Q.1 What is the primary purpose of a cloning vector in gene cloning?
To amplify the DNA fragment of interest
To sequence the entire genome
To destroy unwanted DNA
To edit genes directly in a living organism
Explanation - A cloning vector provides a means to replicate the inserted DNA fragment inside a host cell, resulting in multiple copies of the fragment.
Correct answer is: To amplify the DNA fragment of interest
Q.2 Which enzyme cuts DNA at specific palindromic sequences to create compatible ends for ligation?
DNA polymerase
Ligase
Restriction endonuclease
Helicase
Explanation - Restriction endonucleases recognize specific DNA sequences and cleave the phosphodiester bond, generating sticky or blunt ends for cloning.
Correct answer is: Restriction endonuclease
Q.3 In a plasmid vector, the origin of replication (ori) is essential because:
It determines the antibiotic resistance of the host
It initiates transcription of the inserted gene
It allows the plasmid to be replicated independently of the chromosome
It provides a binding site for restriction enzymes
Explanation - The ori is a DNA sequence that the host's replication machinery recognizes, enabling autonomous plasmid replication.
Correct answer is: It allows the plasmid to be replicated independently of the chromosome
Q.4 Which of the following is a common selectable marker used in bacterial cloning vectors?
Green fluorescent protein (GFP)
Ampicillin resistance gene (bla)
LacZ alpha fragment
His‑tag sequence
Explanation - The bla gene confers resistance to ampicillin, allowing only bacteria that have taken up the plasmid to grow on selective media.
Correct answer is: Ampicillin resistance gene (bla)
Q.5 What does the term "sticky end" refer to in molecular cloning?
A blunt-ended DNA fragment
An overhanging single‑stranded DNA sequence
A DNA fragment that cannot ligate
A region of DNA that binds proteins
Explanation - Sticky ends are produced by certain restriction enzymes, leaving single‑stranded overhangs that can anneal with complementary sequences.
Correct answer is: An overhanging single‑stranded DNA sequence
Q.6 Which technique is commonly used to introduce plasmid DNA into Escherichia coli cells?
Electroporation
Southern blotting
Western blotting
Chromatography
Explanation - Electroporation uses an electric pulse to create temporary pores in the bacterial membrane, allowing DNA uptake.
Correct answer is: Electroporation
Q.7 What is the function of the lacZ gene in many cloning vectors?
To provide resistance to tetracycline
To enable blue‑white screening
To enhance transcription of the insert
To act as a promoter for the cloned gene
Explanation - Insertion of a DNA fragment into the lacZ alpha region disrupts β‑galactosidase activity, allowing colonies with inserts to appear white on X‑gal plates.
Correct answer is: To enable blue‑white screening
Q.8 Which of the following best describes PCR‑based cloning?
Cloning DNA fragments directly into a viral vector
Amplifying a DNA fragment with primers that add restriction sites for ligation
Using RNA polymerase to transcribe DNA into RNA
Inserting DNA fragments without any enzymatic steps
Explanation - PCR primers can be designed to include restriction sites, enabling the amplified product to be digested and ligated into a vector.
Correct answer is: Amplifying a DNA fragment with primers that add restriction sites for ligation
Q.9 Which of the following is NOT a method for removing the original plasmid backbone after successful cloning?
Colony PCR
Restriction digest analysis
Sequencing
Gram staining
Explanation - Gram staining is used for bacterial classification, not for confirming the presence or absence of plasmid DNA.
Correct answer is: Gram staining
Q.10 What is the purpose of adding a poly‑A tail to a cloned eukaryotic gene expressed in mammalian cells?
To facilitate nuclear export of the mRNA
To increase the plasmid copy number
To enable integration into the host genome
To provide a binding site for RNA polymerase
Explanation - A poly‑A tail stabilizes mRNA and aids in its transport from the nucleus to the cytoplasm in eukaryotic cells.
Correct answer is: To facilitate nuclear export of the mRNA
Q.11 In Gateway cloning, what is the role of the attB sites?
They serve as promoters for transcription
They are recognized by restriction enzymes
They mediate recombination with the destination vector
They encode antibiotic resistance
Explanation - attB sites on the PCR product recombine with attP sites in the donor vector via the BP reaction, enabling site‑specific cloning.
Correct answer is: They mediate recombination with the destination vector
Q.12 Which of the following statements about blunt‑end cloning is TRUE?
Blunt ends require ligase but no compatible overhangs
Blunt ends are generated only by Type II restriction enzymes
Blunt‑end ligation is more efficient than sticky‑end ligation
Blunt ends prevent the use of selectable markers
Explanation - Blunt‑end cloning does not need complementary overhangs; ligase can join any blunt ends, though the efficiency is generally lower than with sticky ends.
Correct answer is: Blunt ends require ligase but no compatible overhangs
Q.13 What is the main advantage of using a viral vector (e.g., lentivirus) for gene cloning in mammalian cells?
Higher antibiotic resistance
Stable integration into the host genome
Easier plasmid purification
No need for a promoter
Explanation - Lentiviral vectors can integrate their genetic payload into the host cell's DNA, providing long‑term expression.
Correct answer is: Stable integration into the host genome
Q.14 Which of the following enzymes is required to join DNA fragments after restriction digestion?
DNA polymerase I
RNA ligase
DNA ligase
Topoisomerase
Explanation - DNA ligase catalyzes the formation of phosphodiester bonds between adjacent nucleotides, sealing nicks in the DNA backbone.
Correct answer is: DNA ligase
Q.15 When cloning a gene with an N‑terminal His‑tag, where is the tag usually placed?
Downstream of the stop codon
Upstream of the start codon
Within the promoter region
In the intron of the gene
Explanation - A His‑tag is fused in‑frame at the N‑terminus of the protein coding sequence, typically placed just after the start codon.
Correct answer is: Upstream of the start codon
Q.16 What is the purpose of a 'multiple cloning site' (MCS) in a vector?
To provide a region for high‑copy replication
To house several consecutive restriction sites for flexible cloning
To encode a fluorescent protein
To act as a transcription terminator
Explanation - The MCS contains many unique restriction enzyme sites, allowing the researcher to choose the most convenient enzymes for inserting a fragment.
Correct answer is: To house several consecutive restriction sites for flexible cloning
Q.17 Which of the following best describes the term 'gene library'?
A collection of all possible proteins in a cell
A repository of cloned DNA fragments representing a genome or tissue
A database of gene expression profiles
A set of RNA molecules extracted from a cell
Explanation - Gene libraries consist of cloned DNA fragments stored in vectors, allowing researchers to screen for genes of interest.
Correct answer is: A repository of cloned DNA fragments representing a genome or tissue
Q.18 What is the key difference between a genomic library and a cDNA library?
Genomic libraries contain only exons; cDNA libraries contain introns
Genomic libraries are made from RNA; cDNA libraries are made from DNA
Genomic libraries include both introns and exons; cDNA libraries contain only expressed sequences
Genomic libraries are stored in viruses; cDNA libraries are stored in plasmids
Explanation - cDNA is synthesized from mRNA and therefore lacks introns, representing only the transcribed parts of the genome.
