Problems with synthetic gene synthesis

Gijs van Rooijen vanrooij at ACS.UCALGARY.CA
Tue Feb 11 19:46:53 EST 1997


Dear All,



I am trying to resynthesize a gene with optimized expression in plants.
I am trying to resynthesize this gene in several (150-200 bp blocks. The
strategy I use is as follows:

-Synthesis of top strand 80-120mer (A) and a bottom strand 80 120mer (B)
with a 20-28 bp overlap with the top strand oligo.
-Synthesis of anchor oligo C, which is identical to the first 18 24 bp
of A and synthesis of anchor oligo D, which is identical to the first
18-24 bp of B.
-PCR reaction in which the oligo ratio A:B:C:D is 1:1:100:100
respectively.

-Cloning and sequencing of the PCR product

The problem I am having is that I am not able to obtain a mutation free
PCR product. I am seeing a mutation (almost always a single bp deletion)
almost every 50 bp. This problem occurs in all the blocks. I have varied
annealing temperature, polymerase (Taq, Vent, Pwo and Pfu) and magnesium
concentration. I have also tried primer extension using A and B followed
by PCR of this product using C and D. None of these variations have
given me the right product.

It was suggested  that there could be   something wrong with the
synthesized oligos. According to the oligonucleotide synthesizer manual
is not possible to synthesize an oligo with a deletion (The capping
would prevent this).

I was wondering whether you have experienced a similar problem and/or
could give me some advice on how to solve this problem.

I will summarize all the responses and post them back to this newsgroup

Thank you in advance

Gijs van Rooijen



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