Gene synthesis technologies and techniques are gradually becoming the pillar of molecular biology and play a significant role in synthetic biology. The capability to synthesize new genetic pathways, genes, and even complete genomes is now a reality, and no longer a dream like it was decades ago.
With a thermocycler, DNA polymerases, and commercially synthesized oligonucleotides, a modern molecular biology lab could synthesize numerous kilobase pairs of synthetic genes in just a week by employing existing techniques.
With that said, this article will review the techniques and processes involved in producing synthetic DNA. This article will discuss the custom gene synthesis of oligonucleotides into long sequences and their applications in the real world. Keep reading to find out more about gene synthesis.
What is Gene Synthesis, and How is it Different from Biological Genes?
Gene synthesis is a biological process that involves developing and assembling new unique genes from nucleotides. Also known as artificial gene synthesis, synthetic gene synthesis is quite different from the process of DNA synthesis that takes place in living organisms.
The main difference is that synthetic gene synthesis does not require a prototype DNA. It enables the formation of synthetic DNA molecules that are limitless in the sequence and size of their nucleotides. In addition, you can duplicate and synthesize any sequence you want, regardless of whether it takes place naturally or not.
Steps Involved in Gene Synthesis
Gene synthesis techniques cannot replace each other since every technique covers its niche depending on the project requirements. However, scientists typically employ a five-step process in gene synthesis:
- Oligo design & sequence optimization
- Oligo synthesis
- Entire gene assembly
- Correction & verification
- Preparation of synthetic DNA
Oligo Design & Sequence Optimization
The first stage in gene synthesis is selecting the gene of your preference. When you isolate that gene, you will need to develop the sequence you wish to synthesize. For instance, if you intend to maximize the expression levels of heterologous protein, the best thing to do here is to optimize the codons.
In contrast, following this process may not be effective if you intend to research the internal regulation of DNA expression. These are the most common tools employed in the Oligo design process:
- Gene2Oligo. Its user interface is quite simple and offers both PCR and LCR-oriented assemblies. However, it’s not practical when you have a gene that’s longer than a kilobyte.
- DNA works. It’s straightforward to use and predict if there’ll be Oligo mishybridization. The problem with this tool is that simulation-based outcomes are usually not the exact representation of real success after assembly.
- TMPrime. It provides PCR and LCR-based assemblies and a wide array of annealing and melting temperatures. The problem with this technique is that beginners may find it difficult to operate.
Here, you should begin by creating short oligonucleotides. Usually, scientists do so by employing phosphoramidite chemistry to bring nucleotide monomers together. They blend the phosphoramidite group to 3’O to avoid any branching during the formation process. The phosphoramidite reaction cycle can be broken down into four steps:
Scientists conduct this stage by employing a specialized synthesizer or lab column.
Entire Gene Assembly
The objective of this step is to assemble fragmented Oligos into building blocks for the genomes or entire genes. Four types of assembly are used in gene assembly. They include:
- Ligase Chain Reaction (LCR). This is used to connect the overlapping ends of the assembled Oligos.
- Polymerase Chain Assembly. They blend all one-stranded synthetic Oligos in a single tube, then thermocycling it to enable the blending process.
- In-Vivo homologous recombination. This is great for nucleotides of up to 200 nucleotides.
- Ligation and Sequence independent cloning. Here, a plasmid vector is used to assemble five gene fragments.
Correction & Verification
Here, the developed sequence is verified, and any mistakes found in the process are rectified. This step also involves assessing every sequence before it’s released for use. Any other sequence that has mutations is removed from the pool. Other processes used in quality control include:
- Premature termination
Another process used here is mismatch cleavage, also referred to as mismatch binding. It involves using prokaryotic endonucleases in a process that uses functional assays to rectify incorrect sequences.
Preparing of Synthetic DNA
This is the last step of the gene synthesis process. This is the step where synthetic DNA is prepared for downstream uses. The processes involved in preparing synthetic DNA for user-end applications comprise:
Applications of Gene Synthesis
Presently, researchers can reprogram entire cells and genomes easily and use them in real life. Gene synthesis can be applied in a wide array of applications. They comprise:
- Scientists have used gene synthesis to single out the significant conditions that breast cancer cells thrive in. This has helped create a foundation for developing therapies to treat breast cancer.
- When scientists use artificial gene synthesis to conduct viral sequencing, it helps to produce more effective and safer DNA-based vaccines.
- Plant biologists employ gene synthesis to study developmental reprogramming in the plants’ vascular tissues.
Gene synthesis can be quite advantageous if correctly implemented. The five steps discussed above should guide you through this process to make the most out of it. If you intend to create synthetic genes for whichever use, you may want to contact experts to ensure that you’re following the correct procedure.