Artificial genome synthesis, often known as gene synthesis, is a set of techniques used in synthetic biotechnology to build and integrate genes from scratch. Unlike DNA synthesis in live cells, artificial gene synthesis does not need template DNA, enabling practically any Genetic code produced in the lab. It is divided into two parts: solid-phase DNA synthesis, also known as DNA printing.
This results in oligonucleotide fragments with less than 200 base pairs. The second stage includes utilizing different DNA assembly techniques to link these oligonucleotide segments. Since artificial gene assembly does not need template DNA, a fully synthetic DNA structure with no nucleotide pattern or size restrictions is theoretically conceivable.
But what are the standard methods of gene synthesis? Are there any prominent applications of gene synthesis in modern biology? Dig in to study in between lines of artificial gene synthesis loosely.
What are the steps involved in gene synthesis?
Gene synthesis can create recombinant, altered, or entirely new DNA sequences without the need for a template. It renders it easy to make DNA tools that would otherwise be difficult to make using conventional molecular cloning methods. The steps involved in artificial gene synthesis are:
1. Sequence optimizations and oligo design:
After you’ve chosen your genetic material, you’ll need to create the sequence that will be synthesized. Bear in mind the ultimate objective: for example, codon optimization is suitable if your goal is to optimize heterologous protein expression ratios. Although, it may not be accepted if your goal is to investigate domestic gene expression controls.
Following the completion of the generated sequence, pattern analysis is needed to identify the optimal method for dividing the entire gene into segments that may be synthesized and constructed. For highly significant synthetic genes, you’ll probably want to split the sequence into 500-1000 kB pieces that can be synthesized independently and combined afterward.
2. Oligo synthesis:
Today, all DNA synthesis starts with the phosphoramidite chemical step-by-step addition of nucleotide monomers to create short oligonucleotides. Phosphoramidite chemistry is a method of oligo synthesis that utilizes modified nucleotides termed phosphoramidites to guarantee that nucleotides integrate correctly. It prevents the developing strand from participating in unwanted reactions.
With the assistance of an automated synthesizer, you may conduct oligo synthesis in your lab in a row or plate configuration. Commercial suppliers may also synthesize oligos for a low price and in a short amount of time. Oligos are used as building pieces for synthetic target genes and as primers for assembly and PCR amplification.
3. Gene assembly:
Many techniques for assembling oligos into entire genes or giant genomic building blocks have been devised and tested. The three of which are:
- In a thermocycler, polymerase chain assembly (PCA) is a standard method for polymerase-based oligo construction. This procedure is also known as template-less PCR. The idea is to collect all of the single-stranded oligonucleotides into a single tube, conduct thermocycling to enable multiple rounds of annealing, elongation, and solubilization, and then replicate full-length sequences using the outermost primers.
- Ligase Chain Reaction (LCR) is a technique that utilizes a DNA ligase to connect the matching ends of manufactured oligos in a series of denaturation, melting, and adhesion cycles. Pfu DNA ligase, a thermostable enzyme, enables high-temperature annealing and crucial stages, enhancing strict hybridization.
- Sequence- and Ligation-Independent Cloning (SLIC) is an in vitro homologous recombination technique that uses a T4 DNA polymerase to integrate up to five gene fragments into a recombinant vector simultaneously.
4. Verification and error:
All synthetic genomes should be validated before use, owing to the intrinsic possibility for mistakes in each stage of gene synthesis. Mutant sequences must be detected and either removed from the library or fixed.
Synthetic DNA sequences are prone to internal deformations and deletions, as well as sudden termination. Only approximately 30% of each produced 100-mer is the intended sequence due to the accumulation of mistakes from phosphoramidite enzymatic preparation alone. 53 Heterogeneity in the ultimate pool of synthetic gene products may also be introduced by improper heating during oligo construction.
5. Cloning:
Synthetic genes must be cloned onto suitable vectors for most purposes. It can contain plasmid carriers for translation or electrophoresis into cells or viral vectors for transmission into cells or live animals. Synthetic genes may be constructed with restriction enzyme domains, crossover arms, or additional flanking regions to make cloning easier.
Recombination-based techniques begin with PCR expansion of the gene insert to introduce 15 characters of sequence similarity to the linearized vector, enabling homologous recombination without adding undesired bases as a substitute for providing unique flanking sequences.
Final Thoughts
Gene synthesis technology has transformed current knowledge of how DNA works as the foundation for life and the human capacity to alter DNA for practical, medicinal, and industrial reasons. Although gene synthesis’s skills and efficiency have gradually improved over the past decade, its cost has fallen dramatically, in line with advancements in DNA decoding and chip-based biomarkers. Gene synthesis will become a valuable tool for a growing number of scientific fields and commercially relevant applications as automation, error detection, and cost-effectiveness increase.
