looking for Gene Synthesis suggestions

John E. Smith III jsmith at anri.barc.usda.gov
Wed Mar 5 04:52:47 EST 2003


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 (jsmith at anri.barc.usda.gov)
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