Abstract
Efficient and rapid isolation of nucleic acids is of significant importance in the field of genomics for a variety of applications. Current techniques for the isolation of specific nucleic acids or genes typically involve multiple rounds of amplification of the target sequence using polymerase chain reaction. Described here is a recent development in the fabrication and modification of porous polymer monoliths for the selective concentration and extraction of nucleic acids sequences. The rigid monoliths are cast to shape and are tunable for functionalization using a variety of amine-terminated molecules including oligonucleotide capture probes. Efficient and rapid isolation of nucleic acids can be performed using polymer monoliths in microchannels in a time frame as short as 2 s. The described materials and methods offer the ability to perform concentration of nucleic acids in solution and elute purified samples in volumes as low as 3 µL without the requirement of altering salt concentration in the wash and elution buffers.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Mouradian, S. (2002) Lab-on-a-chip: applications in proteomics. Curr. Opin. Chem. Biol. 6(1), 51–46.
Bilitewski, U., Genrich, M., Kadow, S., and Mersal, G. (2003) Biochemical analysis with microfluidic systems. Anal. Bioanal. Chem. 377(3), 556–469.
Verpoorte, E. (2002) Microfluidic chips for clinical and forensic analysis. Electrophoresis 23(5), 677–712.
de Mello, A. J. and Beard, N. (2003) Dealing with real samples: sample pre-treatment in microfluidic systems. Lab. Chip. 3(1), 11N–19N.
Selvaganapathy, P. R., Carlen, E. T., and Mastrangelo, C. H. (2003) Recent progress in microfluidic devices for nucleic acid and antibody assays. Proc. IEEE 91(6), 954–975.
Jacobsen, N., Nielsen, P. S., Jeffares, D. C., et al. (2004) Direct isolation of poly(A)+ RNA from 4 M guanidine thiocyanate-lysed cell extracts using locked nucleic acid-oligo(T) capture. Nucleic Acids Res. 32(7), e64.
Burgener, M., Candrian, U., and Gilgen, M. (2003) Comparative evaluation of four large-volume RNA extraction kits in the isolation of viral RNA from water samples. J. Virol. Methods 108(2), 165–170.
Aviv, H. and Leder, P. (1972) Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA 69(6), 1408–1412.
Yu, C., Davey, M. H., Svec, F., and Frechet, J. M. (2001) Monolithic porous polymer for on-chip solid-phase extraction and preconcentration prepared by photoinitiated in situ polymerization within a microfluidic device. Anal. Chem. 73(21), 5088–5096.
Throckmorton, D. J., Shepodd, T. J., and Singh, A. K. (2002) Electrochromatography in microchips: reversed-phase separation of peptides and amino acids using photopatterned rigid polymer monoliths. Anal. Chem. 74(4), 784–789.
Shediac, R., et al. (2001) Reversed-phase electrochromatography of amino acids and peptides using porous polymer monoliths. J. Chromatogr. A 925(1–2), 251–263.
West, J. A. A., Hukari, K. W., Hux, G., and Shepodd, T. J. (2004) Microfluidic gene arrays for rapid genomic profiling, in Proceedings of the Society of Photo-Optical Instrumentation Engineers (spie) Conference on Lab-on-a-Chip: Platforms, Devices, and Applications, October 26–28, 2004, Philadelphia, pp. 167–173.
Yu, C., Svec, F. and Frechet, J. M. (2000) Towards stationary phases for chromatography on a microchip: molded porous polymer monoliths prepared in capillaries by photoinitiated in situ polymerization as separation media for electrochromatography. Electrophoresis 21(1), 120–127.
Peterson, D. S., Rohr, T., Svec, F., and Frechet, J. M. (2003) Dual-function microanalytical device by in situ photolithographic grafting of porous polymer monolith: integrating solid-phase extraction and enzymatic digestion for peptide mass mapping. Anal. Chem. 75(20), 5328–5335.
Yu, C., Xu, M. C., Svec, F., and Frechet, J. M. (2002) Preparation of monolithic polymers with controlled porous properties for microfluidic chip applications using photoinitiated free-radical polymerization. J. Polymer Sci. Part A Polymer Chem 40(6), 755–769.
Ngola, S. M., Fintschenko, Y., Choi, W. Y., and Shepodd, T. J. (2001) Conductascast polymer monoliths as separation media for capillary electrochromatography. Anal. Chem. 73(5), 849–856.
Lichtenberg, J., de Rooij, N. F. and Verpoorte, E. (2002) Sample pretreatment on microfabricated devices. Talanta 56(2), 233–266.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
West, J.A.A., Satterfield, B.C. (2007). Fabrication of Porous Polymer Monoliths in Microfluidic Chips for Selective Nucleic Acid Concentration and Purification. In: Floriano, P.N. (eds) Microchip-Based Assay Systems. Methods in Molecular Biology™, vol 385. Humana Press. https://doi.org/10.1007/978-1-59745-426-1_2
Download citation
DOI: https://doi.org/10.1007/978-1-59745-426-1_2
Publisher Name: Humana Press
Print ISBN: 978-1-58829-588-0
Online ISBN: 978-1-59745-426-1
eBook Packages: Springer Protocols