Practice question

Molecular cloning involves several enzymatic manipulations. Klenow fragment, lacking 5' exonuclease activity, can fill 5' overhangs to blunt ends using dNTPs. Isocaudomers XhoI and SalI generate compatible TCGA overhangs permitting cross-ligation. Alkaline phosphatase removes 5'-phosphate groups rather than 3'-phosphates, blocking self-ligation. Blue-white screening depends on alpha-complementation of lacZ; insertion inactivates beta-galactosidase producing white colonies on X-gal. However, white phenotype does not guarantee recombinant insert because vector mutations, primer dimers, or small non-disruptive inserts can also abolish activity, necessitating PCR or restriction verification rather than relying solely on color.