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How to make a synonymous library ??

Prateek Gupta

Monday, 29 Oct 2007 16:43 UTC

Hello,

I am interested in looking at the effect of synonymous codons on the gene expression of a particular protein. Can anyone suggest me how to make a random synonymous codon library.

I know it is relatively easy to make random mutant libraries by error-prone PCR, but I am interested in only those mutants which have synonymous codon changes. If anyone has any ideas, please let me know.

Thanks
-Prateek

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    • Mark Isalan

      05 Nov 2007 | 10:32

      Hi Prateek

      I would try building a gene library of my gene using end-to-end ligation of oligos (see Nature Protocols 1:468-475 (2006)). The synonymous codons would be synthesised by the oligo company, using the universal degenerate base code:

      e.g

      protein Asp-His-Arg-Lys
      oligo1 GAY-CAY-CGN-AAR
      oligo2 GAY-CAY-AGR-AAR

      simply combine and anneal kinased oligos with bridge-oligos, ligate and PCR to build library….

      best wishes,
      Mark Isalan

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