Gene synthesis is the process of synthesizing DNA strands. It follows a base-by-base synthesis process by relying on the single strand molecules to function as a template for which a nucleotide is added systematically. Gene synthesis has become a common procedure in various fields such as research, medicine, and genetic bioengineering. There are multiple steps involved in the synthesis process; however, all the methods involve the first step, which requires the generation of a building block needed for enzymatic assembly. Beyond this stage, the procedures vary based on the chemicals and technologies involved.
There are different gene synthesis methods, and they vary depending on factors such as the complexity and the length of the DNA. Other factors such as automation capabilities and intellectual property rights also determine the methods to avoid further issues such as gene copying. The technologies utilized depend on the chemical synthesis process involved. Here are some of the most common gene synthesis methods.
Polymerase Chain Reaction is one of the most common and unique Gene Synthesis methods. It is unique due to the initial reaction involving DNA polymerase properties used to create a new DNA strand. The DNA polymerase strand needs a primer due to its ability to add a nucleotide to the established 3′-OH group. This allows researchers to target a specific section of the DNA; one can choose a particular gene within the strand and then synthesize it. The researcher can then produce numerous DNA sequences using the PCR method. This method is effective for targeting gene synthesis for large-scale applications.
However, this method has certain shortcomings. The method is vulnerable to various errors due to mutation introduced or can occur during the oligonucleotide synthesis and sequencing. Despite this error, this approach is ideal for the commercial synthesis process; one of its commercial applications is the sequential chain reactions to produce multiple genes. To address some of the errors with the PCR methods, researchers propose the combination of dual asymmetrical PCR and overlap extension PCR.
Total gene synthesis
This process is rapidly becoming one of the most preferred gene synthesizing methods and is suitable for both engineered and natural DNA assembly and sequencing processes. However, setting up the process and creating the final gene requires higher costs. This process also solves other challenges associated with other processes, which suffer from extensive time and cost needed due to changing various nucleotides. It optimizes the codon optimization to promote efficient expression.
The process is different from others due to its reliance and dependence on the universal double-stranded DNA, the main building block. This process is highly automated, and it depends on robotics to assemble desired genes using processes such as standardized biochemical processes. The process is superior to other gene synthesis processes due to the high dependence on technology and automation. Nearly the entire process is computerized and standardized. It also relies on top-quality raw materials to produce quality genes. High dependence on computerization makes this process efficient and reliable and can be applied to complex and time-consuming genes to synthesize.
The process is also referred to as the solid-phase method. It is unique since it uses a 5’ hydroxyl terminus on which nucleotides are attached and added to create an oligonucleotide chain. The process occurs in various stages, such as de-protection, capping, oxidation, and coupling. The capping process ensures only one nucleotide is added to the sequence. The cap ensures the entire process is systematic, and you have the freedom to remove and refresh it before continuing with the synthesis process. The process is ideal for only short oligonucleotides since the probability of errors increases as the length of the oligonucleotide increases.
The process involves the creation of a chain in the 3’ to 5’ direction. At the end of the chain, the researcher will need to remove the protecting groups. Beyond the 5’, the process becomes vulnerable to defects, making the sequence shorter if you anticipate quality outcomes. This process is highly accurate despite being expensive and suitable for low output.
This is the latest gene synthesis method, and it heavily depends on the power of manufactured computer chips to accomplish the synthesis process. Other processes follow the assembling process, which involves assembling the strands to the leading DNA strand. The chip-based method uses a different approach by relying on heat-controlled and electrochemical technologies.
The technologies allow the targeting of various independent temperature control sites to link them with reagents to create oligonucleotides that are highly selective through the Phosphoramidite cycle. This method is relative since the researchers can quickly identify the errors and rectify them during the assembly process. The result leads to a highly effective double-stranded DNA.
Other gene synthesis methods
Other methods include the Fok I method, modified ligase chain reaction method, etc. These processes are less popular since the results are unreliable unless one adds additional elements such as phosphorylated. These methods are also time-consuming, labor-intensive, and expensive; hence they are unsuitable for commercial applications.
There are various applications of gene synthesis processes, and they will become popular in the future due to increased interest in fields such as bioengineering. These gene synthesis methods all have their shortcomings hence the need to select the one that meets your requirement with limited errors. A good synthesis process should allow for large-scale synthesis with few errors and rely heavily on automation to reduce the mistakes and time needed to complete the whole process.