Tools of recombinant DNA technology Questions in English

(D) The first restriction endonuclease to be isolated and characterised was $Hind \;II$.
It was discovered by Werner Arber,Hamilton Smith,and Daniel Nathans in the late $1960s$.
This enzyme was found to always cut $DNA$ molecules at a particular point by recognising a specific sequence of $6$ base pairs,known as the recognition sequence.

(C) The restriction endonuclease $EcoRI$ recognizes the specific palindromic sequence $5'-GAATTC-3'$ in $DNA$.
This sequence is read the same in both the $5' \rightarrow 3'$ and $3' \rightarrow 5'$ directions on the complementary strands.
The correct representation of this double-stranded $DNA$ sequence is:
$5'-GAATTC-3'$
$3'-CTTAAG-5'$
$EcoRI$ cuts the $DNA$ between the $G$ and $A$ bases on both strands,creating sticky ends.

(D) $1$. $Ti$ plasmid of $Agrobacterium \text{ } tumefaciens$: $A. \text{ } tumefaciens$ is a pathogen that can deliver a piece of $DNA$ known as 'T-DNA' to transform normal plant cells into tumor cells.
$2$. Retrovirus: These viruses possess the ability to transform normal cells into cancerous cells in animals.
$3$. Therefore, both $Ti$ plasmid and retroviruses have the ability to transform normal cells into cancerous/tumor cells.

(C) The term 'restriction' refers to the biological function of these enzymes in restricting or preventing the propagation of foreign $DNA$ (such as that of bacteriophages) within the host bacterium. These enzymes act as a defense mechanism for bacteria against viral infections.

(B) In agarose gel electrophoresis,$DNA$ fragments are separated based on their size.
Since $DNA$ molecules are negatively charged,they move towards the anode under an electric field.
The agarose gel acts as a molecular sieve.
Larger $DNA$ fragments encounter more resistance from the gel matrix and move slowly,whereas smaller $DNA$ fragments move through the pores more easily and travel faster.
Therefore,the separation is primarily determined by the size of the $DNA$ molecules.

(D) To isolate $DNA$ from bacterial cells,the cell wall must be broken down and other macromolecules like $RNA$ and proteins must be removed.
$1$. Lysozyme is used to break down the bacterial cell wall.
$2$. Ribonuclease is used to degrade $RNA$.
$3$. Protease is used to degrade proteins.
$4$. Deoxyribonuclease $(DNase)$ is an enzyme that breaks down $DNA$. If $DNase$ is added during the isolation process,it will degrade the $DNA$ we are trying to extract. Therefore,it is not used.

(B) Kary Mullis was an American biochemist who was awarded the Nobel Prize in Chemistry in $1993$ for his invention of the Polymerase Chain Reaction $(PCR)$ technique.
This technique allows for the amplification of specific $DNA$ segments,which revolutionized molecular biology.

(C) Restriction enzymes are a class of enzymes that cut $DNA$ at specific sites. They are naturally found in bacteria,where they serve as a defense mechanism against invading viruses (bacteriophages) by cutting the viral $DNA$. They are not isolated from the bacteriophages themselves. Therefore,the statement that they are isolated from bacteriophages is incorrect. Restriction enzymes recognize specific palindromic sequences and act as endonucleases to produce sticky or blunt ends.

(A) The plasmid $pBR322$ contains two antibiotic resistance genes: $amp^R$ (ampicillin resistance) and $tet^R$ (tetracycline resistance).
When a gene of interest is inserted (cloned) at the $Sal I$ restriction site,it disrupts the $tet^R$ gene.
This process is known as insertional inactivation.
As a result,the recombinant plasmid loses its resistance to tetracycline and becomes susceptible to it,while it remains resistant to ampicillin.

(D) The naming of restriction enzymes follows a specific convention. The first letter of the name comes from the genus and the next two letters come from the species of the prokaryotic cell from which they were isolated. For example,in $BamHI$,$B$ comes from Bacillus and $am$ comes from amyloliquefaciens. The third letter $H$ represents the strain of the bacterium (e.g.,$H$ for strain $H$). The Roman numeral $I$ following the name indicates the order in which the enzyme was isolated from that strain of bacteria.

(B) The 'ori' site stands for the 'Origin of Replication'.
It is a specific $DNA$ sequence within a vector where the replication process begins.
Any piece of $DNA$ linked to this sequence can be made to replicate within the host cells.