Correct answer is: Genomic libraries include both introns and exons; cDNA libraries contain only expressed sequences
Q.19 Which of the following methods can be used to verify that a cloned gene is in the correct orientation?
Restriction digest analysis with two enzymes flanking the insert
Gram staining of bacterial colonies
Measuring optical density of the culture
Performing a Southern blot with a random probe
Explanation - Digesting with enzymes that cut outside the insert yields fragments of predictable sizes that differ based on orientation.
Correct answer is: Restriction digest analysis with two enzymes flanking the insert
Q.20 What is the role of the promoter in a cloning vector designed for protein expression?
To replicate the plasmid in the host
To initiate transcription of the cloned gene
To provide antibiotic resistance
To cut the DNA at specific sites
Explanation - A promoter is a DNA sequence recognized by RNA polymerase, directing the synthesis of mRNA from the cloned gene.
Correct answer is: To initiate transcription of the cloned gene
Q.21 Which technique allows for the rapid insertion of a PCR product into a vector without using restriction enzymes?
Gibson assembly
Northern blotting
DNA fingerprinting
RNA interference
Explanation - Gibson assembly uses exonuclease, polymerase, and ligase in a single reaction to join overlapping DNA fragments seamlessly.
Correct answer is: Gibson assembly
Q.22 In TA cloning, why are Taq‑polymerase‑generated PCR products suitable for ligation into a vector with 3'‑A overhangs?
Taq polymerase adds a single deoxy‑A to the 3' ends of PCR products
Taq polymerase produces blunt ends
Taq polymerase adds a poly‑C tail
Taq polymerase adds restriction sites automatically
Explanation - The non‑templated addition of an adenine by Taq polymerase creates a complementary overhang for ligation with a T‑vector.
Correct answer is: Taq polymerase adds a single deoxy‑A to the 3' ends of PCR products
Q.23 Which of the following is a common host strain of E. coli used for cloning unstable DNA sequences?
DH5α
BL21(DE3)
JM109
Stbl2
Explanation - Stbl2 (and Stbl4) are engineered to reduce recombination events, making them suitable for cloning repetitive or toxic sequences.
Correct answer is: Stbl2
Q.24 What does the term 'copy number' refer to in the context of plasmid vectors?
The number of genes inserted into the plasmid
The number of plasmid molecules maintained per bacterial cell
The number of restriction sites in the vector
The number of antibiotic resistance genes
Explanation - Copy number indicates how many copies of the plasmid are replicated in each host cell, influencing yield of cloned DNA.
Correct answer is: The number of plasmid molecules maintained per bacterial cell
Q.25 Which of the following is a major advantage of using a cosmid vector over a standard plasmid?
Higher copy number in the host cell
Ability to carry larger DNA inserts (up to ~45 kb)
Automatic expression of the cloned gene
Inherent antibiotic resistance without a selectable marker
Explanation - Cosmids combine features of plasmids and bacteriophage λ, allowing them to maintain larger DNA fragments than typical plasmids.
Correct answer is: Ability to carry larger DNA inserts (up to ~45 kb)
Q.26 Which of the following enzymes is used to create a single‑strand break (nick) in double‑stranded DNA at a specific site?
Type I restriction enzyme
Nickase (a mutated restriction enzyme)
DNA polymerase
RNA polymerase
Explanation - Nickases cut only one strand of DNA at a specific recognition site, useful for techniques like strand‑specific labeling or rolling‑circle amplification.
Correct answer is: Nickase (a mutated restriction enzyme)
Q.27 In the context of gene cloning, what does the term "frame shift" refer to?
A change in the reading frame caused by insertion or deletion of nucleotides not in multiples of three
The use of a different promoter for transcription
A mutation that alters the start codon
A modification of the plasmid origin of replication
Explanation - Frameshifts alter the downstream codon grouping, potentially leading to premature stop codons and non‑functional proteins.
Correct answer is: A change in the reading frame caused by insertion or deletion of nucleotides not in multiples of three
Q.28 What is the main purpose of using a shuttle vector?
To move DNA between different host species (e.g., bacteria and yeast)
To increase the plasmid copy number
To provide fluorescence for visualization
To block transcription of the insert
Explanation - Shuttle vectors contain origins of replication and selectable markers for multiple host organisms, facilitating cross‑species cloning.
Correct answer is: To move DNA between different host species (e.g., bacteria and yeast)
Q.29 Which of the following statements about site‑directed mutagenesis is TRUE?
It can only delete entire genes
It introduces specific nucleotide changes using designed primers
It requires viral vectors for delivery
It always results in a frameshift mutation
Explanation - Site‑directed mutagenesis uses primers bearing the desired mutation to amplify the plasmid, producing a targeted change in the DNA sequence.
Correct answer is: It introduces specific nucleotide changes using designed primers
Q.30 Why is the use of a temperature‑sensitive origin of replication advantageous in some cloning strategies?
It allows selective replication at higher temperatures, facilitating plasmid curing
It increases the rate of transcription of the insert
It provides resistance to multiple antibiotics
It eliminates the need for a selectable marker
Explanation - At non‑permissive temperatures, the plasmid cannot replicate, enabling researchers to remove the plasmid from the host after its purpose is fulfilled.
Correct answer is: It allows selective replication at higher temperatures, facilitating plasmid curing
Q.31 What is the purpose of adding an internal ribosome entry site (IRES) in a bicistronic expression vector?
To enable simultaneous translation of two genes from a single mRNA
To provide antibiotic resistance
To increase plasmid copy number
To serve as a restriction site for cloning
Explanation - IRES elements allow ribosomes to initiate translation internally, permitting co‑expression of two proteins from one transcript.
Correct answer is: To enable simultaneous translation of two genes from a single mRNA
Q.32 Which of the following is NOT a typical component of a bacterial expression vector?
Promoter (e.g., T7)
Signal peptide for secretion
Origin of replication
Mitochondrial targeting sequence
Explanation - Mitochondrial targeting sequences are used in eukaryotic cells; bacterial vectors generally lack organelle‑specific targeting signals.
Correct answer is: Mitochondrial targeting sequence
Q.33 What does the term 'recombination cloning' generally refer to?
Cloning using restriction enzymes only
Cloning based on homologous recombination mechanisms
Cloning that requires viral infection
Cloning with random DNA fragments
Explanation - Recombination cloning (e.g., Gateway, Gibson) relies on sequence homology to join DNA fragments without restriction enzymes.
Correct answer is: Cloning based on homologous recombination mechanisms
Q.34 Which of the following best explains why high‑fidelity DNA polymerases are preferred for cloning PCR products intended for protein expression?
They increase the speed of PCR
They have proofreading activity, reducing mutations in the amplified gene
They add a poly‑A tail to the product
They work without a template
Explanation - High‑fidelity polymerases possess 3'→5' exonuclease activity, ensuring the cloned gene retains the correct sequence for functional protein production.
