DETERMINATION OF TELOMERASE ACTIVITY BY REAL TIME DOUBLE TELOMERIC REPEAT AMPLIFICATION PROTOCOL

DOI: https://doi.org/10.29296/24999490-2018-04-08

A.I. Glukhov(1, 2, 3), D.S. Nalobin(1, 3), S.A. Gordeev(1, 2, 3), S.N. Kalabushev(1, 3) 1-Lomonosov Moscow State University, Leninskie Gory, 1, Moscow, 119991, Russian Federation; 2-I.M. Sechenov First Moscow State Medical University, Trubetskaya str., 8–2, Moscow, 119991, Russian Federation; 3-Molecular Biomedicine Scientific Research Center, Malevicha str., 1, Skolkovo Innovation Centre, Moscow, 121205, Russian Federation E-mail: [email protected]

Introduction.The telomerase activity is a universal marker for 85% of malignant tumors. This allows us to develop modern highly sensitive and highly specific kits for the diagnosis of most malignant tumors, including early stages of the disease. Existing methods of the determination the activity of telomerase are considered insufficiently effective when very small amounts of cellular material are analyzed. This makes it impossible to conduct a non-invasive diagnosis of some diseases when the amount of cellular material is very small. In this regard, the development of more sensitive methods for determining the activity of telomerase is an extremely urgent aim of biology and medicine. The aim of study.Development of a highly sensitive and highly specific method for determining telomerase activity in real time. Methods. A new method,Real-Time – Telomeric Repeat Amplification Protocol – 2 Polymerase Chain Reaction (RT-TRAP-2PCR), was proposed for the detection and quantification of telomerase activity in biological samples of eukaryotic organisms by double amplification of telomeric repeats in real time, based on the measurement of the total amount of telomere repeats, newly synthesized by telomerase in the analyzed sample. Results. This approach allows provide in the high sensitivity and specificity of the method, including analysis in biological samples characterized by a low amount of telomerase-positive cells and the presence of telomerase inhibitors. The method refers to biology, medicine, oncology, veterinary medicine, reproductive medicine and can be used, in particular, to determine the activity of telomerase in the diagnosis of malignant neoplasms, at the preparatory stages of assisted reproductive technologies, scientific research. Conclusion. The proposed method makes it possible to carry out not only a qualitative but also a quantitative analysis of the telomerase activity in biological samples. The method allows analyzing telomerase activity in the presence of various impurities of nucleic acids, proteins and other components of cells and tissues characteristic for clinical specimens, at that samples obtained by non-invasive and minimally invasive methods may be suitable for the diagnosis.
Keywords: 
telomere, telomerase, activity, TRAP (Telomeric Repeat Amplification Protocol), RT-TRAP-2PCR (Real-Time – Telomeric Repeat Amplification Protocol – 2 Polymerase Chain Reaction)
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