(D) Normal $E.$ coli cells are wild-type bacteria and do not naturally possess plasmids containing antibiotic resistance genes. Therefore,they do not carry resistance against common antibiotics like Chloramphenicol,Ampicillin,or Tetracycline. These resistance genes are typically introduced into $E.$ coli artificially via recombinant $DNA$ technology using cloning vectors.

(B) chimaeric $DNA$ is a recombinant $DNA$ molecule.
It is formed by joining a foreign $DNA$ fragment (such as $c-DNA$) with a vector $DNA$ (such as a plasmid) using enzymes like restriction endonucleases and $DNA$ ligase.
This process creates a hybrid or chimaeric molecule containing genetic material from two different sources.

(B) plasmid is an extrachromosomal,circular,double-stranded $DNA$ molecule found in many bacteria.
It possesses the ability to replicate autonomously within the bacterial cell,independent of the chromosomal $DNA$ replication control.
While plasmids often carry genes that provide advantages (like antibiotic resistance),they do not contain genes essential for the basic vital activities of the cell,which are located on the main chromosomal $DNA$.

(C) Restriction endonucleases are enzymes that act as molecular scissors.
They recognize specific palindromic nucleotide sequences in $DNA$.
Upon recognizing the specific sequence,they cleave the phosphodiester bonds in the sugar-phosphate backbone of both strands of the $DNA$ double helix.
This cleavage often results in the formation of single-stranded overhangs known as sticky ends or blunt ends,depending on the enzyme used.

(C) In recombinant $DNA$ technology,vector $DNA$ molecules can undergo self-ligation if their $5'$ ends possess phosphate groups.
Alkaline phosphatase is an enzyme that removes the $5'$ phosphate groups from the $DNA$ molecules.
By removing these phosphate groups,the enzyme prevents the vector from re-annealing or self-ligating,thereby ensuring that only the desired insert $DNA$ can be ligated into the vector.

(A) gene library (or genomic library) is a collection of $DNA$ fragments that have been cloned into vectors such as plasmids or phages,representing the entire genetic material of an organism.
Gene pool refers to the total sum of different genes present in an interbreeding population.
Genophore is the term used for the chromosomal $DNA$ of a prokaryote.
Genome refers to the complete haploid set of chromosomes in a gamete or an organism.

(B) Plasmids are widely used in genetic engineering because they can replicate independently of the chromosomal $DNA$.
Plastids are double-membrane organelles found in plants and algae,primarily involved in photosynthesis and storage.
Mitochondria are the powerhouses of the cell,and ribosomes are the sites of protein synthesis,but neither is used as a vector in genetic engineering.

(A) Agarose is a natural polymer extracted from sea weeds (red algae).
It is widely used in $Gel\; electrophoresis$ to separate $DNA$ fragments based on their size.
Agarose gels provide a matrix with a specific pore size that allows the migration of $DNA$ molecules under an electric field.
$Spectrophotometry$ is used for measuring light absorption,$Tissue\; culture$ involves growing cells in artificial media,and $PCR$ is used for $DNA$ amplification,none of which primarily utilize agarose as a separation matrix.

(A) Chromosomal $DNA$ and plasmid $DNA$ differ in several aspects:
$1$. Presence of histones: Chromosomal $DNA$ is associated with histones in eukaryotes,whereas plasmids (found in bacteria) lack histones.
$2$. Nature of histone: Since plasmids lack histones,this is a point of difference.
$3$. Nature of nucleotide: Both chromosomal $DNA$ and plasmid $DNA$ are composed of the same four nucleotides $(A, T, G, C)$. Therefore,the nature of nucleotides is $NOT$ a basis of difference.
$4$. Linear form of genetic material: Chromosomal $DNA$ in eukaryotes is linear,while plasmids are typically circular. Thus,this is a basis of difference.
Since the question asks for what is $NOT$ a basis of difference,only the 'Nature of nucleotide' $(c)$ fits.

(B) Plasmids are small,circular,double-stranded $DNA$ molecules that are distinct from a cell's chromosomal $DNA$.
They are extrachromosomal and capable of autonomous (self) replication.
They do not contain essential genes required for the basic survival of the bacteria; instead,they often carry genes that provide advantages like antibiotic resistance.
Plasmids are not present in all bacteria.
Therefore,the characteristics that are $NOT$ applicable to plasmids are: $(b)$ Linear $DNA$,$(c)$ Present in all bacteria,and $(d)$ Contain essential genes.
Thus,the correct option is $b, c$ and $d$ only.

