Gene Cloning Process

Gene Cloning Process

Which is a direct selection method?

Direct selection eliminates non-recombinant cells at plating stage based on genetic complementation or resistance, without need for secondary screening. Antibiotic resistance gene selection exemplifies this; only bacteria that have acquired plasmid bearing resistance gene such as AmpR survive on antibiotic-supplemented agar, directly selecting transformants. In contrast, blue-white screening requires visual inspection after growth, colony hybridization needs probe overlay and autoradiography, and replica plating is indirect technique transferring colonies between plates to infer phenotype, making them indirect screening procedures rather than immediate survival-based direct 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.

View Details

Gene Cloning Process

Why are inclusion bodies formed in E. coli?

Inclusion bodies represent dense, insoluble cytoplasmic aggregates of misfolded recombinant protein accumulating in Escherichia coli during overexpression. High synthesis rates under strong promoters saturate GroEL, DnaK chaperone pathways, exposing hydrophobic patches that promote intermolecular association. Insufficient time for proper folding, absence of eukaryotic folding catalysts, and reducing environment preventing disulfide formation further exacerbate aggregation. Aggregates are refractile, biologically inactive, and require chaotropic agents for solubilization. Their formation reflects protein quality control failure rather than growth rate, protease action, or DNA recombination deficiency alone.

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.

View Details

Gene Cloning Process

Why is alkaline phosphatase used in cloning?

DNA ligase catalyzes phosphodiester bond formation only between adjacent 3' hydroxyl and 5' phosphate termini. Vector linearized with single restriction enzyme retains complementary sticky ends bearing 5' phosphates, readily self-ligating and producing high background of empty clones. Alkaline phosphatase, including calf intestinal or shrimp isoforms, hydrolyzes 5' phosphate groups leaving 5' hydroxyl termini, rendering the vector ligation-incompetent unless paired with phosphorylated insert DNA supplying phosphates. Subsequent ligation yields circular recombinant product with one nick at each junction, later repaired after transformation into host bacteria.

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.

View Details

Gene Cloning Process

In colony hybridization, what detects hybridization?

After transfer and fixation of colony DNA to membrane, hybridization with radiolabeled probe specific for insert is carried out. To visualize successful annealing, membranes are exposed to X-ray film sensitive to beta radiation emitted by isotopes such as 32P incorporated into probe backbone. Regions where probe hybridized produce dark spots on film due to autoradiographic emission, aligning with original colony positions. This X-ray autoradiography provides high sensitivity and spatial resolution. Detection does not rely on ultraviolet fluorescence, colorimetric X-gal cleavage, or direct fluorescent microscopy observations employed for alternative screening modalities.

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.

View Details

Gene Cloning Process

Which of these does NOT serve as a scorable marker?

Scorable markers produce easily detectable phenotype such as color or fluorescence upon expression, useful for screening recombinants independent of selection pressure. Examples include lacZ producing blue product from X-gal, green fluorescent protein emitting green fluorescence, and luciferase generating bioluminescence catalyzing luciferin oxidation. Tetracycline resistance TetR encodes membrane efflux pump conferring resistance to tetracycline, allowing only selective growth but not visual scoring of insertional events without replica plating. It functions strictly as selectable marker for plasmid retention, not for scoring recombinant status through observable pigment or light emission.

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.

View Details

Gene Cloning Process

Which marker gene helps distinguish recombinant from non-recombinant?

Markers distinguishing recombinant from non-recombinant clones can be scorable reporters whose phenotype alters upon insertional inactivation. Green fluorescent protein provides intrinsic fluorescence detectable under UV without exogenous substrate, making it convenient for colony screening. Vectors containing GFP transcriptional fusion show fluorescence when empty; cloning into gfp disrupts chromophore formation, extinguishing signal in recombinants. Origin of replication governs replication, while AmpR and TetR are selectable markers conferring survival on antibiotics but not visual distinction between recombinant and empty plasmid states. Thus GFP offers rapid optical differentiation.

