Tools of recombinant DNA technology Questions in English

(D) $Probe$ is a single-stranded $DNA$ or $RNA$ molecule that is tagged with a radioactive isotope or a fluorescent molecule.
It is used to detect the presence of complementary sequences in a sample of $DNA$ or $RNA$ by the process of hybridization.
Since it is labeled,it allows for the identification of specific gene sequences in techniques like Southern blotting or colony hybridization.

(D) The plasmid $pBR322$ is a widely used cloning vector in $E. coli$.
$1$. $ori$ stands for the origin of replication,which is the sequence where replication starts.
$2$. $rop$ codes for the proteins involved in the replication of the plasmid.
$3$. $Hind III$ and $Eco RI$ are restriction sites (recognition sites) for specific restriction enzymes,not selectable markers.
$4$. $amp^R$ (ampicillin resistance) and $tet^R$ (tetracycline resistance) are the selectable markers that help in identifying and eliminating non-transformants and selectively permitting the growth of the transformants.

(C) Plasmids are small,circular,extrachromosomal $DNA$ molecules found in bacteria that are widely used as vectors in genetic engineering.
They are ideal for cloning small fragments of $DNA$ (typically up to $10 \ kb$).
In contrast,$BAC$s and $YAC$s are used for cloning large $DNA$ inserts,and cosmids are used for intermediate sizes.
Therefore,the correct option is $C$.

(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. Common examples of vectors include plasmids and bacteriophages.

(B) Restriction enzymes,also known as 'molecular scissors',are enzymes that cut $DNA$ molecules at specific nucleotide sequences called recognition sites. This discovery was a fundamental breakthrough in recombinant $DNA$ technology,allowing scientists to isolate specific genes and insert them into vectors.

(B) $1$. When a foreign $DNA$ fragment and a plasmid vector are cut with the same restriction endonuclease,they produce complementary sticky ends.
$2$. These complementary sticky ends allow the foreign $DNA$ to base-pair with the plasmid vector.
$3$. However,the sugar-phosphate backbone of the $DNA$ remains broken at the nicks.
$4$. The enzyme $DNA$ ligase is responsible for catalyzing the formation of phosphodiester bonds between the adjacent nucleotides,thereby sealing the nicks and creating a stable recombinant $DNA$ molecule.

(D) Restriction enzymes are classified based on the type of ends they produce after $DNA$ cleavage.
$1$. Sticky ends (cohesive ends) are produced by enzymes like $Eco-RI$,$Hind-III$,$Xho-I$,and $Sal-I$,which create staggered cuts.
$2$. Blunt ends (flush ends) are produced by enzymes that cut both strands of $DNA$ at the same position,typically in the center of the recognition site.
$3$. $Eco-RV$ is a well-known restriction enzyme that recognizes the sequence $5'-GATATC-3'$ and cuts between the $T$ and $A$ residues,resulting in blunt ends.
Therefore,the correct option is $D$.

(D) Restriction endonucleases are enzymes that cut $DNA$ at specific recognition sequences.
$Hind-II$ was the first restriction endonuclease to be isolated and characterized.
Proteases are enzymes that break down proteins.
DNase-$I$ and RNase are enzymes that degrade $DNA$ and $RNA$,respectively,but they are not restriction endonucleases.

(C) Plasmids are small,circular,double-stranded $DNA$ molecules that are distinct from a cell's chromosomal $DNA$.
They are found primarily in bacteria and can replicate independently of the chromosomal $DNA$.
They are often used as vectors in genetic engineering because they can be transferred between cells.
Since plasmids are double-stranded,the statement that they are 'single-stranded' is incorrect.

(D) The $Taq$ polymerase enzyme is isolated from a thermophilic bacterium called $Thermus$ $aquaticus$.
This bacterium thrives in high-temperature environments such as hot springs.
Because of its origin,the $Taq$ polymerase enzyme is thermostable,meaning it can withstand the high temperatures required during the denaturation step of the Polymerase Chain Reaction $(PCR)$ process.
Therefore,it is widely used in $PCR$ to amplify $DNA$ segments.

