Genetic engineers and synthetic biologists have valuable tools to tinker with life’s code in the modern age. Furthermore, gene manipulation’s growing range has also made it quite possible for them to accommodate, maneuver, and incorporate. Recently, it has never been faster, cheaper, and easier to embark on the process of manipulating the sequences of nucleic acid. Also, all-new ways of DNA synthesis are changing every dynamic of biology experiments in an extreme way. The technology of de novo synthesis also tends to encapsulate the entire method of gene manipulation. With synthesis gaining more accessibility, it evolves into an ideal alternative to that of cloning. This is because of the work-intensive and conventional system to make sequence constructs.
Are you thinking about which system you should utilize in the next experiment of molecular biology? Let’s do a quick rundown of some of the vital points that you need to consider while opting between traditional cloning and synthesis.
1. Synthesis yields more results and is faster.
If you are familiar with the utilization of cloning for an experiment, you might be aware of how tedious this method is. Amplification and gene isolation through restriction digestion, purification, PCR, and other techniques involved in the cloning process is resource and time-consuming. Let’s take, for instance, with the process of PCR; the primer design often becomes a cumbersome process. You need to know that the primer pairs require to be in the correct location with no reading frame adjustments and the same melting temperatures.
You are required to transform the vector into an E. coli and also need to screen the colonies. Moreover, verifying sequence authenticity is also a vital factor. Such a method happens to be a tiresome process. If you correct or repeat experiments due to the inaccuracy of a sequence, it can compromise the research time you have spent. In contrast, with synthesized DNA, sterility, and sequence precision gets assured into the system. However, extremely low or high GC content, the tendency for hairpin formation, and the sequence repeats can affect the synthesis feasibility. Nevertheless, the synthesis will enable a more natural and faster sequence production, which results in the inclusion of the next vital point.
2. Synthesis enables possibilities in gene engineering.
Since DNA for cloning gets produced through the medium of PCR, it needs the molecular template to be amplified. Conversely, the system of de novo gene synthesis doesn’t need a medium of sequence templates. This provides you with a significant amount of flexibility while designing the target sequence. Furthermore, it also makes synthesis a more straightforward and faster alternative to cloning. The process of synthesis also accelerates the production account of combinatorial DNA variant libraries substantially. And, it also affects the numbers of combinatorial pathway engineering.
When you plan on going beyond gene manipulation, the system of synthesis allows you to construct and design vector fragments tailored to experiments like terminators, promoters, and markers. In the end, synthesis tends to play a vital role in CRISPR as it rapidly produces RNAs that happen to direct Cas9 nucleases towards the direction of a specific locus.
3. Protein expression and codon optimization.
Sequences that are codon-optimized ensure the right heterologous expression. Since classical cloning is a bit limited to other pre-existing templates, sequences found amid nature might yield low protein levels in host organisms. However, with synthesis, you can put entirely new codon-optimized sequences under the process of synthesis.
4. Pricing plays an important role.
Costing plays an essential role in nearly every sector. When it comes down to the options between cloning and synthesis methods, the cost might be a vital factor. Most importantly, selecting one of the most cost-effective ways will highly rely on the type of sequence you want to acquire. At first, the option of cloning might look like a cheaper alternative as you can perform it in your lab internally. Furthermore, when researchers or scientists work on cloning complex sequences, it often gets challenging because of several failed attempts. Such a tedious process further leads to a significant amount of money spent on reagents and the loss of time and investment that goes into ordering synthesized sequences.
Nevertheless, you should take the sequence composition and turnaround time into account. The sequences that are harder to synthesize do cost more to produce or take a bit longer. Moreover, the substantial decrease in the cost of synthesized DNA per base pair has made the synthesis method an ideal and advantageous option.
Cloning is a vital tool in the applications of genetic engineering since the inception of molecular biology. With time and extended systems, it often lacks in meeting the needs of biology’s increasing demand for a much significant sequence complexity and needs to test more combinations and scale-up.