What sequence properties can complicate the synthesis of my oligo? Will a run of Gs or As cause problems? Will repetitive sequences cause problems?

In unmodified oligos, the two factors which will complicate the synthesis of your oligo are the length and the base composition of the oligo. All chemically synthesized oligos are made by coupling the individual bases together using coupling reactions that are only 99% efficient.  The longer the oligo, the more reactions it takes to produce the oligo.  This leads to a decrease in full length product for longer oligos.

The sequence fidelity also drops as an oligo gets longer, again because the oligos are built base by base using chemical reactions.  Any standard desalt oligo will contain a mixture of full length oligo, 5' truncated products, and oligos which are missing internal bases (deletion mutants).  You can remove a great deal of the truncation and deletion mutants by adding PAGE or HPLC purification to your oligo order.  We strongly recommend additional purification for oligos longer than 50nts in length or for any oligo used for cloning, mutagenesis, crystallography, or other more stringent procedures.

The base composition of your oligo will strongly affect the synthesis efficiency.  Runs of g's can be particularly problematic.  Runs of g cause problems because the functional groups on the g base are positioned in a way which allows hydrogen bonding between successive gs, leading to quadruplex formation. This 'knot' causes problems both with the synthesis and with the oligo's ability to hybridize to its target. In general, shorter runs of g (4-6 gs) tend to be less problematic, especially if they are internal, so we should be able to make the oligo, although we would expect a lower yield than usual. If you have more than six gs in a row, or the gs are directly on the 3' end, you might want to consider redesigning the oligo if you can.  G-rich sequences may be unavoidable if you need a GC-clamp for DGGE or are doing mutagenesis in a g/c rich region.  In this case, you can take your chances with your sequence, but we will not be able to make a yield guarantee, there may be synthesis delays, and if the run of g's is too long, we may not be able to make the oligo at all. 

Very long runs of As can also be problematic.  A is the poorest coupler of the four bases, and when you begin to get very long runs (more than 20) it becomes increasingly difficult to ensure a full length product.  We strongly suggest avoiding runs of more than 50 As in a row because it becomes nearly impossible to synthesize the full length oligo.  We will accept shorter runs, but the yield of full length product will be much lower for oligos with more than 20 As than with oligos with a more heterogenous mixture of bases.  Long runs a Ts and Cs do not affect the overall synthesis efficiency as drastically as As and Gs.

Finally, the base composition of your oligo can cause problems with the synthesis if your sequence causes the oligo to form strong secondary structures.  If the oligo knots up into a strong hairpin, it can be much more difficult to add subsequent bases to the oligo, and it can cause problems purifying out the truncation and deletion mutants.  You can check for strong secondary structures using the OligoAnalyzer 3.0 available for free on our website.

To do a hairpin or dimer analysis, begin by entering your sequence into the box provided on the OligoAnalyzer 3.0 website anc click on the Analyze, Hairpin, Self-Dimer, or Heterodimer option to the right of your sequence.  The most valuable piece of information on this screen is the Tm for each of your structures.  If the Tm of the structure is lower than your reaction conditions, then this structure should not cause any problems.  For self dimer analysis, clicking on 'Self-Dimer' will bring up a new window with each possible self dimer your oligo can form.  For each diagram you will be able to see the calculated delta G value for the secondary structure.  If you have a strong delta g (-9kcal/mol or more negative) this oligo could be problematic.  You can also compare the value of the maximum delta g (the delta g for a perfect duplex) to that of each individual self dimer.  If the values are within 10% of each other, you should redesign.  Heterodimer analysis works the same way.  It is beneficial to test all primers for secondary structure before submitting them for synthesis because the same secondary structures which can lead to a low yield and delayed synthesis can also cause problems while trying to use the oligo.

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