Fluorescence Detection of Telomerase Activity in Cancer Cells Based on Isothermal Circular Strand-Displacement Polymerization Reaction

Abstract

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.

Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution

“…Other researchers have attempted to eliminate the PCR step or alter the detection method for the TRAP assay (30–39). Recently, gold plated nanoparticles with a bound telomerase substrate were used in combination with PCR to detect telomerase (38).…”

Advances in the detection of telomerase activity using isothermal amplification

“…The stem-loop primer-mediated exponential amplification reaction can be achieved under isothermal circumstances with a single step and one enzyme (DNA polymerase) ( Figure 6 ). Base on the elongation reaction (telomerase primer), strand-displacement property (polymerase) and inherent signal-transduction mechanism (hairpin fluorescence probe), telomerase activity was converted into fluorescence signal 66.…”

Aptameric Molecular Switch for Cascade Signal Amplification

“…Thus, the development of assay signal amplification paths has become important, and several amplification techniques have been developed, including catalytic nanoparticles (1,2 ), PCR (3,4 ), rolling-circle amplification (5,6 ), enzyme-triggered strand displacement amplification (7)(8)(9)(10), entropydriven signal amplification (11,12 ), and cascade signal amplification (13,14 ). The PCR is currently the most widely used thermal-cycling technique for DNA amplification.…”