Solid-phase synthesis in the amplification and sequencing of nucleic acids

The Oligofastx project covers the different phases of the development of oligonucleotide-based therapies, from design to production, and this is one of the reasons why the consortium is made up of seven companies, each specializing in different phases of the process.

Today we talk about solid-phase synthesis.

Oligonucleotides are short chains of different “building blocks” that are ribonucleosides (a sugar (ribose) and a phosphate group) with different nitrogenous bases. These synthetic oligonucleotides are used in a wide range of applications, from research to therapy. The chemical synthesis of oligonucleotides is based on the solid-phase method, which has been used since the 1970s.

Today, this process is performed by automated equipment that builds an oligonucleotide by adding base by base to complementary ribo¡nucleotide residues in a targeted manner. This technique results in high yields and allows large quantities of material to be produced quickly and cost-effectively. Although there have been many advances in nucleic acid synthesis techniques over the years, many improvements are still being made.

First, chemical synthesis of oligonucleotides has long been used to synthesize oligos for molecular biology research, including the preparation of labeled probes, antisense oligos, aptamers and small interfering RNAs.

In addition, although oligonucleotide synthesis is now considered a mature technology, advances continue to be made in the sequencing process. For example, techniques have been developed to sequence DNA and RNA by chemical means: this method is called chemical amplification because it uses chemically modified nucleotides (nucleotide analogs) that are released from the DNA parent strands during the sequencing reaction, thus enabling correct gene sequencing.

Synthetic chemistry techniques are also used to synthesize other useful molecules, such as peptides and proteins. For example, researchers have devised an efficient chemical method to generate unnatural amino acids and also recycle the by-product for further use (circular economy). This development could enable more agile processes to produce proteins and other biologically active molecules.

Synchronized fluorescence excitation spectroscopy (SFES) is a technique for examining enzymatic reactions through real-time detection provided by fluorescence intensity measurements. It has been successfully used to study various enzymatic reactions as well as nucleic acid hybridization and fusion.


The basic premise of SFES is that an enzyme catalyzes an irreversible reaction at a given rate, which can be followed in real time by changes in the electromagnetic spectrum of light emitted by a fluorophore substrate after being excited by laser light. The reaction occurs on both sides of a double-stranded DNA molecule, resulting in two distinct emission bands on each strand. These emission bands correspond to different base pairs within the DNA helix structure at two different points along its length: one band corresponds to the G residues located near one end of the strand (the 5′ end), while another corresponds to the C residues located near its other end (the 3′ end). This means that if you know how much time has passed since the beginning of the experiment, you can measure these optical properties over time and use them as indicators showing where in the DNA molecule a certain chemical event has taken place during this period and, therefore, what types of chemical reactions have also taken place during this period.

In summary, the chemical synthesis of nucleic acids is an important part of many scientific applications. Oligonucleotide synthesis is used in molecular biology research; for example, it can be used to determine the sequence of DNA molecules. Synchronized fluorescence excitation spectroscopy is a technique for examining enzymatic reactions by monitoring changes in fluorescence intensity as substrates or products are added to or removed from the reaction mixture.