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Low-cost genotyping method based on allele-specific recombinase polymerase amplification and colorimetric microarray detection

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Abstract

The costs of current genotyping methods limit their application to personalized therapy. The authors describe an alternative approach for the detection of single-point-polymorphisms using recombinant polymerase amplification as an allele-specific technique. The use of short and chemically modified primers and locked nucleic acids allowed for a selective isothermal amplification of wild-type or mutant variants at 37 °C within 40 min. An amplification chip platform containing 100 wells was manufactured with a 3D printer and using thermoplastic polylactic acid. The platform reduces reagent consumption and allows parallelization. As a proof of concept, the method was applied to the genotyping of four SNPs that are related to the treatment of tobacco addiction. The target polymorphisms included rs4680 (COMT gene), rs1799971 (OPRM1 gene), rs1800497 (ANKK1 gene), and rs16969968 (CHRNA5 gene). The genotype populations can be well discriminated.

Schematic of the allele-specific recombinase polymerase amplification for genotyping of polymorphisms. The isothermal discrimination reaction occurs in a multi-well amplification chip manufactured with a 3D printer and by using thermoplastic polylactic acid.

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Acknowledgements

The authors acknowledge the financial support received from the Generalitat Valenciana (GVA-PROMETEOII/2014/040 project and GRISOLIA/2014/024 PhD grant) and the Spanish Ministry of Economy and Competitiveness (MINECO CTQ2013-45875-R project).

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Authors and Affiliations

  1. Instituto Inter-universitario de Reconocimiento Molecular y Desarrollo Tecnológico (IDM) - Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n, E46022, Valencia, Spain

    Eric Seiti Yamanaka, Luis A. Tortajada-Genaro & Ángel Maquieira

  2. Unidad Mixta UPV-La Fe, Nanomedicine and Sensors, Instituto de Investigacion Sanitaria La Fe, Valencia, Spain

    Luis A. Tortajada-Genaro & Ángel Maquieira

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  1. Eric Seiti Yamanaka
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  2. Luis A. Tortajada-Genaro
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  3. Ángel Maquieira
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Correspondence to Ángel Maquieira.

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Yamanaka, E.S., Tortajada-Genaro, L.A. & Maquieira, Á. Low-cost genotyping method based on allele-specific recombinase polymerase amplification and colorimetric microarray detection. Microchim Acta 184, 1453–1462 (2017). https://doi.org/10.1007/s00604-017-2144-0

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  • DOI: https://doi.org/10.1007/s00604-017-2144-0

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