Stem-loop structured combined probe and application thereof
A technology of stem-loop structure and combined probes, applied in the field of biological analysis, can solve the problems of lack of signal amplification mechanism, detection sensitivity limitation, high price, etc., achieve huge application potential, easy operation, and overcome time-consuming effects
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Embodiment 1
[0026] Aiming at the telomerase substrate sequence 5′-AATCCGTCGAGCAGAGTT-3′, probe I and probe II with stem-loop structure were designed and synthesized. The base sequence of probe I was 5′-CGACAGCAGAGGATTTGTTGTGTGGAAGTGTGAGCGGATTTTCCTCTGCTGTCGTTTTAATCCGTCGAGCAGAGTT-3′ (by Bao Biological Engineering Co., Ltd. (Dalian) Co., Ltd.); the base sequence of probe II is 5'-ATCGTCGTGACTGTTTGTAATAGGACAGAGCCCCGCACTTCAGTCACGACGATTTTCCCTAACCCTAACCCTAACCC-3' (provided by Treasure Bioengineering (Dalian) Co., Ltd.).
[0027] Adopt above-mentioned probe I and probe II to detect the telomerase activity in cervical cancer cell (Hela), specific method is as follows:
[0028]The cultured Hela cells were digested and centrifuged from the culture medium, washed and centrifuged 3 times with cold D-PBS buffer solution (10mmol / L phosphate, 0.1mol / LNaCl, pH7.4), and then centrifuged at 2000 rpm for 5min . The washed cells were added to cold D-PBS buffer solution to adjust the cell concentration to 100...
Embodiment 2
[0032] The application of the stem-loop structure combination probe of embodiment 1 in the screening of telomerase inhibitors, the specific method is as follows:
[0033] In the method for detecting telomerase activity in cervical cancer cells (Hela) in Example 1, the telomerase inhibitor 3'-azido-3'deoxythymidine (AZT) was added when preparing the telomerase extension product, and the telomere Enzyme inhibitor final concentration is respectively 0mmol / L, 2mmol / L, 5mmol / L, 10mmol / L, other steps are identical with embodiment 1, the result sees image 3 . From image 3 It can be seen from the figure that in the case of a fixed telomerase concentration (100 Hela cells), as the concentration of 3'-azido-3'-deoxythymidine increases, the LAMP reaction speed will gradually slow down, indicating that the inhibitor 3 The increase of '-azide-3' deoxythymidine concentration will gradually inhibit the activity of telomerase, resulting in the gradual reduction of telomerase extension pro...
Embodiment 3
[0035] Targeting the mRNA fragment sequence 5′-GCGUGUGCGGGCCCAGGACCCGCCGCCUGAGCUGUACUUUGUCAAGGACAGGCUCACGGAGGUCAUCGCCAGCAUCAUCAA-3′ near the splicing site of exon 5 and exon 6 of human telomere reverse transcriptase mRNA (hTERT; AccessionNo: NM_198254.1), synthesized by nologTech Probe I and probe II with stem-loop structure were synthesized. The base sequence of probe I was 5′-CGACAGCAGAGGATTTGTTGTGTGGAAGTGTGAGCGGATTTTCCTCTGCTGTCGTTTTCCGTGAGCCTGTCCTTGACA-3′ (provided by Treasure Bioengineering (Dalian) Co., Ltd.); the base sequence of probe II was 5'-ATCGTCGTGACTGTTTGTAATAGGACAGAGCCCCGCACTTTTCAGTCACGACGATTTTTCCCGCCGCCTGAGCTGTA-3' (provided by Treasure Bioengineering (Dalian) Co., Ltd.).
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