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Design of Oligonucleotides for Allele-Specific Amplification Based on PCR and Isothermal Techniques

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PCR Primer Design

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2392))

Abstract

Single-nucleotide variations have been associated to various genetic diseases, variations on drug efficiency, and differences in cancer prognostics. The detection of these changes in nucleic acid sequences from patient samples is particularly useful for accurate diagnosis, therapeutics, and disease management. A reliable allele-specific amplification is still an important challenge for molecular-based diagnostic technologies. In the last years, allele-specific primers have been designed for promoting the enrichment of certain variants, based on a higher stability of primer/template duplexes. Also, several methods are based on the addition of a blocking oligonucleotide that prevent the amplification of a specific variant, enabling that other DNA variants can be observed. In this context, genotyping methods based on isothermal amplification techniques are increasing, especially those assays aimed to be deployed at point-of-care applications. The correct selection of target sequences is crucial for reaching the required analytical performances, in terms of reaction time, amplification yield, and selectivity. The present chapter describes the design criteria for the selection of primers and blockers for relevant PCR approaches and novel isothermal strategies. Several successful examples are provided in order to highlight the main design restrictions and the potential to be extended to other applications.

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Author information

Authors and Affiliations

  1. Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, Universitat de València, Valencia, Spain

    Luis Antonio Tortajada-Genaro

  2. Departamento de Química, Universitat Politècnica de València, Valencia, Spain

    Luis Antonio Tortajada-Genaro

  3. Unidad Mixta UPV-La Fe, Nanomedicine and sensors, Valencia, Spain

    Luis Antonio Tortajada-Genaro

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  1. Luis Antonio Tortajada-Genaro
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Correspondence to Luis Antonio Tortajada-Genaro .

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Editors and Affiliations

  1. Department of Biology, California State University, Northridge, Los Angeles, CA, USA

    Chhandak Basu

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Tortajada-Genaro, L.A. (2022). Design of Oligonucleotides for Allele-Specific Amplification Based on PCR and Isothermal Techniques. In: Basu, C. (eds) PCR Primer Design. Methods in Molecular Biology, vol 2392. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1799-1_3

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  • DOI: https://doi.org/10.1007/978-1-0716-1799-1_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1798-4

  • Online ISBN: 978-1-0716-1799-1

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