A new and improved method for 3'-end labelling DNA using [alpha-32P]ddATP.
We have synthesised dideoxyadenosine-5'-[alpha-32P]triphosphate [( alpha-32P] ddATP ) at a specific activity of 3000 Ci/mmol and directly compared it with cordycepin-5'-[alpha-32P]triphosphate [( alpha-32P] KTP ) as a means to 3'-end label DNA. The [alpha-32P] ddATP was found to be three to five times more efficient than [alpha-32P] KTP . Blunt and 3'-protruding ends were labelled more efficiently with [alpha-32P] ddATP using terminal transferase than were the 5'-ends with [gamma-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 [alpha-32P] ddATP - and [alpha-32P] KTP -labelled DNA in Maxam and Gilbert sequencing procedures and find that both give equally good results.
D001483:Base Sequence; D004247:DNA; D003838:Deoxyadenine Nucleotides; D054306:Dideoxynucleotides; D007700:Kinetics
D003838:Deoxyadenine Nucleotides; D054306:Dideoxynucleotides; C037773:2',3'-dideoxyadenosine triphosphate; C011746:3'-deoxyadenosine 5'-triphosphate; D004247:DNA