Nucleic acid probe preparation method

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...

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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 reaction, liquid phase amino activated ester addition method has extremely low yield

[0022] (1) The solid-phase phosphoramidite triester method is a solid-liquid two-phase reaction, the ratio of the required modification dye to the DNA / RNA substrate is more than 20:1, and the molar yield is about 30-40%, that is, From the perspective of modification dye utilization rate, it is only 1.5-2%, and the waste of material cost is huge

[0023] (2) The reaction process undergoes four-step chemical synthesis, using a large amount of organic solvents and producing harmful waste liquid

For some modified probes with complex structures (such as CY5, ROX, etc.), it is difficult to synthesize active phosphoramidite monomers, or it is limited by the subsequent process, which limits the use of solid-phase phosphoramidite triester method to synthesize modified probes. application

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