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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Royce, R. D., SantaLucia, J., Jr. & Hicks, D. A. (2003). Building an in silico laboratory for genomic assay design. Pharm. Visions 10–12.
SantaLucia, J., Jr. & Hicks, D. (2004). The thermodynamics of DNA structural motifs. Annu. Rev. Biophys. Biomol. Struct. 33, 415–440.
Puglisi, J. &; Tinoco, I., Jr. (1989). Absorbance melting curves of RNA. Methods Enzymol. 180, 304–325.
SantaLucia, J. J. (2000). The use of spectroscopic techniques in the study of DNA stability. In Spectrophotometry and Spectrofluorometry. A Practical Approach (Gore, M. G., ed.), pp. 329–356. Oxford University Press.
SantaLucia, J., Jr. &; Turner, D. H. (1997). Measuring the thermodynamics of RNA secondary structure formation. Biopolymers 44, 309–319.
Press, W. H., Flannery, B. P., Teukolsky, S. A. &; Vetterling, W. T. (1989). Numerical Recipes in C, Cambridge University Press, New York.
SantaLucia, J., Jr. (1998). A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl. Acad. Sci. U. S. A. 95, 1460–1465.
Bommarito, S., Peyret, N. &; SantaLucia, J., Jr. (2000). Thermodynamic parameters for DNA sequences with dangling ends. Nucleic Acids Res. 28, 1929–1934.
Peyret, N., Seneviratne, P. A., Allawi, H. T. &; SantaLucia, J., Jr. (1999). Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A-A, C-C, G-G, and T-T mismatches. Biochemistry 38, 3468–3477.
Allawi, H. T. &; SantaLucia, J., Jr. (1997). Thermodynamics and NMR of internal G-T mismatches in DNA. Biochemistry 36, 10581–10594.
Watkins, N. E., Jr. &; SantaLucia, J., Jr. (2005). Nearest-neighbor thermodynamics of deoxyinosine pairs in DNA duplexes. Nucleic Acids Res. 33, 6258–6267.
Rychlik, W. &; Rhoads, E. R. (1989). A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing, and in vitro amplification of DNA. Nucleic Acids Res. 17, 8543–8551.
Rozen, S. &; Skaletsky, H. (2000). Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132, 365–386.
Li, P., Kupfer, K. C., Davies, C. J., Burbee, D., Evans, G. A. &; Garner, H. R. (1997). PRIMO: a primer design program that applies base quality statistics for automated large-scale DNA sequencing. Genomics 40, 476–485.
Haas, S., Vingron, M., Poustka, A. &; Wiemann, S. (1998). Primer design for large scale sequencing. Nucleic Acids Res. 26, 3006–3012.
Hillier, L. &; Green, P. (1991). OSP: a computer program for choosing PCR and DNA sequencing primers. PCR Methods Appl. 1, 124–128.
Proutski, V. &; Holmes, E. C. (1996). PrimerMaster: a new program for the design and analysis of PCR primers. Comput. Appl. Biosci. 12, 253–255.
Hyndman, D., Cooper, A., Pruzinsky, S., Coad, D. &; Mitsuhashi, M. (1996). Software to determine optimal oligonucleotide sequences based on hybridization simulation data. Biotechniques 20, 1090–1097.
Wallace, R. B., Shaffer, J., Murphy, R. F., Bonner, J., Hirose, T. &; Itakura, K. (1979). Hybridization of synthetic oligodeoxynucleotides to fX174 DNA: the effect of single base pair mismatch. Nucleic Acids Res. 6, 3543–3557.
Sambrook, J., Fritsch, E. F. &; Maniatis, T. (1989). In Molecular Cloning: A Laboratory Manual, 2 edition, Vol. II, pp. 11.46–11.47. Cold Spring Harbor Laboratory Press, New York.
Bolton, E. T. &; McCarthy, B. J. (1962). A general method for the isolation of RNA complementary to DNA. Proc. Natl. Acad. Sci. U. S. A. 48, 1390.
Frank-Kamenetskii, M. D. (1971). Simplification of the empirical relationship between melting temperature of DNA, its GC content and concentration of sodium ions in solution. Biopolymers 10, 2623–2624.
Bonner, T. I., Brenner, D. J., Neufeld, B. R. &; Britten, R. J. (1973). Reduction in the rate of DNA reassociation by sequence divergence. J. Mol. Biol. 81, 123.
Owczarzy, R., Vallone, P. M., Paner, T. M., Lane, M. J. &; Benight, A. S. (1997). Predicting sequence-dependent melting stability of short duplex DNA oligomers. Biopolymers 44, 217–239.
Breslauer, K. J., Frank, R., Blocker, H. &; Marky, L. A. (1986). Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. U. S. A. 83, 3746–3750.
Dimitrov, R. A. &; Zuker, M. (2004). Prediction of hybridization and melting for double-stranded nucleic acids. Biophys. J. 87, 215–226.
Mathews, D., Burkard, M., Freier, S., Wyatt, J. &; Turner, D. (1999). Predicting oligonucleotide affinity to nucleic acid targets. RNA 5, 1458–1469.
Mathews, D. H., Sabina, J., Zuker, M. &; Turner, D. H. (1999). Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288, 911–940.
Innis, M. &; Gelfand, D. H. (1999). Optimization of PCR: conversations between Michael and David. In PCR Applications: Protocols for Functional Genomics (Innis, M., Gelfand, D. H. &; Sninsky, J. J., eds), pp. 3–22. Academic Press, New York.
Henegariu, O., Heerema, N. A., Dlouhy, S. R., Vance, G. H. &; Vogt, P. H. (1997). Multiplex PCR: critical parameters and step-by-step protocol. Biotechniques 23, 504–511.
Ishii, K. &; Fukui, M. (2001). Optimization of annealing temperature to reduce bias caused by a primer mismatch in multitemplate PCR. Appl. Environ. Microbiol. 67, 3753–3755.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press
About this protocol
Cite this protocol
SantaLucia, J. (2007). Physical Principles and Visual-OMP Software for Optimal PCR Design. In: Yuryev, A. (eds) PCR Primer Design. Methods in Molecular Biology™, vol 402. Humana Press. https://doi.org/10.1007/978-1-59745-528-2_1
Download citation
DOI: https://doi.org/10.1007/978-1-59745-528-2_1
Publisher Name: Humana Press
Print ISBN: 978-1-58829-725-9
Online ISBN: 978-1-59745-528-2
eBook Packages: Springer Protocols