(A) $Selectable marker$ is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection.
In recombinant $DNA$ technology, it helps in identifying and eliminating non-transformants and selectively permitting the growth of the transformants.
Examples include genes encoding resistance to antibiotics such as $ampicillin$, $chloramphenicol$, $tetracycline$, or $kanamycin$.

(B) Restriction endonucleases are enzymes that recognize specific palindromic nucleotide sequences in $DNA$ and cut the sugar-phosphate backbone at specific sites on both strands of the $DNA$ molecule.
Option $A$ is correct because these enzymes act at specific internal positions.
Option $B$ is incorrect because restriction endonucleases cut both strands of the $DNA$ double helix,not just one.
Option $C$ is correct as they cleave the phosphodiester bonds in the sugar-phosphate backbone.
Option $D$ is correct as they identify specific palindromic sequences.

(A) palindromic sequence is a nucleic acid sequence ($DNA$ or $RNA$) that is the same when read $5'$ to $3'$ on one strand and $5'$ to $3'$ on the complementary strand.
In the option $A$:
$5' - GAATTC - 3'$
$3' - CTTAAG - 5'$
Reading the top strand from $5'$ to $3'$ gives $GAATTC$. Reading the bottom strand from $5'$ to $3'$ (which is right to left in the representation) also gives $GAATTC$.
This is a classic palindromic sequence recognized by the restriction enzyme $EcoRI$.

(A) Developed in $1984$ by Kary Mullis,$PCR$ is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.
These include $DNA$ cloning for sequencing,$DNA$-based phylogeny,or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases.
In $1993$,Mullis won the Nobel Prize in Chemistry for his work on $PCR$.

(C) hybridization probe is a fragment of $DNA$ of variable length which is used in $DNA$ samples to detect the presence of nucleotide sequences (the $DNA$ target) that are complementary to the sequence in the probe.
The probe hybridizes to single-stranded $DNA$ whose base sequence allows probe-target base-pairing due to complementarity between the probe and the target.
Therefore,$DNA$ probes are specifically used to identify and select genes of interest from a genomic library.

(C) palindromic sequence in $DNA$ is a sequence of base pairs that reads the same on the two strands when the orientation of reading is kept the same (e.g.,$5' \rightarrow 3'$).
Restriction enzymes recognize specific palindromic sequences to cut the $DNA$.
In option $(c)$,the sequence $5'-GAATTC-3'$ on one strand corresponds to $3'-CTTAAG-5'$ on the complementary strand.
When read in the $5' \rightarrow 3'$ direction,both strands yield the sequence $GAATTC$,which is a classic recognition site for the restriction enzyme $EcoRI$.

(B) selectable marker is a gene that facilitates the selection of transformants (cells that have taken up the recombinant vector) by eliminating non-transformants. Option $B$ is incorrect because a selectable marker permits the growth of transformants,not non-transformants.

(A) Plasmids are small,circular,double-stranded $DNA$ molecules that are distinct from a cell's chromosomal $DNA$.
They are naturally found in bacteria and some eukaryotes.
Because they can replicate independently and carry specific genes,they are widely used as vectors in recombinant $DNA$ technology (genetic engineering) to transfer foreign genes into host cells.
Both the assertion and the reason are correct,and the reason explains why plasmids are significant in the context of their biological nature and utility.

(C) Restriction enzymes,a type of endonuclease,function by inspecting the length of a $DNA$ sequence.
Once they find a specific recognition sequence,they bind to it and cut each of the two strands of the double helix at specific points,leaving single-stranded portions at the ends.
This results in overhanging stretches called sticky ends.
These are named so because they form hydrogen bonds with their complementary counterparts; i.e.,they can join similar complementary ends of $DNA$ fragments from other sources with the help of the enzyme $DNA$ ligase.
Therefore,the stickiness of the ends facilitates the action of the enzyme $DNA$ ligase,not $DNA$ polymerase.
Thus,the Assertion is correct,but the Reason is incorrect.

