Gene Synthesis: Cloning of The Future?

I remember the time when elves and wizards walked the Earth and DNA oligonucleotide synthesis was $5 a nucleotide. But the world has changed, nobody thinks twice about ordering an oligo. Whole gene synthesis, which is synthesis of long oligos and their assembly into a very, very long oligo. With prices of around 25–35 cents per nucleotide, gene synthesis at present is still not cheap, but it is within the reach of many labs.

Why would you do it? There are genes and then there are genes. Some are easy to clone, some are almost impossible to PCR, because they have bad AT or GC rich content or are detrimental to E. coli growth. If you compare half a year spent unsuccessfully trying to clone your gene with $1000 spent on a synthetic gene, the second option doesn’t seem too outrageous.

Ordering Your Gene

The procedure for gene synthesis ordering is:

• You ask your boss’ permission, because $1000 spent is unlikely to go unnoticed.
• Talk to several companies about available options and price. Some companies supply just an assembled gene; some clone it in E. coli vector, giving you an unlimited supply of self-correcting template.
• Having chosen a company it is time to design your sequence. This is the time to do some real genetic engineering. If you want to ensure your gene of interest expresses well in a different organism, you have to adjust to the organism’s transcription and translation machinery. For example, you may want to optimize codons (for protein overexpression in E. coli). Some companies offer to do gene optimization for overexpression as a part of the service so check this out before hand.

Things to Consider When Designing Your Gene

Sequence Design Considerations

The sequence design is critical; you should think as far ahead as possible. Things you may wish to consider are:

Vectors

Consider what vectors you have in the lab and what possible uses your gene can have not only in your current, but also in possible future projects. Adding multiple restriction sites to the 3′ and 5′ of your gene in the synthetic gene sequence will allow you to move it around easily, although you may be able to PCR it from the vector.

Mutations

Studying your protein function often means introducing mutations. You may need to check potential sites of posttranslational modifications or replace amino acids in the active centre if your want to ascertain which of them are important for your enzyme function. It is more cost-effective to synthesize a gene with 20 methionines replaced, than to do 20 subsequent mutagenesis reactions.

The Downsides to Gene Synthesis

Cost

Cons? Well, it is not cheap. Each variant of your gene, different by even one nucleotide, will be an independent gene synthesis. Mean protein length (not gene length as you don’t have to have introns is 270 aa for archaea, roughly 330 for bacteria and 500 for eukaryotes, which at 35 cents per nucleotide will give you cost for an average synthetic gene of around $285–530 but note that these are just estimates and you should check with the company you are ordering from for a final cost.

Errors

As with any outsourcing, even commercial, you risk having unforeseen problems with the product. I recommend that you to talk to the company about their policy on correcting their errors beforehand. Also, make sure you sequence the final gene, despite all assurances.

In my experience a lot changes between the time of order and the time you have time to actually use the construct, even if it is just a month. You need to factor in the possibility that your synthesized gene will never be used – that’s potentially hundreds of dollars wasted.

Do you have any advice for gene synthesis? Have you tried it? Leave us a comment below.

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Literature:

Kruglyak S. and Tang H. Protein-length distributions for the three domains of life. TIG (2000), 16:31;107–9.