Phosphorylating the 5 Termini of Oligonucleotides

Synthetic oligonucleotides lacking phosphate groups at their 5' termini are easily radiolabeled by transfer of the ir-32P from [t-32P]ATP in a reaction catalyzed by bacteriophage T4 polynucleotide kinase. The reaction described below is designed to label 10 pmoles of an oligonucleotide to high specific activity. Labeling of different amounts of oligonucleotide can easily be achieved by increasing or decreasing the size of the reaction while keeping the concentrations of all components constant. When the reaction is carried out efficiently, >50% of the oligonucleotide molecules in the reaction become radiolabeled. Similar reaction conditions can be used when adding a nonradiolabeled phosphate to the 5' end of a synthetic oligonucleotide prior to its use in site-directed mutagenesis.

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