Which fragment profile confirms correct orientation in HindIII+PstI screening?
Orientation screening using two enzymes, one cutting in vector backbone and one asymmetrically within insert, generates orientation-specific junction fragments whose sizes differ with insert direction. Correct orientation places internal sites at predicted separation producing characteristic larger and smaller bands calculated from map, while reverse orientation alters distance between sites yielding different fragment sizes. Clone C exhibits size combination consistent with forward transcription direction, intact reading frame, and expected restriction map summed from plasmid and insert sequences, confirming that insert ligated in desired direction for downstream expression studies and functional assays.
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.