Terminal Labeling of DNA for Maxam and Gilbert Sequencing

There are two basic enzymatic activities that are used to end-label DNA with radioactive phosphate (³² P). The enzyme T4-polynucleotide kinase will use the substrate ATP to add a phosphate group (the gamma-phosphate of the ATP molecule) preferentially to the 5′ ends of the molecule. The enzyme DNA polymerase will “fill-in” recessed 3′ ends with the complimentary nucleotides, and can also create recessed 3′ ends from “blunt” ends or even 3′ overhanging ends by its 3′–5′ exonuclease activity, then fill in such ends as well. However, the polymerase enzyme usually used for the fill-in reaction, the Klenow fragment of E . coli DNA Pol I, has very low 3′–5′ exonuclease activity, and therefore its fill-in activity on blunt and 3′ overhanging ends could in principle be ignored for the purpose of end-labeling DNA for sequencing (see Note 1 ).