A nucleic acid aptamer for detecting human uveal melanoma cells

A technology for melanoma cells and nucleic acid aptamers, which can be used in measurement devices, DNA/RNA fragments, instruments, etc., can solve problems such as poor prognosis, and achieve the effects of low preparation cost, small molecular weight, and easy labeling

Pending Publication Date: 2018-05-15
HUNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there have been new advances in the diagnosis and treatment of uveal melanoma in the past few decades, its prognosis is still poo

Method used

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  • A nucleic acid aptamer for detecting human uveal melanoma cells
  • A nucleic acid aptamer for detecting human uveal melanoma cells
  • A nucleic acid aptamer for detecting human uveal melanoma cells

Examples

Experimental program
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Example Embodiment

[0021] Example 1: Screening of specific nucleic acid aptamers for human uveal melanoma cells (OCM-1)

[0022] (1) Design of nucleic acid library and primers used:

[0023] Random nucleic acid library:

[0024] 5’-ATCCAGAGTGACGCAGCA(N25)TGGACACGGTGGCTTAGT-3’

[0025] Upstream primer: 5'-Fluorescein isothiocyanate-ATCCAGAGTGACGCAGCA--3';

[0026] Downstream primer: 5'-Biotin-TGGACACGGTGGCTTAGT-3'.

[0027] (2) Positive screening:

[0028] 2.1 Incubation: Dissolve the above random nucleic acid library with binding buffer (D-PBS, 5 mM magnesium chloride), denature it at 95°C for 5 minutes, quickly put it on ice, and let it stand for 10 minutes; Human uveal melanoma cells (OCM-1) were incubated at 4°C for 1 hour.

[0029] 2.2 Dissociation: After the incubation is completed, remove the solution in the incubation dish, wash the cells in the incubation dish with washing buffer (PBS, containing 0.45% glucose, 5 mM magnesium chloride); scrape the incubation dish with 1 mL of sterile water The midd...

Example Embodiment

[0037] Example 2: Identify the sequence with the strongest specific binding ability to the OCM-1 cell line

[0038] First, the adherent OCM-1 cells were digested from the culture dish with 0.2% EDTA, and the cells were collected in centrifuge tubes, and washed with washing buffer (PBS, containing 0.45% glucose, 5 mM magnesium chloride) by centrifugation Several times; secondly, the 5 sequences and random library with a final concentration of 200nM were added to the OCM soaked in binding buffer (D-PBS, containing 0.45% glucose, 5mM magnesium chloride, 100mg / L tRNA, 1g / L BSA) -1 cells; then placed in a shaker at 4°C and incubated for 30min; after the incubation is completed, washed once with washing buffer (PBS, containing 0.45% glucose, 5 mM magnesium chloride) by centrifugation, and perform fluorescence detection by flow cytometry (results see figure 2 ). The test results show that ZH-12 has the strongest binding ability to OCM-1 cells.

Example Embodiment

[0039] Example 3: Identification of target type and determination of its affinity for OCM-1 cells

[0040] First, take 3 OCM-1 cell dishes that have been inoculated for 24 hours, and digest the cells from the culture dish with 0.2% EDTA, trypsin, and proteinase K (with 0.2% EDTA for 1 minute, trypsin and proteinase K) Digestion for 5 minutes), and then collect them in centrifuge tubes (2 tubes for EDTA digested cells, 1 tube for trypsin and proteinase K digested cells), and then wash buffer (PBS, containing 0.45% glucose, 5 mM magnesium chloride) centrifugally wash, then add 200nM ZH-12 to the cells, add 200nM random library to another tube of EDTA digested cells, incubate in a shaker at 4°C for 30min, and wash with washing buffer after incubation Once, and finally use flow cytometry for fluorescence signal detection (results such as image 3 A). The results of flow cytometry showed that the binding amount of ZH-12 and OCM-1 decreased after proteinase K and trypsin treatment, in...

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Abstract

A nucleic acid aptamer used for detecting human uveal melanoma cells (OCM-1) and applications thereof in preparation of detection preparations are disclosed. Compared with the prior art, the nucleic acid aptamer and the applications are advantageous in that the prepared nucleic acid aptamer can specifically recognize the uveal melanoma cells, has good affinity, no immunogenicity, and a small molecular weight, and can be chemically synthesized in vitro; and different positions of the nucleic acid aptamer can be modified and substituted. In addition, the nucleic acid aptamer has advantages of astable sequence, capability of being convenient to store and mark, and the like. When the nucleic acid aptamer is adopted for detecting the human uveal melanoma cells, operation is simpler and rapider. The nucleic acid aptamer has a synthesis cost lower than antibody preparing costs, a short period and good reproducibility.

Description

technical field [0001] The invention relates to a nucleic acid aptamer and its application, in particular to a nucleic acid aptamer which can be used for detection of human uveal melanoma cells and clinical sample tissues and an application method for preparing a detection reagent. Background technique [0002] Uveal melanoma (Uveal melanoma) is the most common intraocular malignant tumor in adults. Its incidence rate accounts for the first place among intraocular tumors in foreign countries, and it is second only to retinoblastoma in China. the second place. Although there have been new advances in the diagnosis and treatment of uveal melanoma in the past few decades, its prognosis is still poor. About 31% of patients die of uveal melanoma within 5 years after diagnosis, and half of them will die after 25 years. of patients died. Metastasis is common in patients, and the liver is the most likely site of metastasis. The cumulative metastasis rate of uveal melanoma patient...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/574
CPCG01N33/57407C12N15/115Y02A50/30
Inventor 叶茂谭蔚泓张慧熊炜
Owner HUNAN UNIV
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