Primer set and method for detecting telomerase activity

11859,245

16/939098

July 27, 2020

A primer set for detecting telomerase activity, the primer set including a first primer set or a second primer set. The first primer set includes: an upstream primer selected from MTS; and a downstream primer selected from the group consisting of ACX-M4, Beacon ACX62-2C, and Beacon ACX62-10. The second primer set includes: an upstream primer selected from STS or CTS; and a downstream primer selected from the group consisting of ACX, CXT, ACX-M4, Beacon ACX62-2C, or Beacon ACX62-10. The sequences of the primers ACX, CXT, ACX-M4, Beacon ACX62-2C, Beacon ACX62-10, STS, CTS and MTS are shown as SEQ ID NOs: 1 to 8, respectively.

Institute of Zoology, Chinese Academy of Sciences (Beijing, CN)

INSTITUTE OF ZOOLOGY, CHINESE ACADEMY OF SCIENCES (Beijing, CN)

1. A primer set for detecting telomerase activity, the primer set comprising a first primer set or a second primer set; wherein: the first primer set comprises: an upstream primer having a sequence of SEQ ID NO: 8; and a downstream primer selected from the group consisting of having a sequence of SEQ ID NO: 4 or SEQ ID NO: 5; the second primer set comprises: an upstream primer having a sequence of SEQ ID NO: 7; and a downstream primer having a sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

2. The primer set of claim 1 , wherein a 5′-end of the upstream primer of the first primer set and a 5′-end of the upstream primer of the second primer set are both labeled with a fluorescent reporter group.

3. The primer set of claim 2 , wherein the fluorescent reporter group is located on an iso-dC nucleotide or a dCTP nucleotide at the 5′-end of the upstream primer.

4. The primer set of claim 2 , wherein the fluorescent reporter group is selected from the group consisting of fluorescein amidite, boron-dipyrromethene, and tetramethylrhodamine.

5. The primer set of claim 3 , wherein the fluorescent reporter group is selected from the group consisting of fluorescein amidite, boron-dipyrromethene, and tetramethylrhodamine.

6. A method for detecting telomerase activity of a biological sample using the primer set of claim 1 , the method comprising: 1) lysing the biological sample with a 3-((3-cholamidopropyl)dimethylammonio)propanesulfonate (CHAPS) lysis buffer to obtain a cell lysate solution; 2) performing a telomerase extension in the cell lysate solution in the presence of the upstream primer of the first primer set or the second primer set of the primer set to obtain a telomerase template; and 3) conducting a qPCR amplification with the telomerase template in the presence of a corresponding downstream primer of the upstream primer of the first primer set or the second primer set used in the telomerase extension in 2).

7. The method of claim 6 , wherein in 1), obtaining the cell lysate solution comprises: suspending cells of the biological sample in a phosphate buffer saline (PBS) solution, centrifuging the PBS solution comprising the cells at 500×g for 3 min, removing a supernatant of the PBS solution and repeating the centrifuging and removing the supernatant for 3 times; resuspending the cells in an isotonic buffer, dispersing the cells, adding the cells to 2 volumes of CHAPS lysis buffer with respect to a volume of the cells, lysing the cells on an ice for 30 min, centrifuging the cells at 16000×g for 20 min, collecting a supernatant and/or storing the supernatant at −80° C.

8. The method of claim 6 , wherein in 1), obtaining the cell lysate solution comprises: adding 1-175 μL of the CHAPS lysis buffer to a PCR tube, aspirating single or multiple cells of the biological sample into the PCR tube by a flow cytometer or a glass tube under a microscope, and lysing the cells on an ice for 10-30 min to obtain the cell lysate solution.

9. The method of claim 6 , wherein the method is implemented in a PCR tube provided with 20-μL of a reaction solution comprising 10 μL of 2×PCR mix, 0.8 μL of the upstream primer, 0.8 μL of the downstream primer, 2 μL of the cell lysate solution, and 6.4 μL of double distilled water; or the method is implemented in a PCR tube provided with 10 μL of a reaction solution comprising 5 μL of 2×PCR mix, 0.4 μL of the upstream primer, 0.4 μL of the downstream primer, 1 μL of the cell lysate solution, 0.15 μL of dimethylsulfoxide (DMSO), 0.15 μL of 10 mg/mL bovine serum albumin (BSA), and 2.9 μL of double distilled water.

10. The method of claim 9 , wherein the reaction solution is treated under an extension reaction program and a PCR reaction program consecutively; the extension reaction program comprises incubation at 25° C. for 25 min, and denaturation at 94° C. for 2 min; the PCR reaction program comprises: a) denaturation at 94° C. for 30 s; b) annealing at 57-60° C. for 30 s; c) extension at 72° C. for 30 s; d. repeating operations b)-c) 45-50 times; and e) extension at 72° C. for 10 min.

11. The method of claim 6 , wherein the method is implemented in a PCR tube provided with 10 μL of a first reaction system comprising 5 μL of 2×PCR mix, 0.8 μL of the upstream primer, 2 μL of the cell lysate solution, and 2.2 μL of double distilled water, and 20 μL of a second reaction system comprising 5 μL of 2×PCR mix, 0.8 μL of the downstream primers, 10 μL of telomerase template, and 4.2 μL of double distilled water; and the first reaction system and the second reaction system are added to the PCR tube in two steps.

12. The method of claim 11 , wherein the PCR tube is treated under an extension reaction program and a PCR reaction program consecutively; the extension reaction program comprises incubation at 25° C. for 10-25 min, and denaturation at 94° C. for 5 min; the PCR reaction program comprises: a) denaturation at 94° C. for 2 min; b) denaturation at 94° C. for 30 s; c) annealing at 57-60° C. for 30 s; d) extension at 72° C. for 30 s; e) repeating operations b)-d) 45-50 times; and f) extension at 72° C. for 10 min.

13. The method of claim 6 , wherein the CHAPS lysis buffer comprises 15 mM of Tris-HCl (pH 7.5), 2 mM of MgCl 2 , 1.5 mM of ethylene glycol tetraacetic acid (EGTA), 0.75% of CHAPS (m/v), 15% of glycerol (v/v), 7.5 mM of dithiothreitol (DTT), 0.75 mM of a protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 1.5 U/μL of a RNase inhibitor, and 0.6 mg/mL of BSA.

105648051 June 2016

Szatmari et al., Analytical Biochemistry 282, 80-88, (Year: 2000). cited by examiner

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