-GAATTC-3'$ and $3'-CTTAAG-5'$). This is a characteristic feature recognized by restriction enzymes in biotechnology.

(B) $pBR-322$ is one of the most widely used cloning vectors in biotechnology.
It is not a natural plasmid but an engineered (artificial) plasmid constructed by Bolivar and Rodriguez in $1977$ from $E. coli$ plasmids.
It contains specific restriction sites,antibiotic resistance genes ($amp^R$ and $tet^R$),and an origin of replication $(ori)$,which makes it an ideal tool for gene cloning.

(A) Restriction endonucleases are enzymes that cut $DNA$ at specific recognition sequences. They are naturally produced by bacteria as a defense mechanism to destroy foreign $DNA$,such as viral $DNA$,that enters the cell. This process is known as the restriction-modification system. Therefore,option $A$ is the correct statement.

(D) cloning vector is a small piece of $DNA$ that can be stably maintained in an organism and into which a foreign $DNA$ fragment can be inserted for cloning purposes.
For a vector to function,it must possess an $ori$ ($Origin$ of replication) sequence.
This sequence is responsible for initiating replication within the host cell.
Without an $ori$ site,the vector cannot replicate,and the foreign $DNA$ linked to it will not be multiplied.

(D) $DNA$ or $RNA$ molecule that is single-stranded and tagged with a radioactive molecule is known as a $Probe$.
These probes are used in molecular biology to detect the presence of complementary nucleotide sequences in a sample through hybridization.
They are essential tools in techniques like $Southern$ $blotting$ and $Northern$ $blotting$ to identify specific gene sequences.

(A) The $Ti$ plasmid (tumor-inducing plasmid) is naturally found in the soil bacterium $Agrobacterium$ $tumefaciens$.
This bacterium has the natural ability to transfer a portion of its $DNA$,known as $T-DNA$,into the genome of infected plant cells,causing them to form tumors (crown galls).
In biotechnology,scientists have modified this $Ti$ plasmid by removing the tumor-causing genes while retaining the $T-DNA$ border sequences.
This modified plasmid is then used as a cloning vector to introduce desired genes into plant cells,making it a powerful tool for plant genetic engineering.

(A) $Agrobacterium \text{ } tumefaciens$ is a soil bacterium that naturally infects plants.
It contains a large extrachromosomal circular $DNA$ molecule known as the $Ti$-plasmid (Tumor-inducing plasmid).
This plasmid carries the $T-DNA$ (Transfer $DNA$) region, which is integrated into the host plant's genome, causing the formation of crown gall tumors.
Therefore, the correct answer is $Ti$-plasmid.

(A) Restriction enzymes belong to a larger class of enzymes called nucleases. These are of two kinds: exonucleases and endonucleases. Exonucleases remove nucleotides from the ends of the $DNA$ molecule,whereas endonucleases make cuts at specific positions within the $DNA$. These specific positions are known as recognition sequences or restriction sites. Therefore,restriction endonucleases are essential tools in recombinant $DNA$ technology for creating specific fragments of $DNA$.

(B) Sticky ends are short,single-stranded overhangs produced by the action of restriction endonucleases on $DNA$ molecules.
These sticky ends form hydrogen bonds with complementary sequences on another $DNA$ fragment.
However,to permanently join the sugar-phosphate backbones of the two $DNA$ fragments,the enzyme $DNA$ ligase is required.
Therefore,$DNA$ ligase facilitates the covalent joining of the $DNA$ fragments that have been brought together by the base pairing of their sticky ends.

(B) The restriction enzyme $EcoRI$ is derived from $Escherichia$ $coli$ $RY13$.
It recognizes a specific palindromic nucleotide sequence in $DNA$.
The specific recognition sequence for $EcoRI$ is $5'-GAATTC-3'$ and $3'-CTTAAG-5'$.
It cleaves the $DNA$ strand between the bases $G$ and $A$ within this specific recognition site.
Therefore,the correct sequence is $GAATTC$.

(B) Affinity chromatography is a specialized technique used for the purification of specific molecules based on their highly specific biological interactions.
It relies on the reversible interaction between the target molecule (such as an immunoglobulin,enzyme,or $m-RNA$) and a specific ligand immobilized on a solid support matrix.
Because of this high specificity,it is the preferred method for isolating proteins,enzymes,and nucleic acids from complex mixtures.

(B) In bacterial cells, besides the main genomic $DNA$ (nucleoid), there are small, circular, double-stranded, extrachromosomal $DNA$ molecules present.
These molecules are known as $Plasmids$.
$Plasmids$ replicate independently of the chromosomal $DNA$ and often carry genes that provide advantages, such as antibiotic resistance.

