Microchimica Acta

, Volume 184, Issue 5, pp 1453–1462 | Cite as

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

  • Eric Seiti Yamanaka
  • Luis A. Tortajada-Genaro
  • Ángel MaquieiraEmail author
Original Paper


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.


Pharmacogenomics SNP genotyping Isothermal amplification Micro-well plate Microchip Tobacco addiction 3D–printer COMT gene OPRM1 gene ANKK1 gene CHRNA5 gene 



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).

Compliance with ethical standards

The author(s) declare that they have no competing interests.

Supplementary material

604_2017_2144_MOESM1_ESM.docx (4.6 mb)
ESM 1 (DOCX 4659 kb)


  1. 1.
    Manolio TA, Chisholm RL, Ozenberger B, Roden DM, Williams MS, Wilson R et al (2013) Implementing genomic medicine in the clinic: the future is here. Genitourin Med 15:258–267CrossRefGoogle Scholar
  2. 2.
    Scott SA (2013) Clinical pharmacogenomics: opportunities and challenges at point-of-care. Clin Pharmacol Ther 93:33CrossRefGoogle Scholar
  3. 3.
    Limaye N (2013) Pharmacogenomics, Theranostics and personalized medicine-the complexities of clinical trials: challenges in the developing world. Appl Transl Genomics 2:17–21CrossRefGoogle Scholar
  4. 4.
    Abul-Husn NS, Owusu Obeng A, Sanderson SC, Gottesman O, Scott SA (2014) Implementation and utilization of genetic testing in personalized medicine. Pharmacogenomics Pers Med 7:227–240Google Scholar
  5. 5.
    Knez K, Spasic D, Janssen KP, Lammertyn J (2014) Emerging technologies for hybridization based single nucleotide polymorphism detection. Analyst 139:353–370CrossRefGoogle Scholar
  6. 6.
    Shen W, Tian Y, Ran T, Gao Z (2015) Genotyping and quantification techniques for single-nucleotide polymorphisms. TrAC Trends Anal Chem 69:1–13CrossRefGoogle Scholar
  7. 7.
    Milbury CA, Li J, Makrigiorgos GM (2009) PCR-based methods for the enrichment of minority alleles and mutations. Clin Chem 55:632–640CrossRefGoogle Scholar
  8. 8.
    Asari M, Watanabe S, Matsubara K, Shiono H, Shimizu K (2009) Single nucleotide polymorphism genotyping by mini-primer allele-specific amplification with universal reporter primers for identification of degraded DNA. Anal Biochem 386:85–90CrossRefGoogle Scholar
  9. 9.
    Taira C, Matsuda K, Yamaguchi A, Sueki A, Koeda H, Takagi F, Kobayashi Y, Sugano M, Honda T (2013) Novel high-speed droplet-allele specific-polymerase chain reaction: application in the rapid genotyping of single nucleotide polymorphisms. Clin Chim Acta 424:39–46CrossRefGoogle Scholar
  10. 10.
    Tortajada-Genaro LA, Mena S, Niñoles R, Puigmule M, Viladevall L, Maquieira A (2016) Genotyping of single nucleotide polymorphisms related to attention-deficit hyperactivity disorder. Anal Bioanal Chem 408:2339–2345CrossRefGoogle Scholar
  11. 11.
    Woolley CF, Hayes MA (2014) Emerging technologies for biomedical analysis. Analyst 139:2277–2288CrossRefGoogle Scholar
  12. 12.
    Craw P, Balachandran W (2012) Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review. Lab Chip 12:2469–2486CrossRefGoogle Scholar
  13. 13.
    Zhang L, Zhang Y, Wang C, Feng Q, Fan F, Zhang G, Kang X, Qin X, Sun J, Li Y, Jiang X (2014) Integrated microcapillary for sample-to-answer nucleic acid pretreatment, amplification, and detection. Anal Chem 86:10461–10466CrossRefGoogle Scholar
  14. 14.
    Chen F, Zhao Y, Fan C, Zhao Y (2015) Mismatch extension of DNA polymerases and high-accuracy single nucleotide polymorphism diagnostics by gold nanoparticle-improved isothermal amplification. Anal Chem 87:8718–8723CrossRefGoogle Scholar
  15. 15.
    Li J, Macdonald J (2015) Advances in isothermal amplification: novel strategies inspired by biological processes. Biosens Bioelectron 64:196–211CrossRefGoogle Scholar
  16. 16.
    Santiago-Felipe S, Tortajada-Genaro LA, Morais S, Puchades R, Maquieira A (2014) One-pot isothermal DNA amplification–hybridisation and detection by a disc-based method. Sens Actuator B-Chem 204:273–281CrossRefGoogle Scholar
  17. 17.
    Santiago-Felipe S, Tortajada-Genaro LA, Puchades R, Maquieira Á (2016) Parallel solid-phase isothermal amplification and detection of multiple DNA targets in microliter-sized wells of a digital versatile disc. Microchim Acta 183:1195–1202CrossRefGoogle Scholar
  18. 18.
    Tortajada-Genaro LA, Santiago-Felipe S, Amasia M, Russom A, Maquieira A (2015) Isothermal solid-phase recombinase polymerase amplification on microfluidic digital versatile discs (DVDs). RSCAdv 5:29987–29995Google Scholar
  19. 19.
    Li Z, Liu Y, Wei Q, Liu Y, Liu W, Zhang X, Yu Y (2016) Picoliter well Array Chip-based digital recombinase polymerase amplification for absolute quantification of nucleic acids. PLoS One 11:e0153359CrossRefGoogle Scholar
  20. 20.
    Daher RK, Stewart G, Boissinot M, Boudreau DK, Bergeron MG (2015) Influence of sequence mismatches on the specificity of recombinase polymerase amplification technology. Mol Cell Probes 29:116–121CrossRefGoogle Scholar
  21. 21.
    Shin Y, Perera AP, Kim KW, Park MK (2013) Real-time, label-free isothermal solid-phase amplification/detection (ISAD) device for rapid detection of genetic alteration in cancers. Lab Chip 13:2106–2114CrossRefGoogle Scholar
  22. 22.
    NgePN RCI, Woolley AT (2013) Advances in microfluidic materials, functions, integration, and applications. Chem Rev 113:2550–2583CrossRefGoogle Scholar
  23. 23.
    Bhattacharjee N, Urrios A, Kang S, Folch A (2016) The upcoming 3D-printing revolution in microfluidics. Lab Chip 16:1720–1742CrossRefGoogle Scholar
  24. 24.
    Waheed S, Cabot JM, Macdonald NP, Lewis T, Guijt RM, Paull B, Breadmore MC (2016) 3D printed microfluidic devices: enablers and barriers. Lab Chip 16:1993–2013CrossRefGoogle Scholar
  25. 25.
    Bierut LJ, Madden PA, Breslau N, Johnson EO, Hatsukami D, Pomerleau OF, Swan GE, Rutter J, Bertelsen S, Fox L, Fugman D, Goate AM, Hinrichs AL, Konvicka K, Martin NG, Montgomery GW, Saccone NL, Saccone SF, Wang JC, Chase GA, Rice JP, Ballinger DG (2007) Novel genes identified in a high-density genome wide association study for nicotine dependence. Hum MolGen 16:24–35Google Scholar
  26. 26.
    Carpenter MJ, Jardin BF, Burris JL, Mathew AR, Schnoll RA, Rigotti NA, Cummings KM (2013) Clinical strategies to enhance the efficacy of nicotine replacement therapy for smoking cessation: a review of the literature. Drugs 73:407–426CrossRefGoogle Scholar
  27. 27.
    Moody C, Newell H, Viljoen H (2016) FA mathematical model of recombinase polymerase amplification under continuously stirred conditions. Biochem Eng J 112:193–201CrossRefGoogle Scholar
  28. 28.
    Dimitrov RA, Zuker M (2004) Prediction of hybridization and melting for double-stranded nucleic acids. Biophys J 87:215–226CrossRefGoogle Scholar
  29. 29.
    Zhang C, Xing D (2007) Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends. Nucleic Acids Res 35:4223–4237CrossRefGoogle Scholar
  30. 30.
    Liu B, Huang PJJ, Zhang X, Wang F, Pautler R, IpACF LJ (2013) Parts-per-million of polyethylene glycol as a non-interfering blocking agent for homogeneous biosensor development. Anal Chem 85:10045–10050CrossRefGoogle Scholar
  31. 31.
    Wu J, Kodzius R, Cao W, Wen W (2014) Extraction, amplification and detection of DNA in microfluidic chip-based assays. Microchim Acta 181:1611–1631CrossRefGoogle Scholar
  32. 32.
    Li J, Wang L, Mamon H, Kulke MH, Berbeco R, Makrigiorgos GM (2008) Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med 14:579–584CrossRefGoogle Scholar
  33. 33.
    Shen R, Fan JB, Campbell D, Chang W, Chen J, Doucet D, Yeakley J, Bibikova M, Garcia EW, McBride C, Steemers F, Garcia F, Kermani BG, Gunderson K, Oliphant A (2005) High-throughput SNP genotyping on universal bead arrays. Mut Res Fund Mol M 573:70–82CrossRefGoogle Scholar
  34. 34.
    David SP, Strong DR, Leventhal AM, Lancaster MA, McGeary JE, Munafò MR, Bergen AW, Swan GE, Benowitz NL, Tyndale RF, Conti DV, Brown RA, Lerman C, Niaura R (2013) Influence of a dopamine pathway additive genetic efficacy score on smoking cessation: results from two randomized clinical trials of bupropion. Addiction 108:2202–2211CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2017

Authors and Affiliations

  • Eric Seiti Yamanaka
    • 1
  • Luis A. Tortajada-Genaro
    • 1
    • 2
  • Ángel Maquieira
    • 1
    • 2
    Email author
  1. 1.Instituto Inter-universitario de Reconocimiento Molecular y Desarrollo Tecnológico (IDM) - Departamento de QuímicaUniversitat Politècnica de ValènciaValenciaSpain
  2. 2.Unidad Mixta UPV-La Fe, Nanomedicine and SensorsInstituto de Investigacion Sanitaria La FeValenciaSpain

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