Correct answer is: They have proofreading activity, reducing mutations in the amplified gene
Q.35 In a blue‑white screening assay, why do colonies containing an insert appear white?
The insert produces a white pigment
The insertion disrupts the lacZ gene, preventing β‑galactosidase activity
White colonies have a higher copy number
The antibiotic selection turns the colonies white
Explanation - Insertion into the lacZ α‑fragment stops functional β‑galactosidase formation; thus, X‑gal remains unconverted, yielding white colonies.
Correct answer is: The insertion disrupts the lacZ gene, preventing β‑galactosidase activity
Q.36 Which of the following vectors is specifically designed for the expression of secreted proteins in mammalian cells?
pUC19
pBR322
pcDNA3.1 with a signal peptide
pET28a
Explanation - pcDNA3.1 is a mammalian expression vector; inclusion of a signal peptide directs the expressed protein into the secretory pathway.
Correct answer is: pcDNA3.1 with a signal peptide
Q.37 What is the main disadvantage of using a high‑copy plasmid for cloning toxic genes?
The host cells may die due to high expression of the toxic product
The plasmid cannot be isolated easily
High‑copy plasmids are less stable during storage
They cannot be transferred by transformation
Explanation - High copy number leads to abundant transcription and translation, which can be lethal if the gene product is toxic to the host.
Correct answer is: The host cells may die due to high expression of the toxic product
Q.38 In a fosmid vector, the copy number is typically:
Very high ( >200 copies per cell )
Low ( 1–2 copies per cell )
Medium ( 20–30 copies per cell )
Variable depending on the promoter
Explanation - Fosmids are maintained at low copy numbers to increase stability of large DNA inserts.
Correct answer is: Low ( 1–2 copies per cell )
Q.39 Which method is most suitable for cloning a DNA fragment that contains internal restriction sites used for cloning?
Standard restriction‑ligation cloning
TA cloning
Gibson assembly
Blue‑white screening
Explanation - Gibson assembly does not rely on restriction sites; overlapping ends are designed to avoid internal site conflicts.
Correct answer is: Gibson assembly
Q.40 What is the role of the terminator sequence in a cloning vector used for protein expression?
To initiate transcription
To stop transcription and release the mRNA
To replicate the plasmid
To bind restriction enzymes
Explanation - A transcription terminator signals RNA polymerase to disengage, ensuring proper mRNA processing and stability.
Correct answer is: To stop transcription and release the mRNA
Q.41 Which of the following is a common method to increase the solubility of a recombinant protein expressed in E. coli?
Fusion to a maltose‑binding protein (MBP) tag
Increasing the plasmid copy number
Using a stronger promoter
Adding a chloramphenicol resistance gene
Explanation - MBP enhances the solubility of many proteins, reducing inclusion body formation during expression.
Correct answer is: Fusion to a maltose‑binding protein (MBP) tag
Q.42 When cloning a eukaryotic gene into a bacterial expression system, why is it often necessary to remove introns?
Bacteria cannot splice introns from pre‑mRNA
Introns are toxic to bacterial cells
Introns cause plasmid instability
Introns inhibit DNA replication
Explanation - Prokaryotes lack the splicing machinery; thus, intron‑containing genes must be converted to cDNA before bacterial expression.
Correct answer is: Bacteria cannot splice introns from pre‑mRNA
Q.43 Which of the following best describes the principle of "in‑fusion cloning"?
Use of restriction enzymes to cut both vector and insert
Recombination of fragments with overlapping ends using a proprietary enzyme mix
Insertion of DNA fragments via transduction
Random integration into the host genome
Explanation - In‑fusion cloning exploits 15‑bp overlaps between vector and insert, enabling seamless joining without restriction digestion.
Correct answer is: Recombination of fragments with overlapping ends using a proprietary enzyme mix
Q.44 What is the main advantage of using a BAC (bacterial artificial chromosome) for cloning large DNA fragments?
Higher copy number than plasmids
Ability to maintain inserts up to several hundred kilobases
Automatic expression of the cloned gene
Presence of multiple selectable markers
Explanation - BACs are derived from the F‑factor and can stably propagate very large DNA fragments, useful for genome mapping.
Correct answer is: Ability to maintain inserts up to several hundred kilobases
Q.45 Which of the following steps is essential before transforming bacteria with a ligation mixture?
Heat‑shock the mixture at 95 °C for 5 min
Purify the ligated plasmid by gel extraction
Add calcium chloride to make cells competent
Incubate the ligation mixture with RNase
Explanation - Calcium chloride treatment renders bacterial membranes permeable, allowing uptake of DNA during heat‑shock transformation.
Correct answer is: Add calcium chloride to make cells competent
Q.46 What does the term "subcloning" refer to?
Cloning a gene into a different vector after an initial cloning step
Cloning an entire genome into a bacterial host
Directly inserting DNA into the host chromosome
Using a vector to clone multiple genes at once
Explanation - Subcloning moves a DNA fragment from one vector to another, often to change expression characteristics or host range.
Correct answer is: Cloning a gene into a different vector after an initial cloning step
Q.47 Which of the following is a commonly used inducible promoter for protein expression in E. coli?
T7 promoter
SP6 promoter
Lac operon promoter (lacUV5)
CMV promoter
Explanation - The lacUV5 promoter can be induced with IPTG, allowing controlled expression of recombinant proteins.
Correct answer is: Lac operon promoter (lacUV5)
Q.48 What is the purpose of using a 'dual‑origin' plasmid in cloning?
To replicate in both bacterial and yeast hosts
To increase the antibiotic resistance spectrum
To produce two different proteins from the same plasmid
To allow the plasmid to self‑destruct after cloning
Explanation - Dual‑origin plasmids contain origins of replication for multiple hosts, facilitating manipulation in different systems.
Correct answer is: To replicate in both bacterial and yeast hosts
Q.49 Which method would you use to confirm the sequence of a cloned gene after transformation?
Southern blotting
Sanger sequencing
Northern blotting
ELISA
Explanation - Sanger sequencing reads the exact nucleotide order of the cloned insert, verifying that no mutations occurred.
Correct answer is: Sanger sequencing
Q.50 In the context of cloning, what does the term "vector backbone" refer to?
The DNA segment that carries the selectable marker and origin of replication
The gene of interest being cloned
The restriction enzyme used for cloning
The host cell's chromosomal DNA
Explanation - The backbone provides essential functions (replication, selection) but does not include the inserted DNA fragment.
Correct answer is: The DNA segment that carries the selectable marker and origin of replication
Q.51 Which of the following is the most common method for preparing a linearized vector for ligation?
Partial digestion with a restriction enzyme
Heat denaturation at 95 °C
Treatment with RNase A
Incubation with a protease
Explanation - Partial (or complete) digestion creates defined ends on the vector that can be ligated with compatible insert ends.
Correct answer is: Partial digestion with a restriction enzyme
Q.52 What is the purpose of adding a ribosome binding site (RBS) upstream of the cloned gene in a bacterial expression vector?
