The term gene synthesis is actually misleading. Any DNA sequence can be synthesised "de novo" and subsequently subcloned into any plasmid vector. Whether the sequence to be synthesised is a full-length gene, an open reading frame (ORF), a cDNA, a promoter, or a completely new gene (e.g., identified via next generation sequencing)—it does not matter.
Any sequence of any length can be synthesized quickly and reliably.
Cloning
Subcloning
Tedious lab work to get clones for your experiments is no longer necessary. All kinds of required constructs, for any application, can simply be ordered. With our gene synthesis technology, almost any double-stranded DNA sequence can be synthesized and subcloned into any plasmid vector. All synthetic fragments will be sequenced to ensure 100% sequence congruence.
cDNA Cloning
cDNA synthesis is very labor and cost intensive work. It starts with getting the right material that needs to be stored under the correct conditions—often liquid nitrogen is necessary. Extraction of mRNA under RNase-free conditions, challenging reverse transcription, PCR, and subcloning needs to be performed. After DNA sequencing of the subcloned cDNA, the analysis might show that the 5′ or 3′ end of the cDNA is missing, or that mutations have occurred. In such cases, alternative techniques like RACE-PCR or site directed mutagenesis (SDM) would be necessary for getting full length cDNAs.
In contrast, by using our gene synthesis service, you are getting the correct sequence in a much faster, cheaper, and more convenient way. You can even order splice variants.
Generating qPCR and PCR Standards
With gene synthesis, it is possible to design a long sequence consisting of several qPCR/PCR standards. It is no longer necessary to order them separately. Save time and money by having just one standard for all of your qPCR or PCR reactions.
Additionally, with gene synthesis, two alternatives of a SNP position are possible by simply ordering your sequence with IUB codes.
Improvement of Expression in Heterologous Systems
In cooperation with Biolink we have developed GENEius™ sequence optimization software that adapts open reading frames to the codon usage of any heterologous organism. GENEius also optimises the sequence. Repeats and hairpin structures can be avoided; unwanted DNA motifs like restriction sites or artificial splice sites can be excluded; good motifs can be introduced; GC content can be optimized.
Codon Usage Adaptation
The 20 amino acids used by all organisms are encoded by 64 codons. Some amino acids are encoded by up to six different codons. Every species has a different "codon usage" pattern (i.e., some codons, which code for the same amino acid, are used in higher frequencies than others). For example, arginine is encoded by six codons. E. coli uses two of these codons at frequencies of about 40% each. The other four codons are very rarely used in E. coli. If a coding sequence from another organism is introduced into an E. coli expression system, a high frequency of those four rarely used arginine codons will most likely result in very poor expression of the protein.
Codons in the open reading frame to be synthesised can be easily adapted to the codon usage of any organism to ensure optimal translation of proteins in heterologous expression systems. Codons will be used at similar frequencies as they are used in the heterologous expression host and distributed equally over the complete sequence. Very rare codons can even be totally excluded.
Promoter and Enhancer Elements
Ideal promoter or enhancer elements can be chosen and added to the coding sequence to get higher expression levels of mRNA and protein.
Sequence Optimization
Good and Bad Motifs
Unwanted restriction sites or artificial splice sites, potential transcription factor binding sites, or premature polyadenylation signals within your gene sequence can be avoided with synthetic genes. By using GENEius software, we can achieve equal distribution of Gs and Cs or even include unique restriction sites or other motifs (dependent on amino acid sequence) for downstream applications (e.g., introduce restriction sites for easy subcloning of single protein domains in future experiments).
Construction of Hybrid Genes
With synthetic genes you can rearrange protein domains, delete or add introns, or even create completely novel hybrid genes by combining existing gene fragments. Tags and signal sequences can be added to your DNA sequence to ensure proper and easy purification of recombinant proteins.
Creation of Gene Libraries
With our Gene Evolution Library Service, the generation of mutant libraries with single or multiple mutations to screen for improved properties in proteins is no longer a challenge.