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      Nonradioactive direct telomerase activity detection using biotin‐labeled primers

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          Abstract

          Background

          Telomerase is a ribonucleoprotein enzyme responsible for maintenance of telomere length which expressed in more than 85% of cancer cells but undetectable in most normal tissue cells. Therefore, telomerase serves as a diagnostic marker of cancers. Two commonly used telomerase activity detection methods, the telomerase repeated amplification protocol (TRAP) and the direct telomerase assay (DTA), have disadvantages that mainly arise from reliance on PCR amplification or the use of an isotope. A safe, low‐cost and reliable telomerase activity detection method is still lacking.

          Method

          We modified DTA method using biotin‐labeled primers (Biotin‐DTA) and optimized the method by adjusting cell culture temperature and KCl concentration. The sensitivity of the method was confirmed to detect endogenous telomerase activity. The reliability was verified by detection of telomerase activity of published telomerase regulators. The stability was confirmed by comparing the method with TRAP method.

          Results

          Cells cultured in 32°C and KCl concentration at 200 mM or 250 mM resulted in robust Biotin‐DTA signal. Endogenous telomerase activity can be detected, which suggested an similar sensitivity as DTA using radioactive isotope markers. Knockdown of telomerase assembly regulator PES1 and DKC1 efficiently reduced telomerase activity. Compared with TRAP method, Biotin‐DTA assay offers greater signal stability over a range of analyte protein amounts.

          Conclusion

          Biotin‐labeled, PCR‐free, and nonradioactive direct telomerase assay is a promising new method for the easy, low‐cost, and quantitative detection of telomerase activity.

          Abstract

          Telomerase activity can be used as a marker to assist in the early diagnosis of tumors, to determine the prognosis of patients, and to detect recurrence. Two commonly used telomerase activity detection methods, the telomerase repeated amplification protocol (TRAP) and the direct telomerase assay (DTA), have disadvantages that mainly arise from reliance on PCR amplification or the use of an isotope. Herein, we developed a modified direct telomerase activity detection method that employs neither isotope markers nor PCR amplification. It was based on immunoprecipitation and streptavidin–biotin system. Finally, it was proved to be a promising new method for the easy, low‐cost, and quantitative detection of telomerase activity.

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          Most cited references49

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          Protein composition of catalytically active human telomerase from immortal cells.

          Telomerase is a ribonucleoprotein enzyme complex that adds 5'-TTAGGG-3' repeats onto the ends of human chromosomes, providing a telomere maintenance mechanism for approximately 90% of human cancers. We have purified human telomerase approximately 10(8)-fold, with the final elution dependent on the enzyme's ability to catalyze nucleotide addition onto a DNA oligonucleotide of telomeric sequence, thereby providing specificity for catalytically active telomerase. Mass spectrometric sequencing of the protein components and molecular size determination indicated an enzyme composition of two molecules each of telomerase reverse transcriptase, telomerase RNA, and dyskerin.
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            Recent Advances in Photoelectrochemical Sensing: From Engineered Photoactive Materials to Sensing Devices and Detection Modes

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              The structure and function of telomerase reverse transcriptase.

              The structure and integrity of telomeres are essential for genome stability. Telomere dysregulation can lead to cell death, cell senescence, or abnormal cell proliferation. The maintenance of telomere repeats in most eukaryotic organisms requires telomerase, which consists of a reverse transcriptase (RT) and an RNA template that dictates the synthesis of the G-rich strand of telomere terminal repeats. Structurally, telomerase reverse transcriptase (TERT) contains unique and variable N- and C-terminal extensions that flank a central RT-like domain. The enzymology of telomerase includes features that are both similar to and distinct from those characteristic of other RTs. Two distinguishing features of TERT are its stable association with the telomerase RNA and its ability to repetitively reverse transcribe the template segment of RNA. Here we discuss TERT structure and function; its regulation by RNA-DNA, TERT-DNA, TERT-RNA, TERT-TERT interactions, and TERT-associated proteins; and the relationship between telomerase enzymology and telomere maintenance.
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                Author and article information

                Contributors
                yeqn66@yahoo.com
                biolongcheng@outlook.com
                liurong301@126.com
                Journal
                J Clin Lab Anal
                J Clin Lab Anal
                10.1002/(ISSN)1098-2825
                JCLA
                Journal of Clinical Laboratory Analysis
                John Wiley and Sons Inc. (Hoboken )
                0887-8013
                1098-2825
                07 May 2021
                June 2021
                : 35
                : 6 ( doiID: 10.1002/jcla.v35.6 )
                : e23800
                Affiliations
                [ 1 ] Faculty of Hepato‐Pancreato‐Biliary Surgery Chinese PLA General Hospital Beijing China
                [ 2 ] Medical school of Chinese PLA Beijing China
                [ 3 ] Department of Medical Molecular Biology Beijing Institute of Biotechnology Beijing China
                Author notes
                [*] [* ] Correspondence

                Qinong Ye and Long Cheng, Department of Medical Molecular Biology, Beijing Institute of Biotechnology, Beijing100850, China.

                Emails: yeqn66@ 123456yahoo.com ; biolongcheng@ 123456outlook.com

                Rong Liu, Faculty of Hepato‐Pancreato‐Biliary Surgery, Chinese PLA General Hospital, Beijing100853, China.

                Email: liurong301@ 123456126.com

                Author information
                https://orcid.org/0000-0003-1408-8667
                Article
                JCLA23800
                10.1002/jcla.23800
                8183940
                33960443
                0bf619b8-8aff-40b1-909c-a5a23140ef5c
                © 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC

                This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

                History
                : 22 February 2021
                : 24 January 2021
                : 02 March 2021
                Page count
                Figures: 5, Tables: 0, Pages: 10, Words: 5798
                Funding
                Funded by: Key Foundation of PLA
                Award ID: 19SWAQ26
                Funded by: National Natural Science Foundation of China , open-funder-registry 10.13039/501100001809;
                Award ID: 81901957
                Award ID: 82072717
                Funded by: the Beijing Nova Program
                Award ID: Z191100001119020
                Categories
                Research Article
                Research Articles
                Custom metadata
                2.0
                June 2021
                Converter:WILEY_ML3GV2_TO_JATSPMC version:6.0.2 mode:remove_FC converted:07.06.2021

                Clinical chemistry
                biotin‐labeled primers,direct telomerase activity,nonradioactive
                Clinical chemistry
                biotin‐labeled primers, direct telomerase activity, nonradioactive

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