Q.2 The basis for blue-white screening with pUC vectors is
(A) intraallelic complementation (B) intergenic complementation
(C) intragenic suppression (D) extragenic suppression

Blue-white screening in pUC vectors relies on intraallelic complementation to distinguish recombinant clones from non-recombinants. Inserting DNA into the lacZα gene disrupts α-peptide production, preventing β-galactosidase activity and yielding white colonies.

Correct Answer

The correct option is (A) intraallelic complementation. pUC vectors carry the lacZα fragment encoding the α-peptide, which complements the defective ω-peptide in host strains like JM109, restoring β-galactosidase activity for blue colonies on X-gal. Foreign DNA insertion into the multiple cloning site (MCS) within lacZα disrupts this, blocking complementation and producing white colonies.

Option Explanations

Blue-white screening exploits α-complementation of β-galactosidase, where lacZα and lacZω alleles interact within the same protein.

• (A) Intraallelic complementation: Correct; α-peptide (from vector) and ω-peptide (host) are domains of the same lacZ gene, complementing intragenically to form active enzyme.

• (B) Intergenic complementation: Incorrect; involves different genes, unlike lacZα-lacZω from the same gene split.

• (C) Intragenic suppression: Incorrect; refers to second-site mutations restoring function within one gene, not complementation by separate polypeptides.

• (D) Extragenic suppression: Incorrect; suppression by mutations in another gene, unrelated to α-complementation mechanism.

Mechanism Overview

Host cells lack functional β-galactosidase due to lacZω deletion. Non-recombinant pUC provides lacZα for complementation, hydrolyzing X-gal/IPTG to blue pigment. Recombinants disrupt lacZα, yielding white colonies for easy selection in cloning workflows.