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

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.

Keywords: Allele-specific technique; DNA biosensing; Isothermal amplification; PCR; Primer design; Single-nucleotide mutations.