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Molecular beacon probe, kit and in-vitro-transcription fluorescent quantitative real-time tracing detection method and application of molecular beacon probe and kit

A molecular beacon probe, in vitro transcription technology, applied in the fields of molecular biology and nucleic acid detection, can solve the problems of interference beacon fluorescence recovery, limited application of molecular beacons, etc., to achieve real-time quantitative tracking detection, low cost, Easy-to-operate effects

Pending Publication Date: 2021-10-29
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the inventors found that in the in vitro transcription system, DNA-binding proteins such as RNA polymerase and transcriptional regulators can bind to molecular beacon DNA and interfere with the recovery of beacon fluorescence, which limits the application of traditional molecular beacons in in vitro transcription detection.

Method used

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  • Molecular beacon probe, kit and in-vitro-transcription fluorescent quantitative real-time tracing detection method and application of molecular beacon probe and kit
  • Molecular beacon probe, kit and in-vitro-transcription fluorescent quantitative real-time tracing detection method and application of molecular beacon probe and kit
  • Molecular beacon probe, kit and in-vitro-transcription fluorescent quantitative real-time tracing detection method and application of molecular beacon probe and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Comparison of transcriptional activity of T7 promoter and Trc promoter by fluorescent quantitative real-time tracing method

[0059] In this example, the phage T7 promoter and the Escherichia coli Trc promoter were taken as examples to verify the feasibility and reliability of the above fluorescent quantitative real-time tracer detection method in the relative quantitative detection of promoter transcription activity in vitro.

[0060] 1. Synthesis of T7 promoter and Trc promoter template DNA

[0061] A DNA template (T7 template) containing a T7 promoter sequence and a DNA template (Trc template) containing a Trc promoter sequence were synthesized respectively. The sequences of T7 template and Trc template are shown below, the promoter region is underlined, and the probe binding region is marked with lowercase letters.

[0062] T7 template:

[0063] 5′-CGATCCCGCGAAAT TAATACGACTCACTATAGG GGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT...

Embodiment 2

[0085] Example 2: FAM and TAMRA-labeled probe fluorescence quantitative real-time tracking method to detect the in vitro transcription activity of T7 promoter

[0086] In this example, novel molecular beacon probes labeled with FAM fluorescent group and TAMRA quencher group (quenching and self-excitation) were used to detect the in vitro transcription activity of the phage T7 promoter, and the fluorescence quenching and fluorescence excitation signals were compared. Detection efficiency in this novel fluorescent quantitative real-time tracer detection method.

[0087] 1. Synthesis of T7 promoter template DNA

[0088] A DNA template containing the T7 promoter sequence (T7 template, same as in Example 1) was synthesized. The T7 template sequence is shown below, the promoter region is underlined, and the probe binding region is marked with lowercase letters.

[0089] T7 template:

[0090] 5′-CGATCCCGCGAAAT TAATACGACTCACTATAGG GGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAAC...

Embodiment 3

[0109] Example 3: Fluorescent quantitative real-time tracer method to detect the influence of Escherichia coli transcription factor Fur on fhuF gene promoter transcription

[0110] The Escherichia coli iron homeostasis maintenance system depends on the transcription factor Fur. Fur can bind to iron metabolism gene promoters and repress the transcription of target genes. The iron uptake system gene fhuF promoter contains multiple overlapping Fur protein binding sites. This example takes the fhuF promoter as an example to verify the feasibility and reliability of the above fluorescence quantitative real-time tracer detection method in the detection of transcription factor Fur activity.

[0111] 1. Synthesis of fhuF promoter template DNA

[0112] A DNA template (fhuFp template) containing the Escherichia coli fhuF promoter sequence was synthesized. Its sequence is shown below, the promoter region is underlined, and the probe binding region is marked with lowercase letters.

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Abstract

The invention provides a molecular beacon probe, a kit and an in-vitro-transcription fluorescent quantitative real-time tracing detection method and application of the molecular beacon probe and the kit and belongs to the technical field of molecular biology and nucleic acid detection. According to the invention, a novel molecular beacon probe is designed, the molecular beacon probe at least comprises two molecular beacon probes, and the molecular beacon probes are single-stranded DNAs and can be complementarily paired and bound with an in-vitro transcription product RNA, wherein the near 5' end or 5' end of the first molecular beacon probe is marked with a fluorophore, and the near 3' end or 3' end of the second molecular beacon probe is marked with a quenching group. When the molecular beacon probe is used for carrying out in-vitro transcriptional activity detection, real-time quantitative tracing detection on transcriptional activity of a single promoter or multiple promoters can be realized. The method has no need of purifying the product RNA, has no need of independently designing and synthesizing a probe each time, has the advantages of easiness in operation, economy, rapidness, environment friendliness, good result stability and easiness in analysis, and has a good application prospect.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and nucleic acid detection, and specifically relates to a molecular beacon probe, a kit and an in vitro transcription fluorescence quantitative real-time trace detection method and application thereof. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] In vitro transcription refers to the process of using linear DNA containing a promoter region as a template in a cell-free system in vitro, adding conditions such as RNA polymerase and NTP, and imitating the process of in vivo transcription to generate RNA. The in vitro transcription system can conveniently and accurately detect the transc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/6851
CPCC12Q1/6851C12Q2521/119C12Q2563/107
Inventor 谷立川张冯瑜王晓萌张坤迪王宏伟许素娟
Owner SHANDONG UNIV