(A) In genetic engineering,a 'disarmed' vector refers to a vector that has been modified to remove its pathogenic or harmful properties while retaining its ability to transfer $DNA$ into a host cell.
Specifically,in the case of the $Ti$ plasmid (Tumor-inducing plasmid) from $Agrobacterium$ $tumefaciens$,the genes responsible for tumor formation (pathogenicity) are removed or replaced with the gene of interest.
Therefore,'disarmed' means the removal of the pathogenic $T-DNA$ region from the $Ti$ plasmid,rendering it safe for use as a cloning vector.

(A) $DNA$ ligase is an enzyme that acts as a 'molecular glue' by catalyzing the formation of phosphodiester bonds between the $3'$-hydroxyl end of one nucleotide and the $5'$-phosphate end of another.
In recombinant $DNA$ technology,this enzyme is essential for joining the foreign $DNA$ fragment (gene of interest) with the cloning vector (such as a plasmid).
The antibiotic resistance gene is a common marker used in plasmid vectors. To create a recombinant plasmid,the gene of interest and the vector are cut with the same restriction enzyme,and then $DNA$ ligase is used to covalently link them together.
Therefore,both the Assertion and the Reason are correct,and the Reason provides a valid explanation for the importance of $DNA$ ligase in this technology.

(A) $1$. Restriction enzymes are a type of enzyme that cuts $DNA$ at specific sites. They belong to a larger class of enzymes known as nucleases.
$2$. Nucleases are categorized into two types: exonucleases (which remove nucleotides from the ends of $DNA$) and endonucleases (which make cuts at specific positions within the $DNA$).
$3$. Restriction endonucleases are a specific type of endonuclease that recognizes a specific palindromic nucleotide sequence in the $DNA$ and cuts the $DNA$ at that site.
$4$. Therefore,both the Assertion and the Reason are correct,and the Reason provides the correct explanation for the Assertion.

(A) $DNA$ molecules contain a sugar-phosphate backbone where the phosphate groups carry a negative charge.
Due to this negative charge,$DNA$ fragments migrate towards the positively charged electrode,which is the anode,when an electric field is applied during gel electrophoresis.
Therefore,both the Assertion and the Reason are correct,and the Reason is the correct explanation for the Assertion.

(A) The $Ti$ plasmid (Tumor-inducing plasmid) is naturally found in $Agrobacterium$ $tumefaciens$.
By removing the tumor-causing genes,it becomes a 'disarmed' $Ti$ plasmid.
$A$ shuttle vector is defined as a vector that can replicate in two different host organisms (e.g.,both prokaryotes like $E. coli$ and eukaryotes like plants).
Since the disarmed $Ti$ plasmid can be maintained in $E. coli$ (prokaryote) and also used to transform plant cells (eukaryote),it functions as a shuttle vector.
Therefore,both the Assertion and the Reason are correct,and the Reason correctly explains why it is called a shuttle vector.

(A) $Pst I$ is a restriction endonuclease that cuts $DNA$ at specific recognition sequences,creating single-stranded overhanging stretches known as 'sticky ends'.
These sticky ends facilitate the ligation process because they can form hydrogen bonds with their complementary counterparts on another $DNA$ fragment.
Once the hydrogen bonds are formed,the enzyme $DNA$ ligase seals the nicks by forming phosphodiester bonds,resulting in a continuous recombinant $DNA$ molecule.

(A) Ethidium bromide $(EtBr)$ is a fluorescent dye used to stain $DNA$ fragments in agarose gel electrophoresis.
When exposed to $UV$ light,the $DNA$ bands stained with $EtBr$ fluoresce as bright orange bands.
This occurs because $EtBr$ is an intercalating agent that binds between the base pairs of the $DNA$ double helix.
It absorbs light in the $UV$ range,specifically between $260-330 \; nm$,and emits light in the visible spectrum (orange).
Therefore,both the Assertion and the Reason are correct,and the Reason provides the correct explanation for why $DNA$ can be visualized under $UV$ light.