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.

View Details

Gene Cloning Process

Why are eukaryotic genes difficult to express in E. coli?

Eukaryotic gene expression in bacterial system Escherichia coli faces multiple barriers collectively contributing to failure. Bacterial RNA polymerase cannot splice introns as bacteria lack spliceosome, producing nonfunctional transcripts. Post-translational modification systems for glycosylation, phosphorylation, and formation of correct disulfide bonds in endoplasmic reticulum are absent. Cytoplasmic milieu, chaperone repertoire, and codon usage diverge significantly, causing improper folding, codon bias mediated stalling, and proteolysis. Consequently, most complex eukaryotic proteins aggregate or remain inactive, necessitating use of eukaryotic hosts like yeast or mammalian cells for functional production.

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.

View Details

Gene Cloning Process

Which step is done first in map-based sequencing?

Map-based or clone-by-clone sequencing strategy, used for human genome project, first establishes a low-resolution physical map of genome. Initial step involves sizing large genomic fragments created by complete digestion with rare-cutting enzymes using pulsed-field gel electrophoresis, which resolves megabase-sized DNA through alternating electric fields. This allows ordering of yeast artificial chromosome and bacterial artificial chromosome clones into contigs based on overlapping fingerprints before subcloning into smaller vectors and shotgun sequencing. Early mapping prevents misassembly and provides chromosomal anchoring absent in whole-genome shotgun approach.

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.

View Details

Gene Cloning Process

In pBR322, insert in PstI site disrupts which gene?

pBR322, an early synthetic plasmid of 4361 bp, possesses two antibiotic resistance genes: amp encoding β-lactamase and tet encoding tetracycline efflux pump. PstI recognition site CTGCAG lies within coding sequence of amp gene at position 3609, while BamHI and SalI reside within tet. Cloning foreign DNA into PstI site interrupts ampicillin resistance determinant through insertional inactivation, rendering recombinants sensitive to ampicillin but retaining tetracycline resistance. This differential antibiotic phenotype historically enabled recombinant identification before lacZ blue-white system became widespread for easy 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.

View Details

Gene Cloning Process

What change improves protein solubility in E. coli expression systems?

Lowering post-induction temperature to 16-25°C markedly improves solubility of recombinant proteins expressed in Escherichia coli. Reduced temperature slows transcription and translation rates, decreasing nascent chain concentration and allowing more time for co-translational folding assisted by chaperones GroEL and DnaK. Hydrophobic interactions driving aggregation are temperature dependent and weakened at lower temperatures, thus inclusion body formation is minimized. High IPTG or high temperature exacerbates aggregation, while glucose suppresses induction via catabolite repression but does not enhance folding quality of already induced protein.

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.

View Details

Gene Cloning Process

Which of the following is responsible for expression of cloned gene in prokaryotes?

Gene expression in prokaryotes initiates when RNA polymerase holoenzyme containing sigma factor recognizes and binds to promoter elements -35 and -10 upstream of transcription start site, melting DNA and initiating RNA synthesis. Cloning vectors must supply a compatible promoter such as lac, trc, T7, or tac oriented to drive transcription of inserted coding sequence. Origin of replication ensures plasmid maintenance, antibiotic gene provides selection, and lacZ offers screening; none direct transcription of cloned gene. Hence promoter strength and regulation determine mRNA abundance and subsequent recombinant protein yield.

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.

View Details

Gene Cloning Process

What ensures propagation of plasmid in host?

Plasmid propagation depends on origin of replication, a cis-acting DNA element recognized by host replication machinery. Ori contains AT-rich duplex unwinding element, DnaA boxes, and binding sites for host initiation factors that recruit helicase, primase, and DNA polymerase III holoenzyme for autonomous bidirectional replication independent of chromosomal replication. Without functional origin, plasmid cannot duplicate and is lost during cell division. LacZ, promoter, and reporter genes control phenotype or expression but do not confer replication ability. Copy number is also regulated by elements within ori region.

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.

View Details