A method for detecting telomerase activity based on a quencher-free molecular beacon
A technology of molecular beacons and telomerase, which is applied in biochemical equipment and methods, microbiological measurement/inspection, etc., can solve problems that have not been reported before, and achieve low background interference, good fluorescence stability, and good specificity Effect
Active Publication Date: 2021-11-09
SHANDONG NORMAL UNIV
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Problems solved by technology
Since telomerase cannot directly induce the conversion of 2-AP fluorescence, studies using 2-AP MB probes to detect telomerase have not been reported
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Embodiment 1
[0074] 1) Extraction of telomerase, human lung cancer cell line (A549 cell), human cervical cancer cell line (HeLa cell) and human breast adenocarcinoma cell line (MCF-7 cell) contained 10% fetal bovine serum and 1% gluten Culture in amide DMEM medium with 50U / mL penicillin and 50mg / mL streptomycin in a suitable humid environment containing 5% carbon dioxide at 37°C; wash the cells twice with phosphate buffered saline (pH 7.4), and then use trypsin Cells were digested from the bottom of the dish, and the collected cells were centrifuged at 2000 rpm for 10 minutes at 4°C. About 1×10 6 Each cell was resuspended with 200 μL of ice-cold 1×CHAPS lysis buffer solution, incubated on ice for 30 minutes, followed by centrifugation at 4°C, 12,000 g for 20 minutes. After centrifugation, remove the supernatant for quick freezing and store in a -80°C freezer. Control of heat pretreatment, active cell extracts were heated at 85°C and measured 10 minutes in advance;
[0075] 2) Anneal 1 μ...
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The invention belongs to the technical field of biological detection, and in particular relates to a method for detecting telomerase activity based on a non-quencher molecular beacon. The invention provides a 2-aminopurine non-quenching molecular beacon detection method for detecting telomerase activity in cancer cells. Telomerase is extracted from cancer cells, and the catalytic repeat unit (TTAGGG)n is added to the substrate chain to generate the extension product of the telomere repeat. With the addition of KF polymerase, the primer extends along the template to form a double strand, and 2‑ in the double strand The strands of AP MBs can be degraded by T7 exonuclease, releasing free 2‑AP molecules and primer-extended strands, free primer-extended strands can combine with additional 2‑AP MBs to form new double strands, triggering the next round of T7 exonuclease degradation. Compared with the molecular signal method in the prior art, the present invention realizes double signal amplification, has high sensitivity and specificity, and is of great significance for the development of antitumor drugs.
Description
technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for detecting telomerase activity in cancer cells based on a non-quencher molecular beacon. Background technique [0002] Telomerase is an important reverse transcriptase that prevents the shortening of natural telomeres by adding a repeat sequence (TTAGGG)n at the 3' end of chromosomes. In most normal cells, telomerase expression is strictly repressed, resulting in progressive shortening of telomeres as cells divide. In contrast, in more than 85% of human cancer cells, the activity of telomerase is significantly upregulated or activated, including lung cancer, breast cancer, gastric cancer, prostate cancer, and urothelial cancer, which play an important role in cell immortalization. The difference in telomerase activity between normal and cancer cells makes telomerase a potential biomarker for cancer diagnosis. In addition, inhibition of telom...
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IPC IPC(8): C12Q1/48
CPCC12Q1/48
Inventor 张春阳马飞王婷婷
Owner SHANDONG NORMAL UNIV