Benefits of de novo gene synthesis
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 Eurofins Genomics and Blue Heron Biotech 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.
Free DNA Archiving at Blue Heron
Blue Heron Biotech offers free DNA archiving for every gene and vector. This enables us to provide our customers with affordable pricing for additional custom cloning projects or reshipments of any order. Customers pay only the cloning fee for any custom cloning orders of previously synthesized genes. Blue Heron upholds strict security and intellectual property standards, ensuring that your sequences and material are protected.
Vector and Cloning options
Both Eurofins Genomics and Blue Heron Biotech have several vector options for your convenience. Our gene synthesis capacity and cloning expertise allows us to synthesize any desired features to be used for vector modification or De Novo vector synthesis. Providing your own vector for custom cloning is simple with our online ordering tool. Alternatively, clone your synthesis products into any of Blue Heron’s standard high copy vectors at no cost and with no cloning fee. We offer high copy vectors with and without multiple cloning sites allowing you the flexibility to flank your insert with any desired cloning site for your downstream processes. Blue Heron also offers a selection of expression vectors at no additional cost.
Custom Cloning Solutions
Blue Heron Biotech’s custom cloning service provides efficient, low-cost custom cloning into any vector. Blue Heron offers standard pUC derivative vector and a selection of OriGene expression vectors at no additional cost. Alternatively, clone into any vector of your choice using your selected cloning sites.
Adding subcloning to your gene synthesis order can speed up the research process and save you valuable time. Synthesis and cloning products are always sequence verified to ensure quality and sequence fidelity. Custom cloning by Blue Heron adds minimal time to the production of your gene synthesis order.
Free archiving is provided for every order with Blue Heron, so ordering additional DNA or requesting subcloning at a later date is always cost effective. Eliminate the need for multiple vendors with gene synthesis, custom cloning, and plasmid preparation from Blue Heron.
Optimized Process
Traditional Gene assembly: traditional methods of gene assembly requires a template with the target gene and involves tedious protocols to get the same. Besides, the cost of the reagents, the researchers' time is spent on getting the gene or protein. Creation of a fusion proteins or variants of a gene further elongated the time line.
Custom Gene Synthesis by Eurofins Genomics: ordering a new gene from Eurofins Genomics can be as simple as entering the sequence on the web portal and checking the same out. Your gene of interest (<1000 bp long) will be delivered within a week, longer genes or genes with complex segments can take longer.
The protocol for the assembly of the synthetic genes once the order is placed goes through the following steps:
1. Expert Consultation (upon request)
If your sequence shows a high-level of difficulties, e.g., several hairpins, repeats or other difficulties, our Gene Synthesis experts can offer suggestions and/or production feasibility.
2. Sequence Optimization (upon request)
The desired amino acid or the DNA sequence can be optimized and adapted for high-level of protein expression in the host of choice using our proprietary software GENEius. This in-silico optimization tool distributes G's and C's evenly, removes 'bad motiffs' and easily incorporates good motiffs. Try our GENEius light and discover what our optimization software can do for your gene sequence.
3. In silico experiment design
On the receipt of an order for a synthetic gene, GENEius goes to work on the sequence. During this step GENEius does not modify the dsDNA sequence; instead, GENEius breaks down the dsDNA sequence into overlapping single-stranded oligonucleotides. Subsequently, GENEius places the order for the synthesis of these oligonucleotides.
4. Oligonucleotide Synthesis
The Oligo Synthesis lab synthesizes the oligonucleotides required for the assembly of the genes. These oligo are synthesized usign a robust, high-fidelity synthesis protocol that ensures that oligos have a very low sequences error. Such oligonulcoetides ensure higher rate of success in the synthesis of gene and also ensure fast turnaround times.
5. Gene Synthesis
Our Gene Synthesis facility is housed in close proximity to both the Oligo Synthesis Lab and DNA Sequencing Lab and, therefore, facilitates the seamless and automated transfer of materials from one lab to the other. Moreover, our Ph. D. level gene synthesis specialists ensure that the best gene assembly protocols are followed during the, ligation, assembly and amplification of the linear dsDNA strand.
6. Subcloning
There are many different cloning options. Details can be found here. By default, your gene will be subcloned into one of our standard vectors. These are high-copy vectors with resistance to either ampicillin or kanamycin when transformed. You can also select to clone into a custom vector or other commercially available vector.
7. Quality Control
Our in-house DNA sequencing facility uses the Sanger method to read the prepared DNA. Only the plasmid with accurate sequence is taken to the next step.
8. Delivery & Documentation
A standard mini-prep is performed and the resulting plasmid is shipped to you. The complete DNA sequence, the plasmid description, and the restriction map are provided.
Please note that strict confidentiality is ensured for every project. The high quality of our service is overseen by a professional quality management.
Safety and Regulations
Eurofins Genomics screens each gene ordered to determine if the gene contains a regulated pathogen sequence. In the case that the gene contains such a regulated sequence, Eurofins Genomics reserves the right to reject an order and inform regulatory and law enforcement agencies.
Eurofins Genomics also takes necessary steps in ensure that customers who order gene synthesis products are members of bona fide life science research or diagnostic institution. All customers must provide an institution name, billing address, shipping address, e-mail address, and telephone number. Eurofins Genomics does not ship any products to P.O. boxes, and every delivery requires a signature of receipt.
Eurofins Genomics retains records of every quote request and order of gene synthesis products for a minimum of 4 years. These records include both sequence and customer information.
Additional Information
If the synthesis of cDNA, low protein expression levels, or site-directed mutagenesis has slowed down the pace of your research, our molecular biology services has a powerful solution that can save you time as well as money.
The confluece of the capabilities in oligo synthesis and DNA sequencing along with the experience of our molecular biology scientists allows us to offer nearly any double-stranded DNA sequence. These dsDNA sequences are assembled de novo using oligos synthesized at our Oligo Synthesis Lab. The benefits provided by such a de novo gene synthesis include:
- Ready synthesis of fusion proteins.
- Removal of bad motiffs or unwanted restriction sites.
- Availability of cDNA or parent plasmid is not a bottleneck anymore.
- Sequence optimization can lead to higher levels of protein expression.
Order Synthetic Genes from us and get a head start on your project time-table.
While we can assemble a gene with any sequence, the effort for the synthesis and assembly of a gene is dependent on the sequence of the gene. Genes with low sequence variation can prove challenging both in the case of oligo synthesis and gene assembly. Currently, our Molecular Biology services in US is offering gene with the following features:
- Standard genes
- Sequence length: < 3,000 bp
- Mini-prep of the assembled gene
- Sequence verified.
Standard Gene do not included sequences with low variations. The features of the sequence that make the synthesis/assmebly of the gene complex include:
- High (above 75%) or low (below 35%) GC contenct
- Extensive direct or indirect repeats (>18 bp)
- Extensive homopolymeric stretches (>8 bp)
- Critical hairpin structures
Please note that using the GENEius sequence optimization tool, sequences containing segments with these complexities can be optimized to remove these complex features and for higher protein expression in the host organism. However, if it is critical that the gene be prepared with the complex sequence intact, please contact our sales representative at GenomicsUS@eurofins.com. The synthesis of gene containing complex segment takes a longer time.
When you order a Standard Gene by submitting the sequence of the desired insert, you will get:
- A sequence-verified plasmid containing your gene of interest.
- Vector map