Practice question

DNA end-filling uses Klenow fragment or T4 DNA polymerase to incorporate labeled dNTPs at recessed 3' termini using the 5' overhang as template. A molecule with a 5' protrusion provides single-stranded template ahead of a paired 3'-OH, enabling synthesis in 5' to 3' direction with incorporation of alpha-32P nucleotides. Blunt ends lack overhang template, while 3' overhangs cannot be filled because polymerase requires forward template. Labeling 5' overhangs thus highlights specific fragments for restriction mapping, strand polarity determination, and probe preparation in cloning workflows.