Restriction Enzyme and Vector

Restriction Enzyme and Vector

A YAC-derived construct must contain which sequence to survive in yeast?

Functional yeast artificial chromosome must replicate, segregate and be selectable. Autonomously replicating sequence acts as origin where replication complex assembles, licensing DNA duplication once per cell cycle. Without ARS, linear construct fails to duplicate and is lost. Centromere ensures proper segregation, telomeres protect linear ends, but ARS provides essential replication initiation. Bacterial ori functions only in Escherichia coli for shuttling, lac operon regulates bacterial transcription, not yeast survival. Therefore presence of yeast origin ARS is indispensable for autonomous replication and maintenance of YAC construct within Saccharomyces cerevisiae nucleus.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Restriction Enzyme and Vector

Which selectable marker suppresses yeast mutations?

In yeast genetics, suppressor tRNA markers are used to monitor nonsense mutation suppression. SUP4 encodes a tyrosine tRNA with anticodon mutated to recognize ochre stop codon UAA, suppressing ade2-101 and other ochre alleles, leading to phenotypic color change in adenine biosynthesis pathway. In YAC vectors, SUP4 is placed within cloning site; disruption by foreign DNA abolishes suppression. Auxotrophic markers URA3 and TRP1 provide selection for transformation, while LacZ and GFP are bacterial reporter genes. SUP4 thus acts as insertional marker that visibly reports insertion by altering suppression phenotype in yeast.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Restriction Enzyme and Vector

Which is the correct order of increasing insert capacity?

Understanding insert capacity hierarchy guides vector selection for genomic projects. M13 filamentous phage accepts less than 4 kb, plasmid pBR322 or pUC accommodates up to 10 kb, lambda phage 15-25 kb, cosmid 35-45 kb, fosmid and P1 70-100 kb, BAC 100-300 kb, PAC similar, YAC up to 2000 kb and MAC beyond 2000 kb. Incremental order demonstrates progressive increase. Among listed orders, M13 smallest, cosmid intermediate, BAC larger and MAC largest represents logically increasing capacity. Recognizing this gradation helps choose appropriate system for cDNA cloning versus genome walking and positional cloning requiring megabase inserts.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Restriction Enzyme and Vector

Which vector is optimal for 70 kb library?

Library construction requires matching insert size to vector stability and transfer efficiency. Cosmid and fosmid systems accommodate about 40-45 kb, suitable for medium libraries but suffer packaging limits. P1 phage-derived artificial chromosome uses P1 bacteriophage replication and packaging system, stably maintaining inserts around 70-100 kb with less chimerism than YAC systems. BAC also handles 100-300 kb but for 70 kb mid-range genomic libraries, P1 vector offers superior efficiency, lower rearrangement than YAC and single-copy stability. Therefore for approximately 70 kb libraries requiring high fidelity and moderate throughput, P1 system represents optimal choice.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Restriction Enzyme and Vector

Which element distinguishes religated YAC from vector?

YAC cloning involves ligation of insert DNA between two arms each carrying telomere, selection marker and centromere. Linearized vector without insert can recircularize through non-homologous end joining, regenerating empty YAC. SUP4, encoding suppressor tRNA for ochre mutation, serves as distinguishing marker. When SUP4 is interrupted by insert, suppressor phenotype is lost, producing red colonies in ade2-101 background, while religated empty vector retains functional SUP4 giving white colonies. CEN, ARS, URA3, TRP1 present in both forms cannot differentiate. SUP4 inactivation provides positive visual indication of recombinant formation versus vector background.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Restriction Enzyme and Vector

Which statement about YAC is FALSE?

Yeast artificial chromosomes enable cloning of 100 kb to 1 megabase DNA, often maintained singly in yeast and shuttled to Escherichia coli for manipulation. However, characterization reveals significant instability. Because yeast recombination machinery acts on repetitive mammalian DNA, chimerism, internal deletions and rearranged inserts are common, sometimes affecting 20-50 percent of clones. Multiple YACs can coexist per cell and insert can be unstable during mitotic propagation. Therefore claim of complete stability without rearrangements is incorrect. Understanding limitations led to development of BACs and PACs offering greater structural fidelity for genome projects.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Restriction Enzyme and Vector

Which vector includes Cos sites from lambda phage?

