The growing range of gene manipulation methods available today give synthetic biologists and genetic engineers some truly astonishing tools for tinkering with the code of life. It has never been easier, cheaper, and faster to manipulate nucleic acid sequences. In particular, the advent of new ways to synthesize DNA, one base at a time, is extensively changing the dynamics of molecular biology experiments. De novo synthesis technology encapsulates the whole process of gene manipulation, from gene design to actual product, simplifying scientists’ lives. As synthesis becomes more accessible, it also turns into an efficient alternative to cloning - the conventional and work-intensive method to build sequence constructs. So which method should you use in your molecular biology experiment? Here are some of the key points to consider when choosing between synthesis and traditional cloning:
Synthesis is faster and yields more reliable results: Whoever had to use cloning for an experiment knows how tedious it can be. Gene isolation and amplification through PCR, restriction digestion, purification, and other processes involved in cloning are time and resource consuming. For example, with PCR, primer design is often a cumbersome process; the primer pairs need to be in the right location, with similar melting temperatures, and no reading frame adjustments. To make things even trickier, when you are finally done with onloning, your actual work is far from finished as its results have limited predictability and are often susceptible to error. You must then transform your vector into E. coli, screen your colonies, and verify your sequence authenticity. This is a tiresome process and having to repeat or correct experiments due to sequence inaccuracy can compromise precious research time. By contrast, with synthesized DNA, sequence precision and sterility are assured. Not having to worry about the homogeneity of your PCR products and possible contamination during cloning is a major advantage. Moreover, synthesis eliminates the struggle of having to assemble different gene cassettes while making sure that you use the right restriction enzyme cutters and preserve the appropriate reading frames. However, excessive high or low GC content, tendency for hairpin formation, and sequence repeats can impact the feasibility of synthesis. Nevertheless, synthesis will still allow for faster and easier production of a wider variety of sequences, which leads us to the next key point...
Synthesis allows for a greater array of possibilities in gene engineering: As DNA for cloning is most commonly produced through PCR, it requires a molecule template in order to be amplified, i.e., cloning is limited to recombinant sequences found in nature. Conversely, de novo gene synthesis does not require a sequence template to work as sequences are synthesized from scratch. This gives you more flexibility when designing your target sequence and makes synthesis a faster and more straightforward alternative to cloning, which is a multi-step process. Synthesis also accelerates considerably the production of combinatorial DNA variant libraries, and even combinatorial pathway engineering. Beyond gene manipulation, synthesis enables you to design vector fragments tailored to your experiment such as promoters, terminators, and selectable markers. Lastly, synthesis plays an essential role in CRISPR as it is used to rapidly produce guide RNAs that direct Cas9 nucleases to a specific locus.
Codon optimization and protein expression: Codon optimized sequences ensure proper heterologous expression. As traditional cloning is limited to pre existing templates, it may be that the sequence found in nature yields low protein levels in their host organisms. Whereby with synthesis, whole new codon optimised sequences can be synthesized.
Pricing: In the end, the choice between cloning and synthesis might come down to the costs of each method. This is particularly true for academic customers with budget constraints. Essentially, choosing the most cost-effective method will largely depend on the sequence you want to obtain. At first, cloning might seem like the cheaper alternative as it can be done internally in your lab. However, working with sequences that are difficult to clone often equates to months of continuous failed attempts. This can lead to hundreds of dollars spent on reagents and, of course, a loss of your valuable time, making it well worth the investment of ordering a synthesized sequence. Nevertheless, the turnaround time and sequence composition must be taken into account; sequences harder to synthesize can take longer or cost more to produce. Yet, the significant decreases in the price per base pair of synthesized DNA is further reinforcing synthesis as the more advantageous alternative regardless of sequence complexity.
Cloning has been a crucial tool in genetic engineering applications since the dawn of molecular biology. However, it is often insufficient in satisfying biology’s increasing demand for greater sequence complexity as well as requirements to scale up and to test more combinations. Synthesis addresses those needs and, above all, it does so in an efficient manner - the good brains can focus on research rather than on monotonous lab work. To learn more about the amazing potential of synthesized DNA, check out Twist Bioscience.
Happy cloning but more importantly, happy synthesizing!