Table 2.

Brief comparison of the methods for telomerase activity detection

Method for telomerase activity detection	Sensitivity*	Advantages	Drawbacks**	Reference
Direct incorporation of a radioactively labeled substrate	105–106	The absence of artifacts associated with PCR. The telomerase-synthesized DNA can be immediately observed in the gel. Its size and amount can be estimated.	Low sensitivity, the necessity of working with large amounts of a radioactive label, long-term exposition.	[43]
Determination of telomerase-synthesized DNA by the changes in surface plasmon resonance	20–100	The absence of artifacts associated with PCR. No radioactive label and PAAG. Information on the reaction’s kinetics. The possibility of signal detection against 1,000-fold excess of telomerase-negative cells. Telomerase binding and dissociation can be observed.	The BIACORE system and biotin-conjugated primers are required.	[44, 54]
Using oligo-modified magnetic particles and NMR	10	The absence of artifacts associated with PCR. No radioactive label and PAAG. A very high performance microplate format.	A NMR spectrometer and a sample of nanoparticles covalently bound to oligonucleotides are required.	[46]
Using the quartz crystal microbalance technique	3300	The absence of artifacts associated with PCR. High sensitivity. Rapid procedure.	A frequency analyzer and a Au-quartz crystal are required. Identification of artifact signals (in case of emergence) is difficult.	[47]
Electrochemically, using ferrocenyl naphthalene diimide	100–1000	The absence of artifacts associated with PCR. High sensitivity. Rapid procedure.	The number of structures folded into the quadruplex is assessed, which depends not only on the total number of repeats, but also is unpredictably dependent on the length and position of individual DNA fragments.	[48]
Using a biobarcode	10	One of the most sensitive methods today for direct telomerase activity detection without amplification of the telomerase-synthesized DNA	A modified electrode and nanoparticles coated with oligonucleotides of two types are required.	[49]
Using optical biosensors	105	No radioactivity, no PAAG, no PCR.	Phosphorothioate-modified oligonucleotides and a special optosensor are required. The sensitivity is insufficient for clinical materials.	[50]
Based on quantum dots	10,000/270	No radioactivity, no PAAG, no PCR.	Oligonucleotides covalently bound to quantum dots are required. The sensitivity is insufficient for clinical materials.	[51]
Using an on-chip nanowire sensor	100	It is possible to carry out a large number of different analyses of one specimen on a single chip. Telomerase binding and dissociation can be observed.	A transistor chip and the equipment to analyze it are required.	[53]
Bioluminescent method	5	Linear dependence of the signal on the amount of the telomerase-synthesized DNA. No radioactivity, no PAAG, no PCR. High-efficiency format.	The luciferase system for bioluminescence detection and a luminometer are required.	[55]
Electrochemiluminescence method	500	A high signal/noise ratio due to purification by modified magnetic bead extraction. High sensitivity.	It is difficult to synthesize a sample, requirements to the equipment.	[56]
Using FRET and total internal reflection fluorescence microscopy	1	Extremely high sensitivity – it is possible to detect the elongation of individual primers by telomerase. Possibility of combining this method with FRET-based methods for investigating the telomerase structure.	The method determines only the first 1.5 telomerase-synthesized repeats. It was tested only forTetrahymena thermophilatelomerase.	[57]
Using digoxigenin-labeled oligonucleotides complementary to telomeres (ELISA)	37,500	Quantitative determination; the method has been automated.	The sensitivity is insufficient for clinical materials.	[58]
Electrochemically, using avidin­–alkaline phosphatase conjugates	3,300	The absence of artifacts associated with PCR. High sensitivity. Rapid procedure.	A specially prepared electrode is needed. It is difficult to identify artifact signals if they emerge.	[47]
Using a fluorimetric optosensor	106–107	No radioactivity, no PAAG, no PCR.	Phosphotioate-modified oligonucleotides and an optic fiber system for fluorescence detection are required. The sensitivity is insufficient for analyzing clinical materials.	[59]
