Over the last few decades, gene synthesis and assembly technology have developed rapidly. Since the 1960s, our capability to synthesize genes has skyrocketed from less than 100bp to more than 1,000,000bp. Gene synthesis methods and applications have a profound impact in metabolic engineering, genetic network design, and vaccine design. However, existing gene synthesis methods still have their fair share of drawbacks and inconsistencies, such as low yield, high error rate, and lack of scalability both on the size of the project and the size of the gene.
Gene Synthesis Methods
Gene synthesis methods are not able to replace each other, and each occupies its own niche depending on the requirements of the project. The following is a brief overview of several common gene synthesis methods:
- Solid-phase synthesis
- Chip-based DNA synthesis
- PCR based enzyme synthesis
The traditional oligonucleotide synthesis uses a small volume of solution processed in a column full of chemicals. The oligonucleotides are synthesized by attaching nucleotide residues stepwise to the end of the chain, one-by-one. The addition of each oligonucleotide consists of four steps: de-blocking, coupling, capping, and oxidation. The integrity of the sequence and the productivity of the synthesis are hindered for products longer than 200bp, and thus this method is generally limited by DNA sequence length. The primary advantage of this method is its high accuracy, compensating for its high expense and low output.
As the name implies, Chip-based synthesis utilizes microarray chips utilizing a series of electrochemical techniques. Different kinds of oligonucleotides are able to be synthesized in different specific parts of the chips, called assembly subpools. Following this piecewise synthesis, gene fragments in subpools are amplified and then aggregated and assembled into the finished product. Chip-based DNA synthesis is cheaper than solid-phase synthesis and can yield a larger amount of the target gene, but its accuracy suffers in comparison.
PCR-based enzyme synthesis generates gene fragments through a variety of cell systems. Using the Yeast system as an example, by using different incision enzymes and label markers, different kind of genes can be added to Yeast chromosomes. Due to the nature of gene insertion the target gene could have no limit to its length as long as the chromosomes can accommodate. This method performs well in synthesizing large gene fragments, and with the help of the cell systems the accuracy of the gene sequence is guaranteed.
Gene synthesis applications
Synthetic genes have wide implications on a variety of fields, ranging from genetic circuits to metabolic improvement to many more. As our technology advances and our understanding of genetics continues to develop, scientists could modify and design genes, The scientists have already synthesized and assembled a gene fragment of over 100kbp. When inserted into a host bacterium lacking its own genetic material, the bacterium was able to successfully produce new cells. Gene synthesis methods can even be used to construct new metabolic systems in living cells. For example, Jay D. Keasling constructed a new metabolic system in E. coli and S. cerevisiae in the mid2000s to produce artemisinin. An important component in some anti-malarial drugs, Keasling’s construct reduced the cost of artemisinin production by tenfold, showing that gene synthesis has a vast amount of potential in medicine and countless other fields.