Gene Synthesis - Frequently Asked Questions

1. What lengths of synthetic DNA can you synthesize?
   We can synthesize sequences or complete genes from very short fragments up to several thousand base pairs in length. As each synthetic gene project is unique, we’ll be able to provide more information when you contact us for a quote.
   
   
2. How are synthetic genes optimized?
   Eurofins utilizes GENEius sequence optimization software to optimize aspects of your synthetic gene, when requested. You’ll send us the amino acid or DNA sequence and we can optimize your construct to avoid repeated motifs, hairpin structures, and to have a balanced GC content. Also. GENEius software can help with the adaption of codon usage of any organism.
   
   
3. What is the difference between a long oligo and a synthetic gene?
   Due to limitations in oligo synthesis chemistry and other practical considerations, oligos are produced as mixture of single-stranded DNA strands and are limited in length to ~200 bases. Synthetic genes can extend to several thousand, double-stranded base pairs. Usually created as a sequence verified clone, the products from these constructs may be the best tool for your research needs.
   
   
4. What is the difference between genes delivered cloned into a vector and delivered as linear PCR product?
   A Gene Synthesis clone has been fully sequenced and confirmed to have 100% sequence identity to the expected sequence; a PCR product represents a mixture of correct and incorrect sequences due to polymerase incorporation errors. As a consequence of this property of polymerase, subclones created from the original confirmed clone may show variation from the expected sequence. Whenever you use cloning methods that incorporate PCR products for your cloning experiments, we recommend sequencing at least 6-12 subclones to identify one with the expected sequence.
   
   When Eurofins delivers your cloned gene and any additional plasmid preps you order, you will receive materials with the 100% correct sequence and a glycerol stock of a 100% correct clone.
   
   
5. Do you offer cloning into a vector of my choice?
   Yes, Eurofins will create your gene in as many vectors you may choose. One popular example is for customers to order a standard clone, a second construct in a shuttle vector and also a third clone in an expression vector.
   
   
6. What are the quality standards for Eurofins Gene Synthesis services?
   We verify each synthetic gene using DNA sequencing of both strands and ensure 100% sequence accuracy for every synthetic gene. Our gene synthesis labs maintain the highest quality standards throughout the complete synthesis process. Eurofins Gene Synthesis labs adhere to professional Quality Management practices and are certified according to ISO 9001:2000.
   
   
7. How long will it take before I receive my gene?
   The standard delivery times are:
   

   For Standard Genes (up to 950 bp)	12-18 working days
   For every additional 950 bp approx.	5 working days
   For Complex Genes	This varies from 12 working days, and is dependent on gene length and structure
   Additional subcloning into customer vector	5-10 working days

   
   
   
8. How do I order your Gene Synthesis service?
   Whether you will order a standard or complex synthetic gene, they are first quoted based on your construct requirements. Upon submission of your quote request, a Eurofins Customer Service representative will contact you within the hour, and our scientists will have their assessment of the project to you as soon as practical.
   
   
9. In which applications can I use synthetic genes?
   Among the many uses for synthetic genes are adapting codon usage for optimizing gene expression, for protein over-expression and/or protein engineering; as standards for Real Time PCR and standard PCR, mutagenesis studies, to construct hybrid genes, and the production of DNA vaccines.
   
   
10. How should a gene be designed?
    Synthesis of your desired gene is achieved by dividing the sequence into a series of 5´-end overlapping complementary oligonucleotides which we synthesize and assemble. Eurofins MWG Operon uses our proprietary software, GENEius sequence optimization software for the design of the oligonucleotides we’ll create for the construction of your gene however there are some pitfalls to be avoided in order to ensure a successful project.
    
    The formation of potential hairpin structures of 4 or more bases should be avoided within your synthetic gene sequence. Also, sequences that are commonly associated with poor oligo quality such as long stretches of single or di-nucleotide repeats, or high GC content should be avoided. To achieve high levels of gene expression (protein production), we also recommend that you avoid sequences that introduce rarely used codons into a gene.
    
    
11. Are different assembly techniques possible?
    Basically, there are three different approaches for the assembly of synthetic genes. In the approach developed by Khorana (Gupta et al., 1968) a series of sequentially overlapping oligonucleotides are synthesized. As the complementary sequences of the oligos anneal, double-stranded DNA fragments containing nicks on both strands is formed. The nicks are repaired with DNA ligase, an enzyme that catalyzes the formation of a phosphodiester bond between the 5´-phosphate of one double-stranded oligo fragment and the 3´-hydroxyl terminus on an adjacent double-stranded oligo fragment.
    
    Another broadly used approach is the one developed by Narang (Scarpulla et al., 1982) making use of the template-directed and primer-dependent 5´ to 3´-synthesis capability of the large subunit of the enzyme DNA-Polymerase I (Klenow fragment). After end-to-end annealing of the oligos, Klenow uses deoxynucleosidetriphosphates to fill the gaps. Treatment with DNA ligase repairs any nicks in the resulting, double-stranded DNA.
    
    Another broadly used approach is the one developed by Narang (Scarpulla et al., 1982) making use of the template-directed and primer-dependent 5´ to 3´-synthesis capability of the large subunit of the enzyme DNA-Polymerase I (Klenow fragment). After end-to-end annealing of the oligos, Klenow uses deoxynucleosidetriphosphates to fill the gaps. Treatment with DNA ligase repairs any nicks in the resulting, double-stranded DNA.
    
    
    An alternative strategy has been developed, using of synthesizing very long oligonucleotide chains. In this approach (Rossi et al., 1982). In this approach, two long oligos are synthesized and upon annealing, their 3´-ends will overlap. The construct is completed to a full length double-strand using a DNA polymerase to filling-in missing bases. After treatment with the polymerase, overhanging ends are generated on the double-stranded fragment by digestion with the appropriate restriction enzyme.
    
    
    Typically all of these methods are followed by the molecular cloning of the gene into an appropriate vector and so technical limitations of subsequent cloning steps must be considered while developing the assembly strategy. For example, assemblies for larger genes may be divided into sub-assemblies which are sequenced to confirm 100% identity and then brought together to complete the full construct which receives a second round of sequencing to verify the full assembly.
    
    
12. Is de novo synthesis or site-directed mutagenesis preferable if I need to create variant constructs for my experiment?
    When you need the mutations to be distributed across the whole gene, we recommend de novo synthesis. De novo synthesis allows you an opportunity to also optimize features of the gene such as codon usage, GC content, restriction sites etc.
    
    Site-directed mutagenesis is best used when the modifications are few, or are clustered in a small area of the gene.

GENEius software designed and developed for Eurofins MWG Operon by BioLink Informationstechnologie GmbH.