(B) The correct matching is as follows:
$(a)$ Restriction endonuclease: These enzymes cut $DNA$ at specific recognition sequences within the molecule. Thus, $(a) - (iii)$.
$(b)$ Restriction exonuclease: These enzymes remove nucleotides from the ends of $DNA$ molecules. Thus, $(b) - (iv)$.
$(c)$ $DNA$ ligase: This enzyme acts as molecular glue, joining $DNA$ fragments by forming phosphodiester bonds. Thus, $(c) - (i)$.
$(d)$ $Taq$ polymerase: This is a thermostable enzyme used in $PCR$ to extend primers on a genomic $DNA$ template. Thus, $(d) - (ii)$.
Therefore, the correct sequence is $a-iii, b-iv, c-i, d-ii$.

(A) The plasmid vector $pBR322$ is one of the most commonly used cloning vectors in biotechnology.
It contains two distinct antibiotic resistance genes,which serve as selectable markers.
These genes are the $amp^R$ gene,which provides resistance to Ampicillin,and the $tet^R$ gene,which provides resistance to Tetracycline.
These markers allow for the identification and selection of recombinant cells from non-recombinant ones during the cloning process.

(A) selectable marker is a gene introduced into a cell,especially a bacterium or to cells in culture,that confers a trait suitable for artificial selection.
In recombinant $DNA$ technology,selectable markers (such as antibiotic resistance genes) are used to identify and eliminate non-transformants (cells that have not taken up the recombinant $DNA$) and selectively permit the growth of transformants (cells that have successfully taken up the recombinant $DNA$).
This ensures that only the desired transformed cells are regenerated or cultured.

(C) Exonucleases are enzymes that remove nucleotides from the ends of $DNA$ molecules.
In contrast,endonucleases make cuts at specific positions within the $DNA$ molecule.
$DNA$ ligase is an enzyme that joins $DNA$ fragments together.
Proteases are enzymes that break down proteins.
Therefore,the correct enzyme for removing nucleotides from the ends of $DNA$ is Exonuclease.

(A) $E. coli$ is a bacterium whose plasmid is widely used as a cloning vector to introduce foreign $DNA$ segments into host cells.
$Bacillus thuringiensis$ is a bacterium from which a specific gene is isolated and inserted into cotton plants to produce insect-resistant plants,commonly known as $Bt$ cotton.

(A) The restriction endonuclease $\text{EcoRI}$ is derived from the bacterium $\text{Escherichia coli}$ strain $\text{RY13}$.
In the name $\text{EcoRI}$,the first letter '$E$' comes from the genus $\text{Escherichia}$,and the next two letters 'co' come from the species $\text{coli}$.
The letter '$R$' is derived from the strain name $\text{RY13}$,and the Roman numeral '$I$' indicates the order in which the enzyme was isolated from that bacterial strain.

(N/A) The restriction endonuclease enzyme used is $BamHI$.
It recognizes the specific palindromic sequence $5'-GGATCC-3'$.
The enzyme cleaves the $DNA$ between the $G$ and $G$ residues on both strands.
Diagrammatic representation:
$5'-G \downarrow GATCC-3'$
$3'-CCTAG \uparrow G-5'$
Products produced:
$5'-G-3'$ and $5'-GATCC-3'$
$3'-CCTAG-5'$ and $3'-G-5'$
This results in the formation of 'sticky ends' or 'cohesive ends' which are essential for recombinant $DNA$ technology.