To promote plasmid replication
To facilitate translation initiation of the mRNA
To act as an antibiotic resistance marker
To serve as a transcription terminator
Explanation - The RBS (Shine‑Dalgarno sequence) aligns the ribosome with the start codon, enhancing translation efficiency.
Correct answer is: To facilitate translation initiation of the mRNA
Q.53 Which of the following vectors would you choose to express a protein with a C‑terminal FLAG tag in mammalian cells?
pET28a
pcDNA3.1‑FLAG‑C
pBR322
pUC19
Explanation - pcDNA3.1 is a mammalian expression vector; the C‑terminal FLAG tag allows detection/purification of the expressed protein.
Correct answer is: pcDNA3.1‑FLAG‑C
Q.54 When cloning a gene that encodes a secreted protein, why might you include a signal peptide sequence in the expression construct?
To direct the protein to the cytoplasm
To target the protein to the endoplasmic reticulum for secretion
To increase plasmid copy number
To make the protein fluorescent
Explanation - Signal peptides are recognized by the secretory pathway, guiding the nascent polypeptide into the ER lumen and eventually outside the cell.
Correct answer is: To target the protein to the endoplasmic reticulum for secretion
Q.55 Which of the following is a major drawback of using random (shotgun) cloning for constructing a genomic library?
The library size is limited to <1 kb inserts
Clones may contain overlapping fragments, requiring extensive screening
The method cannot be used for eukaryotic DNA
It requires viral vectors
Explanation - Random fragmentation leads to many overlapping clones, making the identification of unique genes more labor‑intensive.
Correct answer is: Clones may contain overlapping fragments, requiring extensive screening
Q.56 What is the function of the lacI gene in vectors that contain the lac promoter?
It provides resistance to ampicillin
It encodes the repressor protein that blocks transcription until IPTG is added
It codes for β‑galactosidase enzyme
It initiates replication of the plasmid
Explanation - LacI produces the lac repressor which binds the operator, preventing transcription; IPTG inactivates the repressor, inducing expression.
Correct answer is: It encodes the repressor protein that blocks transcription until IPTG is added
Q.57 Which of the following statements about the pBR322 plasmid is FALSE?
It contains both ampicillin and tetracycline resistance genes
It has a high copy number in most E. coli strains
It was one of the first widely used cloning vectors
It possesses a multiple cloning site
Explanation - pBR322 is a low‑copy-number plasmid (~15–20 copies per cell), unlike modern high‑copy vectors.
Correct answer is: It has a high copy number in most E. coli strains
Q.58 In a CRISPR‑based cloning approach, what is the role of the guide RNA (gRNA)?
To cut the plasmid backbone
To direct the Cas9 nuclease to a specific genomic location for insertion
To provide antibiotic resistance
To act as a promoter for transcription
Explanation - gRNA pairs with target DNA, guiding Cas9 to create a double‑strand break at a precise site, facilitating homology‑directed repair with a donor template.
Correct answer is: To direct the Cas9 nuclease to a specific genomic location for insertion
Q.59 What does the term "homologous recombination" imply in the context of gene targeting in mammalian cells?
Integration of DNA at random loci
Insertion of DNA only at the plasmid origin
Exchange of DNA between similar sequences, allowing precise gene replacement
Duplication of the entire genome
Explanation - Homologous recombination uses sequence similarity to replace or insert DNA at a specific chromosomal location.
Correct answer is: Exchange of DNA between similar sequences, allowing precise gene replacement
Q.60 Which of the following is an advantage of using a 'self‑splicing intron' in a cloning vector?
It eliminates the need for a promoter
It allows the vector to replicate without an origin of replication
It can be removed during transcription, restoring the open reading frame without additional enzymes
It provides antibiotic resistance
Explanation - Self‑splicing introns excise themselves from the pre‑mRNA, ensuring correct translation of the downstream gene.
Correct answer is: It can be removed during transcription, restoring the open reading frame without additional enzymes
Q.61 When using a bacterial artificial chromosome (BAC) for cloning, why is it important to grow the host strain at a lower temperature (e.g., 30 °C) rather than 37 °C?
Lower temperature reduces plasmid copy number, increasing stability of large inserts
It increases the speed of bacterial growth
It enhances antibiotic resistance expression
It activates the lac promoter
Explanation - BACs are low‑copy vectors; growing at cooler temperatures minimizes stress and recombination, preserving large DNA fragments.
Correct answer is: Lower temperature reduces plasmid copy number, increasing stability of large inserts
Q.62 Which of the following best describes the purpose of a 'chromatin immunoprecipitation (ChIP)‑cloning' approach?
To clone DNA fragments that are bound by a specific protein in vivo
To amplify plasmid DNA directly from bacterial colonies
To insert a gene into a viral genome
To perform site‑directed mutagenesis
Explanation - ChIP isolates DNA-protein complexes; the recovered DNA can be cloned and sequenced to identify binding sites.
Correct answer is: To clone DNA fragments that are bound by a specific protein in vivo
Q.63 What is a primary benefit of using a 'dual‑promoter' vector for co‑expression of two proteins in a single host cell?
Both proteins are expressed from a single mRNA, ensuring stoichiometric production
The vector can be maintained in both bacteria and yeast simultaneously
It reduces the overall size of the vector
It allows independent regulation of each gene's expression
Explanation - Dual‑promoter vectors contain separate promoters for each gene, permitting independent control of expression levels.
Correct answer is: It allows independent regulation of each gene's expression
Q.64 In the context of cloning, what does the term "phage display" refer to?
Displaying cloned DNA fragments on the surface of bacteriophages for screening
Using bacteriophages as vectors for large genome cloning
A method of visualizing plasmids under a microscope
A technique for amplifying RNA molecules
Explanation - Phage display presents peptides or proteins fused to phage coat proteins, enabling selection of binders from large libraries.
Correct answer is: Displaying cloned DNA fragments on the surface of bacteriophages for screening
Q.65 Which of the following is a key reason to use a 'low‑copy' plasmid when cloning DNA that is toxic to the host cell?
Low‑copy plasmids produce more protein per cell
Low‑copy plasmids limit expression levels, reducing toxicity
Low‑copy plasmids are easier to extract
Low‑copy plasmids confer resistance to multiple antibiotics
Explanation - Reduced plasmid copy number translates to lower transcription/translation of the toxic gene, improving host viability.
Correct answer is: Low‑copy plasmids limit expression levels, reducing toxicity
Q.66 What is the purpose of a 'poly‑cistronic' construct in bacterial cloning?
To enable expression of multiple genes from a single promoter and mRNA
To increase the plasmid size
To provide antibiotic resistance against three different drugs
To create a circular DNA molecule
Explanation - In bacteria, poly‑cistronic mRNA can be translated into several proteins using ribosome binding sites for each open reading frame.
Correct answer is: To enable expression of multiple genes from a single promoter and mRNA
Q.67 Which of the following techniques is commonly used to insert a DNA fragment into a vector without the need for ligase?
