Biomedical Microdevices

, Volume 12, Issue 1, pp 1–12 | Cite as

Microfluidic gradient PCR (MG-PCR): a new method for microfluidic DNA amplification

Article

Abstract

This study develops a new microfluidic DNA amplification strategy for executing parallel DNA amplification in the microfluidic gradient polymerase chain reaction (MG-PCR) device. The developed temperature gradient microfluidic system is generated by using an innovative fin design. The device mainly consists of modular thermally conductive copper flake which is attached onto a finned aluminum heat sink with a small fan. In our microfluidic temperature gradient prototype, a non-linear temperature gradient is produced along the gradient direction. On the copper flake of length 45 mm, width 40 mm and thickness 4 mm, the temperature gradient easily spans the range from 97 to 52°C. By making full use of the hot (90–97°C) and cold (60–70°C) regions on the temperature gradient device, the parallel, two-temperature MG-PCR amplification is feasible. As a demonstration, the MG-PCR from three parallel reactions of 112-bp Escherichia coli DNA fragment is performed in a continuous-flow format, in which the flow of the PCR reagent in the closed loop is induced by the buoyancy-driven nature convection. Although the prototype is not optimized, the MG-PCR amplification can be completed in less than 45 min. However, the MG-PCR thermocycler presented herein can be further scaled-down, and thus the amplification times and reagent consumption can be further reduced. In addition, the currently developed temperature gradient technology can be applied onto other continuous-flow MG-PCR systems or used for other analytical purposes such as parallel and combination measurements, and fluorescent melting curve analysis.

Keywords

Microfluidic gradient PCR (MG-PCR) Microfluidic DNA amplification Continuous-flow Temperature gradient Nature convection 