(N/A) No,eukaryotic cells do not possess restriction endonucleases. In eukaryotic cells,the $DNA$ is heavily methylated by specific enzymes known as methylases. This methylation process serves as a protective mechanism,preventing the $DNA$ from being cleaved by restriction enzymes. Restriction endonucleases are naturally occurring enzymes found exclusively in various strains of bacteria or prokaryotic cells,where they function as part of a defense mechanism against invading viral $DNA$.

(N/A) Palindrome nucleotide sequences in $DNA$ molecules are base pairs that read the same in both the $5^{\prime} \rightarrow 3^{\prime}$ and $3^{\prime} \rightarrow 5^{\prime}$ directions. These sequences serve as recognition sites for restriction endonucleases.
Five examples of palindromic $DNA$ sequences are:
$(i)$ $5^{\prime}-GAATTC-3^{\prime}$ / $3^{\prime}-CTTAAG-5^{\prime}$
$(ii)$ $5^{\prime}-GGATCC-3^{\prime}$ / $3^{\prime}-CCTAGG-5^{\prime}$
$(iii)$ $5^{\prime}-AAGCTT-3^{\prime}$ / $3^{\prime}-TTCGAA-5^{\prime}$
$(iv)$ $5^{\prime}-ACGCGT-3^{\prime}$ / $3^{\prime}-TGCGCA-5^{\prime}$
$(v)$ $5^{\prime}-ACTAGT-3^{\prime}$ / $3^{\prime}-TGATCA-5^{\prime}$

(N/A) $PCR$ - Polymerase Chain Reaction (in vitro method) is a molecular biological technique for the enzymatic amplification of a single small strand or a few copies of a segment of $DNA$ into millions of copies of that specific $DNA$ sequence in a short time period (approx $2$ hours).
$3$ steps in $PCR$ are:
$(i)$ Denaturation (at $96\,^{\circ}C$): Separation of desired double-stranded $DNA$ into single-stranded $DNA$ $(ssDNA)$.
$(ii)$ Annealing (at $55-65\,^{\circ}C$): Binding of primers to the $ssDNA$ template.
$(iii)$ Extension (at $72\,^{\circ}C$): Synthesis of new $DNA$ strands by Taq $DNA$ polymerase,isolated from the bacterium $Thermus$ $aquaticus$.
Uses: Amplification of a desired gene or gene cloning.
$(b)$ Restriction enzymes and $DNA$ - Restriction enzymes are a group of enzymes used to cleave or cut $DNA$ strands. Each enzyme recognizes a specific characteristic base sequence known as a recognition site or restriction site.
$(i)$ They restrict foreign $DNA$ from entering a cell by digesting it at specific recognition sites. These sites are typically palindromic.
$(ii)$ They include both endonuclease and exonuclease types.
$(iii)$ They often produce sticky ends. Restriction enzymes are believed to be a defense mechanism evolved by bacteria to resist viral attacks.
$(c)$ Chitinase - Chitinase is a hydrolytic enzyme that digests or breaks down glycosidic bonds in chitin. It is used to break down the cell walls of fungal cells to facilitate the extraction of $DNA$ or other cellular components.

(N/A) Plasmid $DNA$ and Chromosomal $DNA$

$1$. Plasmid $DNA$ is autonomously replicable, whereas Chromosomal $DNA$ replicates under nuclear control.

$2$. Plasmid $DNA$ is double-stranded and circular, while Chromosomal $DNA$ can be double-stranded, circular, or linear.

$3$. Plasmid $DNA$ is not associated with histones, whereas Chromosomal $DNA$ is associated with histone proteins.

$4$. Plasmid $DNA$ contains few genes for traits like antibiotic resistance, while Chromosomal $DNA$ contains genes for essential metabolic functions.

$(b)$ $RNA$ and $DNA$

$1$. $RNA$ contains ribose sugar, whereas $DNA$ contains deoxyribose sugar.

$2$. $RNA$ is typically single-stranded, while $DNA$ is double-stranded.

$3$. $RNA$ contains uracil, whereas $DNA$ contains thymine.

