08. Neuroscience

Gene Synthesis: 5 Different Methods And Applications

February 13, 2023

Chemical gene production or synthesis is a crucial pillar in modern molecular biology, helping to produce whole native genes and novel genes (those that don’t occur naturally). Moreover, the process is the basis for producing entire genomes (the complete set of genetic instructions present in a living cell).

Advances in gene synthesis technologies mean diverse gene production methods are available to interested parties. However, each method has its niche application, synthesizing specific genes, and one technique cannot substitute another. Therefore, below is an overview of the common gene synthesis techniques and their characteristics to guide your method choice for different projects.

Gene Synthesis Protocol?

Synthetic gene production is a stepwise process that facilitates the production of a gene and other gene products without relying on a DNA template. Therefore it facilitates the production of diverse genes, including custom genes with modified sequences or base pairs.

As stated above, advances in biotechnology mean that multiple synthetic gene production techniques exist. However, all the techniques borrow from a living organism's native gene production process as the basis for chemical gene production, with slight modifications here and there.

Therefore, understanding the basic gene synthesis process helps appreciate the nuances among gene synthesis techniques. Below is an overview of the steps involved in gene synthesis.

Oligonucleotide synthesis

Oligonucleotides are short nucleic acid (DNA or RNA) strands and function as the building block for any gene product production, including peptide and protein synthesis. Different gene synthesis methods employ different reagents and techniques to initiate oligonucleotide synthesis. However, the process moves in a 3’ to 5’ direction in all the methods.

Oligonucleotide annealing

Annealing entails heating molecules like oligonucleotides before cooling them gradually to facilitate hybridization or the formation of a chemical bond between two molecules. Different gene synthesis methods employ unique annihilation techniques to form a complete gene sequence.

Gene sequence cloning

Cloning entails replicating copies of the newly-formed gene sequence using a cloning vector.

Clone screening

Gene synthesis is not a perfect process. Therefore, clone screening is necessary to identify the target gene within the clones. Popularscreening tools include ELISA kits and chromatography.

Sequence analysis and error correction

Besides identifying the target gene, a thorough analysis of the base pairs in the sequence is necessary. Moreover, corrective measures to rectify replication errors like base deletion and substitution ensure desirable plasmid placement.

Methods and Applications

Below is an overview of the most popular gene synthesis methods and applications.

1. Solid-phase Synthesis

Solid-phase synthesis is a classic gene synthesis method and entails using chemically-modified nucleosides, including locked nucleic acids(LNAs), to synthesize target oligonucleotides. A reagent column containing a deblocking acid holds the nucleosides that gradually form an oligonucleotide chain with the deprotection of subsequent nucleosides.

The enzymatic assembly process entails de-blocking (deprotection) of the nucleosides, followed by coupling, capping, and oxidation to form a gene sequence from the newly-formed oligonucleotides. Solid-phase synthesis is a fully-automated process, and researchers collect the genes at the end. Its advantages include a remarkably high gene sequence accuracy.  

However, the deprotection process increases the chances of side reactions, and the risks increase with increasing length. Therefore, solid-phase synthesis only produces genes of 15-25 bases in length (200 nucleotide residue maximum). Such genes have applications in molecular biology and medicine, including as antisense in protein synthesis or as probes for detecting complementary genetic matter.

2. Chip-based DNA Synthesis

Chip-based DNA synthesis is a next-generation gene synthesis process. Unlike solid-phase synthesis, which is a chemical process, chip-based synthesis is an electrochemical process.

The method utilizes microarray semiconductor chips fitted with temperature controls to generate multiple oligonucleotides within one setting. Chip-based synthesis complements the traditional phosphoramidite cycle chemical process by creating pockets of temperature-controlled zones called virtual wells/ islands, facilitating selectivity.

Moreover, it facilitates error detection and correction during the oligonucleotide assembly process and does not require a separate sequence analysis and error correction stage. Chip-based synthesis’ advantages include high throughput and the capacity to generate gene fragments with longer base pairs. The technique produces gene sequences for applications requiring a high target DNA volume and low accuracy.

3. PCR-based Enzyme Synthesis

PCR (polymerase chain reaction) gene synthesis is a classic process that produces millions of gene fragments in two stages that utilize primers. The first stage is assembling overlapping nucleotides through a self-priming chain reaction to generate a 60bp oligonucleotide, covering the entire sequence.

Second, a subsequent PCR reaction generates DNA fragments 400-500bp long. An additional primer amplifies the target DNA fragment. The method is ideal for applications that require high-accuracy, long gene fragments.

4. Gene Synthesis From Array-derived Oligonucleotide Pools

Array-derived gene synthesis is arguably the most affordable gene production process due to its low reagent consumption. Second, the method accommodates a multiplex capacity, producing thousands to tens of thousands of oligonucleotide sequences.

However, while the diverse oligonucleotide sequences are a plus, assembling the oligonucleotides into viable gene fragments is challenging due to sequence homology. Therefore, the method is ideal for custom gene synthesis processes requiring much lower quantities of gene fragments.

5. Liquid-phase Gene Synthesis

Liqui-phase gene synthesis is also a classic technique, similar in many elements to solid-phase synthesis. However, unlike solid-phase synthesis, oligonucleotide generation occurs in a solution rather than in column support. Also, liquid-phase gene synthesis has a lower side-chain reaction risk and can generate long DNA fragments with minimal errors, albeit slower.

Conclusion

Gene synthesis techniques are constantly evolving to meet the rising demand for high-quality genes, cost-effectiveness, and scalability. The methods highlighted above are the primary gene production methods, and you can consult your service provider on the ideal method for your project and budget.

Witness the benefits of NeuroTrackerX. Start Today!