(A) $Agrobacterium$ $tumefaciens$ is a soil bacterium that naturally infects plants.
Its $Ti$-plasmid $(Tumor$ $inducing$ $plasmid)$ is widely used in genetic engineering to transfer foreign $DNA$ into the genome of higher plants.
Therefore, it acts as a natural vector for gene transfer.

(A) Restriction endonuclease is the enzyme that recognizes a specific $DNA$ base sequence and cleaves both strands of a $DNA$ molecule at a particular site called the restriction site,which typically contains a palindromic sequence.
Because these enzymes cut $DNA$ at specific locations,they are commonly referred to as molecular scissors.

(D) $Ti-$ plasmid (Tumour-inducing plasmid) is found in $Agrobacterium \; tumefaciens$,which produces crown gall (tumour) in a large number of dicot species.
$Agrobacterium \; tumefaciens$ is a Gram-negative soil bacterium that infects a wide range of plants and causes crown galls.

(C) The $T_{i}$ plasmid (Tumor-inducing plasmid) is a circular $DNA$ molecule found in the soil bacterium $Agrobacterium$ $tumefaciens$.
It is widely used in plant genetic engineering as a cloning vector because it has the natural ability to transfer a specific segment of its $DNA$, known as $T$-$DNA$, into the genome of host plants.
This property is exploited to introduce foreign genes into dicotyledonous plants.

(A) The polymerase chain reaction $(PCR)$ is a technique by which small samples of $DNA$ can be quickly amplified.
This repeated amplification is achieved by the use of a thermostable $DNA$ polymerase,known as $Taq$ polymerase.
$Taq$ polymerase is isolated from the bacterium $Thermus$ $aquaticus$.
It remains active during the high-temperature induced denaturation step of double-stranded $DNA$ in the $PCR$ cycle.

(C) $T-DNA$ (Transfer $DNA$) is a segment of $DNA$ found in the $Ti$ (Tumor-inducing) plasmid of the soil bacterium $Agrobacterium$ $tumefaciens$.
This bacterium naturally infects plants and transfers its $T-DNA$ into the host plant genome, causing crown gall disease.
In biotechnology, scientists have modified this system by removing the disease-causing genes and replacing them with genes of interest, making $Agrobacterium$ $tumefaciens$ an excellent vector for plant genetic engineering.

(D) Restriction endonucleases are enzymes that recognize specific base sequences in $DNA$ and cut the $DNA$ at specific sites. These enzymes are produced by bacteria as a defense mechanism to protect themselves against the invasion of foreign $DNA$,such as that from bacteriophages (viruses that infect bacteria).

(C) molecular probe is a single-stranded fragment of $DNA$ or $RNA$ that is tagged with a radioactive isotope,such as $P^{32}$.
These probes are used to detect specific sequences of nucleic acids by hybridizing with their complementary strands.
Molecular probes are widely used in the diagnosis of various genetic disorders,including Duchenne muscular dystrophy,cystic fibrosis,and Tay-Sachs disease.

(C) Plasmids,cosmids,or bacteriophages can be used as vectors in genetic engineering.
Plasmids are the most widely used circular,extrachromosomal $DNA$ segments found in bacterial cells.
The primary reason they are used as vectors is their ability to carry a foreign gene or desired gene into the host organism.
The size of plasmids ranges from $1 \times 10^{6}$ to $200 \times 10^{6}$ daltons.
Common examples of vector plasmids include $pBR322$,those of the $pUC$ series,and $Ti$ and $Ri$ plasmids of $Agrobacterium$.

(A) Restriction endonucleases are enzymes that recognize a specific $DNA$ base sequence,known as the recognition site or restriction site,which typically consists of a palindromic sequence.
These enzymes cleave both strands of the $DNA$ molecule at or near these specific sites.
This process results in the generation of restriction fragments,which may have overhanging ends (sticky ends) or blunt ends.

(D) plasmid is an extrachromosomal,self-replicating,circular $DNA$ molecule found in bacteria and some eukaryotes.
It is not part of the chromosomal $DNA$ and does not contain genes essential for the host's vital survival activities.
Due to its ability to replicate independently and carry foreign genes,it is widely used as a vector in genetic engineering and gene transfer experiments.

(D) Plasmids are extrachromosomal molecules of $DNA$ that replicate independently of the bacterial chromosomes.
They are typically closed,circular,and supercoiled structures.
Due to their ability to replicate autonomously and carry foreign genes,they are widely used as vectors for cloning and genetic engineering.

