Fluorescence detection of telomerase activity in cancer cell extracts based on autonomous exonuclease III-assisted isothermal cycling signal amplification.
Based on the extension reaction of a telomerase substrate (TS) primer in the presence of the telomerase, strand-displacement process to perform more stable longer duplex chain, and stepwise hydrolysis of mononucleotides from the blunt or the recessed 3'-hydroxyl termini of duplex DNA in the presence of Exonuclease III (Exo III), an amplified fluorescence detection of telomerase activity in the cancer cells was described in this manuscript. A fluorescence probe DNA, a quencher DNA, and a TS primer were mixed to construct a three-chain DNA structure and a two-chain DNA structure because the amount of the TS primer was less than the other two DNA. In the presence of the telomerase, the quencher DNA was replaced from the probe DNA and the telomerase activity could be determined with the fluorescence enhancement. The telomerase activity in HeLa extracts equivalent to 6-2000 cells was detected by this method. Moreover, the strategy was further proved by using telomerase extracted from Romas cells. With the multiple rounds of isothermal strand displacement and the hydrolysis process, constituted consecutive of signal amplification for the novel detection paradigm that allowed measuring of telomerase activity in crude cancer cell extracts confirmed the reliability and practicality of the protocol, which reveal this platform holds great promise in the biochemical assay for the telomerase activity in early diagnosis for cancers.
Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China. Electronic address: dingcaifeng2003@163.com.
Caifeng
C
Ding
Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China.
Xiaoqian
X
Li
Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China.
Wei
W
Wang
Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China.
Yaoyao
Y
Chen
D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D015342:DNA Probes / Q000737:chemistry; D057075:Enzyme Assays / Q000379:methods*; D005090:Exodeoxyribonucleases / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D057230:Limit of Detection; D009369:Neoplasms / Q000201:enzymology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000379:methods; D019098:Telomerase / Q000032:analysis / Q000378:metabolism*
D015342:DNA Probes; D019098:Telomerase; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III
Biosensors; Cancer detection; Exo-III-assisted; Fluorescence detection; Isothermal cycling signal amplification; Telomerase activity