$4$. $RNA$ is less stable due to the $2'-OH$ group, while $DNA$ is more stable.

$(c)$ Exonuclease and Endonuclease

$1$. Exonucleases remove nucleotides from the ends of $DNA$, whereas Endonucleases cut $DNA$ at specific internal sites.

$2$. Exonucleases often produce blunt ends, while Endonucleases can produce sticky ends.

(N/A) In the year $1963$,two enzymes responsible for restricting the growth of bacteriophage in $Escherichia$ $coli$ were isolated. One of these added methyl groups to $DNA$,while the other cut $DNA$.
The enzymes which cut $DNA$ are called restriction endonucleases.
$Hind-II$ was the first restriction endonuclease enzyme discovered.
$Hind-II$ always cuts $DNA$ molecules at a particular point by recognizing a specific sequence of six base pairs. This specific base sequence is known as the recognition sequence for $Hind-II$.
Besides $Hind-II$,today we know more than $900$ restriction enzymes that have been isolated from over $230$ strains of bacteria.
Nomenclature: The naming of these enzymes is based on the bacteria from which they are isolated. The name consists of three or four letters.
For example,in $EcoRI$:
- The first letter '$E$' comes from the genus $Escherichia$.
- The next two letters '$co$' come from the species $coli$.
- The letter '$R$' is derived from the name of the strain $(RY13)$.
- The Roman numeral '$I$' indicates the order in which the enzymes were isolated from that strain of bacteria.

(N/A) Restriction enzymes belong to a larger class of enzymes called nucleases.
$(i)$ Exonuclease enzymes: They remove nucleotides from the ends of the $DNA$.
$(ii)$ Endonuclease enzymes: Endonucleases make cuts at specific positions within the $DNA$.
Each restriction endonuclease functions by inspecting the length of a $DNA$ sequence. Once it finds the specific recognition sequence,it will bind to the $DNA$.
It then cuts each of the two strands of the double helix at specific points in their sugar-phosphate backbones.
- Each restriction endonuclease recognizes a specific palindromic nucleotide sequence in the $DNA$.
- The palindrome in $DNA$ is a sequence of base pairs that reads the same on the two strands when the orientation of reading is kept the same.

(N/A) The process of cutting $DNA$ involves the following steps:
$1$. Restriction enzyme digestions are performed by incubating purified $DNA$ molecules with specific restriction enzymes under optimal conditions (temperature,$pH$,and buffer).
$2$. Agarose gel electrophoresis is employed to check the progression and completion of the restriction enzyme digestion.
$3$. Since $DNA$ is a negatively charged molecule,it moves towards the positive electrode (anode) during electrophoresis.
$4$. The same procedure is repeated with the vector $DNA$ using the same restriction enzyme to ensure complementary sticky ends.
$5$. Finally,the cut 'gene of interest' from the source $DNA$ and the linearized vector $DNA$ are mixed,and the enzyme $DNA$ ligase is added to join them,resulting in the formation of recombinant $DNA$.

(N/A)

Restriction Endonuclease	Restriction Exonuclease
$(1)$ Endonucleases cut $DNA$ molecules at specific internal positions.	$(1)$ Exonucleases remove nucleotides sequentially from the ends of the $DNA$ molecule.
$(2)$ They often produce sticky or blunt ends depending on the enzyme.	$(2)$ They generally do not produce sticky ends as they act on the termini.
$(3)$ They are essential tools in recombinant $DNA$ technology for creating gene fragments.	$(3)$ They are primarily involved in $DNA$ repair and degradation processes.
$(4)$ Examples include $BamHI$, $HindIII$, $EcoRI$.	$(4)$ Examples include $Exonuclease III$, $RecJ$.

