Seven PCR
Design "Myths"
What you need to know for Optimal PCR
Design |
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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/
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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;
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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
>> CLICK Here for Full Text Sample
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
>>
CLICK Here for Full Text Sample
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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