Practice question

Nick translation creates labeled DNA by mimicking cellular DNA repair synthesis. DNase I at low concentration introduces single-strand breaks by cleaving phosphodiester bonds, leaving 3'-hydroxyl and 5'-phosphate ends. Escherichia coli DNA polymerase I then binds at nicks; its 5' to 3' exonuclease activity removes nucleotides ahead while its polymerase activity incorporates new nucleotides, including labeled deoxynucleotides, behind. The nick thus translates along the duplex. This coordinated excision and synthesis yields uniformly labeled double-stranded probes with high specific activity for in situ hybridization and membrane blotting.