Labeling the 5′ Termini of DNA with Bacteriophage T4 Polynucleotide Kinase

Abstract

The removal of 5′ phosphates from nucleic acids with phosphatases and their readdition in radiolabeled form by bacteriophage T4 polynucleotide kinase is a widely used technique for generating ³²P-labeled probes. When the reaction is performed efficiently, 40%–50% of the protruding 5′ termini in the reaction becomes radiolabeled. However, the specific activity of the resulting probes is not as high as that obtained by other radiolabeling methods because only one radioactive atom is introduced per DNA molecule. Nevertheless, the availability of [γ-³²P]ATP with specific activities in the 3000–7000 Ci/mmol range allows the synthesis of probes suitable for many purposes. This protocol includes procedures for labeling the 5′ ends of dsDNA and oligonucleotides.