A kind of deoxynucleotide primer synthesis method

Abstract

The invention provides a synthesis method of a deoxynucleoside acid primer. The synthesis method of the deoxynucleoside acid primer comprises the following steps: step S1: removing protective radicals; step S2: performing activation and coupling; step S3: performing intermediate oxidation; step S4: performing capping; step S5: washing a residual solution; step S6: repeating the step S1 to the stepS5; and step S7: separating a deoxynucleoside acid primer from a carrier to obtain the deoxynucleoside acid primer. Compared with the prior art, the synthesis method has the following advantages: (1)the primer mutation rate is reduced and quality of the synthesized primer is improved by changing sequence of an oxidation reaction and a capping reaction; (2) only existing synthetic reagents and instruments of deoxynucleoside acid are used, and then the synthesis method can be easily popularized; (3) the method can be used for synthesis of extremely micro-products, and waste of the products isavoided; and (4) a use amount of synthetic reagents is low, and the period of synthesis is shortened.

Description

technical field [0001] The invention belongs to the field of biomolecules, and in particular relates to a method for synthesizing a deoxynucleotide primer. Background technique [0002] At present, the primer synthesis basically adopts the solid-phase phosphoramidite triester method. There are many kinds of DNA synthesizers, no matter what machine is used for synthesis, they are all synthesized by solid-phase phosphoramidite method: the phosphoramidite monomer in the solution forms a 3'-5' phosphodiester bond through condensation reaction, and connects to the solid phase on the carrier. And extend in sequence until the last 5' base of the sequence is connected to the end of synthesis. The whole synthesis process is automatically completed by the instrument, and each cycle is completed in sequence according to five steps of deprotection, activation, coupling, capping and oxidation. [0003] In the existing reaction procedures, the activation and coupling reactions will gen...

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Problems solved by technology

[0003] In the existing reaction procedures, the activation and coupling reactions will generate unstable nucleoside phosphite intermediates. Due to their unstable chemical properties, this step is often the cause of primer mutations; in order to reduce the probability of mutations caused by coupling reactions, Patent CN110115963 reports a new coupling activator

But adopting tetrazolium as coupling activator to participate in the reaction has been very ripe in existing synthesizer application, adopts new coupling activator to be difficult to adapt to existing synthesis instrument and new reagent is also difficult to buy on the market, therefore Difficult to promote further

[0004] Not only that, most of the currently used synthetic primer solid-phase carriers are powdered controllable microporous glass beads (CPG). The process of making the carrier is cumbersome, and the use of CPG synthesis requires a large amount of filling, insufficient reaction with reagents, and low synthesis efficiency. , the amount of synthetic target primers is small, the amount of failed primers is large, the effect is not good, and the cost is high

When using CPG powder as a carrier to synthesize, the carrier produced is generally a fixed amount of powder (usually 4mg), and the synthesized primer is about 5OD, while the general experimental primer only needs 1-2 OD, and some even only need 0.1OD , and it is difficult to sell it again after being preserved as a product, so it seems that this synthesis method will cause a lot of waste

[0005] And the time needed to synthesize a base in the synthesizer is about 8 minutes. If 192 30nt primers are synthesized, the required synthesis time is about 4 hours, and the synthesis efficiency is low.

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