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Question regarding extensibility settings in Visual OMP:

I noticed one thing when I was playing the VisualOmp. When I tried to predict the binding of a probe to a single strand DNA, the software thinks the heterodimer is extensible species.  There is a clearly AA mismatch at  3' end of probe. Can you explain why it is considered as extensible specie(s)? 

 

Visual OMP provides the ability to define what types of hybridization are extensible.  You can set the "Extensibility Settings" by opening the "Sequences" table clicking on the "Experiment Conditions" tab, and thee clicking the button "Extensibility Settings". 

The "Min Template length" is the number of nucleotides on the template beyond the position where the 3'-end of the primer hybridizes.  This basically accounts for the fact that a polymerase can only extend a primer if there is template available (i.e. polymerase do not extend blunt end hybridizations).

The "Min Basepairs" is thee minimum number of base pairs that must be present in the last 8 positions of the primer in order to be considered a helix that is long enough for the polymerase to extend.  Generally polymerases require at least 5 base pairs to be extended.

The "target overhang" is the number of terminal mismatches of the primer hybridized to the target that are tolerated.  We set this to a default of 1 meaning that we consider a single 3'-mismatch to still be extensible (so that is why the hybridization that you sent us was displayed).  If you only want to see primer binding with perfect complements at the 3'-end, then set Target overhang = 0.  Some polymerases such as Vent and Pfu have 3'-exonuclease activity (which means that they can chew off the primer mismatches and then the polymerase will extend them perfectly fine. Taq polymerase does not have that activity and is better for Allele discrimination.  However, even Taq polymerase will extend some terminal mismatches more than others.  Here are two references about primer extension efficiencies for different mismatches:
Huang, Arnheim, Goodman (1992) NAR 20, 4567-4573.  
Latorra et al. (2003) Hum. Mutat. 22: 79-85.  

I believe that the Latorra study is the more reliable of the two papers.   Basically, it shows that CC and AG are among the least efficently extended mismatches, but AC, CA, CT, and GT are extended quite efficiently.  AA, which is what is observed in the primer that you sent a picture of, is a low efficiency extension mismatch.  Currently, Visual OMP does not account for the sequence dependence, but we do have plans to incorporate this in the future.

Hope this is help,
Sincerely,
John SantaLucia

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