A new and improved method for 3′-end labelling DNA using [α-32P]ddATP

We have synthesised dideoxyadenosine-5′-α-³²P]triphosphate (α-³²P]ddATP) at a specific activity of 3000 Ci/mmol and directly compared it with cordycepin-5′-α-³²P]triphosphate (α-³²P]KTP) as a means to 3′-end label DNA. The α-³²P]ddATP was found to be three to five times more efficient than α-³²P]KTP. Blunt and 3′-protruding ends were labelled more efficiently with α-³²P]ddATP using terminal transferase than were the 5′-ends with γ-³²P]ATP using polynucleotide kinase by standard methods. This improvement in efficiency of labelling DNA and the simplicity of the method allows 3′-end labelling of DNA to become a realistic alternative to 5′-end labelling. We have also compared α-³²P]ddATP- and α-³²P]KTP-labelled DNA in Maxam and Gilbert sequencing procedures and find that both give equally good results.