Cosmid vectors combine plasmid origin and lambda phage cos site required for packaging into phage heads. Cos sites are 12 base sticky cohesive ends that direct concatemer cleavage during lambda packaging. Incorporating cos sequence enables in vitro packaging of recombinant DNA flanked by cos sites into infectious lambda particles, allowing transduction into Escherichia coli with high efficiency. This permits cloning of 37-52 kb inserts larger than standard plasmids but smaller than BACs. Cosmid retains plasmid replication and antibiotic selection, merging phage delivery with plasmid maintenance, widely used for genomic library construction and large insert screening.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Restriction Enzyme and Vector

What allows visual detection in GFP-based vectors?

Green fluorescent protein from Aequorea victoria emits green fluorescence upon excitation with blue to ultraviolet light, requiring no exogenous substrates or cofactors. When integrated into cloning or expression vectors as reporter, GFP allows direct visualization. Transformants expressing GFP fluoresce under UV or blue light transilluminator, quantified by fluorimetry, flow cytometry or microscopy. Unlike lacZ system requiring X-gal hydrolysis for color development or enzymatic assays involving substrates, GFP detection is non-destructive, real-time and quantitative, enabling monitoring of expression, localization and promoter activity in living cells without cell lysis or staining protocols.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Restriction Enzyme and Vector

Which vector has >2000 kb capacity?

Insert capacity scales with vector complexity. Plasmids up to 10 kb, cosmids about 45 kb, fosmids and P1 about 70-100 kb, BAC and PAC up to 300 kb, YAC up to 1000-2000 kb. Mammalian artificial chromosomes and human artificial chromosomes constructed with mammalian centromeres and alphoid DNA can maintain inserts exceeding 2000 kb, sometimes several megabases, as independent chromosomes in host cells. This ultra-large capacity enables transfer of entire genomic loci with intact long-range regulatory elements, modeling aneuploidy and providing platforms for large transgene delivery where bacterial and yeast systems are insufficient.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Restriction Enzyme and Vector

pBR322 carries resistance genes for:

pBR322 is one of earliest low-copy artificial cloning plasmids derived from pMB1, constructed by Bolivar and Rodriguez. It contains pMB1 origin replicating in Escherichia coli, restriction sites clustered in antibiotic genes. Antibiotic resistance determinants include bla gene encoding beta-lactamase conferring ampicillin resistance and tet gene encoding tetracycline efflux pump conferring tetracycline resistance. Inactivation mapping allows selection: insertion in one resistance gene renders recombinant sensitive to corresponding antibiotic while retaining resistance to the other, enabling insertional marker selection. This dual marker system was foundational for recombinant DNA technology teaching.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Restriction Enzyme and Vector

In blue-white screening, white colonies contain:

Blue-white screening exploits insertional inactivation of lacZ alpha peptide within vector multiple cloning site. Functional beta-galactosidase cleaves X-gal chromogenic substrate producing blue colonies. When foreign DNA inserts into MCS, it disrupts lacZ alpha reading frame, preventing alpha complementation, so active enzyme is not formed. Colonies harboring recombinant plasmid therefore remain white on indicator plates containing IPTG and X-gal. Non-recombinant vectors retain intact lacZ and give blue colonies. White phenotype thus indicates successful insertion event, providing rapid visual screening without antibiotic sensitivity assays or colony PCR for primary selection.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Restriction Enzyme and Vector

Which vector is virus-based and integrates into the host genome?

Viral vectors are divided by integration capability and host range. Adenovirus vectors are non-integrating, episomal and transient. Herpes simplex virus and adeno-associated virus mostly remain episomal with low random integration. Lentivirus, a subclass of retroviridae derived from HIV, possesses reverse transcriptase and integrase that stably inserts proviral cDNA into host chromatin, including non-dividing cells. Integrated provirus is permanently maintained and transmitted to daughter cells, providing long-term expression. This property makes lentiviral vectors preferred for stable gene transduction, hematopoietic stem cell modification and creation of transgenic cell lines requiring persistent expression.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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