The vast majority of biological and bioengineering research relies on customized synthetic DNA sequences. The design, synthesis, and testing of DNA sequences will help to study the interaction between DNA and proteins, signal pathways, enzyme functions, and rebuild new expression systems.

Figure 1. The synthetic biology test cycle

Single-Stranded DNA Synthesis
Single-stranded DNA (ssDNA) is a basic nucleic acid raw material, which has a wide range of application potential, such as homologous arm sequences used in CRISPR gene editing systems, scaffolds for DNA nanotechnology, carriers for drug delivery, molecular diagnosis, and DNA data storage. To date, the common synthesis methods of ssDNA include chemical synthesis, enzyme synthesis, and bacteria-based synthesis.

Chemical Synthesis
Chemical synthesis is a method to generate ssDNA without a template. Single-stranded DNA fragments smaller than 200 nt are mainly produced by chemical synthesis, such as column-based oligo synthesis and microarray synthesis.

1) Column-Based Synthesis
In the early 1980s, the solid-phase phosphoramidite method and automatic instruments significantly improved the stability and accuracy of oligonucleotide synthesis, and can be used in commercial production. The standard column-based synthesis is a cyclical process, in which the nucleotide chain is extended between the 3′-end and 5′-end, including four processes: deprotection, coupling, blocking, and oxidation. At the same time, for different applications, many different strategies can be used to introduce modifications into oligonucleotides to obtain different functions.

2) Array-Based Synthesis
The yield of oligonucleotide synthesis based on microarrays is usually within the fmol scale, which is two to four orders of magnitude lower than the traditional column synthesis. This low synthesis scale leads to the reduction of the amount of required reagents for synthesis. In addition, each chip has a synthesis density of tens of thousands, which reduces the cost of synthesis per base by a factor of 100-5000 compared to column synthesis.

Figure 2. Methods for solid-phase synthesis of oligonucleotides

Enzymatic Synthesis
Enzymatic synthesis is a fast, stable, and low-cost method of ssDNA synthesis, including the production of ssDNA by ligase or polymerase.

1)ssDNA Synthesis Based on Terminal Deoxynucleotidyl Transferase (TDT)
Terminal deoxynucleotidyl transferase (TDT) is a polymerase with low specificity for nucleotide substrates and does not rely on templates. Deoxynucleotidyl triphosphates (dNTPs) can be added to the 3’end of ssDNA without selection. It is a relatively cheap and rapid method to synthesize ssDNA. A major challenge in TDT synthesis is to control the addition of a single base, because TDT enzymes tend to catalyze the addition of multiple bases per cycle.

2) ssDNA Synthesis Based on Transcription and Reverse Transcription
In vitro transcription and reverse transcription (ivTRT) is a three-step method, which involves preparation of dsDNA templates, transcription of RNA from the dsDNA, and preparation of ssDNA from the RNA. The ivTRT method can be used to synthesize 0.5-2 kb ssDNA. The use of the nuclease in this method will limit its yield and will require a high quality DNA template.

3) Asymmetric PCR (aPCR)
In the aPCR reaction system, there are two kinds of amplification primers with different concentrations, which go through two amplification stages to produce the required ssDNA. The first stage is the exponential amplification of the dsDNA template, and the second stage is the linear amplification of ssDNA. Although aPCR easily produces non-specific amplification and requires a lot of experiments to optimize reaction conditions, it has been successfully utilized to generate ssDNA sequences ranging from hundreds to thousands of nucleotides.

4) Isothermal Amplification of ssDNA
Isothermal amplification technology for ssDNA production depends on enzyme activity or designed primers to avoid thermal denaturation of dsDNA template, including primer exchange reaction, rolling circle amplification, etc. The difference between different isothermal amplification techniques lies in the use of different amplification initiation methods. Therefore, the initiation step is also a key factor limiting the speed and efficiency of the isothermal reaction. Isothermal amplification has the advantages of high sensitivity and simple operation, with excellent performance in the field of DNA detection.

Figure 3. Schematic representation of enzymatic ssDNA synthesis

(a) The mechanism of terminal deoxynucleotide transferase (TdT)-based ssDNA synthesis
(b) The mechanism of transcription and reverse transcription
(c) The mechanism of asymmetric polymerase chain reaction (aPCR)
(d) The primer exchange reaction (PER) cycle and mechanism for ssDNA synthesis
(e) The mechanism of rolling circle amplification (RCA).

dsRNA Synthesis
dsDNA is synthesized by a heat-stable DNA polymerase with complementary overlapping sequences between adjacent oligonucleotides. After sequence verification, the obtained dsDNA fragments can continue to be spliced and assembled into longer DNA sequences, such as large enzyme complexes, the whole metabolic pathway, or genome. In vitro double-stranded DNA synthesis benefits from the development of DNA assembly technology. At present, common DNA cloning and assembly technologies include restriction endonuclease cloning technology, Gibson assembly, Golden Gate assembly, sequence and ligation-independent cloning (SLIC), ligation cycling reaction, paper-clip assembly, yeast assembly, and circular polymerase extension cloning (CPEC). For example, using Gibson assembly, a 16.3 kb mouse mitochondrial genome can be assembled from a 60 mer oligonucleotide group, or multiple 0.5-1 Mbp bacterial genomes can be assembled by using the homologous recombination ability in yeast.

Figure 4. Polymerase chain assembly

One of the standards of gene synthesis is to apply reasonable design principles to the design and assembly of biological components. Therefore, the modular design of DNA components can obtain more kinds of potential constructs through the hybrid combination of DNA components. In addition to simplifying the assembly process, reducing time, labor, and costs associated with construction and testing, it also makes automation possible.

Gene Synthesis | Synbio Technologies
As a leader in synthetic biology technologies, we have independently developed multiple biological intelligent analysis platforms by using leading algorithms and AI technology. Our existing technologies include Complexity Index (CI), AI-TAT, NG Codon, and DNA Studio. These advanced platforms, combined with our annual synthesis rate of millions of base pairs, promote smarter, faster, and more precise gene synthesis. Our professional DNA technology services have enabled the development of synthetic biology, antibody drug screening, genetic engineering vaccine research and development, molecular breeding, and DNA information storage.

[1]Hughes RA, Ellington AD. Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology. Cold Spring Harb Perspect Biol. 2017; 9(1): a023812.
[2]Hao M, Qiao J, Qi H. Current and Emerging Methods for the Synthesis of Single-Stranded DNA. Genes (Basel). 2020; 11(2): 116.