6 Key Steps Involved in Gene Synthesis
The process of gene and DNA synthesis has become crucial in several fields, replacing the classical cloning procedures. For example, the approach facilitates high levels of recombinant protein expression and forms the foundation for synthetic biology. New gene synthesis technologies have transformed biology by making it possible to manipulate a single gene using several methods.
Today, more people are interested in the gene synthesis process. Unlike DNA replication, the gene synthesis process doesn’t require a template strand to work. Instead, it involves adding nucleotides to a single-stranded module. This guide can help you understand the steps involved in the synthesis.
- Sequence optimization
This is the bioinformatics stage of gene synthesis. It is the strategy design that comes after selecting your gene sequence. Sequence optimization entails designing a progression depending on the desired end application. For example, if you intend to maximize heterologous protein expression, then you will use codon optimization. However, the same may not work when seeking to study a different aspect of gene expression.
In most cases, maintain the intended reading frame during the coding region while ensuring it doesn’t include any sequence that interferes with downstream workflow. During sequence optimization, consider crucial features such as using high or low GC content. They can affect the accuracy of the synthesis process and create errors.
- Oligo design
Once you are done with sequence optimization, focus on designing the oligo sequences. The design entails adjusting the oligo length, GC content, and sequence repeats. For example, changing the GC content helps in stabilizing the DNA strand and its melting temperature. You will find several design tools to help with this process with advantages and limitations. For example, people use DNA Work in oligo design since it’s easy to use. However, the tool is limited to PCR-based methods and won’t work with other models.
These oligo design methods rely on having equal melting temperatures to achieve perfect sequences. It also helps avoid any errors in the process that are costly to correct. You can dodge improper hybridization through careful oligo design.
- Oligo Synthesis
Upon confirmation, the next step is to add nucleotide monomers to form short oligonucleotides. The oligo synthesis focuses on assembling nucleotides and preventing any strand from engaging in other reactions during the synthesis. For example, it inhibits any unwanted branching until the entire process is complete.
Usually, the synthesis occurs in the 3’ to 5’ direction. This is the opposite of DNA synthesis that moves from the 5 to 3’ direction. This process moves from deprotection, coupling, capping, and oxidation.
Oligo synthesis refers to the chemical synthesis of short fragments of nucleic acid that can be done in a lab. You use automated synthesizers or commercial vendors to complete the process. This step is necessary for the gene synthesis process in producing long stretches of the nucleotide sequence. The oligo serves as building blocks and primers to facilitate gene assembly.
- Gene assembly
In the next phase of the process, you focus on assembling the oligo into complete genes. For example, the polymerase cycling assembly is used during gene assembly by exploiting single-stranded templates. This is a standard technique in gene assembly that focuses on combining single-stranded oligo into a singular tube. It relies on accurate oligo synthesis and similar melting and annealing temperatures.
You can also use ligase chain reaction in gene assembly, which focuses on joining overlapping ends of synthetic oligos using DNA ligase. Other methods include sequence-and-ligation-independent cloning that allows assembling up to five gene fragments. It facilitates the recombination of these fragments without sequence restrictions. The accuracy of oligo synthesis and gene assembly can help avoid errors.
- Sequence verification and error correction
After gene assembly, focus on quality control and error correction. Since no process is complete without errors, this stage ensures that any insertion or deletion mutations are corrected. Quality control is significant considering the errors can affect up to 70percent of the desired sequence. Therefore, focus on identifying and removing or correcting harboring mutations from the pool. Also, amend any premature termination in DNA sequencing.
The easiest way to verify is by cloning the newly synthesized sequences into plasmid vectors. It allows primers to bind to the vector regions and ensure correct sequencing. If you can’t achieve it, use other methods for error correction, such as stringent hybridization and extensive purification using electrophoresis. Other methods include mass spectrometry, mismatch-binding, and using prokaryotic endonucleases.
- Preparing synthetic DNA for downstream application
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The last step is the downstream application of the gene synthesis. Researchers achieve this by developing synthetic DNA through cloning and propagating synthetic genes.
Most applications need to clone synthetic genes to specific vectors. For example, plasmid vectors allow transfection into cells, while viral vectors facilitate transduction into animal cells. You may need to design synthetic genes to facilitate this cloning. Alternatively, use recombination-based methods to facilitate homologous recombination.
After cloning them, you need to propagate the gene constructs. While this can be a challenge, focus on creating the best host for propagation. Sometimes the plasmid constructs may not be amplified in a bacteria propagation host. Also, the long genes may be difficult to maintain since they cause an energetic burden on the host cell. If genes are toxic in the host cell, they may affect the propagation.
The Bottom Line
There have been several gene synthesis methodologies developed in recent years that have revolutionized DNA functions. The technology is helping scientists manipulate the DNA for industrial, medical, or experimental purposes. Today, the capabilities of gene synthesis have risen in parallel with DNA sequencing, making it a powerful tool in several scientific disciplines. More industries will rely on the process to solve problems in food supply and environmental protection.
Thank you,
Glenda, Charlie and David Cates
