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 synthesised fast and reliable.
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 synthesised and subcloned into any plasmid vector. All synthetic fragments will be sequenced double stranded to ensure 100% sequence congruence.
cDNA Cloning
cDNA synthesis is a very labour- and costintensive 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 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 if required.
Generating qPCR and PCR Standards
With gene synthesis it is possible to design a long sequence consisting several qPCR/PCR standards. It is not necessary anymore to order them separately. Save time and money by having just one standard for all 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.
In cooperation with Biolink we have developed a software called GENEius* that adapts open reading frames to the codon usage of any heterologous organism. GENEius also optimises the sequence. It avoids repeats and hairpin structures; unwanted DNA motifs like restriction sites or artificial splice sites can be excluded; good motifs can be introduced.
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, Arginin 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 Arginin codons will most likely result in very poor expression of the protein.
Codons in the open reading frame to be synthesised, can easily be 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 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.
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 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 to your DNA sequence can be added to ensure proper and easy purification of recombinant proteins. With our optimisation service you can create new hybrid genes by rearranging optimised sequences for higher expression levels, for convenient protein purification and much more.
Creation of Gene Libraries
The generation of mutant libraries with single or multiple mutations in order to to screen for improved properties in proteins is not a challenge anymore with our Gene Evolution Library Service.
