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Preparation of a DNA quadrangular pyramid for visual detection of tumor-associated mRNAs in living cells

A tumor-related, quadrangular pyramid technology, applied in the field of biosensing, can solve the problems of time-consuming and labor-intensive, difficult to achieve real-time detection of single cells, and a large number of sample cells, so as to avoid false positive signals, save costs, and achieve good biocompatibility. Effect

Active Publication Date: 2022-04-08
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are not only time-consuming and laborious, but also require a large number of sample cells, making it difficult to achieve real-time detection of single cells

Method used

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  • Preparation of a DNA quadrangular pyramid for visual detection of tumor-associated mRNAs in living cells
  • Preparation of a DNA quadrangular pyramid for visual detection of tumor-associated mRNAs in living cells
  • Preparation of a DNA quadrangular pyramid for visual detection of tumor-associated mRNAs in living cells

Examples

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Embodiment 1

[0026] A method for preparing a DNA pyramid for visual detection of tumor-associated mRNA in living cells, comprising the following steps:

[0027] 1) Take equimolar S1 and CP, heat in a metal bath at 95ºC for 5 min, and naturally cool to room temperature to obtain mixture 1; then take equimolar S3 and CP to prepare as above to obtain mixture 2;

[0028] 2) After mixing mixture 1 and mixture 2, add equimolar S2 and S4, and anneal in the PCR instrument according to the following procedure: heat at 50ºC for 10 minutes, cool to 25ºC (3min / ºC), and hold at 4ºC for 60 minutes to obtain DNA pyramid at a final concentration of 1 μM;

[0029] 3) Add different concentrations of c-myc target to measure fluorescence;

[0030] The specific operation method in step 3) is: in the buffer system containing 1×TAE / Mg2+, mix different amounts of the target substance with DNA at a concentration of 200 nM, so that the final concentrations of the target are 0, 10, 20, 30, 40 and 50 nM. Put all t...

Embodiment 2

[0032] A method for preparing a DNA pyramid for visual detection of tumor-associated mRNA in living cells, comprising the following steps:

[0033] Step 1), 2) are the same as in Example 1

[0034] 3) The ability of DNA pyramids to avoid false positive signals. Add DNase I to the buffer containing DNA pyramids so that the final concentrations are 0.5 U / mL and 5 U / mL respectively, and incubate at 37 ºC in the dark for 0, After 10, 20, 30, 40, 50, and 60 min, measure the fluorescence intensity of the sample in the range of 540-570 nm under the excitation light wavelength of 525 nm, and the results are as follows image 3 shown. It is a well-established fact that DNA is easily degraded by nucleases ubiquitous in biological systems to generate false positive signals. Compared with single-intensity-based sensing probes, FRET-based fluorescence ratiometric signals can effectively avoid false positive signals due to nuclease digestion, protein binding, or thermodynamic fluctuations...

Embodiment 3

[0036] A method for preparing a DNA pyramid for visual detection of tumor-associated mRNA in living cells, comprising the following steps:

[0037] Step 1), 2) are the same as in Example 1

[0038] 3) In cell imaging experiments, MCF-7 cells were inoculated with DMEM medium in culture dishes and incubated in 5 % CO 2 Incubate for 24 hours at 37 ºC in an incubator. Add 100 nM of DNA to MCF-7 cells at 37 ºC and 5 % CO 2 Incubate for 4 h. Wash multiple times with PBS to remove excess sample. Under a confocal microscope, confocal fluorescence images of cells were obtained by laser scanning, the results are as follows Figure 4 shown. A clear FRET signal was observed in MCF-7 cells, indicating the presence of the target C-myc mRNA.

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Abstract

The invention discloses a method for preparing a DNA quadrangular pyramid for visually detecting tumor-associated mRNA in living cells. The DNA quadrangular pyramid is composed of two capture probes and four DNA support probes with fluorescent labels through a two-step annealing reaction Self-assembled. When the target is not present, the rigid double strands supporting the probe keep the DNA pyramid in an open three-dimensional structure; when the target exists, the target strand triggers a strand displacement reaction, and the capture probe leaves and captures the target. At the same time, the single strand at the bottom of the DNA pyramid automatically folds to form a more stable hairpin structure, which drives the overall spatial structure of the DNA pyramid to change. Cy3 and Cy5 labeled at the two ends of the base of the DNA quadrangular pyramid approach with the folding process until fluorescence resonance energy transfer occurs. This work presents another dimension to the visual detection of specific mRNAs in cells and may also open new avenues for the detection of many other biomolecules.

Description

technical field [0001] The invention belongs to the technical field of biosensing, and in particular relates to a method for preparing a DNA pyramid for visually detecting tumor-associated mRNA in living cells. Background technique [0002] Tumor-associated mRNAs are widely used as specific biomarkers to reveal information about the occurrence, development and metastasis of malignant tumors. Therefore, the detection of tumor-associated mRNA in cells is of great significance for the early diagnosis, treatment and prognosis of cancer. To date, many methods have been developed to detect these mRNAs, such as Northern-blots, microarray analysis, and reverse transcription polymerase chain reaction (RT-PCR). However, these methods are not only time-consuming and laborious, but also require a large number of sample cells, making it difficult to achieve real-time detection of single cells. Fluorescence microscopy-based molecular imaging, characterized by high selectivity, high reso...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6818C12N15/11
CPCC12Q1/6818C12Q2563/107C12Q2565/101
Inventor 陈宪文俊梅陈果
Owner FUZHOU UNIV
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