Artificial gene assembly using single-strand oligonucleotides to create recombinant DNA is revolutionizing DNA technology. The process transcends the standard procedure of cloning DNA from a template strand and facilitates the creation of unique, currently unavailable DNA sequences.
Gene synthesis is at the heart of De novo DNA technology and helps facilitate research in diverse crucial fields. Although the first record of chemical gene synthesis was in the 1950s, the commercialization of the process is pretty recent and marred by inaccurate myths.
To better understand recombinant gene production, check out these six facts:
Complete Plasmid Synthesis is Possible
Plasmids are genetic material found primarily in bacteria; you can refer to them as DNA equivalents. The primary distinguishing factor between a DNA strand and a plasmid is that plasmids feature a circular shape, while DNA strands feature a double helix structure.
Besides carrying genetic material, plasmids also give bacterial cells genetic advantages, with the most prominent benefit being antibiotic resistance. In a nutshell, plasmids are a treasure trove of genetic knowledge waiting to be unearthed, and biotech has already made tremendous headway studying them.
One of the most significant biotech developments regarding plasmids is synthesizing complete plasmids. While DNA strands and plasmids differ in structure, both of them have base pairs. However, an entire plasmid can have several hundred thousand base pairs; hence the somewhat popular yet misleading notion that complete plasmid synthesis is impossible.
On the contrary, gene synthetic procedures can facilitate complete plasmid synthesis in a two-step process. The first is using overlapping oligonucleotides to create the ideal number of base pairs for a specific plasmid. Once researchers achieve the desired oligonucleotide length, they use a circulation procedure to achieve the round shape synonymous with complete plasmids.
Complete plasmids have numerous genetic advantages and are beneficial as expression vectors . So, if you want a whole custom plasmid for your project, it is 100% attainable.
Non-coding Gene Sequences Cannot Be Optimized
A gene sequence typically features coding and non-coding sequences. The coding sequence contains codon sequences, three nucleotides that correspond to a specific amino acid and are used in protein transcription. In contrast, the non-coding sequences do not correspond to any amino acids but serve a regulatory role in protein translating, signaling when translation begins and ends.
Although coding sequence repetition in DNA structures is common, research shows that organisms often prefer specific coding sequences. This phenomenon is called gene usage bias, and its extent varies among species.
Codon usage bias is not necessarily a bad thing; biotech uses it as a channel to enhance coding composition, enhancing protein efficacy. Gene optimization entails the introduction of preferred gene sequences to improve translational efficiency without altering protein function.
Coding sequences, particularly the redundant codons, are the primary focus during gene optimization. In contrast, non-coding sequences serve no use in gene optimization since they do not correspond to any amino acid and only initiate and conclude translation.
Gene Synthesis’ Advantages Transcend Codon Optimization
Codon Optimization plays an invaluable role in biotechnology, facilitating research and treatment developments like gene therapy and protein drugs. However, codon optimization is not the only gene synthesis advantage. For one, gene synthesis has substantially reduced the time and labor resources necessary to obtain complementary DNA (cDNA). Reverse transcription to obtain cDNA via gene synthesis takes roughly 48 hours, way quicker than using RT-PCR on an mRNA.
Besides fast delivery, utilizing gene synthesis for subcloning eliminates the hassle of dealing with unnecessary restriction sites. However, it allows you to easily add beneficial components to your expressed vector, including desirable restriction sites.
A High Codon Adaptation Index (CAI) Is Not The Gold Standard of Gene Optimization Tools
One of the primary functions of gene optimization (discussed above) is to facilitate optimum protein expression. It begins by studying the DNA sequence to establish the most suitable codons for optimization using CAI.
However, while CAI serves an invaluable role, an efficiently programmed gene optimization tool features other considerations. It is the harmonization of the codons selected by CA to facilitate efficient translation and stable protein creation.
Secondly, it filters out undesirable elements, including restriction enzymes and splice sites that impede translation and proper vector expression. Finally, it balances out GC distribution and prevents the formation of GC-rich templates. GC-rich templates have a reinforced structure and a unique temperature, making them extremely difficult to sequence and amplify. Therefore, consider all the above factors when deciding on an optimization toolkit.
It Is Possible to Subclone An Insert Into A Vector Even If The Insert Lacks Restriction Vector Sites From The Multiple Cloning Site
An MCS (multiple cloning site) is a DNA segment containing approximately 20 restriction sites that occur to facilitate the insertion of DNA. However, your vector does not need to have all the 20 or so restriction enzymes for successful subcloning to occur.
All you need is one or two restriction enzymes that align with the restriction sites on your vector’s MCS. Afterward, you can amplify your gene insert using a PCR, creating sites on the insert that share the same relative position as those on your vector.
Vectors Featuring Restriction Enzyme Sites Can Undergo Downstream Cloning Without Issue
Restriction enzymes only occur along a specific portion of the DNA. The restriction sites are minute in size due to the enzyme digestion that vectors undergo beforehand. Therefore, restriction enzyme presence does not impact any vital process.
Gene synthesis remains a somewhat novel practice, even in the biotech field, which explains the misinformation on the subject. However, these facts should help you debunk any unfounded myths you encounter while researching gene synthesis.