Problems with synthetic gene synthesis

The Great American Gene Company geneco at ix.netcom.com
Tue Feb 11 20:46:22 EST 1997


In article <33011225.7CC0 at acs.ucalgary.ca>,
	vanrooij at ACS.UCALGARY.CA (Gijs van Rooijen) wrote:

>
>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
>
>
>
>******************************************************************
>* Gijs van Rooijen PhD        *  Phone:(403) 220-8903 (office)   *
>* Dept of Biological Sciences *        (403) 220-3742 (lab)      *
>* University of Calgary       *   Fax:          (403) 220-0704   *
>* 2500 University Dr NW       * E-mail: vanrooij at acs.ucalgary.ca *
>* Calgary, Alberta            *                                  *
>* Canada T2N 1N4              *                                  *
>******************************************************************

 Dear Gijs:

Read the following two papers.  You will understand the problem:

Farrance et al. 1989. Nucl. Acids Res. 17:1231
Sonveaux, E. 1994. Meth. Mol. Biol. 26:1

The bottom line is synthetic oligos that are homogenous by HPLC and/or PAGE
nevertheless contain randomly distributed defects.  Farrance found that
nearly 10% of 35-mers could not be cleaved enzymatically.  The problem is
that there is a small frequency of spurious chemical modification with each
coupling cycle.  These include the reaction products phosphoesters, abasic
sites, and products arising from the deprotection of exocyclic amines during
ammonolysis and acid hydrolysis.  Transaminination of cytidine and some
small frequency of phosphitylation of O6 in guanine also contribute to the
problem.  

The answer is that long oligos are going to be marginal, at best.  The
underlying chemistry poorly supports the synthesis of oligos longer than
about 65 - 70 bases.  Modern synthesis techniques can give you good coupling
efficiencies, but it cannot eliminate the residual damage.  

-Mike



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