TOPO cloning
Restriction digestion
Southern blotting
Electrophoretic separation
Explanation - TOPO cloning utilizes topoisomerase I covalently attached to the vector ends, enabling ligase‑free insertion of PCR products with compatible ends.
Correct answer is: TOPO cloning
Q.68 What is the main advantage of using a 'reporter gene' (e.g., GFP) downstream of a cloned promoter in a vector?
It allows visualization of promoter activity in living cells
It provides antibiotic resistance
It stabilizes the plasmid DNA
It enhances transcription of the cloned gene
Explanation - Reporter genes generate measurable signals (fluorescence, enzymatic activity) reflecting the activity of the upstream regulatory elements.
Correct answer is: It allows visualization of promoter activity in living cells
Q.69 During a cloning workflow, why is it important to verify the orientation of the insert after ligation?
The insert orientation determines whether the gene will be transcribed correctly
Orientation affects plasmid copy number
Only one orientation allows antibiotic resistance
Orientation influences the size of the plasmid
Explanation - If the insert is reversed relative to the promoter, transcription may not occur or produce a non‑functional product.
Correct answer is: The insert orientation determines whether the gene will be transcribed correctly
Q.70 Which of the following is NOT a typical feature of a mammalian expression vector designed for stable integration?
A viral integrase gene
A selectable marker for mammalian cells
A bacterial origin of replication
An origin of replication that functions only in yeast
Explanation - Stable integration vectors for mammalian cells usually contain elements for bacterial propagation (origin of replication) and mammalian selection, not yeast‑specific origins.
Correct answer is: An origin of replication that functions only in yeast
Q.71 In the context of cloning, what does the term "synthetic biology" often involve?
Creating entirely new genetic parts and circuits not found in nature
Using only natural DNA fragments
Cloning only viral genomes
Avoiding the use of any enzymes
Explanation - Synthetic biology designs and assembles novel DNA sequences and pathways to achieve new functions beyond natural systems.
Correct answer is: Creating entirely new genetic parts and circuits not found in nature
Q.72 Which of the following is a common method for removing the selection marker after successful integration of a transgene into the mouse genome?
Cre‑LoxP recombination
Southern blotting
PCR amplification
Blue‑white screening
Explanation - Cre recombinase recognizes LoxP sites flanking the marker and excises it, leaving a clean integration.
Correct answer is: Cre‑LoxP recombination
Q.73 What is the principle behind "inverse PCR" in the context of cloning unknown flanking regions?
Amplifying DNA outward from a known internal sequence using primers oriented in opposite directions
Using restriction enzymes to cut DNA at random sites
Sequencing the entire plasmid directly
Cloning DNA into a viral vector
Explanation - Inverse PCR uses outward‑facing primers on a known segment to amplify unknown adjacent DNA after circularization.
Correct answer is: Amplifying DNA outward from a known internal sequence using primers oriented in opposite directions
Q.74 Which of the following vectors is specifically designed for the production of recombinant adeno‑associated virus (AAV) particles?
pUC19
pAAV‑MCS
pET21a
pBluescript
Explanation - pAAV vectors contain the inverted terminal repeats (ITRs) necessary for AAV packaging and a multiple cloning site for the transgene.
Correct answer is: pAAV‑MCS
Q.75 When cloning a gene into a yeast expression vector, why might a researcher include a 2‑micron (2µ) origin of replication?
To enable high‑copy-number maintenance of the plasmid in Saccharomyces cerevisiae
To confer antibiotic resistance
To provide a promoter for transcription
To allow replication in bacterial cells
Explanation - The 2µ origin ensures the plasmid replicates to high copy numbers in yeast, enhancing expression levels.
Correct answer is: To enable high‑copy-number maintenance of the plasmid in Saccharomyces cerevisiae
Q.76 Which of the following is an essential component of a CRISPR‑Cas9 based gene knock‑in strategy?
A donor DNA template with homology arms
A ribosomal RNA gene
A viral capsid protein
An antibiotic resistance gene only
Explanation - After Cas9 creates a double‑strand break, the donor template with flanking homology arms enables precise insertion via homology‑directed repair.
Correct answer is: A donor DNA template with homology arms
Q.77 What is the main purpose of using a 'self‑cleaving ribozyme' downstream of a transcribed RNA in a cloning construct?
To remove the 5' cap structure
To generate precise 3' ends of the RNA transcript without additional processing
To increase plasmid copy number
To provide antibiotic resistance
Explanation - Ribozymes catalyze site‑specific cleavage, producing defined RNA ends useful for functional studies or RNA therapeutics.
Correct answer is: To generate precise 3' ends of the RNA transcript without additional processing
Q.78 In the context of gene cloning, what does the term "synthetic promoter" refer to?
A promoter derived from viral DNA
A promoter artificially designed by combining regulatory elements to achieve desired expression levels
A promoter that confers antibiotic resistance
A promoter that is only active in bacterial cells
Explanation - Synthetic promoters are engineered sequences that provide precise control over transcription strength and regulation.
Correct answer is: A promoter artificially designed by combining regulatory elements to achieve desired expression levels
Q.79 Which of the following best describes the use of a "split‑intein" system in cloning?
A method to ligate two protein fragments post‑translationally, allowing protein engineering without scars
A technique for DNA sequencing
A way to increase plasmid copy number
A system for antibiotic selection
Explanation - Split inteins catalyze protein splicing, joining two polypeptide fragments inside the cell, useful for constructing large or multi‑domain proteins.
Correct answer is: A method to ligate two protein fragments post‑translationally, allowing protein engineering without scars
Q.80 When designing a cloning strategy for a gene that contains internal EcoRI sites, which of the following is the most suitable approach?
Use EcoRI for cloning and hope the internal sites are ignored
Mutate the internal EcoRI sites without altering the amino‑acid sequence, then use EcoRI
Switch to a different restriction enzyme that does not cut within the gene
Perform blunt‑end cloning with EcoRI‑digested vector
Explanation - Choosing enzymes that do not cut inside the gene avoids unwanted fragmentation; mutating internal sites can be labor‑intensive and may affect expression.
Correct answer is: Switch to a different restriction enzyme that does not cut within the gene
Q.81 Which of the following is the most common reason for using a 'His‑tag' at the N‑terminus versus the C‑terminus of a recombinant protein?
N‑terminal tags guarantee correct folding of the protein
Some proteins lose activity if the C‑terminus is modified
N‑terminal tags increase plasmid copy number
C‑terminal tags are not recognized by purification columns
Explanation - The functional domain of some proteins resides at the C‑terminus; placing the tag at the N‑terminus preserves activity.
Correct answer is: Some proteins lose activity if the C‑terminus is modified
Q.82 In a cloning experiment, why is it advisable to perform a 'control ligation' without insert DNA?
To test the efficiency of the restriction enzymes
To check for background colonies arising from vector self‑ligation
To increase the number of transformants
To verify the antibiotic resistance gene works
Explanation - A control ligation helps assess the rate of vector recircularization without an insert, which can lead to false positives.
Correct answer is: To check for background colonies arising from vector self‑ligation
Q.83 Which of the following best explains why a gene cloned into a plasmid under the control of a strong promoter may be toxic to the host cell?
