Isothermal-based dna biosensors for application in pharmacogenetics

• Autores: Eric Seiti Yamanaka
• Directores de la Tesis: Luis Antonio Tortajada-Genaro (dir. tes.), Ángel Maquieira Catala (dir. tes.)
• Lectura: En la Universitat Politècnica de València ( España ) en 2020
• Idioma: español
• Tribunal Calificador de la Tesis: Ciara K. O'Sullivan (presid.), José Miguel Avellà Oliver (secret.), María Isabel Pividori (voc.)
• Programa de doctorado: Programa de Doctorado en Técnicas Experimentales en Química por la Universitat de València (Estudi General) y la Universitat Politècnica de València
• Materias:
  • Química
    • Química analítica
      • Análisis bioquímico
    • Bioquímica
      • Química clínica
      • Ácidos nucleicos
• Enlaces
  • Tesis en acceso abierto en: RiuNet
• Resumen

  • The determination of genetic biomarkers is progressively becoming more extended and popular, being commercialized even in kits for personalized medicine. Establishing specific genotype variations for each patient, such as single nucleotide polymorphisms (SNPs), could be a fundamental tool in the field of diagnosis, prognosis and therapy selection. However, the use of DNA testing is not fully implemented in general healthcare, mainly due to technical and economic barriers associated to the current technologies, which are limited only to specialized centers and large hospitals.

    In this thesis, the main goal was to overcome these obstacles by developing simpler, faster and more affordable point-of-care (POC) genotyping systems. Allele discrimination was achieved by employing isothermal enzymatic reactions, like recombinase polymerase amplification (RPA), ligation of oligonucleotides and loop-mediated isothermal amplification (LAMP). These processes were integrated to colorimetric indicators and immunoenzymatic assays, in a microarray format. Using compact discs and polycarbonate chips as platforms, the detection was achieved through widespread electronics, like disc-reader, flatbed scanner and smartphone. To demonstrate their capacities, the resulting systems were applied for identifying SNPs in human samples, associated to therapies for tobacco smoking cessation, major depression disorder and blood clotting-related diseases.

    After selecting the proper conditions, all studied strategies discriminated SNPs in samples containing as low as 100 copies of genomic DNA, with an error rate below 15%. Most importantly, the developed methods have reduced assays times varying between 70 and 140 minutes, at a cost similar to a conventional PCR-based analog, but maintaining or raising amplification efficiency and eliminating the need of specialized temperature cyclers and fluorescence scanners.

    In conclusion, the biosensors based in isothermal reactions and consumer electronics devices greatly improve the competitivity of POC DNA analysis. It was demonstrated that the technologies developed in this thesis could support genotyping assays in low-resource areas, such as primary healthcare centers and emerging countries. Through this democratization of genetic testing and by performing adequate association studies, molecular diagnostics and personalized medicine practices could have their application extended to the clinical routine.