Artificial genome formulation, often defined as gene synthesis, is a collection of artificial biotechnology techniques for creating and integrating genes from scratch. Unlike DNA synthesis in living cells, synthetic gene replication does not need model DNA, allowing almost any genetic code generated in the lab. However, there is a lot of misinformation on the internet relating to gene synthesis.
Many of the facts you’ll find on the web aren’t true. In reality, the information behind the myth is just as easy to find as the myth itself, yet many people will accept whatever they see first.
So how can you identify truth from myths regarding gene synthesis? Is there any way to identify myths and save yourself from false knowledge? Well, if you are in search of correct information about gene production, you’re at the perfect place. Read on to know the factually accurate things about gene synthesis and how you can tell what a myth is and what isn’t.
What are the essential myths and facts that you must know about gene synthesis?
On various online platforms, there are many misconceptions and misunderstandings regarding gene synthesis. Here are the most prominent claims about gene expression and molecular genetics that are false with appropriate reasons and proof:
1. The manufacture of entire plasmids is not feasible:
This statement is a myth. You can replicate genes of any sequence and entire plasmids using several gene synthesis processes. Synthetic genes are often made by putting together overlapping DNA oligonucleotides. This technique allows for the creation of lengthy segments that will eventually combine into a single significant synthetic gene.
It is, therefore, feasible to construct entire plasmids of any length by incorporating a circularisation process. Since ELISA kit manufacturers often deal with E. coli, your “genotype” (i.e., the novel plasmid) must, of course, include an E.coli preference flag and a duplication origin.
2. Non-coding sequences are impossible to optimize
This is a true statement. Due to the apparent repetitive nature of the DNA code, one amino acid may be coded by more than one triplet codon, for example, arginine – CGT, CGA, CGC, CGG, AGA, AGG. The occurrence of these codons varies depending on the species. As a result, a DNA sequence may be optimized to have the same density of codons as the creature in which the gene will be translated. The optimization also aids in the lowering of the gene’s high GC and repetitive sections.
However, this kind of optimization is only feasible when the gene to be manufactured (or a portion of it) codes for a protein chain and therefore contains codons that may be modified. Because a non-coding length of DNA does not include codons that code for an amino acid, an optimization based on DNA redundancy is not possible.
3. The sole benefit of gene synthesis is codon optimization
This statement is a myth. Yes, most scientists utilize gene synthesis to enhance the expression of their chosen genes in heterologous systems. You may accomplish this by modifying codon use. However, there are numerous other benefits to utilizing gene synthesis. Assume your NGS results show a particularly intriguing DNA sequence, which you now wish to investigate further.
You may easily arrange your DNA or RNA sequence using gene synthesis; all you need is the in vitro sequence. Another benefit of gene synthesis is quick and dependable access to cDNA sequences, which was previously only possible via labor-, time-, and expensive cDNA synthesis.
When utilizing gene synthesis, no RNA extraction, RT-PCR, or RACE is required to get flawless full-length cDNA. Unwanted restriction sites may be avoided during adaptation, and sub-cloning into your expression system is also an option. Of course, you may add promoter and enhancer regions, start codon signals, restriction sites, and other components to your gene sequence.
4. Even if your insert lacks constriction enzyme locations from your vector’s various cloning sites (MSC), it may still be sub-cultured into the vector
This information is accurate. You need one or two constriction enzymes that address the MCS to slice your vector. Your gene insert may then be PCR amplified using primers to produce vector-homologous sections at the extremities of the PCR result. These homologous sections are subsequently sub-cloned into your vector using sequence and independent junction cloning (SLIC).
Since the PCR output will not be broken during sub-cloning, any restriction sites in your insert are irrelevant. SLIC is just as quick and efficient as conventional restriction enzyme site sub-cloning. The molecular biology specialists will guarantee that all of your criteria are fulfilled.
5. There will be problems with subsequent cloning if the conventional vector includes locations for your restriction proteins
Everything about the above statement is false. The standard vectors are created with just a few restriction endonucleases sites in the MCS. Even if a restriction enzyme site is accessible, this will not cause any issues. The resultant fragment will be so tiny that it will not appear on an agarose gel after constraint enzyme digestion. It will not harm the effectiveness of the digestion since all restriction sites in the MCS are at least 4bp (in most instances >10bp) away.
Even if one of your selected restriction enzyme domains is included in the vector’s backend and you discover a band the same or comparable size as your gene after processing, there is a way to get around the problem. You can create an extra cut in the vector backend and therefore decrease the size of the undesired band, using a third constraint enzyme for digestion. You may then remove the necessary band from the agarose plate in most instances.
While genetic modification now primarily refers to recombinant DNA methods, it used to have a broader meaning. Taking advantage of this, many people claim several things about gene synthesis that aren’t true. The information given above will help you identify facts from heaps of false claims.
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