Telomerase activity assay experiment
Telomerase activity assay experiment
Telomerase is a reverse transcriptase capable of synthesizing telomeric DNA using its own RNA as a template. It is generally accepted that telomerase consists of a protein fraction and an RNA fraction, with the RNA fraction (telomerase RNA, abbreviated as TR or TERC) serving as the template for telomere synthesis, and a protein fraction consisting of at least two subunits, the large subunit of telomerasereverse transcriptase (TERT), which has internal substrate-telomere binding sites, catalytic sites for dNTP binding, and sites for template alignment. telomerasereverse transcriptase (TERT), which has an internal substrate-telomere binding site, a catalytic site for dNTP binding, and a site for template alignment. The small subunit proteins consist of small nucleolar complexes such as dyskerin (DKC1) and other members of the telomerase-associated H+ACA box that are assembled into active telomerase during TR treatment, and a schematic representation of telomerase-catalyzed telomere synthesis is shown in Figure 11-7. Since TR and TERT are the active groups of telomerase, the detection of telomerase activity is usually realized by detecting the activities of TR and TERT. Quantitative PCR and other methods can be used to detect the expression level of TR, but since TR is not actually the rate-limiting factor of telomerase activity, the research of detecting the activity of telomerase is mainly designed for the detection of the activity of TERT. Traditionally, TERT activity can be measured by three methods: PCR, in situ hybridization, and immunofluorescence/immunohistochemistry/immunoblotting. Currently, there is one main method used for the detection of telomerase activity: the TRAP assay.
Principle
The basic principle of the TRAP assay for telomerase activity is the addition of a telomeric repeat (GGTTAG) to the 3' end of an oligonucleotide substrate TS catalyzed by active telomerase, followed by 30-33 cycles of PCR amplification, labeling of the TS primer with the radioisotope y-32P-ATP, and detection of the PCR products by polyacrylamide electrophoresis. The PCR products are detected by polyacrylamide electrophoresis, and finally, the amplification results are detected and analyzed by autoradiography.
Alternatively, instead of radioisotope labeling of the primers, the PCR products can be stained with a non-radioactive SYBRGreen dye to analyze and quantify telomerase activity.
Operation method
Detection of telomerase activity by TRAP assay
Principle
The basic principle of the TRAP assay for telomerase activity is the addition of a telomeric repeat (GGTTAG) to the 3' end of an oligonucleotide substrate TS catalyzed by active telomerase, followed by 30-33 cycles of PCR amplification, labeling of the TS primer with the radioisotope y-32P-ATP, and detection of the PCR products by polyacrylamide electrophoresis. The PCR products are detected by polyacrylamide electrophoresis, and finally, the amplification results are detected and analyzed by autoradiography. Alternatively, instead of radioisotope labeling of the primers, the PCR products can be stained with a non-radioactive SYBRGreen dye to analyze and quantify telomerase activity.
Materials and Instruments
Equipment: Move The basic procedure of the TRAP assay for telomerase activity can be divided into the following steps: B TRAP reaction setup, add 2 μl of template, 5 μl of 10x TRAP reaction buffer, 1 μl of 50x dNTP Mix, 1 μl of TS primer, 1 μl of TRAP assay primer (including RP primer, K1 primer, TSK1 primer), 0.4 μl of Taq DNA polymerase (5 U/μl), and 39.6 μl of ddH2O to each reaction. C PCR amplification, PCR reaction conditions: incubation at 30 ℃ for 30 min, followed by two steps of 30 cycles, including denaturation at 94 ℃ for 30 s, and annealing/extension mixing at 59 ℃ for 30 s. The reaction was actually a co-buffer, two-enzyme system reaction, and in the reaction of the first enzyme system, telomerase transferred a certain number of telomeric repeats (GGTTAG) to the 3' end of the oligonucleotide substrate TS. TS to the 3' end of the oligonucleotide substrate, whereas in the reaction of the second enzyme system, the TS and RP primers amplify a product gradient in 6-base increments, e.g. starting at 50 nucleic acids: 50, 56, 62, 68 ---- -The fragmentation of the reaction products is shown in the following table. D Reaction product electrophoresis and color development reaction, PCR reaction products were sampled into 10% polyacrylamide gel, electrophoresed in 1x TBE buffer, and the gel was stained with 1:10,000 SYBR Green for 15 min and then photographed. Caveat 1 1x CHAPS buffer lysis of cells should be carried out on ice, tissue samples are more difficult to be lysed, may need to use an electric homogenizer, note that it should be ground on ice to avoid too high a temperature, centrifugation should be used in a freezer centrifuge in order to prevent the degradation of telomerase in the presence of heat.2 SYBR Green staining time should not be too long, the staining should be protected from light, and the shooting time should not be too long, in order to prevent the fluorescent dye from decomposing in the presence of light, so that a clear image cannot be obtained. For more product details, please visit Aladdin Scientific website.
① Tissue grinder
② Ultraviolet spectrophotometer
③ PCR instrument
④ Vertical electrophoresis tank
⑤ UV gel imaging system
⑥ Cells or tissue sample cells to be tested.
Reagents:
① Polyacrylamide gel reagent
① Taq DNA polymerase; ③ dNTP Mix.
① Polyacrylamide gel reagent; ② Taq DNA polymerase; ③ dNTP Mix.
④ SYBR Green dye
⑤ Primers for TRAP assay (including TS primer, RP primer, K1 primer, TSK1 primer).
⑥ 1x CHAPS buffer (10 mmol/L Tris-HCI, pH 7.5, 1 mmol/L MgCl
2, 1 mmol/L EGTA, 1 mmol/L MgCl
1 mmol/L EGTA, 0.1 mmol/L benzenecarboximidamide, 5 mmol/L β-mercaptoethanol, 0.5% CHAPS, 10% glycerol)
(vii) 10x TRAP reaction buffer (200 mmol/L Tris-HCI (pH 8.3), 15 mmol/L MgCl
2
(200 mmol/L Tris-HCI (pH 8.3), 15 mmol/L MgCl2, 630 mmol/L KCI, 0.5% Tween-20, 10 mmol/L EGTA)