Elegance in science is a somewhat abstract concept, difficult to explain to individuals within and outside the scientific research arena. However, it refers to scientific models, experiments, and models that embody precision/ neatness and simplicity or a self-explanatory nature.
Scientists across various fields, including biotechnology, have described select project projects as elegant. One such project or model is the recombinant gene production. Recombinant gene production is an alternative to molecular cloning that entails producing reproducible genes in vitro without an original template.
So, what makes recombinant gene production a beautiful/ elegant scientific technique? Keep reading to learn more about this contemporary scientific method.
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What Is Gene Synthesis?
As highlighted above, recombinant gene production or gene synthesis is a technique that facilitates DNA production without a template. It utilizes chemical reactions rather than a template to form gene sequences, including sequences that do not exist naturally.
Its advantages over molecular cloning include enhanced efficiency due to lower labor intensity, high throughput, faster production/ low turnaround time, lower costs, and overcoming the constraints of limited cloning vector options. Moreover, cell-free cloning allows researchers to develop genetic cDNA libraries featuring all possible natural and novel combinations. cDNA libraries allow researchers to reference potential genes and build on each other’s work, saving research time and other resources.
Gene synthesis qualifies as an elegant biological science technique due to its simplicity. Nonetheless, the multi-step process has revolutionized live science research and industrial applications. Below is an overview of the steps involved in gene synthesis.
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Step-by-step Guide To Gene Synthesis
Below is an overview of the basic steps involved in gene synthesis
- Oligo Design/ Optimization/ Sequence Optimization
Oligos or oligonucleotides are the building blocks for numerous biotechnology processes, including chemical gene synthesis. Gene synthesis begins with creating specific oligonucleotides based on the target DNA. Non-specific oligonucleotides create incorrect gene sequences during assembly.
Ideally, the oligonucleotides used in a gene sequence must have similar thermodynamic properties and be target-specific to facilitate accurate sequence optimization. Consequently, gene synthesis companies use integrated algorithms to decipher the ideal oligos for a specified gene sequence.
The algorithm functions by receiving the information on the target DNA molecule and dividing it into oligo sequences representing complementary DNA strands in the double helix structure. Sequence optimization occurs via oligonucleotide elongation to enhance specificity and thermodynamic temperature realization. Moreover, oligonucleotide optimization considers specific DNA characteristics, including repetitive sequences, structure complexity, and length.
Oligonucleotide design goes hand-in-hand with a sequence analysis to determine how to break down the sequence into synthesizable fragments. Gene synthesis companies typically use phosphoramidite synthesis to generate target oligonucleotides up to 200 base pairs in the 3’ to 5’ directionality. Phosphoramidite chemistry/synthesis eliminates errors in oligonucleotide synthesis, especially for molecules featuring shorter base pairs.
- Oligonucleotide Synthesis
The target oligonucleotide design and optimization for length and temperature sets the pace for the synthesis process. As highlighted above, phosphoramidite chemistry is the primary technique employed in in vitro gene synthesis.
Phosphoramidite chemistry utilization entails using nucleotides or DNA monomers to grow an oligonucleotide chain in the 3’ to 5’ directionality. The technique begins with phosphoramidites or modified nucleotides attached to polymeric support to facilitate elongation via sequential addition, adding new nucleotides to the chain to form an oligonucleotide.
As highlighted above, specificity is key to successful oligonucleotide synthesis. Therefore, phosphoramidite chemistry entails using PCR (polymerase chain reaction) primers like methylating phosphate groups and protective di-isopropylamine to prevent side reactions that could compromise the final oligonucleotide’s integrity.
The primers facilitate protection and deprotection during elongation, whereby the protecting group prevents side reactions before elongation. On the other hand, the deprotecting group deprotects one nucleotide at a time to allow covalent bond formation.
Various automated synthesizers facilitate oligonucleotide design in a lab setting. You can consult your service provider on the synthesizers they use, including each synthesizer’s advantages and disadvantages.
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- Gene Assembly
Besides assembling nucleotides into oligonucleotides, gene synthesis entails assembling the newly formed oligonucleotides into the target gene. The techniques vary depending on the target gene’s physical characteristics, meaning each technique has its pros and cons based on the target gene.
For example, shorter gene sequence assembly is achievable via chain reactions like PCR (explained above) and ligase chain reaction. In contrast, longer sequences are more successful when assembled via in-vivo techniques such as cloning vectors. Yeast cloning vectors are ideal for assembling gene sequences with 500-1000 base pairs.
- Characterization/ Gene Sequence Verification
Although chemical gene synthesis has numerous benefits over traditional cloning, it is yet to become 100% error-free. Therefore, the generated gene sequences must be verified to eliminate elements with base pairs other than those stipulated in the target gene.
Moreover, verification eliminated sequences with anomalies like side chains from non-specific bonding and mutations. Depending on the error level, the service provider may decide to correct the error or eliminate the defective genes. Plausible error correction techniques employed in gene synthesis include hybridization, mismatch cleavage, and functional assays.
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Chemical gene synthesis fits the bill as an elegant scientific method based on its ease of execution, precision, and ease of explanation. The technique has numerous applications in biotechnology, revolutionizing live sciences research and applications.