The plasmid will replicate too slowly
High transcription and translation can overload the host's metabolic machinery
The antibiotic resistance gene will be over‑expressed
The plasmid will be lost during cell division
Explanation - Overexpression can deplete resources, produce misfolded proteins, or generate toxic products, leading to reduced growth or cell death.
Correct answer is: High transcription and translation can overload the host's metabolic machinery
Q.84 Which of the following methods allows for the precise insertion of a gene into a specific site of the E. coli chromosome?
Lambda Red recombineering
Random transposon mutagenesis
Blue‑white screening
Plasmid transformation
Explanation - Lambda Red recombination uses recombinases to facilitate homologous recombination of linear DNA into a defined chromosomal locus.
Correct answer is: Lambda Red recombineering
Q.85 In the context of cloning, what does the term "gene synthesis" refer to?
Extracting genes from natural sources
Using chemical methods to assemble a gene from oligonucleotides without a template
Cloning a gene into a viral vector
Amplifying a gene by PCR
Explanation - Gene synthesis allows the creation of custom DNA sequences de novo, enabling codon optimization or the incorporation of mutations.
Correct answer is: Using chemical methods to assemble a gene from oligonucleotides without a template
Q.86 Which of the following is a key advantage of using a 'single‑cell cloning' step after transformation?
It ensures that each colony originates from a single transformed cell, guaranteeing clonal purity
It increases the copy number of the plasmid
It eliminates the need for antibiotic selection
It speeds up bacterial growth
Explanation - Single‑cell isolation guarantees that downstream analyses are performed on a uniform population derived from one transformation event.
Correct answer is: It ensures that each colony originates from a single transformed cell, guaranteeing clonal purity
Q.87 What is the main purpose of a 'poly‑histidine (His) tag' in recombinant protein purification?
To increase protein solubility
To enable affinity purification using nickel‑NTA resin
To act as a transcriptional terminator
To provide antibiotic resistance
Explanation - His‑tags bind strongly to nickel or cobalt ions immobilized on a resin, allowing selective purification of the tagged protein.
Correct answer is: To enable affinity purification using nickel‑NTA resin
Q.88 When cloning a gene that contains a strong secondary structure at its 5' end, which strategy can improve expression in E. coli?
Add a ribosome binding site upstream of the start codon
Introduce synonymous mutations to reduce the secondary structure
Use a lower temperature during bacterial growth
All of the above
Explanation - Optimizing the RBS, reducing secondary structure via codon changes, and lowering growth temperature can all enhance translation initiation and protein yield.
Correct answer is: All of the above
Q.89 Which of the following is a common method to create a 'conditional knockout' mouse using gene cloning techniques?
Insertion of LoxP sites flanking essential exons, followed by Cre‑mediated recombination
Random integration of the transgene
Overexpression of the gene under a strong promoter
RNA interference (RNAi) delivery
Explanation - The Cre‑Lox system enables tissue‑specific or inducible deletion of the target gene when Cre recombinase is expressed.
Correct answer is: Insertion of LoxP sites flanking essential exons, followed by Cre‑mediated recombination
Q.90 What does the term "codon optimization" refer to in the context of gene cloning for heterologous expression?
Changing the gene's start codon to a stronger one
Altering codons to match the host organism’s preferred codon usage without changing the amino‑acid sequence
Removing all restriction sites from the gene
Adding extra stop codons at the end of the gene
Explanation - Optimizing codons improves translation efficiency and protein yield in the host by matching its tRNA pool.
Correct answer is: Altering codons to match the host organism’s preferred codon usage without changing the amino‑acid sequence
Q.91 In a cloning experiment, why might a researcher add a 'KpnI' site at the 5' end and a 'XhoI' site at the 3' end of a PCR primer pair?
To create compatible sticky ends for directional cloning into a vector digested with the same enzymes
To make the PCR product blunt-ended
To increase the melting temperature of the primers
To prevent the PCR product from being ligated
Explanation - Including different restriction sites on each end forces the insert into a single orientation during ligation.
Correct answer is: To create compatible sticky ends for directional cloning into a vector digested with the same enzymes
Q.92 Which of the following is an advantage of using a 'baculovirus expression system' for protein production?
It provides high‑level expression in insect cells with proper eukaryotic post‑translational modifications
It works directly in bacterial cells
It does not require any selectable markers
It produces circular DNA that can replicate in mammalian cells
Explanation - Baculovirus vectors infect insect cells, enabling expression of complex eukaryotic proteins with appropriate folding and modifications.
Correct answer is: It provides high‑level expression in insect cells with proper eukaryotic post‑translational modifications
Q.93 When cloning a gene into a vector that contains a C‑terminal FLAG tag, which of the following must be ensured?
The gene lacks a stop codon so translation continues into the FLAG sequence
The gene is placed downstream of a promoter
The vector contains an origin of replication
All of the above
Explanation - Proper expression requires a promoter, a downstream ORF without an early stop codon (so the tag is added), and an origin of replication for plasmid maintenance.
Correct answer is: All of the above
Q.94 Which of the following cloning techniques relies on the natural ability of bacteriophage λ to package large DNA fragments?
Cosmid cloning
Plasmid cloning
BAC cloning
Fosmid cloning
Explanation - Cosmids contain the λ cos site, enabling the packaging of inserts up to ~45 kb into phage capsids for efficient delivery.
Correct answer is: Cosmid cloning
Q.95 In the context of gene cloning, what does "gateway cloning" primarily utilize?
Site‑specific recombination between att sites mediated by BP and LR reactions
Traditional restriction enzyme digestion and ligation
CRISPR‑Cas9 mediated insertion
Electroporation of linear DNA
Explanation - Gateway cloning uses recombination of attB, attP, attL, and attR sites to transfer DNA fragments between vectors without restriction enzymes.
Correct answer is: Site‑specific recombination between att sites mediated by BP and LR reactions
Q.96 Which of the following is a primary reason to use a 'dual‑selection' system (e.g., two different antibiotics) in a cloning vector?
To increase plasmid copy number
To ensure that only cells containing both the vector and an inserted gene survive
To make the colonies appear colored
To speed up bacterial growth
Explanation - Dual selection can be used when the insert carries a second resistance marker, ensuring retention of both vector and insert.
Correct answer is: To ensure that only cells containing both the vector and an inserted gene survive
Q.97 When designing a cloning experiment for a gene that will be expressed in chloroplasts of plants, which element is essential in the vector?
A chloroplast transit peptide sequence
A mitochondrial targeting sequence
A bacterial origin of replication
A yeast selectable marker
Explanation - The transit peptide directs the expressed protein into the chloroplast, where it can function or accumulate.
Correct answer is: A chloroplast transit peptide sequence
Q.98 What is the purpose of using a 'terminator' downstream of a transgene in a plant expression vector?
To stop transcription and provide a poly‑A signal for mRNA stability
To increase plasmid replication
To confer herbicide resistance
To promote translation initiation
Explanation - A terminator ensures proper termination of transcription and often includes a poly‑adenylation signal that stabilizes the mRNA.
