DNA structure

Gene synthesis is an application of genetic engineering using oligonucleotides to generate stretches of double-stranded DNA. The Gene synthesis process involves DNA replication directed by an RNA template. 

However, some myths and misunderstandings about gene synthesis exist on the web. This article looks at the five things you should know about Custom Gene Synthesis. 

1. You can synthesize complete plasmids

One of the common myths about gene synthesis is that you cannot synthesize complete plasmids. This is a lie because it can synthesize genes of any length, including whole plasmids. You can assemble the overlapping DNA oligonucleotides into one large synthetic gene during the gene synthesis process. When you add a secularization procedure, creating complete plasmids of any length is possible.

The oligonucleotides are designed to cover the complete sequence and generate the full-length molecule progressively. Since artificial gene synthesis doesn’t require template DNA, making a synthetic DNA molecule possible without limiting the nucleotide size or sequence is possible. 

There are several benefits of synthesizing a complete plasmid. For example, you can choose to include a multiple cloning site that is tailor-made for your project. It also allows you to select elements such as selection markers, promoter and enhancer, the origin of replication, and restriction sites. The nucleotide sequence of the vector is optimized for the host organism to regulate the expression level of genes. 

2. You cannot optimize non-coding sequences. 

Due to the redundant nature of the DNA code, you can use over one triplet codon to encode for the same amino acid. The differing frequency of these codons across species makes it possible to optimize a DNA sequence so it contains the same frequency. Therefore, it’s possible to optimize non-coding lines during gene synthesis. A non-coding stretch of DNA doesn’t have codons coding for amino acids, which makes it possible to perform an optimization exploiting the redundancy.

Codon optimization leads to a reduction of high GC content and repeated regions in the gene. This optimization will be possible when the gene is synthesized and coded for a protein sequence. Optimizing a gene for the same frequency of codons where the gene will be expressed is possible when the target gene encodes a protein.

3. Codon optimization is just one advantage of gene synthesis. 

Gene synthesis is a valuable codon optimization tool to circumvent codon bias issues during heterologous protein expression. The gene synthesis with codon optimization improves the gene of interest in a heterologous system. Because of the codon bias, the adaptation of condom usage is used to enhance gene expression since the expression rates of transfected genes are lower in the transformed organism. This makes it the best optimization software in gene synthesis.

However, codon optimization is not the only advantage of using gene synthesis. For example, you can order a DNA or RNA sequence if your subsequent generation sequencing data shows a DNA sequence you would want to investigate further. You can also use gene synthesis to produce full-length cDNA. You can also use gene synthesis with codon optimization to create and deliver genes in less than 48 hours, making it time and cost-efficient. The codon optimization can help you avoid unwanted restriction sites that occur during subcloning into an expression vector. 

4. A high codon adaptation index is not the best codon for gene optimization. 

The codon adaptation index measures the synonymous codon usage bias for a DNA or RNA sequence. It assesses the extent to which selection has influenced the molding of the codon usage pattern. However, choosing the most suited codons does not yield the highest protein expression.  

Codon adaptation index (CAI) ranges from 0 to 1, with higher values indicating a higher gene expression potential. The gene tool randomly assembles the DNA sequence and analyses it with codon usage. It considers frequently used codons from highly expressed genes in gene synthesis and checks for unsuitable motifs like restriction enzyme sites.

However, a higher codon adaptation index doesn’t make it the best codon for gene optimization. A good codon adaptation index should not reach extreme levels to avoid GC-rich sequences within the gene. Extreme values in the nucleotides or the amino acid composition can have a significant impact on a differential preference for synonymous codons.

5. A standard vector with restriction enzymes will not cause issues with downstream cloning.

The classic technique of cloning genomic DNA fragments into a plasmid involves digesting with one or more restriction enzymes. You use restriction enzymes and DNA ligase when inserting genes and other pieces of DNA into plasmids during DNA cloning. The standard vectors are designed to contain only a few restriction enzymes. 

The restriction enzyme cloning takes advantage of the site specificity of these enzymes. However, using a standard vector with restriction enzymes will not affect downstream cloning. Even when this site for your restriction enzyme is present, it won’t cause any problem. After restriction enzyme digestion, the resulting fragment will be so small that it won’t be visible.

This means you can use restriction enzymes for high-efficiency cloning. You can use a third restriction enzyme for digestion to make an additional cut in the vector and reduce the size of the unwanted band. If there is no suitable restriction enzyme site, you can amplify the gene with PCR primers. 

Bottom line

DNA synthesis has been incorporated into molecular biology during genetic engineering. This article has debunked some myths with myths and misunderstandings regarding gene synthesis. We have looked at some elements that complicate gene synthesis and reduce instability, transcription, and translation efficiency. Include a promoter sequence upstream of a gene during the gene design to save time and money. For example, you can use the pEX vector as an expression vector for protein expression. It is a standard cloning vector for E. coli as it does not contain a promoter or terminator for the subcloned gene expression. 

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