Orientation of a cloned DNA fragment (gene) in a plasmid vector can be checked by
Determining insert orientation in a plasmid requires direction-sensitive detection. Amplification using one primer annealing within the insert and another annealing in the vector backbone produces a product only when the insert is in a defined orientation and yields size indicating forward or reverse cloning. Using two insert-specific primers merely confirms insert presence, independent of vector context. Single-site restriction digestion linearizes plasmid regardless of orientation, while two vector sites excise insert but do not distinguish its direction. This vector-insert PCR strategy is q
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.
