Gene synthesis is the process primarily implemented in synthetic biology. It involves a group of methods to construct and assemble genes from nucleotides. This process is different from the synthesis of genes from living cells. In this, there is no need for the template DNA i.e.; the artificial synthesis can create any DNA sequence.
The process of gene synthesis consists of two primary steps:
- DNA Printing: This step involves solid-phase DNA synthesis. As a result of this step, there is a formation of several oligonucleotides fragments that are under 200 pairs.
- The second step involves connecting these oligonucleotides using specific DNA assembly methods. In artificial gene synthesis, it is possible to create a DNA molecule with any number of nucleotides because it doesn’t involve the need for a template DNA.
Gene synthesis is one of those branches of synthetic biology that is quickly picking up pace in research. There have been many discoveries in this field so far, and many are in progress. To get a better understanding of gene synthesis, here are five lesser-known facts about it:
Fact 1 – It is impossible to optimize the noncoding sequence effectively.
Due to the properties of DNA code, it allows for more than one triplet codon as an encoder for the same type of amino acid. As an example, codons CGC, CGA, CGG, AGA, and AGG are responsible for encoding Arginine. But, the frequency in which they occur in different species varies and is known as codon bias.
It has been found that the optimization of codon reduces the high GC content and the repeated regions present in the gene. This means that this process is only possible when the target gene encodes a specific protein. Furthermore, this optimization cannot occur in noncoding stretches of DNA. This is because they do not contain codons that can encode amino acids.
Fact 2 – It is possible to synthesize complete plasmids.
According to research, genes of any length can undergo artificial synthesis to form complete plasmids. In this process, DNA oligonucleotides are assembled to long fragments. Then, these fragments undergo assembly to create a sizeable synthetic gene. Further, using a circularisation process results in the formation of plasmids of varying lengths.
- There are many advantages of synthesizing a plasmid using gene synthesis:
- There is plenty of room to choose elements, including promoter and enhancer, selection makers, the origin of replication, restriction sites, polyadenylation signals, and many others.
- There is a choice to choose a tailor-made multiple choosing site (MCS) as per your project requirements.
If you want to make use of plasmid commercially, there is no potential licensing issue.
Fact 3 – Codon optimization is possible in gene synthesis.
Codon optimization is used with gene synthesis to achieve improvement in the gene present in a heterologous system. The expression rate of the transfected genes in transformed organisms can be lower than that of the organism of origin. This is due to the property of codon bias.
It is also possible to produce full-length cDNA through gene synthesis. A long time ago, the process of gene synthesis required intensive labor, cost, and time. But, nowadays, the advanced systems and technology enable production in less than 48 hours.
Fact 4 – Subcloning of the insert into the vector even if enzyme sites are missing from the multiple cloning site (MCS) is possible.
This is a fact that in gene synthesis, you only need a vector with only two restriction enzymes that match the MCS. Further, this gene insert can be PCR-simplified with the help of primers producing regions at the end of PCR products that are generally homologous to the vector. Then the subcloning of these regions via sequence and ligation is done into the vector.
It is worth noting that a restriction site present in the insert does not affect subcloning because there will be no cutting of the PCR product during the process. The process of SLIC is as fast and efficient as the traditional process of subcloning using restriction enzyme sites.
Fact 5 – Even if the standard vector contains sites for restriction enzymes, there will be no problem in downstream cloning.
Modern methods of gene synthesis have scientific designs that use only a few restriction enzyme sites in the MCS. This means that even if a vector for your restriction enzymes occurs within the system, there will be no problem in cloning. The resulting fragment will be so tiny that it won’t be visible on an agarose gel after the restriction enzyme digestion process.
If the restriction site in the MCS is at least 4bp (in most cases >10bp) away from the gene, no issue in restriction enzyme digestion will occur.
