Why does recombinant DNA disrupt LacZ activity?
The lacZα peptide encoded within the plasmid multiple cloning region provides the N-terminal fragment for α-complementation to produce active β-galactosidase. When foreign DNA is ligated into restriction sites located within this coding sequence, the continuity of lacZ reading frame is physically interrupted. The insertion introduces extraneous nucleotides, causing frameshift or domain disruption that prevents functional α-peptide synthesis. Consequently, enzymatic activity is lost through insertional inactivation. This loss is not due to toxicity, introduction of stop codons alone, or effects on separate antibiotic resistance cistrons.
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.