Navigation Links
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.


The following protocol corresponds to use of OptiKinase for labeling of oligonucleotides by the forward reaction. The protocol is similar to that for T4 PNK, with the exception that there is no need to use excess radiolabeled ATP.

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.


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).


OptiKinase outperforms T4 PNK in a variety of 5'-labeling reactions, in terms of labeling uniformity, efficiency, and ease of use. For these reasons, OptiKinase is highly recommended for 5'-phosphorylation applications in general, and for 5'-labeling applications in particular.


1. GALBURT, E. A., PELLETIER, J., WILSON, G., AND STODDARD, B. L. (2002) Structure 10, 1249- 1260.

2. WANG, L. K., LIMA, C. D., AND SHUMAN, S. (2002) EMBO J. 21, 3873-3880.

3. RICHARDSON, C. C. (1981) The Enzymes , 3rd Edition, Ed. P. D. Boyer, (Academic Press, New York) 14, 299-314.

4. MAXAM, A. M. AND GILBERT, W. (1980) Methods in Enzymology 65, 499-560.

5. VAN HOUTEN, V., DENKERS, F., VAN DIJK, M., VAN DEN BREKEL, M. AND BRAKENHOFF, R. (1998) Anal. Biochemistry 265, 386-389.

6. LILLEHAUG, J. R. AND KLEPPE, K. (1975) Biochemistry 14, 1221-1225.



Page: All 1 2 3

Related biology technology :

1. New Microarray Labeling Kit for Preparing cDNA Probes
2. aRNA Synthesis and Labeling for Array Analysis
3. Amino Allyl Labeling for Array Analysis
4. Fluorescently Labeling Long dsRNA with the Silencer siRNA Labeling Kit
5. Efficient Europium Labeling of Lymphokine-Activated Killer Cells Using the Gene Pulser II with RF Module, Rev C
6. Oligonucleotide Purity Analysis by Capillary Electrophoresis
7. Using the OligoGreen Oligonucleotide Quantitation Reagent in the Gemini XS, Gemini EM and SpectraMax M2 Microplate Readers (MaxLine Application Note #21)
Post Your Comments:

(Date:10/8/2015)... ... October 08, 2015 , ... ... fibrosis transmembrane conductance regulator (CFTR) gene and results in dysfunction of the lungs ... accumulates in the lungs. This mucus is very difficult to clear, blocks the ...
(Date:10/7/2015)... , Oct. 7, 2015 Boston ... the start of the Boston Scientific Connected Patient ... advancements in the use of remote patient monitoring to ... designers and entrepreneurs can submit their ideas and collaborate ... ( ). Cambridge, MA ...
(Date:10/7/2015)... ... October 07, 2015 , ... ... will present a public educational seminar on the latest advances in adult stem ... 1:00 pm – 3:00 pm at the Hilton Dallas/Southlake Town Square in Southlake, ...
(Date:10/7/2015)... N.J. , Oct. 7, 2015  ContraVir Pharmaceuticals, ... a biopharmaceutical company focused on the development and commercialization ... an underwritten public offering of 5,000,000 shares of its ... shares of the Company,s common stock at a fixed ... of common stock and warrants will be issued separately.  ...
Breaking Biology Technology:
... , Purpose , ... the use of chloramphenicol (CAP) as a veterinary drug for food ... for the identification and quantification of CAP in milk and milk ... many analytical procedures are described in the literature, the presented method ...
... , ... and detailed structural data of highlyknotted spider toxins , ... The primary structure determination ... cLC 492 sequencing system and the QSTAR Pulsar hybrid quadrupole time-of-flight mass spectrometer. ...
... Abstract , The generation of hybrid ... production is a fundamental and well-established technique. However, , ... , has varied little over the years. ... antigens is particularly problematic. We have developed a robust , ...
Cached Biology Technology:
(Date:10/2/2015)... DUBLIN , Oct. 02 2015 ... addition of the "Enforcing the Law Using ... ) has announced the addition of ... report to their offering. --> Research ... addition of the "Enforcing the Law Using ...
(Date:9/30/2015)... GARDENS, Fla. , Sept. 30, 2015  The ... this month issued another key ruling in favor of ... determination that Korean fingerprint scanner company Suprema and its ... Act of 1930, a trade provision that declares it ... import trade, by infringing two of Crossmatch,s patents, the ...
(Date:9/29/2015)... MILWAUKEE , Sept. 29, 2015  iDAvatars is excited ... partners to introduce its product to market. The official announcement was ... Cognitive Economy event in San Francisco ... Avatar platform powered by IBM Watson. "It is ... the first 100 companies to bring to market the cognitive ...
Breaking Biology News(10 mins):
... 16 issue of the journal Cell Host & ... this immune response in mice infected with lymphocytic choriomeningitis ... of drug treatments for immunosuppressive diseases in humans. ... of the interferon response in vivo," says the paper,s ...
... at the University of Delaware have discovered that when the ... it can send out an S.O.S. to the roots for ... that brings beneficial bacteria to the rescue. The finding ... of passing pathogens--and sheds new light on a sophisticated signaling ...
... TALLAHASSEE, Fla. -- The discovery of a protein involved ... a researcher at the Florida State University College of ... Institutes of Health. Branko Stefanovic, associate professor in ... Medicine, hopes his discovery could lead to treatment methods ...
Cached Biology News:
Collected from 8-12 week old sexually mature rats. Pricing: $100/unit for 1 - 10 units...
... Kit is one of the most ... of RNA in solution. The ... than absorbance measurements. As little as ... fluorescence microplate readers and fluorometers using ...
... serum is extensively scrutinized, including ... All horses are of ... worming are controlled and documented ... consistency. Pricing: $12/unit ...
Biology Products: