SantaLucia, J., Jr. (2007)
"Physical Principles and Visual-OMP Software for Optimal PCR Design", Methods in Molecular Biology: PCR Primer Design, Anton Yuryev, Ed., Humana Press, Totowa, New Jersey, Methods Mol. Biol.
The original publication is available at http://www.springerlink.com/
Summary: The physical principles of DNA hybridization and folding are described within the context of how they are important for designing optimal PCRs. The multi-state equilibrium model for computing the concentrations of competing unimolecular and bimolecular species is described. Seven PCR design “myths” are stated explicitly, and alternative proper physical models for PCR design are described. This chapter provides both a theoretical framework for understanding PCR design and practical guidelines for users. The Visual-OMP (oligonucleotide modeling platform)
package from DNA Software, Inc. is also described.
Key Words: Thermodynamics; nearest-neighbor model; multi-state model;
Visual-OMP; secondary structure; oligonucleotide design; software
Myth 1:
PCR Nearly Always Works and Design Is Not that Important
Myth 2:
Different Methods for Predicting Hybridization Tm Are Essentially Equivalent in Accuracy
Myth 3:
Designing Forward and Reverse Primers to Have Matching Tm’s Is the Best Strategy to Optimize for PCR
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Myth 4:
"Primer Dimer" Artifacts Are Due to Dimerization of Primers
Myth 5:
A BLAST Search Is the Best Method for Determining the Specificity of a Primer
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Myth 6:
At the End of PCR, Amplification Efficiency Is Not Exponential Because the Primers or NTPs Are Exhausted or the Polymerase Looses Activity
Myth 7:
Multiplex PCR Can Succeed by Optimization of Individual PCRs
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