looking for Gene Synthesis suggestions

Lei Young lyoung at student.usyd.edu.au
Tue Mar 18 07:08:08 EST 2003


According to Stemmer et al, you should be able to assemble the whole gene
in one reaction, have you tried that? The only thing that is of great
concern is the error rate, which I think is due to the inefficiencies in
the decapping of DMT and the blocking reactions during oligo synthesis,
which would results in series of n-1 oligos with the deletions at
different points. As the deletions should be in different points, it's
worthwhile to introduce restriction sites into the gene, so that after
sequencing, it's possible to reassemble the correct pieces into one.

Alternatively, you can try ozex.netfirms.com, they offer gene synthesis
service, for your gene, it cost merely US$3/bp, and you can be certain
that you get the correct sequence.

John Smith wrote:

> Hello, All
>
> I am planning a gene synthesis strategy and have a few questions.  I
> am trying to build a codon-optimized gene 1287bp long.
>
> To get rid of some of the errors associated with synthesis of long
> oligos, I have planned a series of 60mers with 20 nt overlap at each
> end (aid of the DNABuilder program found online.  I planned to pool
> groups of 6 or so  overlapping oligos together, PAGE purify and
> phosphorylate the groups, and use Pfu pol to fill in the 20bp gaps and
> then use Pfu ligase to seal the ends.  However, I have not found a
> protocol/description of filling in and sealing the gaps.  Does anyone
> have experience doing this?  Can anyone point me to a protocol?
>
> Alternatively, I have found protocols for LCR-based gene synthesis
> which requires abutting phosphorylated oligonucleotides on at least
> one strand.  This approach would get rid of the requirement to fill-in
> gaps.  However, there is the increased cost of  synthesizing extra
> nucleotides.
>
> With long oligonucleotides one could synthesize essential one strand
> of phosphorylated oligos with short overlapping nonphosphorylated
> oligos on the otherstrand.  This approach would require good quality
> long oligos.  Can anyone recommend a source of unusually high quality
> long oligos?
>
> Any comments about considerations/personal experiences and advice will
> be greatly appreciated.
>
> Thanks,
>
> -John Smith (drjes3 at yahoo.com or jsmith at anri.barc.usda.gov)




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