6 Important Things To Know About Custom Gene Synthesis
Custom Gene synthesis refers to chemically synthesizing a strand of DNA base-by-base. Gene synthesis does not require a template strand, unlike DNA replication in cells or by Polymerase Chain Reaction (PCR). On the other hand, Gene synthesis entails the step-by-step addition of nucleotides to a single-stranded molecule that serves as a template for other molecules, a pattern for making a complementary strand. Gene synthesis is the most fundamental step in the process.
The discipline of synthetic biology is based on this technology. Biology research has been transformed by gene synthesis technology. Scientists no longer have to limit themselves to modifying a single gene at a time; they can now create or reprogram entire genomes and cells. To speed vaccine research, you can swiftly manufacture newly discovered viral genomes. Further, you can create new enzymes to fight cancer and manufacture long-lasting biofuels. The following article will zoom up on six critical things you have to know about custom gene synthesis.
Sequence Optimization
It would help design the sequence you wish to synthesize during custom gene synthesis. However, your selection should be guided by your final goal in gene synthesis. For example, codon optimization is suitable if you aim to increase heterologous protein expression levels, but it may not be appropriate when investigating endogenous gene expression regulation. Ensure your desired reading frame is maintained throughout the entire coding region for constructs with multiple segments.
Short flanking sequences are frequently added to assist later excision or recombination using restriction enzymes or similar techniques. Make sure your sequence doesn’t contain any restriction enzyme recognition sites or other sequences that could disrupt your operation.
Oligo Design
Once you select the appropriate sequence optimization, you have to understand and choose the oligo design that will help determine the optimum method for dividing the entire gene into segments generated and assembled. For numerous synthetic genes, you’ll probably want to split the sequence into smaller portions that can be synthesized individually and combined later. There are various oligo design software tools available to help in oligo design.
The oligo length differs and is affected by several factors such as individual preference, sequence complexity, and the assembly method applied. Shorter oligos have a lower error rate but require more overlaps making them very expensive on the other hand; longer oligos overlaps reduce the rate of nonspecific annealing, increasing the likelihood of proper assembly. Even lengths and melting temperatures can easily be achieved with the oligos.
Oligo Synthesis
Oligo synthesis is a significant process in custom gene synthesis. Oligo synthesis is usually carried out via phosphoramidite, which generally uses chemically modified nucleotides known as phosphoramidites. These nucleotides ensure that all the genes are assembled correctly and deter the growing strands from getting into undesired reactions during the synthesis process.
To prevent undesired branching, the phosphoramidite group is linked to the 3′ O and comprises a methylation phosphite as well as a protective di-isopropylamine. Because phosphate reacts more quickly than phosphate, it is employed. To protect against undesired reactions until oligonucleotide synthesis is finished, methyl groups are connected to the phosphite, and amino-protecting groups are added to the bases.
Gene Assembly
During the custom gene synthesis process, gene assembly is a significant step to understand. There are different methods of assembling oligos into complete genes. The approach to use will be determined by the length of the sequence you used. You can use the polymerase-based method to create the oligos for shorter sequences. On the other hand, a more extended sequence Vivo- recombination-based method is recommended. To ensure quality and optimum gene synthesis, you must select the correct gene assembly method with a high-fidelity enzyme.
Sequence Verification and Error Correction
While carrying out a gene synthesis process, there are likely possible errors in each step. Therefore, all the synthetic sequences have to be verified before applying. You must identify the various harboring mutations and effectively remove them from the pool or otherwise correct them. Synthetic DNA sequences are prone to internal insertions and deletions, as well as premature termination.
Only around 30% of any synthesized 100-mer is the required sequence due to the accumulation of mistakes from phosphoramidite chemical synthesis alone. Heterogeneity in the final pool of synthetic gene products can also be introduced by improper annealing during oligo construction.
Cloning
Synthesized genes have to be cloned into appropriate vectors. These vectors may include plasmids for transfection technology or electroporation into cells. Synthetic genes may be created with restriction enzyme sites, recombination arms, or other flanking regions to make cloning easier. Recombination-based approaches begin with PCR extension of the gene insert to introduce 15 bases of sequence similarity to the linearized vector, promoting homologous recombination without adding undesired grounds as an alternative to introducing unique flanking sequences.
Wrapping up
With improvements in technology, custom gene synthesis is likely to increase steadily and the whole process simplified. The progress in automation is expected to lead to better and more effective error correction methods and reduce the cost of the entire process. Gene synthesis is also becoming an essential tool in the scientific discipline and has both scholarly and economic importance.