(A) $DNA$ ligase is an enzyme that facilitates the joining of $DNA$ strands together by catalyzing the formation of a phosphodiester bond.
It acts as a molecular glue to seal nicks or join fragments of $DNA$ during replication,repair,and recombinant $DNA$ technology.
Ligase enzymes catalyze the condensation of $ATP$ or other similar triphosphates to provide the energy required for this joining process.

(A) The isolation of restriction endonucleases by Nathans and Smith $(1970)$ made it possible to cut $DNA$ at specific sites.
Restriction enzymes can cut both strands of $DNA$ when foreign nucleotides are introduced into the cell.
They cleave $DNA$ to generate a nick with a $5'$ phosphoryl and $3'$ hydroxyl terminus,which is the fundamental step in recombinant $DNA$ technology.

(C) probe is a single-stranded $DNA$ or $RNA$ molecule that is tagged with a radioactive isotope or a fluorescent marker.
It is used to identify specific sequences by hybridizing to its complementary $DNA$ or $RNA$ strand in a sample.
Since both $DNA$ and $RNA$ can act as probes depending on the experimental requirement,the correct answer is that a probe can be either $RNA$ or $DNA$.

(A) The restriction endonuclease Eco $RI$ is isolated from the bacterium Escherichia coli $RY13$.
It recognizes the specific palindromic nucleotide sequence $5'-GAATTC-3'$ and cuts between $G$ and $A$ on both strands.
Therefore,$GAATTC$ is the recognition site for Eco $RI$.

(D) Restriction endonucleases are the enzymes that cleave $DNA$ molecules at specific nucleotide sequences known as restriction sites.
$DNA$ ligase is an enzyme that acts as a 'molecular glue' to join bits of $DNA$ fragments together.
Therefore,the statement that $DNA$ ligases are used to cleave a $DNA$ molecule is incorrect.

(D) Restriction endonucleases are enzymes that cut $DNA$ molecules at specific recognition sites,which have specific base sequences. Examples include Hae $III$,Eco $RI$,Bam $HI$,Hind $II$,and Pst $I$.
$DNAse$ $I$ is a deoxyribonuclease enzyme that degrades $DNA$ by breaking phosphodiester bonds,but it is not a restriction endonuclease.
Therefore,$DNAse$ $I$ is the correct answer.

(A) $Escherichia$ $coli$ and $Agrobacterium$ $tumefaciens$ are microbes found to be very useful in genetic engineering.
$E.$ $coli$ is a motile,Gram-negative,rod-shaped bacterium which is a normal inhabitant of the human colon. It is most extensively used in bacterial genetics and molecular biology.
$Agrobacterium$ $tumefaciens$ is a soil bacterium. It contains a $T_{i}$ plasmid (Tumour-inducing plasmid),which is used as a vector for the transfer of a desired gene into dicot plants.

(C) Restriction endonucleases are enzymes that recognize specific base pair sequences in $DNA$ and cut the $DNA$ at those sites.
These specific sequences are known as palindromic nucleotide sequences.
$A$ 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.,$5' - GAATTC - 3'$ and $3' - CTTAAG - 5'$).
Therefore,the correct option is $C$.

(A) Restriction enzymes recognize specific palindromic nucleotide sequences in $DNA$ to perform cleavage.
$A$ 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.,$5' \rightarrow 3'$).
Option $A$ $(5'-GAATTC-3'; 3'-CTTAAG-5')$ represents the recognition site for the restriction enzyme $EcoRI$.
This sequence is a perfect palindrome because the sequence $GAATTC$ read from $5' \rightarrow 3'$ on one strand is identical to the sequence $GAATTC$ read from $5' \rightarrow 3'$ on the complementary strand.
Options $B$,$C$,and $D$ do not represent standard palindromic sequences recognized by common restriction enzymes.

(D) Statement $I$ is correct: Restriction endonucleases scan the $DNA$ molecule for specific recognition sequences,which are palindromic nucleotide sequences (sequences that read the same forward and backward on the two strands).
Statement $II$ is correct: Most restriction enzymes do not cut exactly in the middle of the palindromic site; instead,they cut the $DNA$ strands a little away from the centre,often between the same two bases on opposite strands,leaving single-stranded portions at the ends known as sticky ends.