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John SantaLucia Blog

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,
John SantaLucia

ThermoBLAST CE Blog

DNA Software introduces the full commercial release of ThermoBLAST Cloud Edition (TB-CE). 

TB-CE provides a new standard for evaluating the target specificity of oligonucleotides and includes the following features: 

  • Automatic detection of all thermodynamically stable hybridizations against huge genome databases.
  • Automatic detection of PCR amplicons for all combinations of multiplex primers against every GenBank accession in the playlist.
  • Increased speed and database management using the computational capacity of Cloud computing
  • Huge repository of formatted and curated sequence databases
  • Create and format custom sequence playlists in minutes
  • Visualize hits in a new Genome viewer
  • Archive past results in your secure personal account on the Cloud  

CopyCount Blog

The Problem:

Obtaining absolute PCR quantification currently requires the laborious preparation of standards and acquiring a standard curve, thereby wasting reagents and using valuable plate real estate. In addition, the traditional analysis method for qPCR determines the "cycle threshold", Ct or Cq, which varies for different assays, different machines, and varies from plate to plate, thereby making the Ct value hard to interpret.

The Solution:

DNA Software has made a breakthrough in understanding the mechanism of PCR amplification. Our new product, qPCR CopyCount™, allows for any qPCR curve to be analyzed to directly determine the absolute number of copies of DNA at cycle zero. The DNA copy count is the quantity that every biologist wants and the results provided by qPCR CopyCount have unprecedented relative and absolute accuracy.  Click here to watch a video seminar on Copy Count.

  • DNA copy number results that are 3-4 X more precise than Ct method
  • Eliminates two common sources of user error, namely the quantification of standards and the running of standard curves
  • Accelerate your workflow with existing qPCR instrumentation
  • Cost effective alternative to Digital PCR
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Thursday, 06 November 2014 22:15

DNA Software introduced the full commercial release of ThermoBLAST Cloud Edition (TB-CE). TB-CE provides a new standard for evaluating the target specificity of oligonucleotides.  Click here to learn more.  

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