( he protocol is similar to that for T4 P...) USB OptiKinase Eliminates Base Bias in Labeling of Oligonucleotide 5'-Ends

2. Mix the contents well and centrifuge briefly. Incubate at 37C for 30 min.

3. Terminate the reaction by heating at 65C for 10 min.

Optional performance assay: The amount of radioactive phosphate incorporated into 5'-ends may be determined by separating 5'-end-labeled oligonucleotide from precursor ATP by binding to DE81 filter paper and washing the filter with a solution of 5% Sodium Phosphate dibasic.

* Either [γ-³²P]ATP or [γ-³³P]ATP (≥ 3000 Ci/mmol) may be used. Whether OptiKinase or T4 PNK is used, best results are obtained by using very fresh radiolabeled ATP. Even a few days (less than one half life) can dramatically decrease the specific activity of the final product.

Results

OptiKinase offers a variety of advantages over T4 PNK. Primary among these is that OptiKinase overcomes base bias, allowing uniform labeling of oligonucleotides (Fig. 2). Additionally, OptiKinase shifts the reaction equilibrium toward phosphorylation, allowing efficient labeling with relatively lower amounts of radiolabel (Fig. 3). It performs well across a range of enzyme concentrations, eliminating the need for careful enzyme titrations (Fig. 4). OptiKinase works well for labeling of double-stranded oligonucleotides (Fig. 5). It can also be used for phosphorylation of oligonucleotides with non-labeled ATP and for labeling of variety of double-stranded DNA ends after restriction digestion and removal of the 5'-phosphate by phosphatase treatment (data not shown).

Discussion

OptiKinase outperforms T4 PNK in a variety of 5'-labeling reactions, in terms of labeling uniformity, efficiency, and ease of use. For these rea
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