DNA synthesis technology is to synthesize any artificially designed sequence without relying on a DNA template. In recent years, with the rapid development of synthetic biology-related technologies, the artificial de novo synthesis of DNA has been of great significance in the field of biology, providing strong support for the design and transformation of various biological systems, and the elucidation of gene functions. It has more and more extensive applications in scientific research at different levels, promoting the further development of bioinformatics, genomics, precision medicine and other disciplines, and promoting the progress of life science research.
In the 1960s and early 1970s, chemists developed a range of DNA synthesis techniques. Khorana’s group carried out the earliest enzyme-based gene synthesis, such as tRNA gene synthesis in the 1960s. By the 1970s, the chemical synthesis of short genes (<1.0 kilobase pairs, kb), such as the lac operon and the gene encoding somatostatin, were reported one after another. Beginning in the 1980s, with the development of oligonucleotides with the development of synthesis technology (>100 bases (nt)), the chemical synthesis efficiency of DNA has been further improved. Some oligonucleotide-based DNA synthesis and assembly methods have been reported successively, including enzymatic ligation assembly methods, polymerase chain reaction-dependent assembly methods assembly method and ligase chain reaction (LCR)-dependent assembly method.
The development of PCR technology in the 1990s led to the rapid development of chemical synthesis and assembly technology based on PCR technology. In 2003, Gao et al. described a gene synthesis method that is thermodynamically balanced inside-out (TBIO). In 2004, Young and Dong et al. developed a two-step gene synthesis method that combined the dual asymmetrical PCR (dual asymmetrical PCR) method and the overlap extension PCR (OE-PCR) method. In the same year, Xiong et al. established a PCR-based two-step DNA synthesis (PTDS) method. In addition, the development of DNA synthesis equipment has also led to the continuous development of the high-throughput synthesis of oligonucleotides.
After 2000, the scale of gene synthesis has risen to the genome level. In 2002, a functional poliovirus genome (about 7,500 kilobase pairs, bp) was successfully synthesized by chemical methods. In 2003, Smith et al. completed the synthesis of the phi X174 phage genome (5,386 bp) in two weeks, which would have taken months. In 2010, Venter and Smith successfully replaced the genome of Mycoplasma capricolum with the artificially synthesized Mycoplasma mycoides genome. Through genome replacement, cells of one species were changed into cells of another species, representing scientists in the creation of new cells based on artificially synthesized genes. A historic step on the path of cells. In 2019, Halpain et al. reported a DNA polymerase-based method that also enables DNA synthesis using transient oligonucleotide hybridization.
The current artificial DNA synthesis technology can synthesize oligonucleotides with tens to hundreds of bases and synthesize Mb-level microbial genomes by chemical synthesis, combined enzymatic splicing and microbial cloning.
Applications of DNA Synthesis
Synthetic DNA can be used to construct gene networks, metabolic pathways, and even complete genomes, and even artificially create a new living system. J.Craig Venter’s research group has completed the artificial synthesis of a megabase pair bacterial genome and replaced the genetic information of the bacterial cell host itself, successfully creating an artificial living cell. This sets a new milestone in the field of synthetic biology. In the future, researchers can use artificially constructed biological systems to benefit mankind, such as the production of green energy fuels, cheaper and more effective new drugs, and the development of new gene therapy methods for some difficult diseases (such as cancer).
The same amino acid can be represented by different synonymous codons, and different organisms use different codons to synthesize amino acids. Different organisms will have their corresponding preferred codons, and some even use rare codons, so the use of different codons plays a very important role in protein heterologous expression. Therefore, when heterologous expression of proteins, researchers will select codon-optimized DNA as genetic information to optimize expression levels in the new host.
The DNA synthesized in situ on the chip can be directly used in protein engineering to optimize protein expression. Quan et al. used an in-situ gene synthesis strategy to optimize codons and improve protein expression. They synthesized an oligonucleotide library encoding the same gene but with different codon versions, and assembled the full-length gene directly on the chip, followed by protein expression screening. Through this approach, Quan et al. optimized the expression of 74 transcription factors from Drosophila melanogaster.
A very valuable application area of DNA synthesis is metabolic engineering. By engineering metabolic pathways in cells, researchers can reprogram cells to obtain valuable metabolites, such as low-cost drugs or prodrugs. Currently, when engineering complex gene regulatory circuits or entire metabolic pathways, de novo DNA is only a small fraction of the total sequence required, and most protein-coding genes are cloned from natural sources. However, Keasling and his colleagues successfully constructed a metabolic pathway for the biosynthesis of artemisinin precursors in yeast by synthesizing DNA de novo, reducing the cost of this antimalarial to ten times the cost of plant extraction methods. one part.
Existing DNA synthesis techniques allow researchers to construct complete genomes, and viral genomes are relatively small and are very useful engineering targets for developing vaccines. It is worth noting that compared to other codon optimization strategies used to optimize heterologous protein expression, the virus was developed using an opposite strategy, through the codon elimination optimization process, to create an attenuated virus. Coleman and his colleagues developed a method called SAVE (synthetic attenuated virus engineering) and succeeded in producing an attenuated strain of the influenza virus.
Future of DNA Synthesis
The development of DNA synthesis technology has made life sciences move from the observable, understandable, and descriptive digital life era brought by sequencing to the predictable, quantifiable, and creatable synthetic biology engineering era. The development of DNA synthesis technology has promoted the development of metabolic engineering, enzyme engineering, antibody engineering, IVD diagnosis, oligonucleotide drugs, DNA data storage and other fields of synthetic biology. DNA synthesis technology for specific applications will bring revolutionary changes to downstream applications.
However, the cost of DNA synthesis is still high at present, the synthesis cycle of large DNA fragments is still relatively long, and the failure rate is high. The development of more efficient large-scale DNA synthesis technology at the genome level will be the key to the application of DNA synthesis from local genetic modification of organisms to large-scale global life reconstruction. More innovative research and continuous efforts are needed.
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