Correct answer is: To stop transcription and provide a poly‑A signal for mRNA stability
Q.99 Which cloning method is best suited for assembling many DNA fragments (≥5) simultaneously in a defined order?
Gibson assembly
TA cloning
Blue‑white screening
Southern blotting
Explanation - Gibson assembly can join multiple overlapping fragments in a single isothermal reaction, allowing complex construct assembly.
Correct answer is: Gibson assembly
Q.100 Why is it often necessary to include a 'ribosome binding site (RBS)' upstream of the coding sequence in a bacterial expression construct?
To allow the ribosome to recognize the start codon and initiate translation efficiently
To provide antibiotic resistance
To replicate the plasmid
To act as a promoter
Explanation - The RBS (Shine‑Dalgarno sequence) aligns the ribosome with the start codon, facilitating translation initiation.
Correct answer is: To allow the ribosome to recognize the start codon and initiate translation efficiently
Q.101 Which of the following best describes the concept of "modular cloning" (MoClo)?
A standardized system using type IIS restriction enzymes to assemble DNA parts in a hierarchical fashion
Cloning DNA fragments randomly into any vector
Using viral vectors for gene delivery
Cloning only mitochondrial DNA
Explanation - MoClo employs Type IIS enzymes (e.g., BsaI) that cut outside their recognition sites, enabling scar‑less, standardized assembly of multiple DNA modules.
Correct answer is: A standardized system using type IIS restriction enzymes to assemble DNA parts in a hierarchical fashion
Q.102 When cloning a gene that encodes a membrane protein, which of the following strategies can improve successful expression in E. coli?
Co‑express molecular chaperones
Use a lower temperature induction
Add a solubility‑enhancing fusion tag
All of the above
Explanation - Membrane proteins often misfold; chaperones, lower temperature, and solubility tags can each help improve proper folding and yield.
Correct answer is: All of the above
Q.103 What is the primary function of a 'terminator' sequence in a plasmid used for bacterial expression?
To halt transcription downstream of the gene
To increase plasmid copy number
To provide antibiotic resistance
To initiate translation
Explanation - Terminator sequences signal RNA polymerase to stop transcription, preventing read‑through into downstream vector regions.
Correct answer is: To halt transcription downstream of the gene
Q.104 Which of the following is a key feature of a 'self‑replicating RNA vector' used for gene expression?
It contains an RNA-dependent RNA polymerase gene enabling autonomous replication in the cytoplasm
It integrates into the host genome
It requires a bacterial origin of replication
It cannot be transcribed
Explanation - Self‑replicating RNA vectors (e.g., alphavirus replicons) replicate their RNA genome in the cytoplasm, leading to high levels of transient expression.
Correct answer is: It contains an RNA-dependent RNA polymerase gene enabling autonomous replication in the cytoplasm
Q.105 In cloning, why might a researcher choose to use a 'temperature‑sensitive' promoter?
To allow induction of gene expression simply by shifting the growth temperature
To increase plasmid copy number at high temperatures
To confer resistance to heat shock proteins
To make the plasmid fluorescent
Explanation - Temperature‑sensitive promoters (e.g., cI857) are repressed at low temperatures and become active at higher temperatures, enabling controlled expression.
Correct answer is: To allow induction of gene expression simply by shifting the growth temperature
Q.106 Which of the following is a common method to generate a library of random point mutations across an entire gene?
Error‑prone PCR
Southern blotting
Northern blotting
Restriction digestion
Explanation - Error‑prone PCR uses low‑fidelity polymerases and imbalanced dNTPs to introduce random mutations during amplification.
Correct answer is: Error‑prone PCR
Q.107 What is the main advantage of using a 'bicistronic' vector for co‑expression of a protein and its chaperone in bacteria?
Both genes are expressed from a single mRNA, ensuring similar expression levels
It reduces the size of the plasmid dramatically
It eliminates the need for a promoter
It allows the chaperone to be secreted
Explanation - A bicistronic construct includes two open reading frames under one promoter, allowing coordinated expression of the target protein and its folding assistant.
Correct answer is: Both genes are expressed from a single mRNA, ensuring similar expression levels
Q.108 When cloning a gene into a vector for expression in mammalian cells, why is it important to include a Kozak consensus sequence upstream of the start codon?
To enhance translation initiation efficiency in eukaryotes
To provide antibiotic resistance
To act as a terminator
To increase plasmid copy number
Explanation - The Kozak sequence (gccRccAUGG) optimizes ribosome binding and start codon recognition in eukaryotic translation.
Correct answer is: To enhance translation initiation efficiency in eukaryotes
Q.109 Which of the following best describes the purpose of a 'poly‑adenylation signal' in a eukaryotic expression vector?
To signal transcription termination and add a poly‑A tail to the mRNA
To increase plasmid stability
To provide a start codon for translation
To confer resistance to antibiotics
Explanation - A poly‑A signal ensures proper 3' end processing of mRNA, which enhances stability and translation efficiency.
Correct answer is: To signal transcription termination and add a poly‑A tail to the mRNA
Q.110 What is the main function of a 'selectable marker' in a cloning vector?
To enable identification of cells that have successfully taken up the vector
To increase the size of the plasmid
To facilitate DNA sequencing
To enhance transcription of the insert
Explanation - Selectable markers (e.g., antibiotic resistance genes) allow growth only of transformed cells on selective media.
Correct answer is: To enable identification of cells that have successfully taken up the vector
Q.111 In the context of gene cloning, what does the term "heterologous expression" mean?
Expression of a gene in a different host species than its origin
Expression of a gene within its native organism
Expression of multiple genes from the same vector
Expression of a gene without any promoter
Explanation - Heterologous expression involves producing a protein in a host that does not naturally contain the gene, often to simplify purification or modify expression levels.
Correct answer is: Expression of a gene in a different host species than its origin
Q.112 Which of the following strategies can be employed to reduce the formation of inclusion bodies when expressing a recombinant protein in E. coli?
Induce expression at lower temperatures
Use a weaker promoter
Co‑express molecular chaperones
All of the above
Explanation - Lower temperature, weaker promoters, and chaperone co‑expression each lessen aggregation and improve soluble protein yield.
Correct answer is: All of the above
Q.113 What is the purpose of a 'multiple cloning site' (MCS) in a plasmid vector?
To provide several unique restriction sites for flexible insertion of DNA fragments
To act as a transcription terminator
To increase plasmid copy number
To encode a fluorescent protein
Explanation - An MCS contains a series of distinct restriction sites, making it easier to clone inserts using various enzymes.
Correct answer is: To provide several unique restriction sites for flexible insertion of DNA fragments
Q.114 Which of the following is a characteristic of a 'low‑copy' plasmid?
It typically carries a high‑affinity origin of replication resulting in ~500 copies per cell
It often contains the p15A origin, resulting in ~10–20 copies per cell
It cannot be transformed into bacteria
It lacks an antibiotic resistance gene
Explanation - Low‑copy plasmids use origins like p15A that replicate fewer times per cell, which can be beneficial for toxic gene expression.
