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.