( Wild-type T4 Polynucleotide K...) USB OptiKinase Eliminates Base Bias in Labeling of Oligonucleotide 5'-Ends

Wild-type T4 Polynucleotide Kinase (PNK)^(1,2,3) is commonly used for phosphorylation and labeling of 5'-ends of DNA and oligonucleotides. Labeling, in particular, can be accomplished by two approaches, the forward reaction and the exchange reaction (Fig. 1)⁽³⁾. Historically, T4 PNK has played a crucial role in molecular biology and it continues to be important in applications such as labeling of probes for hybridization, sequencing or mapping of transcripts and in phosphorylation of DNA ends for cloning^(3,4). Although T4 PNK continues to be widely used, the enzyme exhibits two limitations. First, T4 PNK exhibits base bias; the effectiveness of phosphorylation depends on the base at the 5'-end of the oligonucleotide target, with 5'-C exhibiting the lowest extent of phosphorylation(5). Second, T4 PNK can be challenging to use; precise enzyme titrations and careful optimization of reaction times are often required in order to achieve desired results^(3,6). USB OptiKinase overcomes these limitations, greatly simplifying and improving phosphorylation and labeling reactions.

OptiKinase is a recombinant version of T4 PNK that has been genetically modified to accomplish uniform and consistent phosphorylation and labeling of 5'-ends of DNA and oligonucleotides. Base bias is dramatically reduced, which should greatly increase uniformity of labeling of diverse templates. The need for careful optimization of reaction conditions is also reduced, which should improve consistency of results between experiments. Finally, lower amounts of radioactive ATP can be used without sacrificing labeling efficiency, resulting in reduced reagent costs. Thus, USB OptiKinase offers substantial advantages over wild-type T4 PNK.

Method

The following protocol corresponds to use of OptiKinase for labeling of oligonucleotides by the forward reaction. T
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