Gene synthesis using long oligos

brett brett at BORCIM.WUSTL.EDU
Thu Apr 10 12:20:25 EST 1997


>Dear netters,
>
>I'm in the process of producing a synthetic gene using overlap extension
>PCR with long oligos (85 nt) and an enzyme mix containing Taq and a
>small amount of a DNA polymerase with proofreading activity. Sequencing
>of the cloned products showed that they contained many errors. Many of
>the errors differed between different clones suggesting incorporation in
>the later stages of synthesis or random errors present in the oligos
>used as starting material. Previously, the first attempts at producing
>another synthetic gene using only DNA polymerase with proofreading
>activity and oligos from another supplier also yielded products with
>many errors, which could be eliminated by combining correct segments of
>different clones.
>Normally, PCR with pure Pwo or Pfu or a mixture of Taq and one of these
>is quite good and very few errors are encountered. Therefore, I am
>inclined to blame the long oligos I used for most of the errors I see.
>If so, are there alternative (affordable) ways of producing long oligos
>with low error rates?
>
>Thank you very much in advance for your comment,
>

This came up a few months ago. The longer your oligos, the greater chance
they will contain errors, most probably small deletions, as the coupling rate
is usually only 99% or so. Also, purification via electrophoresis or HPLC is
practically a necessity for 80-mers. Good luck.


Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             




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