(D) The given $DNA$ sequence $5'-GAATTC-3'$ and $3'-CTTAAG-5'$ represents a palindromic nucleotide sequence.
$A$ palindromic sequence is a sequence of base pairs in $DNA$ that reads the same in both directions ($5'$ to $3'$ direction) on the two complementary strands.
These specific sequences are recognized by restriction endonucleases,which cut the $DNA$ at these sites.

(C) : Palindromic nucleotide sequences in the $DNA$ molecule are groups of bases that form the same sequence when read in both forward and backward direction.
In the given question,only option $(c)$ represents a palindromic sequence,which is the recognition site for the restriction enzyme $EcoRI$.
It reads $GAATTC$ from $5' \rightarrow 3'$ on both strands,allowing it to be cut at the middle by the enzyme.

(D) : Direct gene transfer is the transfer of naked $DNA$ into plant cells. Since the rigid plant cell wall acts as a barrier to $DNA$ uptake,protoplasts are the preferred target for this process. Polyethylene glycol $(PEG)$ mediated $DNA$ uptake is a direct gene transfer method that utilizes the interaction between $PEG$,naked $DNA$,salts,and the protoplast membrane to facilitate the transport of $DNA$ into the cytoplasm.

(C) : $Ti$ plasmid (tumor-inducing) from the soil bacterium $Agrobacterium \text{ } tumefaciens$ is effectively used as a vector for gene transfer to plant cells.
The part of the $Ti$ plasmid transferred into the plant cell $DNA$ is called the $T-DNA$.
This $T-DNA$, with the desired $DNA$ spliced into it, is inserted into the chromosomes of the host plant.
It integrates into the host genome, allowing the foreign gene to be expressed throughout the new plant.
Such transformed plant cells are then cultured and induced to differentiate to form plantlets, which grow into mature transgenic plants.

(C) Restriction endonucleases are known as molecular scissors because they cut $DNA$ at specific recognition sequences.
They recognize a specific base pair sequence and cleave the phosphodiester backbone of the $DNA$ molecule.
This property is fundamental in recombinant $DNA$ technology for isolating and inserting genes of interest.

(A) $Taq$ polymerase is a thermostable enzyme isolated from the thermophilic bacterium $Thermus$ $aquaticus$.
This enzyme is widely used in the Polymerase Chain Reaction $(PCR)$ technique because it can withstand the high temperatures required for the denaturation of $DNA$ strands without getting denatured itself.

(D) The first restriction endonuclease to be discovered and isolated was $Hind II$.
It was isolated by Hamilton Smith and his colleagues in $1970$.
$Hind II$ always cuts $DNA$ molecules at a particular point by recognizing a specific sequence of $6$ base pairs,known as the recognition sequence.

(D) $1$. $Agrobacterium$ $tumefaciens$ is a plant pathogen used as a vector for plants,not animals.
$2$. $Bam$ $HI$ is a restriction enzyme; it is not a gene.
$3$. $Pst$ $I$ is a restriction enzyme; it is not a gene.
$4$. Agarose is a natural polymer extracted from seaweeds (red algae like $Gelidium$ and $Gracilaria$),which is used to form the gel matrix for gel electrophoresis. Therefore,the pair 'Agarose gel - Seaweeds' is correct.

(C) To isolate $DNA$ from different types of cells,specific enzymes are used to break down the cell walls or membranes:
$1$. Bacterial cells have a cell wall that can be broken down by $Lysozyme$ $(1-r)$.
$2$. Plant cells have a cell wall made of cellulose,which is broken down by $Cellulase$ $(2-s)$.
$3$. Fungal cells have a cell wall made of chitin,which is broken down by $Chitinase$ $(3-p)$.
$4$. $RNA$ molecules are removed by using the enzyme $Ribonuclease$ $(4-q)$.
Therefore,the correct matching is $(1-r), (2-s), (3-p), (4-q)$.

(B) Restriction enzymes,also known as molecular scissors,are enzymes that cut $DNA$ molecules at specific nucleotide sequences known as recognition sites.
These enzymes are essential tools in genetic engineering as they allow scientists to isolate specific genes or $DNA$ fragments for cloning or further analysis.
Unlike ligases,which join $DNA$ fragments,restriction enzymes specifically cleave the phosphodiester backbone of $DNA$ at precise locations.

(A) The restriction endonuclease $Hind-II$ was the first restriction enzyme to be isolated.
It always cuts $DNA$ molecules at a particular point by recognizing a specific sequence of $6$ base pairs.
This specific base sequence is known as the recognition sequence.

