Abstract
PCR with degenerate primers can be used to identify the coding sequence of an unknown protein or to detect a genetic variant within a gene family. These primers, which are complex mixtures of slightly different oligonucleotide sequences, can be optimized to increase the efficiency and/or specificity of PCR in the amplification of a sequence of interest by the introduction of mismatches with the target sequence and balancing their position toward the primers 5′- or 3′-ends. In this work, we explain in detail examples of rational design of primers in two different applications, including the use of specific determinants at the 3′-end, to: (1) improve PCR efficiency with coding sequences for members of a protein family by fully degeneration at a core box of conserved genetic information, with the reduction of degeneration at the 5′-end, and (2) optimize specificity of allelic discrimination of closely related orthologous by 5′-end degenerate primers.
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Acknowledgments
This study had the support of Fundação para a Ciência e Tecnologia (FCT), through the strategic project UID/MAR/04292/2013 granted to MARE, the Department of Economy and Infrastructure of the Regional Government of Extremadura (Group CTS001) and the University of Extremadura (Group MIVET).
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Campos, M.J., Quesada, A. (2017). Strategies to Improve Efficiency and Specificity of Degenerate Primers in PCR. In: Domingues, L. (eds) PCR. Methods in Molecular Biology, vol 1620. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-7060-5_4
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DOI: https://doi.org/10.1007/978-1-4939-7060-5_4
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