Practice question

Distinguishing cohesive versus blunt termini relies on substrate specificity of labeling enzymes. DNA polymerase I Klenow fragment and T4 DNA polymerase require a single-stranded template to incorporate labeled dNTPs, a condition fulfilled by 5' overhanging sticky ends with recessed 3'-OH. Blunt ends lack overhangs for templated fill-in. Therefore only sticky-ended fragments become radioactive upon incubation with labeled nucleotides, while blunt fragments stay unlabeled. Agarose pattern shows total DNA, while western blot and RT-PCR detect proteins and RNA, not relevant for terminus typing.