Correct answer is: It often contains the p15A origin, resulting in ~10–20 copies per cell
Q.115 When constructing a gene library, why is it important to use a host strain that is 'recA‑'?
recA‑ strains have reduced homologous recombination, preserving insert integrity
recA‑ strains grow faster
recA‑ strains provide higher antibiotic resistance
recA‑ strains produce more plasmid DNA
Explanation - recA deficiency limits recombination events that could rearrange or delete cloned DNA fragments, maintaining library stability.
Correct answer is: recA‑ strains have reduced homologous recombination, preserving insert integrity
Q.116 Which of the following is a common method to verify the size of an inserted DNA fragment after cloning?
Agarose gel electrophoresis of a restriction digest
Measuring optical density of bacterial culture
Performing a western blot
Using a spectrophotometer to assess DNA purity
Explanation - Digesting the plasmid with enzymes flanking the insert and running on an agarose gel reveals the insert size based on band migration.
Correct answer is: Agarose gel electrophoresis of a restriction digest
Q.117 In a cloning workflow, which step is performed to ensure that only plasmids with the desired insert survive after transformation?
Blue‑white screening using X‑gal and IPTG
Incubation at 95 °C for 5 min
Addition of RNase A
Electrophoretic separation of plasmid DNA
Explanation - Blue‑white screening differentiates colonies with functional lacZ (white) versus disrupted lacZ (blue), indicating insertion of the DNA fragment.
Correct answer is: Blue‑white screening using X‑gal and IPTG
Q.118 What is the purpose of adding a 'poly‑His tag' to the C‑terminus of a cloned protein?
To facilitate purification using metal affinity chromatography
To increase the gene's transcription rate
To provide antibiotic resistance
To act as a promoter
Explanation - A poly‑His tag binds nickel or cobalt ions, allowing selective purification of the tagged protein from cell lysates.
Correct answer is: To facilitate purification using metal affinity chromatography
Q.119 Which of the following cloning strategies is best suited for assembling a synthetic pathway consisting of multiple enzymes?
Modular cloning (MoClo) using Type IIS restriction enzymes
TA cloning of a single gene
Blue‑white screening
Southern blotting
Explanation - MoClo enables the hierarchical and scar‑less assembly of multiple genetic modules (promoters, ORFs, terminators) into a single construct.
Correct answer is: Modular cloning (MoClo) using Type IIS restriction enzymes
Q.120 When cloning a gene for expression in yeast, which of the following elements is typically required?
A yeast promoter (e.g., GAL1), a yeast terminator, and a yeast selectable marker
A bacterial origin of replication only
A viral capsid protein
A chloroplast transit peptide
Explanation - Yeast expression vectors need promoters and terminators recognized by yeast transcription machinery and markers for yeast selection.
Correct answer is: A yeast promoter (e.g., GAL1), a yeast terminator, and a yeast selectable marker
Q.121 Which of the following is a common reason to use a 'fusion tag' (e.g., MBP, GST) in a cloning construct?
To enhance solubility and facilitate purification of the recombinant protein
To increase plasmid copy number
To provide resistance to antibiotics
To act as a transcription terminator
Explanation - Fusion tags improve protein solubility and enable affinity purification via specific ligands (e.g., amylose for MBP, glutathione for GST).
Correct answer is: To enhance solubility and facilitate purification of the recombinant protein
Q.122 In a cloning experiment, why might a researcher use a 'high‑fidelity' DNA polymerase for PCR amplification of the insert?
To minimize the introduction of unwanted mutations during amplification
To increase the speed of PCR
To add restriction sites automatically
To make the DNA resistant to nucleases
Explanation - High‑fidelity polymerases possess proofreading activity, reducing error rates and preserving the correct sequence of the insert.
Correct answer is: To minimize the introduction of unwanted mutations during amplification
Q.123 What is the main advantage of using a 'self‑replicating' viral vector (e.g., adenovirus) for gene delivery in mammalian cells?
It enables high levels of transient expression without genomic integration
It integrates into the host genome permanently
It requires no selectable marker
It is resistant to all antibiotics
Explanation - Adenoviral vectors replicate episomally, delivering genes that are expressed robustly but do not integrate, reducing insertional mutagenesis risk.
Correct answer is: It enables high levels of transient expression without genomic integration
Q.124 Which of the following is a typical feature of a 'phagemid' vector used for phage display libraries?
A f1 origin of replication enabling packaging into filamentous phage particles
A mitochondrial targeting sequence
A chloroplast promoter
A yeast origin of replication
Explanation - The f1 origin allows the phagemid to be packaged into M13 phage capsids, displaying the fused peptide on the phage surface.
Correct answer is: A f1 origin of replication enabling packaging into filamentous phage particles
Q.125 When performing site‑directed mutagenesis using overlapping primers, what is the crucial design aspect of the primers?
Primers must contain the desired mutation in the middle of the overlap
Primers must be completely complementary with no mismatches
Primers should have no GC content
Primers must be longer than 200 bp
Explanation - The mutation is introduced within the overlapping region; during PCR the two fragments anneal and extend, incorporating the change.
Correct answer is: Primers must contain the desired mutation in the middle of the overlap
Q.126 Which of the following is the most appropriate method to clone a large (>150 kb) genomic fragment?
Using a bacterial artificial chromosome (BAC) vector
Using a standard pUC plasmid
Using TA cloning
Using a high‑copy‑number plasmid
Explanation - BACs can stably maintain very large DNA inserts (up to ~300 kb) that are unsuitable for standard plasmids.
Correct answer is: Using a bacterial artificial chromosome (BAC) vector
Q.127 In the context of gene cloning, what does 'inverse PCR' allow researchers to do?
Amplify DNA flanking a known internal sequence by using outward‑facing primers
Introduce random mutations across a gene
Directly insert DNA into a viral genome
Perform high‑throughput sequencing
Explanation - Inverse PCR circularizes the template DNA and uses primers that face outward from a known region to amplify adjacent unknown sequences.
Correct answer is: Amplify DNA flanking a known internal sequence by using outward‑facing primers
Q.128 Which of the following best explains why a researcher would use a 'dual‑promoter' vector for expressing two proteins in a bacterial system?
To enable independent regulation of each gene’s expression level
To reduce plasmid size
To avoid the need for a selectable marker
To increase the antibiotic resistance spectrum
Explanation - Separate promoters allow each gene to be expressed at desired levels, which is essential for balanced multi‑protein systems.
Correct answer is: To enable independent regulation of each gene’s expression level
Q.129 When cloning a gene into a vector for expression in mammalian cells, which of the following signals is essential for nuclear export of the mRNA?
A poly‑adenylation signal
A mitochondrial targeting sequence
A bacterial origin of replication
A Shine‑Dalgarno sequence
Explanation - The poly‑A tail and associated signal facilitate nuclear export and stability of the mature mRNA in eukaryotic cells.
Correct answer is: A poly‑adenylation signal