The nucleic acid synthesizer is a core equipment for modern molecular biology, genomics, and biopharmaceutical research and development. It can efficiently and accurately synthesize specific sequences of DNA or RNA oligonucleotides (oligonucleotides) in vitro.Its technical principle integrates solid-phase synthesis technology, organic chemistry, and precision automation control, achieving "on-demand writing" of the genetic code of life.

　　1、 Core principle: Solid phase phosphoimine chemical method

The current mainstream nucleic acid synthesis technology is based on the principle of solid-phase synthesis, with the core being the phosphoimine chemical method. This method has the characteristics of high efficiency, high yield, and easy automation, replacing the early phosphodiester and phosphodiester methods. The entire synthesis process is carried out in the synthesis column of the instrument, and the synthesis direction of the target chain is from the 3 'end to the 5' end.

A synthesis cycle mainly includes four basic steps:

1. Unprotected:

The synthesis begins with the first nucleotide covalently attached to a solid support. The 5 '- hydroxyl group of the nucleotide is blocked by a protective group. The first step of the cycle is to rinse the synthesis column with an acid solution to remove the DMT protecting group at the 5 'end of the first nucleotide, exposing the active 5' - hydroxyl group and preparing for the next coupling reaction. The detached DMT cation appears orange and can be monitored for synthesis efficiency online using photometry.

2. Activation and coupling:

Next, the instrument pumps the next nucleotide monomer to be connected and the activator into the synthesis column simultaneously. The nucleotide monomer itself is highly activated, with its 3 'end protected by a phosphoimine group and its 5' end protected by DMT, and corresponding protective groups on the base.

The activator will activate the 3 'phosphoimine group of the nucleotide monomer, making it a good electrophilic reagent. Subsequently, it rapidly undergoes nucleophilic substitution reaction with the exposed 5 '- hydroxyl group on the solid-phase carrier, forming a triphosphate bond, thereby adding new nucleotides to the elongating oligonucleotide chain.

3. Cap:

The coupling step is not 100% effective, and there are always very few 5 '- hydroxyl groups in the chain that fail to participate in the reaction. If these failed sequences are not sealed, they will continue to participate in the reaction in subsequent cycles, producing by-products of missing sequences.

The "capping" step uses a mixture of two reagents to acetylate and permanently inactivate the unreacted 5 '- hydroxyl group. These blocked short chains will be removed during final purification, greatly improving the sequence accuracy and purity of the final product.

4. Oxidation:

The previous coupling formed an unstable triphosphate bond, which is easily decomposed by acid. In this step, a mixed solution of iodine/water/pyridine is introduced to oxidize it into a more stable phosphate triester bond, thus completing a complete nucleotide addition cycle.

Afterwards, the instrument automatically repeats the four steps of "deprotection → coupling → capping → oxidation", adding a new nucleotide in each cycle until the entire target sequence is synthesized.

　　2、 Post synthesis processing

After the nucleic acid synthesizer completes the assembly of the chain, the resulting product is still a crude DNA product connected to the solid-phase carrier and all functional groups are protected. Therefore, further processing is required:

1. Cutting and deprotection: Treatment with concentrated ammonia solution, on the one hand, the synthesized oligonucleotide chain is cut off from the CPG carrier, and on the other hand, all protective groups on the bases and DMT groups at the end of the chain are synchronously removed.

2. Purification: According to the application requirements, it can be purified by high performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE) to remove the failed sequence and by-products and obtain a high-purity final product.

　　3、 Summary

The essence of a nucleic acid synthesizer is a highly automated and precise organic chemical reactor. It decomposes complex synthetic chemical reactions into a series of programmable and standardized steps, and precisely controls the liquid flow rate, reagent dosage, and reaction time by a computer, thereby achieving fast, high-throughput, and precise synthesis of DNA/RNAoligonucleotides. The maturity and popularization of this technology have greatly promoted the rapid development of cutting-edge technologies such as gene editing, PCR detection, nucleic acid drugs, DNA storage, etc.