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Detection of activity of telomerase in tumor cells using fiber optical biosensors

Item Type:Article
Title:Detection of activity of telomerase in tumor cells using fiber optical biosensors
Creators Name:Schmidt, P.M. and Lehmann, C. and Matthes, E. and Bier, F.F.
Abstract:Human telomerase plays an important role in the cancerogenesis as it is up-regulated in 80-90% of malignant tumors. Thus, it is considered as a potential cancer marker and relevant target in oncology. Its task is the extension of guanine-rich strands of the telomere using an intrinsic RNA as the template. In this paper we developed a new biosensoric assay based on total internal reflection fluorescence measuring the activity of the telomerase on sensor surface. Two alternatives to determine the telomeric activity are demonstrated without the use of amplifying steps as e.g. PCR. The enzymatic inclusion of FITC-labeled dUTPs should reveal the synthesis process in real-time indicating the elongation of a phosphothioate telomeric substrate (PS/TS)-modified primer. Additionally the elongated strand was detected by hybridization with a FITC-labeled complementary linear DNA probe. As the telomeric guanine-rich single-stranded DNA adopts intramolecular quadruplex structures, it was necessary for the hybridization to linearize the telomeric DNA by increasing the reaction temperature to 48°C. The comparison of the telomerase activity using labeled and unlabeled nucleotides indicated the inhibition effect of the FITC-labeled nucleotides slowing down the synthesis rate of the enzyme. It is shown with the modified biosensor that the PS/TS primer binds the telomerase from the HL-60 cell lysates, effectively elongating the immobilized primer. Furthermore no more purification steps were required as all measurements were performed with crude cell extract.
Keywords:Telomerase, TRAP-assay, Fiber Optic, G-Quartettes
Source:Biosensors and Bioelectronics
Publisher:Elsevier Advanced Technology
Page Range:1081-1087
Date:December 2002
Official Publication:https://doi.org/10.1016/S0956-5663(02)00102-1
PubMed:View item in PubMed

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