(N/A) In recombinant $DNA$ technology,the fragment of desired $DNA$ must be joined with a vector or amplified. For this,precise cutting of both the vector and the desired $DNA$ is essential.
Endonucleases cut $DNA$ at specific internal sites,often producing 'sticky' or cohesive ends.
Exonucleases remove nucleotides from the ends of $DNA$ molecules,typically resulting in 'blunt' ends.
Sticky ends are necessary for the efficient annealing and ligation of the desired $DNA$ fragment into the vector.
Therefore,endonucleases are more suitable than exonucleases for recombinant $DNA$ technology.

(B) No,an exonuclease would not be chosen. Exonucleases act on the free ends of a linear $DNA$ molecule by removing nucleotides one by one.
Instead of producing $DNA$ fragments with sticky ends suitable for ligation,an exonuclease would shorten or completely degrade the $DNA$ fragment containing the gene of interest.
Furthermore,a circular plasmid (vector) lacks free ends,so an exonuclease would not be able to cut it at all.
Therefore,restriction endonucleases are preferred as they cut $DNA$ at specific internal sites to produce sticky or blunt ends.

(A) The naming convention for restriction enzymes follows specific rules:
$1$. The first letter '$H$' is derived from the genus name,which is $\text{Haemophilus}$.
$2$. The next two letters '$in$' are derived from the species name,which is $\text{influenza}$.
$3$. The letter '$d$' is derived from the specific strain of the bacteria,which is $\text{Rd}$.
$4$. The Roman numeral '$III$' indicates the order in which the enzyme was isolated from that specific strain of bacteria.

(N/A) vector is designed to have a unique recognition site for a specific restriction enzyme within its cloning site. If a restriction enzyme has multiple recognition sites within the vector,the vector $DNA$ will be cleaved into multiple fragments upon treatment with that enzyme. This would disrupt the integrity of the vector,making it impossible to insert the gene of interest and successfully create a recombinant $DNA$ molecule.

(A) The tumor-inducing $(Ti)$ plasmid of $Agrobacterium$ $tumefaciens$ is modified by removing the $T-DNA$ region,which is responsible for tumor formation in plants.
This process is known as 'disarming' the plasmid.
By removing the $T-DNA$ and replacing it with the gene of interest,the plasmid becomes a non-pathogenic cloning vector.
It retains the ability to transfer the desired gene into plant cells using its natural $vir$ $(virulence)$ gene machinery.

(N/A) Plasmids are small,circular,double-stranded $DNA$ molecules that are distinct from a cell's chromosomal $DNA$.
They are found in the cytoplasm of bacterial cells and are capable of autonomous replication.
Role in bacteria:
$1$. Antibiotic Resistance: Plasmids often carry genes that provide resistance to antibiotics,allowing bacteria to survive in hostile environments.
$2$. Genetic Exchange: They facilitate horizontal gene transfer between bacteria through processes like conjugation.
$3$. Virulence Factors: Some plasmids carry genes that enhance the pathogenicity or virulence of the bacteria.
$4$. Metabolic Functions: They may contain genes for the degradation of complex organic compounds,aiding in bacterial metabolism.

(B) Restriction enzymes,specifically Type $II$ endonucleases,recognize and cut $DNA$ at specific palindromic sequences.
This process generates fragments with single-stranded overhangs known as 'sticky ends'.
These sticky ends are crucial because they allow the $DNA$ fragment to base-pair with a complementary vector $DNA$ cut by the same enzyme.
If the enzymes cut at random sites,the resulting fragments would lack these specific sticky ends,making it impossible to join the gene of interest with the vector to form a recombinant $DNA$ molecule.

(N/A) The reason for this observation is the structural difference between the two $DNA$ molecules:
$1$. $A$ plasmid is a circular $DNA$ molecule. When it is cut with a restriction enzyme at a single site,it becomes linearized but remains a single continuous molecule. Therefore,it appears as a single band on the agarose gel.
$2$. $A$ linear $DNA$ molecule,when cut at a single site,is cleaved into two separate fragments. Since these fragments have different sizes,they appear as two distinct bands on the agarose gel.
Thus,the circular nature of the plasmid prevents fragmentation upon a single cut,whereas the linear $DNA$ is physically separated into two pieces.

