Abstract
Continuous-flow thermal gradient PCR is a new DNA amplification technique that is characterized by periodic temperature ramping with no cyclic hold times. The device reported in this article represents the first demonstration of hold-less thermocycling within continuous-flow PCR microfluidics. This is also the first design in which continuous-flow PCR is performed within a single steady-state temperature zone. This allows for straightforward miniaturization of the channel footprint, shown in this device which has a cycle length of just 2.1 cm. With a linear thermal gradient established across the glass device, the heating and cooling ramp rates are dictated by the fluid velocity relative to the temperature gradient. Local channel orientation and cross-sectional area regulate this velocity. Thus, rapid thermocycling occurs while the PCR chip is maintained at steady state temperatures and flow rates. Glass PCR chips (25 × 75 × 2 mm) of both 30 and 40 serpentine cycles have been fabricated, and were used to amplify a variety of targets, including a 181-bp segment of a viral phage DNA (ΦX174) and a 108-bp segment of the Y-chromosome, amplified from human genomic DNA. With this unique combination of hold-less cycling and gradient temperature ramping, a 40-cycle PCR requires less than 9 min, with the resulting amplicon having high yield and specificity.
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Notes
The primer sequences for the selected targets are as follows: ΦX174, 110-bp (F - GGTTCGTCAAGGACTGGTTT, R - TTGAACAGCATCGGACTCAG) ΦX174, 181-bp (F - GCTTCCATGACGCAGAAGTT, R - GCGAAAGGTCGCAAAGTAAG) Y-chromosome, 108-bp (F - ATTACACTACATTCCCTTCCA, R - AGTGAAATTGTATGCAGTAGA)
References
W. Cao, C.J. Easley, J.P. Ferrance, J.P. Landers, Anal. Chem. 78, 7222–7228 (2006)
J. Chiou, P. Matsudaira, A. Sonin, D. Ehrlich, Anal. Chem. 73, 2018–2021 (2001)
C.J. Easley, J.M. Karlinsey, J.M. Bienvenue, L.A. Legendre, M.G. Roper, S.H. Feldman, M.A. Hughes, E.L. Hewlett, T.J. Merkel, J.P. Ferrance, J.P. Landers, PNAS 103, 19272–19277 (2006)
T. Fukuba, T. Yamamoto, T. Naganuma, T. Fujii, Chem. Eng. J. 101, 151–156 (2004)
P. Garstecki, M.J. Fuerstman, M.A. Fischbach, S.K. Sia, G.M. Whitesides, Lab Chip 6, 207–212 (2006)
M. Hashimoto, P.C. Chen, M.W. Mitchell, D.E. Nikitopoulos, S.A. Soper, M.C. Murphy, Lab Chip 4, 638–645 (2004)
R.M. Jendrejack, E.T. Dimalanta, D.C. Schwartz, M.D. Graham, J.J. de Pablo, Phys. Rev. Lett. 91, (2003)
W.M. Kays, M. E. Crawford. Convective Heat and Mass Transfer (McGraw-Hill, New York, 1993), pp. 110–116
M.U. Kopp, A.J. de Mello, A. Manz, Science 280, 1046–1048 (1998)
J. Lapham, J.P. Rife, P.B. Moore, D.M. Crothers, J. Biomol. Nmr. 10, 255–262 (1997)
S.F. Li, D.Y. Fozdar, M.F. Ali, H. Li, D.B. Shao, D.M. Vykoukal, J. Vykoukal, P.N. Floriano, M. Olsen, J.T. McDevitt, P.R.C. Gascoyne, S.C. Chen, Journal of Microelectromechanical Systems 15, 223–236 (2006)
H.B. Mao, M.A. Holden, M. You, P.S. Cremer, Anal. Chem. 74, 5071–5075 (2002)
T. Nakayama, Y. Kurosawa, S. Furui, K. Kerman, M. Kobayashi, S.R. Rao, Y. Yonezawa, K. Nakano, A. Hino, S. Yamamura, Y. Takamura, E. Tamiya, Anal. Bioanal. Chem. 386, 1327–1333 (2006)
P.J. Obeid, T.K. Christopoulos, Anal. Chim. Acta 494, 1–9 (2003a)
P.J. Obeid, T.K. Christopoulos, H.J. Crabtree, C.J. Backhouse, Anal. Chem. 75, 288–295 (2003b)
K. Pappaert, J. Biesemans, D. Clicq, S. Vankrunkelsven, G. Desmet, Lab Chip 5, 1104-1110 (2005)
K.M. Ririe, R.P. Rasmussen, C.T. Wittwer, Anal. Biochem. 245, 154–160 (1997)
M.G. Roper, C.J. Easley, L.A. Legendre, J.A.C. Humphrey, J.P. Landers, Anal. Chem. 79, 1294–1300 (2007)
I. Schneegass, R. Brautigam, J.M. Kohler, Lab Chip 1, 42–49 (2001)
P.C. Simpson, A.T. Woolley, R.A. Mathies, Biomed. Microdevices 1, 7–25 (1998)
K. Sun, A. Yamaguchi, Y. Ishida, S. Matsuo, H. Misawa, Sens. Actuators, B: Chem. 84, 283–289 (2002)
H. Wang, J.F. Chen, L. Zhu, H. Shadpour, M.L. Hupert, S.A. Soper, Anal. Chem. 78, 6223–6231 (2006)
C.T. Wittwer, M.G. Hermann, in PCR Applications: Protocols for Functional Genomics, eds. by M.A. Innis, D.H. Gelfand, J.J. Sninsky (Academic, San Diego, 1999), pp. 211–229
C.T. Wittwer, G.B. Reed, K.M. Ririe, in “Rapid Cycle DNA Amplification.” The Polymerase Chain Reaction, eds. by K.B. Mullis, F. Ferre, R. Gibbs (Springer, Deerfield Beach, 1994), pp. 174–181
M. Yang, R. Pal, M. A. Burns, J Micromechanics Microengineering 15, 221–230 (2005)
Acknowledgement
Authors acknowledge the Utah State Center of Excellence Grants, the University of Utah Synergy Program, and the NSF IGERT Program for the funding of this work. Authors also wish to thank the respective members of the Wittwer and Gale research labs, whose valued contributions have allowed for this project to advance at a welcome pace.
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Crews, N., Wittwer, C. & Gale, B. Continuous-flow thermal gradient PCR. Biomed Microdevices 10, 187–195 (2008). https://doi.org/10.1007/s10544-007-9124-9
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DOI: https://doi.org/10.1007/s10544-007-9124-9