Abstract

Measurement of thymidine kinase-1 (TK1) and deoxycytidine kinase (dCK) activity may be useful in cancer disease management. Therefore, a one-step homogeneous assay for real-time determination of TK1 and dCK was developed by combining enzyme complementation with fluorescent signal generation using primer extension and a quenched probe oligodeoxyribonucleotide system at 37 degrees C. Complementation, for producing dCTP and TIP from nucleoside substrates, was carried out by dTMP kinase and/or UMP/CMP kinase and nucleoside diphosphate kinase. dNTP was continuously incorporated into a fixed oligodeoxyribonucleotide primer, template, and probe system, and the fluorescent signal was generated by using the combined actions of primer extension and 5' exonuclease activity of Thermophilus aquaticus (Taq) DNA polymerase for specific relief of fluorescent quenching. Fluorescence was captured at 1-min intervals using a real-time polymerase chain reaction (PCR) instrument. A horizontal threshold line, crossing all sample relative fluorescent units (RFU) values at the level of the RFU of the blank sample at the end of the assay (i.e., 90 min), was drawn, obtaining RFU measurement data in minutes for each sample. Duplex proof of principle was demonstrated by the independent determination of different amounts of dCK and TK1 in combination. R-2 values of 0.90 were demonstrated with Prolifigen TK-REA U/L reference values obtained from pathological canine and human serum samples. (C) 2012 Elsevier Inc. All rights reserved.

Keywords

Homogeneous assay; Deoxyribonucleoside activity assay; Real-time; Quenching; Primer extension; Enzyme complementation

Published in

Analytical Biochemistry
2013, Volume: 432, number: 2, pages: 155-164
Publisher: ACADEMIC PRESS INC ELSEVIER SCIENCE

SLU Authors

• Wang, Liya

  • Department of Animal Biosciences, Swedish University of Agricultural Sciences
    

• Saellström, Sara

  • Department of Clinical Sciences, Swedish University of Agricultural Sciences
    

• Rönnberg, Henrik

  • Department of Clinical Sciences, Swedish University of Agricultural Sciences
    

• Eriksson, Staffan

  • Department of Animal Biosciences, Swedish University of Agricultural Sciences
    

Sustainable Development Goals

Ensure healthy lives and promote well-being for all at all ages


UKÄ Subject classification

Biochemistry and Molecular Biology


Publication identifier

DOI: https://doi.org/10.1016/j.ab.2012.08.004

Permanent link to this page (URI)

https://res.slu.se/id/publ/42615