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

Inactive Publication Date: 2016-04-27
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it should be pointed out that this method lacks a signal amplification mechanism, which greatly limits the detection sensitivity, and the molecular beacon needs to be double-labeled, which is expensive

Method used

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  • Stem-loop structured combined probe and application thereof
  • Stem-loop structured combined probe and application thereof
  • Stem-loop structured combined probe and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a stem-loop structured combined probe and application thereof. The probe is constituted by two DNA sequences of stem-loop structures; from 5' terminals to 3' terminals, probe I and probe II sequentially include stem-loop zones and detection object recognition zones, and are applicable to telomerase activity, RNA and DNA detection and telomerase inhibitor screening; when a detection object is telomerase, the detection object recognition zone of the probe I is a telomerase substrate sequence, and the detection object recognition zone of the probe II is a sequence which is complementary with 8-40 bases in a telomerase extension sequence; and when the detection object is RNA or DNA, the detection object recognition zone of the probe I is a sequence which is complementary with 8-40 bases, close to the 3' terminal, of to-be-detected RAN or DNA, and the detection object recognition zone of the probe II is a sequence which is equal to 8-40 bases, close to the 5' terminal, of the to-be-detected RAN or DNA. The combined probe disclosed by the invention is unnecessary to make use of fluorescence labeling and radioactive element labeling, and the combined probe, with the specific stem-loop structure, provides a specific amplification template for a nucleic acid amplification reaction, so that rapid signal amplification under constant temperature is achieved; and the combined probe is high in sensitivity and amplification efficiency.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, and in particular relates to a combined probe with a stem-loop structure and the application of the combined probe in the detection of telomerase activity and the quantitative detection of RNA and DNA. Background technique [0002] Telomerase (Telomerase) is a reverse transcription DNA synthetase that extends telomeres, and is a ribonucleic acid-protein complex composed of RNA and protein. Telomerase compensates for the progressive shortening of telomere length with each cell division. However, the activity of telomerase is quite tightly regulated in normal somatic cells, so telomerase activity is inhibited in normal human cells, but activated in tumor cells, which is closely related to the continuous proliferation of cancer cells. Therefore, the detection of telomerase activity can realize the diagnosis of cancer, and at the same time, using telomerase as a target molecule to study ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/25C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/136C12Q2600/158C12Q2545/113C12Q2561/113C12Q2563/107C12Q2521/113
Inventor 李正平王洪红王辉刘成辉
Owner SHAANXI NORMAL UNIV
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