(C) selectable marker is a gene that helps in identifying and eliminating non-transformants and selectively permitting the growth of the transformants.
If a plasmid lacks a selectable marker,it becomes impossible to distinguish between cells that have taken up the plasmid (transformants) and those that have not (non-transformants).
Therefore,the experiment is negatively affected because the researcher cannot isolate the successfully transformed host cells from the population.

(N/A) Based on the standard map of the cloning vector $pBR322$:
$A$ represents the restriction site for $Bam HI$.
$B$ represents the restriction site for $Pst I$.
$C$ represents the gene for ampicillin resistance,denoted as $amp^{R}$.

(N/A) The tumor-inducing $(Ti)$ plasmid of $Agrobacterium$ $tumefaciens$ has been modified into a cloning vector. This modified plasmid is no longer pathogenic to plants but retains the ability to use its natural mechanism to deliver genes of interest into a variety of plant cells. This process is widely used in genetic engineering to create transgenic plants.

(N/A) $(iii)$ Cloning Sites: To link the alien $DNA$,the vector needs to have very few,preferably single,recognition sites for the commonly used restriction enzymes. Presence of more than one recognition site within the vector will generate several fragments,which will complicate the gene cloning process.
The ligation of alien $DNA$ is carried out at a restriction site present in one of the two antibiotic resistance genes. For example,you can ligate a foreign $DNA$ at the $BamHI$ site of the tetracycline resistance gene in the vector $pBR322$.
The recombinant plasmids will lose tetracycline resistance due to the insertion of foreign $DNA$,but can still be selected out from non-recombinant ones by plating the transformants on an ampicillin-containing medium.
The transformants growing on the ampicillin-containing medium are then transferred to a medium containing tetracycline. The recombinants will grow in the ampicillin-containing medium but not on that containing tetracycline. But non-recombinants will grow on the medium containing both the antibiotics.
In this case,one antibiotic resistance gene helps in selecting the transformants,whereas the other antibiotic resistance gene gets 'inactivated' due to the insertion of alien $DNA$,and helps in the selection of recombinants.

(A) The correct matching is as follows:
$(a)$ Ligase: It acts as a molecular glue that connects parts of $DNA$ by forming phosphodiester bonds $(ii)$.
$(b)$ Endonuclease: These enzymes cut $DNA$ at specific internal sites,often producing sticky ends $(iv)$.
$(c)$ Exonuclease: These enzymes remove nucleotides one by one from the ends of $DNA$ molecules $(i)$.
$(d)$ $DNA$ Polymerase: This enzyme is essential for synthesizing new $DNA$ strands and is used in $PCR$ for repeated amplification of $DNA$ sequences $(v)$.
Therefore,the correct sequence is $(a-ii, b-iv, c-i, d-v)$.

(N/A) The five key tools for recombinant $DNA$ technology are:
$1$. Restriction endonucleases: These enzymes are used to cut $DNA$ molecules at specific recognition sequences.
$2$. Gel electrophoresis: This technique is used for separating $DNA$ fragments based on their size and charge.
$3$. Ligase enzymes: These enzymes are used to join or ligate $DNA$ fragments to create recombinant $DNA$ molecules.
$4$. $DNA$ delivery systems: Methods such as electroporation,microinjection,and the gene gun method are used to introduce recombinant $DNA$ into host cells.
$5$. Competent host: Organisms like bacteria or yeast are used as hosts to take up and replicate the recombinant $DNA$.

(B) The restriction endonuclease $EcoRI$ recognizes the specific palindromic sequence $5^{\prime}-GAATTC-3^{\prime}$ and $3^{\prime}-CTTAAG-5^{\prime}$.
This sequence is read the same in both directions when the orientation of the strands is $5^{\prime}$ to $3^{\prime}$.