Gene synthesis is a new concept in molecular biology, accelerating preliminary and applied research. The process is also less labor-intensive than traditional gene cloning, yet it facilitates a high-quality yield and consistency in DNA and protein synthesis.
Essentially, custom peptide synthesis allows biologists to source genes further away in the tree of life by making it easier to study gene structure and facilitating gene modification. Such research has facilitated significant strides in application areas like therapeutics, cancer treatment, genetic engineering, and ecosystem sustainability.
If you want to know more about gene synthesis, here are six crucial things to understand:
Gene Synthesis Begins With Oligonucleotides
Gene synthesis is a synthetic biology branch that allows researchers to assemble unique gene sequences that typically don’t exist in nature. Note that genes contain the coding information that functional molecules like enzymes and body cells require to perform life-sustaining functions.
While natural gene variants occur primarily due to gene mutation, gene synthesis facilitates in vitro creation of gene variants using an oligonucleotide. An oligonucleotide is a short DNA or RNA molecule comprising approximately three to 20 nucleotides (basic DNA structural units) and can be either single-stranded or double-stranded.
However, the gene synthesis service company must determine the oligonucleotide concentration in a DNA sample and have a recommended minimum quantity for each oligonucleotide. Alternatively, it is also possible to synthesize an oligonucleotide from scratch using a solid-phase chemical synthesis process if you lack a DNA sample.
Oligonucleotides function as probes to help identify genetic sequences in De Novo DNA samples. It is also possible to fuse multiple oligonucleotides via the tandem oligonucleotide synthesis process, joining them to the 5” end of a DNA sequence. Therefore, Oligonucleotides’ flexibility forms the basis for gene manipulation in gene synthesis.
There Are Different Synthetic DNA Assembly Methods
Oligonucleotides form the foundation for gene synthesis and can be fused into a single strain. Gene synthesis provides different processes (phosphoramidite techniques) to fuse the oligonucleotides into other DNA compounds, including genes, synthons (primary segment on a target molecule), and genomes. Below are the main phosphoramidite methods employed during oligonucleotide synthesis.
Column-based oligonucleotide synthesis
Column-based Oligonucleotide Synthesis is the most common oligonucleotide process that utilizes a synthesis column as its support matrix. The actual phosphoramidite process is a four-step cycle featuring deprotection, coupling (regulation), capping, and oxidation.
Additional oligonucleotides join the lengthening sequence as the cycles proceed. Also, the column-based phosphoramidite cycles elongate the oligonucleotide sequence on the 3’end and the 5’end. The technique’s main advantage is scalability.
Microarray-based oligonucleotide synthesis
The microarray phosphoramidite method is among the recent oligonucleotide synthesis techniques. It substitutes the column as its matrix support with a microchip. The technique’s advantages are that it is cost-effective and facilitates better control and specification where hybridization is involved.
More oligonucleotide synthesis processes keep emerging thanks to technological advancement. Each process has its pros and cons in specific situations.
Gene Synthesis Facilitates Multiple Downstream Applications
Besides gene expression, gene synthesis also creates more options for downstream applications. Downstream applications refer to gene manipulation and analytical processes that help understand different gene functions and improve or modify them. Such applications include recombinant DNA subcloning, mutagenesis, and genotyping; gene synthesis service companies charge downstream applications separately from gene synthesis and sequencing.
Before the advent of recombinant DNA production, nucleic acid samples containing genetic matter were the test samples used in downstream applications. However, nucleic acid samples are challenging when used for downstream applications because they have a higher degradation risk.
Secondly, the nucleic acid’s quality depends on the purification process, making research results standardization somewhat challenging. Therefore, thanks to gene synthesis, downstream applications currently supersede conventional PCRs. The emergence of CRISPR (clustered regularly interspaced short palindromic repeats), a gene-editing tool, is perhaps the most advanced development in downstream application.
Gene Synthesis Does Not Require A DNA Template
In vivo DNA expression begins with a pre-existing DNA strand. The target DNA strand unfolds to reveal its base pairs to facilitate transcription and translation processes.
However, as explained above, gene synthesis begins with an oligonucleotide, excluding the making of the target DNA’s copy to facilitate transcription and translation. The oligonucleotides function as probes helping direct the sequencing by identifying the most compatible base pairs in a gene sequence.
Consequently, gene synthesis uses De novo gene prediction systems that use statistics and probes to identify and synthesize De novo genes. De novo gene sequences are novel sequences that don’t occur naturally.
Gene Synthesis is Cost-effective
Natural in vitro gene expression is costly, considering the lengthy turnaround time and the intensive labor required to monitor the process. A gene expression process that may have taken several weeks or a month to complete takes seven to ten days with gene synthesis.
Although in vitro gene expression using cell cultures may yield a higher DNA volume in some circumstances, low yield makes it costly in others. Therefore, gene synthesis techniques are more affordable and efficient, facilitating the undertaking of various researches.
Error Correction is Possible in Gene Synthesis
Like any artificial process, gene synthesis is not without flaws, and unfortunately, these flaws may result in errors during the gene and genome sequencing processes. Some methods and stages have a higher error probability, especially during chemical oligonucleotide creation and synthesis.
Fortunately, error correction techniques like DNA mismatch recognition and repair help overcome the cause. Other corrective techniques keep emerging to make large-scale recombinant and DNA production error-free and cost-effective.
Gene synthesis and synthetic biology as a whole hold immense promise for researchers in various biotechnology fields. Although the process has imperfections, its pros often outweigh its cons. Regardless, the essential thing is adhering to biology ethics when using gene synthesis technology.