(D) The naming convention for restriction enzymes follows specific rules:
$1$. The first letter of the name comes from the genus $(Escherichia)$.
$2$. The next two letters come from the species $(coli)$.
$3$. The fourth letter comes from the strain of the bacterium ($R$ for strain $RY\, 13$).
$4$. The Roman numeral $(I)$ indicates the order in which the enzyme was isolated from that strain.
Therefore,$EcoRI$ is derived from Escherichia coli $RY\, 13$.

(C) The nomenclature of $Restriction$ $Endonuclease$ enzymes follows a specific convention.
$1$. The first letter represents the genus name (e.g.,$E$ for $Escherichia$).
$2$. The next two letters represent the species name (e.g.,$co$ for $coli$).
$3$. The fourth letter represents the strain of the organism (e.g.,$R$ for $RY13$).
$4$. The Roman numeral indicates the order in which the enzymes were isolated from that specific strain of the source organism.
For example,in $EcoRI$,the Roman numeral $I$ indicates that it was the first enzyme isolated from the $RY13$ strain of $Escherichia$ $coli$.

(B) Restriction endonucleases are enzymes that recognize specific nucleotide sequences in $DNA$ and make cuts at these specific positions,known as recognition sites.
These enzymes are essential tools in recombinant $DNA$ technology for creating $DNA$ fragments.
$DNA$ ligase is used to join $DNA$ fragments.
$DNA$ polymerase is used for $DNA$ replication.
Reverse transcriptase is used to synthesize $cDNA$ from $RNA$.

(D) The restriction endonuclease $EcoRI$ is derived from the bacterium $Escherichia$ $coli$ $RY13$.
It recognizes a specific palindromic nucleotide sequence in $DNA$.
The specific recognition sequence for $EcoRI$ is $5'-GAATTC-3'$.
It cuts the $DNA$ strand between the bases $G$ and $A$ within this sequence.
Therefore,the correct sequence is $GAATTC$.

(C) In recombinant $DNA$ technology, the enzyme $DNA$ ligase is used to join foreign $DNA$ fragments to a vector, such as a plasmid.
$DNA$ ligase acts as a 'molecular glue' by catalyzing the formation of phosphodiester bonds between the $3'-OH$ end of one nucleotide and the $5'-phosphate$ end of another.
This process is essential for creating recombinant $DNA$ molecules.

(A) Agarose is a linear polysaccharide that is extracted from marine red algae (seaweeds) such as $Gelidium$ and $Gracilaria$.
It is widely used in biotechnology for gel electrophoresis to separate $DNA$ fragments based on their size.
Therefore,the correct source of agarose is sea weeds.

(B) Agarose is a natural polymer extracted from seaweeds (marine red algae like Gelidium and Gracilaria).
It is widely used in gel electrophoresis to separate $DNA$ fragments based on their size.
During the process,the $DNA$ fragments move towards the anode under an electric field through the agarose gel matrix,which acts as a molecular sieve.

(D) In gel electrophoresis, $DNA$ fragments are separated based on their size through a gel matrix (usually agarose).
Since $DNA$ is colorless, it cannot be seen directly.
To visualize the separated $DNA$ fragments, the gel is stained with a fluorescent dye called $Ethidium bromide$ $(EtBr)$.
After staining, when the gel is exposed to $UV$ radiation, the $DNA$ bands appear as bright orange-colored bands.

(B) $pBR322$ is a well-known artificial cloning vector constructed from $E. coli$ plasmids.
It contains unique recognition sites for several restriction enzymes,an origin of replication $(ori)$,and two antibiotic resistance genes ($amp^R$ and $tet^R$).
Cla $I$,Hind $III$,and BamH $I$ are names of restriction enzymes,not cloning vectors.

(C) The polymerase chain reaction $(PCR)$ is a technique used to amplify a specific segment of $DNA$.
This process requires a heat-stable $DNA$ polymerase enzyme that can withstand the high temperatures used during the denaturation step of the reaction.
$Taq$ polymerase is isolated from the thermophilic bacterium $Thermus$ $aquaticus$.
It remains active at high temperatures and is essential for the extension step of $PCR$.

(C) The correct pair is $C$ (Plasmid $DNA$ - vector).
$A$ is incorrect because Biolistics (gene gun) is a method for direct gene transfer,not a bioreactor.
$B$ is incorrect because $Thermus$ $aquaticus$ is the source of $Taq$ polymerase,not $T-DNA$.
$C$ is correct because plasmids are small,circular,extrachromosomal $DNA$ molecules used as vectors to carry foreign $DNA$ into host cells.
$D$ is incorrect because $EcoRI$ is a restriction endonuclease,not an exonuclease.