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#recombinant selection

2 public questions tagged with this topic.

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.

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Gene Cloning Process

In blue-white screening, what do white colonies indicate?

Blue-white screening exploits α-complementation of lacZ encoding β-galactosidase in Escherichia coli. Plasmids carrying lacZα produce functional enzyme when induced by IPTG, cleaving X-gal to 5,5'-dibromo-4,4'-dichloro-indigo yielding intensely blue colonies on indicator plates. Insertion of gene of interest within the multiple cloning site located inside lacZα causes insertional inactivation, abolishing enzyme activity and preventing complementation. No cleavage of X-gal occurs. Therefore white colonies harbor recombinant plasmids with insert, while blue colonies retain empty vector, allowing rapid visual identification of successful cloning events.

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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