Why use gene synthesis instead of PCR cloning?
PCR cloning, the most common method of obtaining genes, may not be able to produce a gene of sufficient quality, or even at all. First, a cDNA library for a specific type of tissue has to be prepared or purchased, which requires either time or expense. Second, the gene must be abundant in that cDNA library or it will be very difficult to clone in any useful way. Third, when PCR is successful, its products may have mutations or single nucleotide polymorphisms (SNP), which can cause problems during experimentation or other applications. Finally, with PCR cloning you are limited to copying sequences that exist in nature and employing time-consuming, error-prone methods of mutagenesis and recombination to create custom constructs such as reporter genes, fusion proteins, etc; de novo gene synthesis offers complete flexibility to design your sequence, including codon optimization to improve protein expression levels. Pepmic guarantees 100% sequence fidelity and fast delivery of workable quantities of precise, unmutated genes at a far more economical price than PCR cloning.
Is there a length limit for Pepmic gene synthesis?
There is no length limit. Pepmic routinely synthesizes genes of 10 kb or longer. Although gene synthesis method has no theoretical limits, longer genes require extra planning and have a longer turnaround time, and can sometimes be very difficult to synthesize. We encourage our customers to synthesize no more than 10kb in length. It costs much more, and takes much longer time to synthesize longer genes than 10kb.
Does codon optimization really matter?
It matters as much as protein expression matters. Different organisms have different codon usage preferences. For example, the preferred codons used in a human gene may be rare in bacteria. This can cause problems when researchers attempt to express human genes in E. coli. Using our optimized synthetic genes, many of our customers have reported dramatic increases in protein expression.
What are the applications of gene synthesis?
Gene synthesis has a variety of applications, including the creation of specialized cDNA libraries, large-scale production of microarray-ready cDNA, the design of gene therapy vectors, and the synthesis of gene variants. For a long list of gene synthesis applications, please click here.
What is the standard vector for Pepmic gene synthesis?
Pepmic standard cloning vectors include pUC57, pUC57-Kan and pUC57-Simple. In general, synthetic genes will be cloned into EcoR V site of the standard vector, or any cloning sites you desired.
Can Pepmic subclone the gene into the vector of my choice?
Pepmic can subclone the synthetic gene into the vector of your choice for a reasonable additional fee. The vector (commercial vector or non-commercial vector) must be provided by the customer with a full-length sequence and other requested info.
How can I place my order?
You can give Pepmic your sequence and payment information via a variety of means. If you have a PO number or credit card, you can place the order directly online. Alternatively, you can send us the sequence by e-mail and fax us the PO.
What is the time frame for delivery?
For genes up to 1.5 kb, the delivery time is 10-12 business days. One week is added for each additional 1.5 kb. Express service is available at an additional charge, offering delivery in as few as 4 business days.
For subcloning into a vector provided by customer, our typical turnaround is about 5 business days for < 3 kb genes if the gene template is synthesized at Pepmic at the same time.
What kinds of payments do you accept?
Pepmic accepts credit cards (Visa, MasterCard, American Express), checks and wire transfer. You may send us a PO. For certain orders, Pepmic may require a deposit.
Why is gene synthesis more expensive than oligo synthesis?
During gene synthesis, oligos have to be assembled together in the correct order, which takes time and expertise.
Can you use gene synthesis to finish my partial clone?
Yes. Provided that you have the sequence, Pepmic can synthesize the 5'-end or 3'-end of the gene and anneal it to your partial clone to obtain the full-length gene.
If I do not have the sequence, can you synthesize the gene for me?
No. We need the sequence to synthesize a gene.
What are the steps in the process of gene synthesis?
The steps in gene synthesis include designing and synthesizing oligos, assembling oligos to obtain full-length genes, correcting mutations, and confirming the sequence.
Do you provide protein expression services for codon-optimized genes?
Yes! Pepmic provides protein expression and purification services using bacteria, yeast, baculovirus, and mammalian cell systems.
Can you explain more about codon optimization?
Codon optimization is recommended to achieve optimal expression of recombinant proteins. Pepmic can help re-code the gene sequences according to scientifically researched algorithms, free of charge. Different genes encoding the same protein can have expression in totally different levels. Most proteins are often difficult to express outside their original species or to over-express even within their native species. Altering the coding sequence by codon optimization to increase protein expression is highly recommended.
How do you guarantee the sequence accuracy of my genes? What QC data will be provided?
We guarantee 100% accuracy by sequencing. You will receive complete QC data. Each gene comes with complete QC data: project report, construct map, complete sequence, sequence verifying data (chromatograms) and alignment file.
What is the delivery package?
The delivery specifications for all gene synthesis orders are:
4 µg of lyophilized plasmid containing your gene insert
Sequence chromatograms covering your gene (electronic)
Construct map for the plasmid (electronic)
Quality assurance certificate
Can Pepmic optimize a gene for a specific organism or even for two specific organisms?
Expression in different organisms or tissue types is often enhanced by the use of a special subset of codons. For many organisms and tissues, codon usage tables have been computed from genes with high expression in these target organisms and tissues. By adjusting the codon usage of your synthetic gene to the codon usage of your host organism, you will likely increase its expression efficiency, and consequently, the yield of the expressed protein. We can also optimize a gene using a mixed codon table computed from the codon usage tables of the two organisms.
Which is preferable between gene synthesis and gene modification?
It depends on the complexity of the necessary modifications. If they are scattered across the whole gene and abundant, a complete synthesis is probably advisable. A synthesis also gives you the opportunity to optimize many other features of the gene such as codon usage, restriction sites, introns adapted to the expression host, etc. Modification of an existing gene, on the other hand, is preferable if the modifications are few or are clustered in a small part of the gene. Simple hybrids should also be made by conventional modification procedures.
What is the difference between gene synthesis and fragment synthesis?
With gene synthesis, you receive a plasmid - the target gene is subcloned into a vector of your choice. With fragment synthesis, you receive a ligation product without being subcloned into a vector. The ligation product is immediate product of gene synthesis, which is sequenced and check for any errors. You must ligate the ligation product into a vector in your own lab. Since a small amount of incorrect bases at any position cannot be seen during sequencing (it is overshadowed by the large number of correct bases at that position), you have to transform a host with the vector and screen for correct clones. This is compensated by the much lower price of the ligation product. In our gene synthesis service, we handle the ligation, transformation and screening. You receive the complete plasmid with your gene insert.