Nucleic acid probe preparation method

A nucleic acid probe and nucleic acid technology, applied in the field of molecular biology, can solve the problem that the molar yield of the liquid-phase amino-activated ester addition method is not high, the liquid-phase amino-activated ester addition method is low in yield, and difficult to synthesize active phosphorous. amide monomer and other problems, to achieve the effect of shortening the reaction time, improving the utilization rate of materials, and improving the use efficiency

Active Publication Date: 2022-06-28
百力格生物科技(上海)股份有限公司
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  • Summary
  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

Generate more waste of organic solvents
[0016] (3) The ratio of modified dye to DNA/RNA substrate is about 3:1, but because the reaction is a water-containing reaction, although succinimide activated ester modified dye can preferentially react with DNA/RNA with amino structure, succinimide Imide-activated ester modified dyes can also undergo side reactions with water quickly, and the final molar yield of the liquid-phase amino-activated ester addition method is still not high, most of which are between 20-30%, that is, the use of modified dyes rate, only 7-10%
Material costs are still too high
[0017] (4) Due to the complex package of the internal structure of DNA/RNA, when DNA contains more than 50 deoxynucleotides and RNA contains more than 40 nucleotides, it is difficult to combine the amino structure with succinimide-activated ester-modified dyes Contact

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0042] Example 1 The basic principle of the method of the present invention

[0043] Amorphous flake-like solids of DNA / RNA with amino groups and succinimide-activated ester-modified dyes of crystalline solid-like solids are mixed and ground through solid mixing, which can make the two chemicals fully contact and provide during the grinding process. Sufficient heat; because there is no solvent involved, the reaction has the best concentration effect; at the same time, because the water phase is not used in the reaction, the succinimide activated ester modified dye will not be degraded, and the side reactions are greatly reduced, so that the reaction can be fully carried out.

[0044] The reaction chemical equation of the present invention is as follows:

[0045]

Example Embodiment

[0046] The specific implementation is as follows:

[0047] Step 1, mix 20umol DNA / RNA with amino active group and 60umol succinimide activated ester modified dye solid, perform mechanical grinding at room temperature, fully grind the two solids for 20 minutes, the solid becomes gel during the process shape.

[0048] Step 2, directly dissolving the colloidal substance in 9 ml of ultrapure water, transferring it to a high performance liquid chromatograph, and using 0.1 M triethylamine acetate and acetonitrile for HPLC purification to obtain a pure product.

[0049] The principle of the present invention is:

[0050] (1) Friction generates heat during the grinding process, which makes the Brownian motion of the substance more intense at the molecular level, which helps the reaction to proceed in a positive direction.

[0051] (2) The crystal form of the solid is destroyed during the simultaneous grinding process, and a uniform colloid is gradually formed during the reaction pro...

Example Embodiment

[0052] Example 2. Mixed grinding ratio experiment of DNA / RNA solid with amino active group and succinimide activated ester modified dye solid

[0053] The present invention also screened the relative ratio of the solid-phase amino-activated ester grinding addition method reaction substrate, and the specific embodiments are as follows:

[0054] 1. Get the DNA / RNA solid of 10umol amount and put it into the mortar of 4cm inner diameter;

[0055] 2. Add 10-40umol different amounts of succinimide activated ester modified dye solids in the above-mentioned mortar;

[0056] 3. At room temperature, grind with a mortar rod with a diameter of 1.8 cm for 20 minutes, and the solid forms a viscous gel during the process;

[0057] 4. Add 9ml of ultrapure water in batches on the mortar, dissolve and transfer the above gelatinous substance to a 10ml centrifuge tube;

[0058] 5. The dissolved colloid was directly loaded on the high performance liquid chromatography purifier, and 0.1M triethyl...

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Abstract

The invention provides a preparation method of a nucleic acid probe, which comprises the following steps: mixing and grinding a nucleic acid solid with an amino active group and an activated ester modified dye solid capable of reacting with the amino group, and carrying out one-step reaction to obtain the nucleic acid probe. The nucleic acid solid and the dye solid are mechanically mixed and ground into jelly, and no reaction solvent or other reaction reagent is added in the process. The preparation method of the nucleic acid probe is a solvent-free reaction, does not need a toxic organic solvent, and is more green and pollution-free; and the production process is greatly simplified, and production can be amplified by combining the ball mill.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for preparing a nucleic acid probe. Background technique [0002] Single-stranded DNA and RNA are composed of a certain number of deoxynucleotides or nucleotides. Some modifications to the structure of DNA or RNA form DNA modified probes and RNA modified probes. DNA-modified probes and RNA-modified probes are widely used in molecular diagnostics QPCR (real-time fluorescence quantification), STR (short tandem repeats), and FISH (fluorescence in situ hybridization). [0003] Generally, there are two common methods available for the engineering of single-stranded DNA and RNA. One is the solid-phase phosphoramidite triester method, and the other is the liquid-phase amino-activated ester addition method. The traditional liquid-phase amino-activated ester addition method is to dissolve DNA or RNA containing amino-active groups in an alkaline buffer, and then add 3...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/11
CPCC12N15/10C12N15/11
Inventor 李星豪蔡晶晶苏敏李俊
Owner 百力格生物科技(上海)股份有限公司
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