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

• Published in: Mikrochimica acta (1966) Vol. 184; no. 5; pp. 1453 - 1462
• Main Authors: Yamanaka, Eric Seiti, Tortajada-Genaro, Luis A., Maquieira, Ángel
• Format: Journal Article
• Language: English
• Published: Vienna Springer Vienna 01.05.2017; Springer; Springer Nature B.V
• Subjects: Amplification; Analysis; Analytical Chemistry; Characterization and Evaluation of Materials; Chemical reduction; Chemistry; Chemistry and Materials Science; Colorimetry; Economic aspects; Genetic aspects; Genetic polymorphisms; Methods; Microengineering; Nanochemistry; Nanotechnology; Nucleic acids; Original Paper; Polylactic acid; Populations; Reagents; Thermoplastics; Three dimensional printing; Tobacco; Isothermal amplification; gene; Tobacco addiction; 3D–printer; ANKK1 gene; Micro-well plate; Microchip; SNP genotyping; Pharmacogenomics
• ISSN: 0026-3672; 1436-5073
• DOI: 10.1007/s00604-017-2144-0

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. Graphical abstract 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.