Using DNAzyme-labeled oligonucleotides	1,000	No radioactivity, no PAAG, no PCR. Simple procedure and short analysis time.	The specificity of the method is not very high; a background signal is present.	[61]
On the basis of the telomerase substrate that refolds in DNAzyme	10,000	No radioactivity, no PAAG, no PCR. A very simple procedure and short analysis time.	The sensitivity and specificity of the method are not very high; a background signal is present.	[60]
TRAP	10	Amplificates of telomerase-synthesized DNA can be observed. PAAG makes it possible to identify some PCR artifacts. High specificity.	Working with the radioactive label, necessity of using PAAG. PCR artifacts are possible.	[11]
TRAP from a single cell	1	High sensitivity. PAAG makes it possible to identify some PCR artifacts and qualitatively judge the processivity.	Working with the radioactive label, working with individual cells, necessity of using PAAG. PCR artifacts are possible.	[13]
TRAP with fluorescent primers	100	No radioactive labeling. PAAG makes it possible to identify some PCR artifacts and qualitatively judge the processivity.	Necessity of fluorescently labeled primers, necessity of using PAAG. PCR artifacts are possible.	[20]
TRAP with fluorescent staining of DNA in gel	10	No radioactive labeling. PAAG makes it possible to identify some PCR artifacts and qualitatively judge the processivity.	Necessity of using PAAG. PCR artifacts are possible. Most intercalating fluorescent dyes are mutagenic.	[16, 18]
TRAP with DNA staining with silver nitrite in gel	10	No radioactive labeling. PAAG makes it possible to identify some PCR artifacts and qualitatively judge the processivity.	Necessity of using PAAG. Possibility of PCR artifacts. High labor input.	[17]
TRAP with analyzing by scintillation proximity assay	10	No radioactivity, no PAAG. Highly efficient microplate format. Simple estimation of the amounts.	Necessity of using [3H]TTР and biotin-conjugated primers. Working with tritium. PCR artifacts are possible.	[27]
TRAP with detection by “hybridization protection”	10	No radioactivity, no PAAG. Highly efficient microplate format. Simple estimation of the amounts.	Acridine-labeled samples are required. PCR artifacts are possible.	[28]
TRAP with amplifluore primers	10-50	No radioactivity, no PAAG. Highly efficient microplate format. Quantitative assessment. Different labeling of amplificates by the telomerase-synthesized DNA and PCR control.	PCR artifacts are possible. Amplifluores are required.	[22]
TRAP combined with ELISA	10	No radioactivity, no PAAG. Highly efficient microplate format.	PCR and ELISA artifacts are possible.	[29]
TRAP with primers with fluorescence resonance energy transfer(FRET)	10	No radioactivity, no PAAG. Highly efficient microplate format.	Unequal consideration of the first and subsequent repeats synthesized by telomerase when carrying out the quantitative assessment. PCR artifacts are possible. Special fluorophores are required.	[26]
TRAP on microchips	10	No radioactivity, no PAAG. Small reaction volumes. Highly efficient microplate format. Simple estimation of the amounts.	Chips and an instrument to read them are necessary.	[35]
TRAP with real-time PCR	50	No radioactivity, no PAAG. Highly efficient microplate format. Simple estimation of the amounts. Different labeling of amplificates of the telomerase-synthesized DNA and PCR control with amplifluores is possible.	An amplifier for real-time PCR is required. The identification of PCR artifacts is complicated.	[32, 33]
In situTRAP	1	The method provides information on the telomerase activity in individual cells and some information on endocellular localization of the activity.	A complicated procedure. Low efficiency. A fluorescence microscope is required. The method has been optimized only for analyzing clinical samples and cell lines.	[36]
TRAP with transcriptional amplification	10	Isothermal amplification, no PCR artifacts, PAAG-free detection, highly efficient format.	Acridine-labeled samples are required. Artifacts of transcriptional amplification are possible. Higher requirements of purity (absence of RNases) because of the transcriptional amplification.	[42]

* The minimum number of cells of telomerase-positive cell lines for which activity can be detected.

** In PAAG-free methods (with the exception of determination of telomerase activity using FRET and total internal reflection fluorescence microscopy), polymerase activity (but not processivity) is assessed.