Abstract
Primer design is a crucial initial step in any experiment utilizing PCR to target and amplify a known nucleotide sequence of interest. Properly designed primers will increase PCR amplification efficiency as well as isolate the targeted sequence of interest with higher specificity. Many factors that may limit the success of a primer pair can be detected a priori with computational methods. For example, primer dimer detection, amplification of alternative products, stem loop interference, extreme melting temperatures, and genotype-specific variations in the target sequence can all be considered computationally to minimize subsequent PCR failures. The use of computational sequence analysis tools to select the best primer pair from the available candidates will not only reduce experimental rates of failure but also avoid the generation of misleading results arising from the amplification of alternative products.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
SourceForge.net: Open Source Software, http://sourceforge.net/
Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmer. Met Mol Biol 132:365-386
Rice P, Longden I, Bleasby A (2000) EMBOSS: The European molecular biology open software suite. Trends Gen 16:276-277
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403-410
Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31:3497-3500
Rychlik W, Spencer WJ, Roads RE (1990) Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res 18:6409-6412
Breslauer KJ, Frank R, Blocker H, Marky LA (1986) Prediction DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A 83:3746-3750
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY
Bolton ET, McCarthy BJ (1962) A general method for the isolation of RNA complementary to DNA. Proc Natl Acad Sci U S A 48:1390-1397
Li K, Brownley A, Stockwell T, Beeson K, McIntosh TC, Busam D, Ferriera S, Murphy S, Levy S (2007) Novel computational methods for increasing PCR primer design effectiveness in directed sequencing. BMC Bioinformatics 9:191
Chen SH, Lin CY, Cho CS, Lo CZ, Hsiung CA (2003) Primer design assistant (PDA): a web-based primer design tool. Nucleic Acids Res 31:3751-3754
Weckx S, Rijk PD, Van Broeckhoven C, Del-Favero J (2005) SNPbox: a modular software package for large-scale primer design. Bioinformatics 21:385-387
Yao F, Zhang R, Zhu Z, Xia K, Liu C (2006) MutScreener: primer design tool for PCR-direct sequencing. Nucleic Acids Res 34:W660-W664
Wu X, Munroe D (2006) EasyExonPrimer: automated primer design for exon sequences. Appl Bioinformatics 5(2):119-120
Tsai MF, Lin YJ, Cheng YC, Lee KH, Huang CC, Chen YT, Yao A (2007) PrimerZ: streamlined primer design for promoters, exons and human SNPs. Nucleic Acids Res 35:W63-W65
Acknowledgements
The authors would like to thank colleagues at the J. Craig Venter Institute for their contributions that led to the success of the software implementation of the primer design methods described in this chapter. Sean Murphy and Samuel Levy helped to conceive the project and participated in its design. Tim Stockwell aided in the analysis of data, evaluation of the results, and the management of the authors’ time. Karen Beeson, Tina C. McIntosh, Dana Busam, and Steve Ferriera designed the laboratory protocols, ran the experiments, and generated the data. These concerted efforts were necessary to bring the empirical results closer with the theoretical calculations.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Li, K., Brownley, A. (2010). Primer Design for RT-PCR. In: King, N. (eds) RT-PCR Protocols. Methods in Molecular Biology, vol 630. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-629-0_18
Download citation
DOI: https://doi.org/10.1007/978-1-60761-629-0_18
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-628-3
Online ISBN: 978-1-60761-629-0
eBook Packages: Springer Protocols