Gene synthesis using long oligos

Gerard Rouwendal g.j.a.rouwendal at ato.dlo.nl
Thu Apr 10 09:01:42 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,


-- 
Gerard J.A. Rouwendal
Tel. +31-(0)317-475315/Fax. +31-(0)317-475347
E-mail: g.j.a.rouwendal at ato.dlo.nl (inst.)
        rouwenda at worldonline.nl (priv.)
Agrotechnological Research Institute ATO-DLO
P.O. Box 17, 6700 AA Wageningen, The Netherlands



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