References

  1. P. Baaske, S. Duhr, D. Braun, Appl. Phys. Lett. 91, 133901 (2007)CrossRefGoogle Scholar
  2. K.M. Balss, D. Ross, H.C. Begley, K.G. Olsen, M.J. Tarlov, J. Am. Chem. Soc 126, 13474–13479 (2004)CrossRefGoogle Scholar
  3. D. Braun, N.L. Goddard, A. Libchaber, Phys. Rev. Lett. 91, 158103 (2003)CrossRefGoogle Scholar
  4. J.S. Buch, C. Kimball, F. Rosenberger, W.E. Highsmith Jr. D.L. DeVoe, C.S. Lee, Anal. Chem. 76, 874–881 (2004)CrossRefGoogle Scholar
  5. J.S. Buch, F. Rosenberger, W.E. Highsmith Jr. C. Kimball, D.L. DeVoe, C.S. Lee, Lab Chip 5, 392–400 (2005)CrossRefGoogle Scholar
  6. M. Chabert, K.D. Dorfman, P. de Cremoux, J. Roeraade, J.L. Viovy, Anal. Chem. 78, 7722–7728 (2006)CrossRefGoogle Scholar
  7. M. Chang, H.J. Lee, Anal. Biochem. 340, 174–177 (2005)CrossRefGoogle Scholar
  8. J.Y. Cheng, C.J. Hsieh, Y.C. Chuang, J.R. Hsieh, Analyst 130, 931–940 (2005)CrossRefGoogle Scholar
  9. N. Crews, T. Ameel, C. Wittwer, B. Gale, Lab Chip 8, 1922–1929 (2008a)CrossRefGoogle Scholar
  10. N. Crews, C. Wittwer, B. Gale, Proc. SPIE 6465, 646504 (2007)CrossRefGoogle Scholar
  11. N. Crews, C. Wittwer, B. Gale, Biomed. Microdevices 10, 187–195 (2008b)CrossRefGoogle Scholar
  12. N. Crews, C. Wittwer, R. Palais, B. Gale, Lab Chip 8, 919–924 (2008c)CrossRefGoogle Scholar
  13. N. Crews, C.T. Wittwer, J. Montgomery, R. Pryor, B. Gale, Anal. Chem. 81, 2053–2058 (2009)CrossRefGoogle Scholar
  14. M. Curcio, J. Roeraade, Anal. Chem. 75, 1–7 (2003)CrossRefGoogle Scholar
  15. K.D. Dorfman, M. Chabert, J.H. Codarbox, G. Rousseau, P. de Cremoux, J.L. Viovy, Anal. Chem. 77, 3700–3704 (2005)CrossRefGoogle Scholar
  16. S. Duhr, D. Braun, Proc. Natl. Acad. Sci. U. S. A. 103, 19678–19682 (2006)CrossRefGoogle Scholar
  17. H.C. Fan, S.R. Quake, Anal. Chem. 79, 7576–7579 (2007)CrossRefGoogle Scholar
  18. J. Grover, R.D. Juncosa, N. Stoffel, M. Boysel, A.I. Brooks, M.P. McLoughlin, D.W. Robbins, IEEE Sens. J. 8, 476–487 (2008)CrossRefGoogle Scholar
  19. T. Kajiyama, Y. Miyahara, L.J. Kricka, P. Wilding, D.J. Graves, S. Surrey, P. Fortina, Genome Res. 13, 467–475 (2003)CrossRefGoogle Scholar
  20. D.J. Kinahan, T.M. Dalton, M.R.D. Davies, Biomed. Microdevices 11(4), 747–754 (2009)Google Scholar
  21. M.M. Kiss, L. Ortoleva-Donnelly, N.R. Beer, J. Warner, C.G. Bailey, B.W. Colston, J.M. Rothberg, D.R. Link, J.H. Leamon, Anal. Chem. 80, 8975–8981 (2008)CrossRefGoogle Scholar
  22. M.U. Kopp, A.J. de Mello, A. Manz, Science 280, 1046–1048 (1998)CrossRefGoogle Scholar
  23. M. Krishnan, V.M. Ugaz, M.A. Burns, Science 298, 793 (2002)CrossRefGoogle Scholar
  24. Y.Y. Li, D. Xing, C.S. Zhang, Anal. Biochem. 385, 42–49 (2009)CrossRefGoogle Scholar
  25. H. Mao, M.A. Holden, M. You, P.S. Cremer, Anal. Chem 74, 5071–5075 (2002a)CrossRefGoogle Scholar
  26. H. Mao, T. Yang, P.S. Cremer, J. Am, Chem. Soc. 124, 4432–4435 (2002b)CrossRefGoogle Scholar
  27. T. Morrison, J. Hurley, J. Garcia, K. Yoder, A. Katz, D. Roberts, J. Cho, T. Kanigan, S.E. Ilyin, D. Horowitz, J.M. Dixon, C.J. Brenan, Nucleic Acids Res. 34, e123 (2006)CrossRefGoogle Scholar
  28. H. Nagai, Y. Murakami, K. Yokoyama, E. Tamiya, Biosens. Bioelectron 16, 1015–1019 (2001)CrossRefGoogle Scholar
  29. P.J. Obeid, T.K. Christopoulos, H.J. Crabtree, C.J. Backhouse, Anal. Chem. 75, 288–295 (2003)CrossRefGoogle Scholar
  30. T. Ohashi, H. Kuyama, N. Hanafusa, Y. Togawa, Biomed. Microdevices 9, 695–702 (2007)CrossRefGoogle Scholar
  31. V.C. Padmakumar, R. Varadarajan, Anal. Biochem. 314, 310–315 (2003)CrossRefGoogle Scholar
  32. N. Park, S. Kim, H. Hahn, Anal. Chem. 75, 6029–6033 (2003)CrossRefGoogle Scholar
  33. D. Ross, L.E. Locascio, Anal. Chem. 74, 2556–2564 (2002)CrossRefGoogle Scholar
  34. W. Rychlik, W.J. Spencer, R.E. Rhoads, Nucleic Acids Res. 18, 6409–6412 (1990)CrossRefGoogle Scholar
  35. Y. Schaerli, R.C. Wootton, T. Robinson, V. Stein, C. Dunsby, M.A. Neil, P.M. French, A.J. Demello, C. Abell, F. Hollfelder, Anal. Chem. 81, 302–306 (2009)CrossRefGoogle Scholar
  36. Y.H. Shim, C.D. Park, D.H. Kim, J.H. Cho, M.H. Cho, H.J. Kim, Biol. Pharm. Bull. 28, 671–676 (2005)CrossRefGoogle Scholar
  37. N. Stoffel, A. Fisher, S.S. Tan, M. Boysel, Proceedings of 57th Electronic Components & Technology Conference, Reno, NV, 2007, pp, 1561-1566 (2007)Google Scholar
  38. Y. Sun, N.T. Nguyen, Y.C. Kwok, Anal. Chem. 80, 6127–6130 (2008)CrossRefGoogle Scholar
  39. W. Sybesma, J. Hugenholtz, I. Mierau, M. Kleerebezem, Biotechniques 31, 466, 468, 470, 472 (2001)Google Scholar
  40. P. Wilding, M.A. Shoffner, L.J. Kricka, Clin. Chem. 40, 1815–1818 (1994)Google Scholar
  41. D.Y. Wu, L. Ugozzoli, B.K. Pal, J. Qian, R.B. Wallace, DNA Cell Biol. 10, 233–238 (1991)CrossRefGoogle Scholar
  42. C.S. Zhang, D. Xing, Nucleic Acids Res. 35, 4223–4237 (2007)CrossRefGoogle Scholar
  43. C.S. Zhang, D. Xing, Y.Y. Li, Biotechnol. Adv. 25, 483–514 (2007a)CrossRefGoogle Scholar
  44. C.S. Zhang, J.L. Xu, W.L. Ma, W.L. Zheng, Biotechnol. Adv. 24, 243–284 (2006)CrossRefGoogle Scholar
  45. C.S. Zhang, J.L. Xu, J.Q. Wang, H.P. Wang, Anal. Lett. 40, 497–511 (2007b)CrossRefGoogle Scholar
  46. H.D. Zhang, J. Zhou, Z.R. Xu, J. Song, J. Dai, J. Fang, Z.L. Fang, Lab Chip 7, 1162–1167 (2007c)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.MOE Key Laboratory of Laser Life Science & Institute of Laser Life ScienceCollege of Biophotonics, South China Normal UniversityGuangzhouPeople’s Republic of China

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