title: A PCR-free fluorescence strategy for detecting telomerase activity via double amplification strategy.
abstract: As a universal tumor biomarker, research on the activity and inhibition of telomerase is of great importance for cancer diagnosis and therapy. Although the telomeric repeat amplification protocol (TRAP) has served as a powerful assay for detecting telomerase activity, its application has been significantly limited by amplification related errors and time-consuming procedure. To address the limitations of PCR-based protocol, a dual amplification fluorescence assay was developed for PCR-free detecting telomerase activity. Briefly, we designed an arch-structure DNA probe to specifically control strand displacement reaction and subsequent enzyme-aided amplification. Telomerase substrate (TS) primer was extended by telomerase to form long elongation products which contain several TTAGGG repeat units. So, one elongation product can release more than one trigger DNA (t-DNA) via strand displacement reaction to realize first amplification. Subsequently, t-DNA specifically opened molecular beacon (MB) to restore the fluorescence of MB. Meanwhile, t-DNA was recycled by the aid of nicking endonuclease to continuously open more and more MBs, leading to a second amplification. Owing to the double amplification strategy, the proposed method allowed the measurement of telomerase activity in crude cell extracts equivalent to 5 HeLa cells and 10 CCRF-CEM cells without PCR amplification. Besides, the influence of telomere-binding ligands on the telomerase activity demonstrated that the proposed method holds the potential to evaluate the inhibition efficiency of telomerase inhibitors.
authors: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors: Xiafei
authors: X
authors: Zhang
authors: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors: Rui
authors: R
authors: Cheng
authors: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors: Zhilu
authors: Z
authors: Shi
authors: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China. Electronic address: jinyan@snnu.edu.cn.
authors: Yan
authors: Y
authors: Jin
mesh_terms: D014408:Biomarkers, Tumor / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015374:Biosensing Techniques; D004247:DNA / Q000737:chemistry / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009369:Neoplasms / Q000175:diagnosis*; D016133:Polymerase Chain Reaction; D019098:Telomerase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D016615:Telomere / Q000737:chemistry / Q000235:genetics; D034501:Telomere-Binding Proteins / Q000737:chemistry
chemical_list: D014408:Biomarkers, Tumor; D034501:Telomere-Binding Proteins; D004247:DNA; D019098:Telomerase
keywords: Dual amplification; Fluorescence; PCR-free; Telomerase