Preparation method of high-efficiency low-toxicity fluorescent nucleic acid probe

Abstract

The invention relates to the technical field of preparation of nucleic acid detection materials, and in particular relates to a preparation method of a high-efficiency low-toxicity fluorescent nucleicacid probe, and the method comprises the following steps: preparing a bromo-3,8-diamino-5-ethyl-6-phenylphenanthridine methylene chloride solution A with a certain concentration; preparing a tetraphenylboron salt or tetraphenylboron derivative salt solution B with a certain concentration; mixing the solution A and the solution B according to a certain volume ratio for shading reaction until the reaction is complete; after the reaction is completed, adding water, performing liquid separation, extracting a dichloromethane phase with a dichloromethane solution, and collecting; performing columnchromatography separation on the collected dichloromethane phase after water is removed, collecting a first red fluorescent color band, and drying to obtain the high-efficiency low-toxicity fluorescent nucleic acid probe. The high-efficiency low-toxicity fluorescent nucleic acid probe prepared by the preparation method makes up for the defects of ethidium bromide in light stability and cytotoxicity, and meets the requirements of scientific researches on DNA fluorescent probes with low toxicity, high sensitivity, high stability and low price.

Description

technical field [0001] The invention relates to the technical field of preparation of nucleic acid detection materials, in particular to a method for preparing a highly efficient and low-toxic fluorescent nucleic acid probe. Background technique [0002] Fluorescence sensors have received great attention in recent years due to their fast response, high sensitivity, and easy operation. The characteristics of the fluorescent sensor make it possible to make up for the shortcomings of instrumental analysis, and have obvious advantages for timely detection and on-site emergency monitoring, especially the application of filter paper strips, which has an attractive prospect. However, due to the rigid planar structure of traditional fluorescent sensors, the formation of π-π interactions in the molecules leads to aggregation-induced fluorescence quenching (ACQ), and the fluorescence intensity decreases sharply or even disappears at high concentrations or in solid state. Scientists h...

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Problems solved by technology

However, these commercialized molecules still have many insurmountable shortcomings and cannot fully meet the needs of nucleic acid detection. [2-4]

For example, the SYBR series still has cytotoxicity, poor photostability, no staining ability for DNA fragments below 50bp, and low detection sensitivity for DNA fragments below 100bp; the acridine orange (AO) molecule used by Goldview has high cytotoxicity and mutagenicity, and has high staining background and low sensitivity; the Gelred series is expensive, which greatly increases the detection cost; although the double EB (EthD) can greatly improve the detection limit of EB, it also increases The molecular toxicity of EB, and the existence of cross-linking coordination, affects the accuracy of DNA fragment detection, etc.

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