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Synthesizing an arbitrary DNA sequence for total gene synthesis is in demand now more than ever. One of the most significant issues in total gene synthesis is that it comes with quite a hefty cost, due to the high-cost of oligonucleotides synthesis as well as post‐synthesis sequencing.

The evolution in this biological process is relatively slow, but a new method promises to cut down on the costs while maintaining the quality of total gene synthesis. 

The affordable two-step gene synthesis method combines dual asymmetrical PCR and overlap extension PCR, allowing DNA sequence synthesis without any present issues. Since it’s a relatively straightforward process, in theory, it’s effortless to automate it, further cutting down on the costs. 

What is Total Gene Synthesis

Total gene synthesis is the process of DNA or RNA replications in its whole. While gene synthesis means reproducing one or the other in their own respective right, total gene synthesis means replicating the whole gene. 

Since DNA isn’t always readily available within an organism, the optimal expression has to be achieved through codon optimization. More often than not, this results in an incomplete DNA or RNA chain, the process, and issues that need to be solved with site‐directed mutagenesis. 

That isn’t only expensive and unpredictable – it’s very time-consuming. All these techniques ramp up the costs of the process. A new process called Total Gene Synthesis is gaining popularity, but it’s still costly. It has to be performed in a couple of different ways, such as:

  • The Fok 1 method
  • An altered form of a ligase chain reaction used for gene synthesis
  • The ligation of preformed duplexes of phosphorylated overlapping oligonucleotides 

What ties all of these methods together is the requirement of phosphorylated, polyacrylamide gel-purified oligonucleotides to achieve the best results. 

The PCR and OE‐PCR Approach

The newer, better, and faster technology includes combining the dual asymmetric DA‐PCR and OE‐PCR to reduce the length of oligonucleotides that are used for the gene assembly process under 25nt, not even requiring a primer sequence or any reaction condition optimization.

When it comes to this method, one of the essential things is enzyme screening. The enzyme screening process solves the mutation problem, which occurs with all other gene synthesis methods, and is the main reason behind the price of the technology.

This method isn’t only the most successful in producing a total gene synthesis promptly – it’s one of the most effective methods of synthesizing genes from 470 bp to 1.2 kb in length. The range associated with this method is up to standard with the other methods but includes a multitude of benefits unique to this approach. 

The whole process is accomplished in a very timely manner, as it takes up to a week from start to finish. Since it’s a relatively simple process in nature, it doesn’t require much human interaction, making it very applicable to automatization. 

In Conclusion

The total gene synthesis process is integral to the advancement of many different forms of biology, specifically synthetic and molecular biology. It aids the exchange of material, provides for more accurate results, and enables the unlimited creation of modular and reusable DNA, RNA, and their subsequent parts.

Since the equipment required to do this whole process is quite expensive and requires an on-site laboratory, many professionals choose to outsource their total gene synthesis needs to professional laboratories that utilize this method. This way, researchers are guaranteed an affordable and timely product with the utmost quality, accuracy, and speed.