Fluorescence detection of telomerase activity in cancer cells based on isothermal circular strand-displacement polymerization reaction.
On the basis of the extension reaction of a telomerase substrate (TS) primer in the presence of the telomerase, the inherent signal-transduction mechanism of the hairpin fluorescence probe, and the strand-displacement property of polymerase, an amplified fluorescence detection of telomerase activity in the cancer cells was described in this manuscript. A hairpin fluorescence probe was used as not only the fluorescence signal carrier but also a template of the telomere elongation reaction. In the presence of the telomerase, the stems of the hairpin probes were opened and the telomerase activity could be determined with the fluorescence enhancement. The telomerase activity in the HeLa extracts equivalent to 40-1000 cells was detected by this method, with the multiple rounds of isothermal strand replication, which led to strand displacement, and constituted consecutive signal amplification for the novel detection paradigm that allowed measurement of telomerase activity in crude cell extracts equivalent to 4 cultured HeLa cells. Using magnetic beads as both the separation tool and the immobilization matrix of the aptamer of Ramos cells (CRL-1596, B-cell, human Burkitt's lymphoma), the detection of the amount of the Ramos cell with the low concentration of 100 cells mL(-1) confirmed the reliability and practicality of the protocol, which reveal a good prospect of this platform for analysis.
Key Laboratory of Eco-chemical Engineering, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China.
Caifeng
C
Ding
Xiangling
X
Li
Ying
Y
Ge
Shusheng
S
Zhang
D014408:Biomarkers, Tumor / Q000032:analysis* / Q000378:metabolism; D045744:Cell Line, Tumor; D004270:DNA, Circular / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D016133:Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence / Q000379:methods*; D019098:Telomerase / Q000032:analysis* / Q000378:metabolism
D014408:Biomarkers, Tumor; D004270:DNA, Circular; D005456:Fluorescent Dyes; D019098:Telomerase