Home » 5 Steps involved in Gene Synthesis

5 Steps involved in Gene Synthesis

Gene synthesis is the biological process of the construction and assembly of new genes from nucleotides de novo. Artificial gene or synthetic gene synthesis is different from the DNA synthesis that happens inside living cells. The significant difference is that it does not need a prototype DNA. It allows the creation of synthetic molecules of DNA that do not have a limit in size or sequence of their nucleotides. Also, you can copy and synthesize any sequence, whether it occurs naturally or not. The study and advancement in gene synthesis have led to great strides in biology, whereby scientists can now easily reprogram whole genomes and cells.

The scientific benefits of gene synthesis include:

  • Gene synthesis has been used by researchers to single out the conditions under which breast cancer tumor cells multiply. This has formed a basis for the creation of therapies that can effectively treat cancer.
  • When scientists do viral sequencing using artificial gene synthesis, it produces safer and more effective DNA based vaccines.
  • The Chanel rhodopsin variant makes it possible to activate deep-brain neurons through non-invasive optical activation.
  • Plant biologists can use gene synthesis to study developmental reprogramming in vascular tissues of the plants.

Steps Followed in Gene Synthesis

Scientists follow a five-step process when they are carrying out gene synthesis. The five steps are:

  • Oligo design and sequence optimization
  • Oligo synthesis
  • Gene assembly
  • Verification and correction
  • Preparation of the synthetic DNA

1. Oligo Design and Sequence Optimization

The first step in the process is choosing the gene that interests you. After isolating the gene, you will have to create the sequence you want to synthesize. For example, if you aim to maximize heterologous protein expression levels, the ideal path to follow is to optimize the codons. On the other hand, this process might not be useful if you aim to study the internal regulation of gene expression. Here are the most popular tools used for the Oligo design process, and their pros and cons:

  • DNA works: it is easy to use and can predict the outcome of Oligo mishybridization. However, the simulation-based scores might not offer an accurate representation of actual success in assembly.
  • Gene2Oligo: The interface is easy to use and has both LCR and PCR-based assemblies. On the other hand, it will not be useful if your gene is longer than one kilobyte.
  • TMPrime: TMPrime: It offers both LCR and PCR-based assemblies and a wide range of melting and annealing temperatures. The only shortcoming is that inexperienced users could find the interface complicated. The users need to understand various input parameters for accurate submissions.

When you finalize the sequence to synthesize, the next step is dividing the gene into smaller fragments to synthesize and assemble. Factors that affect this step include Oligo length, the tendency of hairpin formation, and sequence repeats.

2. Oligo Synthesis

The first step in DNA synthesis is forming short oligonucleotides. Scientists achieve this by using phosphoramidite chemistry to add nucleotide monomers together. Phosphoramidite chemistry is beneficial because it prevents the growing strand from engaging in unwanted reactions during the formation process. They combine the phosphoramidite group to the 3’O to prevent unwanted branching during the formation. The phosphoramidite group contains di-isopropylamine, which offers protection and methylated phosphite. The four-step phosphoramidite reaction cycle is as follows:

  • Deprotection: Here, they remove DMT by washing using a mild acid such as trichloroacetic acid. This exposes the 5’O for reactions.
  • Coupling: The 3’O of the second nucleotide bonds with the 5’O of the first nucleotide.
  • Capping: Here, all the 5’OHs of any nucleotide that did not acolyte helps in such a way that it does not engage in unwanted reactions further down the process.
  • Oxidation: Here, they add iodine, forming a phosphodiester bond out of the phosphate triester bond, which leads to creating the backbone of the DNA.

Experts carry out this step using a lab column or a specialized synthesizer.

3. Gene Assembly

The step aims to assemble the fragmented Oligos into either complete genes or building blocks for genomes. Four types of assembly are useful in gene assembly. The effectiveness of the gene assembly method depends on the length of the sequence they want to combine. The most common assembly methods are:

  • Ligase chain reaction or LCR: They use DNA Ligase to join the overlapping ends of synthetic Oligos.
  • Polymerase Chain Assembly: they combine all one stranded Oligos in one tube, and then, they do thermocycling to facilitate joining.
  • In-Vivo homologous recombination in yeast: Ideal for long nucleotide strands of up to 200 nucleotides and 1000Bp.
  • Sequence and Ligation independent cloning: They use a plasmid vector to assemble up to five gene fragments. They do this by incorporating it into the plasmid vector.

4. Verification and Correction

The fourth step involves verifying the sequence that has been created and rectifying any mistakes that could have occurred during the process. It also involves testing all the sequences before releasing them for use. Any sequences that have mutations are removed from the collected pool. Other processes that are part of quality control include:

  • Insertions
  • Deletions
  • Premature termination

Sometimes, the experts cannot obtain the correct sequence from the pool of synthesized DNA for amplification. Here, they use various methods to detect and correct errors. The scientists may decide to use stringent hybridization of keenly designed Oligos. They could also opt for electrophoresis as an exhaustive purification method. Another viable option is mass spectrometry. The biochemical method they use depends on how successful they predict it will be in purifying the DNA.

Other methods that they use include mismatch binding, which is also known as mismatch cleavage. Here, they use prokaryotic endonucleases, a process where functional assays are used to correct the correct sequences. 

Before going into the process, it is advisable to understand that only about one-third of the total product synthesized will be of the desired quality. To simplify the process, they only clone strands that are found to have no errors. Working with a lab or program that understands

5. Preparation of the synthetic DNA

This is the last step in the process. Here the synthetic DNA is made ready for downstream applications. Several processes form part of this preparation for user-end applications:

  • Cloning: They clone the synthetic genes into useful vectors that include plasmid or viral vectors. Sometimes, the experts will design synthetic genes that restrict enzyme sites, recombinant arms, and other adjoining sequences.
  • Propagation: The synthetic genes are difficult to propagate because of several reasons. First, experts find it hard to amplify low copy number plasmid constructs using bacterial hosts. Also, some genes alter the physiology of their hosts, and finally, long genes exhaust the host cell because of the energy burden they exert on it. At times, the experts have to deal with genes that are toxic in particular hosts and harmless in others. Scientists will try and identify the ideal bacterial host for the success of the propagation process.
  • Biosafety: Finally, the relevant bodies will receive reports of the process used, assess the evidence, and determine whether the resultant product is safe for the general population. The Recombinant DNA Advisory Committee, for instance, reviews the protocols to ensure that safety was upheld and that the product will pose no risk to the environment. 

Conclusion

Gene synthesis is very beneficial when done correctly. These five steps will guide you through the process. If you are thinking about creating a synthetic gene for any purpose, make sure to contact experts in order to ensure that you follow the right step in the most accurate order.

Follow: