DNA Extraction Using a Tetramethyl Orthosilicate-Grafted

Anal. Chem. 2006, 78, 1673-1681

DNA Extraction Using a Tetramethyl Orthosilicate-Grafted Photopolymerized Monolithic Solid Phase Jian Wen,†,‡,§ Christelle Guillo,‡.§ Jerome P. Ferrance,‡ and James P. Landers*,‡,|

Departments of Molecular Physiology and Biological Physics, and the Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, and the Department of Pathology, University of Virginia Health Science Center, Charlottesville, Virginia 22908

A novel high-capacity, high-efficiency DNA extraction method is described using a photopolymerized silicabased monolithic column in a fused-silica capillary. Development involved investigation of the composition of the sol-gel monomer, fabrication conditions, and surface modifications in order to optimize the binding capacity. Extraction capacity and efficiency with the 3-(trimethoxysilyl)propyl methacrylate (TMSPM) monolith formulations fabricated in capillaries were investigated using a simple three-step procedure consisting of sample loading, washing of the solid phase, and elution of the DNA using a low ionic strength Tris buffer at pH 8. Once the TMSPM monomer concentration was optimized to yield a monolith with maximum test stability (robustness) and minimum back pressure, the monolith surface was modified by the grafting of tetramethyl orthosilicate (TMOS) for increased DNA binding capacity. After the examination of a variety of TMOS concentrations, 85% v/v TMOS was found to be optimal for DNA extraction without any obvious changes to the monolith structure. The reduction of time allowed for TMSPM hydrolysis prior to UV polymerization from 20 to 5 min led to a lower back pressure of the monolith, enabling better TMOS derivatization and therefore higher binding capacity. Minimal buffer volume (as low as 1 µL) was required to elute DNA from the solid phase, providing a DNA concentrating effect potentially important for downstream processes. While experimentation employed monolithic columns that were 12 cm in length, reduction of the length to 2 cm still allowed for a DNA binding capacity of at least 100 ng of prepurified human genomic DNA and extraction efficiencies greater than 85%. Extraction of low sample volumes (submicroliter) of human whole blood were successfully performed, with extraction efficiencies from the 2-cm monolithic column higher than those obtained from a commercial DNA extraction kit. These results position this novel matrix as an attractive * To whom correspondence should be addressed. Phone: 434-243-8658. Fax: 434-243-8852. E-mail: [email protected]. † Department of Molecular Physiology and Biological Physics, University of Virginia. ‡ Department of Chemistry, University of Virginia. § Contributed equally to this work. | University of Virginia Health Science Center. 10.1021/ac051796t CCC: $33.50 Published on Web 01/20/2006

© 2006 American Chemical Society

alternative for solid-phase extraction of DNA and other biologically active molecules in microscale devices. The last two decades have seen significant effort invested in reducing the amount of sample required for analysis, often to address the needs of clinical and forensic communities. The development of new techniques such as capillary electrophoresis, micro-HPLC, and, more recently, the transfer of traditional analytical methodologies to the microscale format, have enabled the development of assays designed for small-volume applications (typically in the nano- to microliter range). In particular, the miniaturization of sample preparation techniques, including analyte purification and enrichment, has been extensively investigated to minimize sample handling, reduce contamination, and speed up analysis time.1-7 DNA purification and preconcentration, a requirement for almost all genetic analysis applications, is a crucial step in clinical/forensic laboratories due to the small amount of sample available for analysis. Conventional DNA extractions were commonly performed by liquid-liquid extraction,8,9 but the tedious precipitation steps and difficulties in automating for highthroughput DNA purification led to the development of solid-phase extraction (SPE) methods.10,11 SPE is now the sample purification technique of choice allowing for the extraction of DNA from crude biological matrixes, including blood. The advantageous characteristics of microscale analyses, including small sample volume requirements, fewer processing steps, and shorter analysis times, (1) Saito, Y.; Jinno, K. J. Chromatogr., A 2003, 1000, 53-67. (2) Guzman, N. A. Electrophoresis 2003, 24, 3718-3727. (3) Marko-Varga, G. A.; Nilsson, J.; Laurell, T. Electrophoresis 2004, 25, 34793491. (4) Ekstrom, S.; Wallman, L.; Malm, J.; Becker, C.; Lilja, H.; Laurell, T.; MarkoVarga, G. Electrophoresis 2004, 25, 3769-3777. (5) Chung, Y. C.; Jan, M. S.; Lin, Y. C.; Lin, J. H.; Cheng, W. C.; Fan, C. Y. Lab Chip 2004, 4, 141-147. (6) Horsman, K. M.; Barker, S. L. R.; Ferrance, J. P.; Forrest, K. A.; Koen, K. A.; Landers, J. P. Anal. Chem. 2005, 77, 742-749. (7) Bienvenue, J. M.; Duncalf, N.; Marchiarullo, D.; Ferrance, J. P.; Landers, J. P. Accepted to J. Forensic Sci. (8) Rudin, L.; Albertsson, P. A. Biochim. Biophys. Acta 1967, 134, 37-44. (9) Henry, J. M.; Raina, A. K.; Ridgway, R. L. Anal. Biochem. 1990, 185, 147150. (10) Boom, R.; Sol, C. J. A.; Salimans, M. M. M.; Jansen, C. L.; Wertheim-van Dillen, P. M. E.; Van der Noordaa, J. J. Clin. Microbiol., 1990, 28, 496503. (11) McCormick, R. M. Anal. Biochem. 1989, 181, 66-74.

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have led to the development of micro-SPE devices in both capillaries12 and microdevices.13 The most widely used solid supports for SPE of DNA have been silica-based particles, where the interaction of DNA with silica is thought to involve some combination of intermolecular electrostatic forces, DNA dehydration, and intermolecular hydrogenbonding forces.14 Biologically inert silica nanoparticles, covalently modified to create a cationic surface, have also been shown to bind plasmid DNA electrostatically, and thus could be used as a solid-phase matrix for DNA purification.15 In the microscale format, DNA purification on a glass microchip using silica resins has been achieved with good efficiency; however, reproducibility was a problem as a result of the inability to completely immobilize the silica beads.13 This problem has been addressed using the dual weir-type approach described by Oleschuk et al.16 or the method described in Breadmore et al.17 to immobilize the beads. With the latter case, the silica beads were packed into the glass microchip and then immobilized with a nanoglue, in the form of a tetraethoxyorthosilicate (TEOS)-based sol-gel, to provide a continuous and stable solid phase for DNA extraction. Incorporation of existing SPE methods into microdevices has several disadvantages, most notably, the fabrication of the solid phase within the device through bead packing. The additional processes involved in filling the microchannel with beads or particles and the chip-to-chip extraction reproducibility are significant problems. In addition, while the surface area available for DNA binding is enhanced by decreasing the bead diameter, smaller diameter beads (e.g., 5 µm) are more difficult to contain and result in higher back pressures, thus, limiting SPE microdevices to relatively low flow rates and low binding capacity. It has already been demonstrated that the packing problem can be eliminated by providing a high surface area-to-volume ratio in the SPE chamber through the etching of pillars in the chamber during fabrication.18,19 While this increases the surface area for DNA binding and provides a regular array for reproducible chromatography, the complex fabrication requirements and cost make these devices less attractive. In addition, a large volume of elution buffer (greater than 50 µL) is required to elute the bound DNA, potentially creating difficulties with downstream processing (e.g., PCR). The work of Svec, Frechet, and co-workers in the 1990s has truly pioneered the use of rigid, porous monoliths based on organic polymers as alternative column materials for HPLC,20-22 and the efforts from this group have provided new possibilities (12) Tian, H.; Hu ¨ hmer, A. F. R.; Landers, J. P. Anal. Biochem. 2000, 283, 175191. (13) Wolfe, K. A.; Breadmore, M. C.; Ferrance, J. P.; Power M. E.; Conroy J. F.; Norris P. M.; Landers J. P. Electrophoresis 2002, 23, 727-733. (14) Melzak, K. A.; Sherwood, C. S.; Turner, R. F. B.; Haynes, C. A. J. Colloid Interface Sci. 1996, 181, 635-644. (15) Kneuer, C.; Sameti, M.; Haltner, E. G.; Schiestel, T.; Schirra, H.; Schmidt, H.; Lehr, C. M. Int. J. Pharm. 2000, 196, 257-261. (16) Oleschuk, R. D.; Shultz-Lockyear, L. L.; Ning, Y.; Harrison, D. J. Anal. Chem. 2000, 72, 585-590. (17) Breadmore, M. C.; Wolfe, K. A.; Arcibal, I. G.; Leung, W. K.; Dickson, D.; Giordano, B. C.; Power, M. E.; Ferrance, J. P.; Feldman, S. H.; Norris, P. M.; Landers, J. P. Anal. Chem. 2003, 75, 1880-1886. (18) Cady, N. C.; Stelick, S.; Batt, C. A. Biosens. Bioelectron. 2003, 19, 59-66. (19) Christel, L. A.; Petersen, K.; McMillan, W.; Northrup, M. A. J. Biomed. Eng. 1999, 121, 22-27. (20) Svec, F.; Fre´chet, J. M. J. Science 1996, 273, 205-211. (21) Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1992, 54, 820-822. (22) Petro, M.; Svec, F.; Fre´chet, J. M. J. J. Chromatogr., A 1996, 752, 59-66.

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for microscale support materials. These materials are prepared in situ by thermal or photoinduced polymerization of a solution of monomer, initiator, and porogenic solvent, providing pores ranging from nanometer to micrometer size with a continuous interconnected network of channels. Advantages of in situ polymerization, including pore size control, high flow rate, and large mass transfer, have allowed them to be successfully used in capillary electrochromatography23,24 and preconcentration applications, (e.g., chemical compounds,25,26 peptides,27,28 and proteins29-31). Thermally induced monoliths have also been recently demonstrated as a functional medium for DNA separations where supercoiled plasmid DNA32 and oligonucleotides33-36 were separated by HPLC on commercial flat-disk CIM (BIA Separations) monolith matrixes. DNA purification and separation were also performed on bacterial and yeast genomic DNA in these matrixes, but they showed low extraction efficiencies and required a highsalt and high-pH buffer for DNA release.37 The advantage of UV-induced photopolymerization is that it enables the accurate placement of monolithic matrixes within the architecture of microscale devices.38 Both silica- and organic polymer-based photopolymerizable monoliths have been reported, with some incorporation into microdevices presented in the literature.28,38 However, no work has been reported on the use of UV-initiated monoliths as extraction matrixes for nucleic acids. The aim of the work described in this report was to provide an initial investigation of the utility of photopolymerizable silica monoliths for DNA extraction, with preliminary optimization of the monolith formulation for increased column capacity and extraction efficiency. The optimized monolith was evaluated for DNA extraction of prepurified human genomic DNA and from more complex biological matrixes such as whole blood. The results of this work illustrate the potential for DNA extraction in microscale devices using this material. EXPERIMENTAL SECTION Materials and Reagents. Fused-silica capillary (250 µm i.d. × 365 µm o.d.) was purchased from Supelco, Inc. (Bellefonte, PA). 3-(Trimethoxysilyl)propyl methacrylate (TMSPM, minimum 98%) (23) Dulay, M. T.; Quirino, J. P.; Bennett, B. D.; Zare, R. N. J. Sep. Sci. 2002, 25, 3-9. (24) Chirica, G. S.; Remcho, V. T. J. Chromatogr., A 2001, 924, 223-232. (25) Quirino, J. P.; Dulay, M. T.; Bennett, B. D.; Zare, R. N. Anal. Chem. 2001, 73, 5539-5543. (26) Quirino, J. P.; Dulay, M. T.; Zare, R. N. Anal. Chem. 2001, 73, 5557-5563. (27) Hilder, E. F.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 2004, 76, 3887-3892. (28) Yu, C.; Davey, M. H.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 2001, 73, 5088-5096. (29) Peterson, D. S.; Rohr, T.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 2003, 75, 5238-5335. (30) Kato, M.; Sakai-Kato, K.; Jin, H.; Kubota, K.; Miyano, H.; Toyo’oka, T.; Dulay, M. T.; Zare, R. N. Anal. Chem. 2004, 76, 1896-1902. (31) Peterson, D. S.; Rohr, T.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 2002, 74, 4081-4088. (32) Giovannini, R.; Freitag, R. Anal. Chem. 1998, 70, 3348-3354. (33) Sy´kora, D.; Svec, F.; Fre´chet, J. M. J. J. Chromatogr., A 1999, 852, 297304. (34) Premstaller, A.; Oberacher, H.; Huber, C. G. Anal. Chem. 2000, 72, 43864393. (35) Podgornik, A.; Barut, M.; Jaksa, S.; Jancar, J.; Strancar, A. J. Liq. Chromatogr. Relat. Technol. 2002, 25, 3099-3116. (36) Branovic, K.; Forcic, D.; Ivancic, J.; Strancar, A.; Barut, M.; Gulija, T. K.; Zgorelec, R.; Mazuran, R. J. Chromatogr., B 2004, 81, 331-337. (37) Bencˇina, M.; Podgornik, A.; Sˇ trancar, A. J. Sep. Sci. 2004, 27, 801-810. (38) Morishima, K.; Bennett, B. D.; Dulay, M. T.; Quirino, J. P.; Zare, R. N. J. Sep. Sci. 2002, 25, 1226-1230.

and tetramethyl orthosilicate (TMOS, 98%) were obtained from Sigma-Aldrich (Milwaukee, WI). Toluene (99.9%), 2-propanol (HPLC grade), guanidine hydrochloride (Gu-HCl, electrophoresis grade), ethanol (95%), and tris(hydroxymethyl)aminomethane (Tris) were purchased from Fisher Scientific (Fairlawn, NJ). EDTA was obtained from American Research Products (Solon, OH). Photoinitiator Irgacure 1800 was generously donated by Ciba (Tarrytown, NY). All solutions were prepared with Nanopure water (Barnstead/Thermolyne, Dubuque, IA). Labeled DNA Fragment Generation. A 380-bp β-globin DNA fragment was amplified using the polymerase chain reaction (PCR) in a Bio-Rad MyCycler thermal cycler (Hercules, CA) using forward primer tagged with the fluorescence dye 5-FAM (λex 488 nm, λem 520 nm) (MWG Biotech, High Point, NC). Thermocycling conditions were as follows: 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s (30 cycles) with a 3-min preincubation at 95 °C and a final extension of 1 min at 72 °C. The purity of the PCR product was assessed by capillary electrophoresis (Beckman P/ACE MDQ, Fullerton, CA) using LIF detection. The fluorescently tagged PCR product was used in preliminary solid-phase extraction experiments to allow sensitive on-line detection of the profiles achieved using a capillary electrophoresis instrument equipped with LIF detection. Preparation of Photopolymerized Monolith. The internal wall surface of a 250-µm-i.d. fused-silica capillary was first treated with TMSPM as reported previously.39 Briefly, the capillary was rinsed with 1 M NaOH for 15 min, water for 15 min, 0.1 M HCl for 15 min, and finally 95% ethanol for 5 min at 0.1 mL/min. A 30% (v/v) TMSPM solution (in 95% ethanol), adjusted to pH 4 with acetic acid, was flushed through the capillary at 3 µL/min for 60 min using a syringe pump (KD Scientific, Holliston, MA) and subsequently dried under a stream of nitrogen overnight. The treated capillary was left at room temperature for at least 1 day before use. The monomer solution was prepared as previously reported by Dulay et al.40 A monomer solution consisting of 85% (v/v) TMSPM and 15% (v/v) 0.1 M HCl was stirred at room temperature in the dark for 20 min. The sol-gel solution was prepared with 10% (v/v) monomer solution, 90% (v/v) toluene, and 5% (w/v) photoinitiator Irgacure 1800 and stirred in the dark at room temperature for 5 min. The treated capillary was then filled with the sol-gel solution and exposed to UV light using a 100-W broadUV wavelength lamp (model RSM100W, Regent Lighting Corp. Burlington, NC) for 5 min to initiate polymerization. The monolith length was controlled by removing a portion of the capillary polyimide coating. The detection window fabricated on the capillary by removal of the polyimide coating was covered with a black cloth during UV exposure to prevent polymerization at this particular location. After polymerization, the capillary was installed into the capillary electrophoresis instrument cartridge and ethanol was flushed through the column to visualize the monolith and remove excess monomer reagent. The minimum pressure required to flow solution through the monolith is referred to as the back pressure through the column in the following sections. (39) Rohr, T.; Hilder, E. F.; Donovan, J. J.; Svec, F.; Fre´chet, J. M. J. Macromolecules 2003, 36, 1677-1684. (40) Dulay, M. T.; Quirino, J. P.; Bennett, B. D.; Kato, M.; Zare, R. N. Anal. Chem. 2001, 73, 3921-3926.

TMOS Derivatization of Monolith. Once the monolith was formed, the surface was modified by treatment with various concentrations of TMOS. For example, an 85% TMOS solution was prepared with 85% (v/v) TMOS and 15% (v/v) 0.1 M HCl. The TMOS/HCl ratio was varied accordingly for different TMOS concentrations. The solution was vortexed at room temperature for 1 min. The monomer solution was then pressure-flushed through the monolithic capillary (12 psi) at room temperature for the indicated period of time. Solid-Phase Extraction Procedure. PCR-amplified 380-bp human genomic β-globin fragment products were diluted 5-fold in 7.5 M Gu-HCl prepared in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). Human genomic DNA, purified from human blood (60 µg/mL, A260/A280 ) 1.795), was diluted 20-fold in 6 M Gu-HCl buffer (pH 6). It is noteworthy that the samples were diluted in Gu-HCl solutions of different starting concentrations due to the difference in dilution factors, to achieve a final guanidine concentration of 5.7 M. Before extraction, the SPE column was equilibrated with 6 M Gu-HCl prepared in TE buffer for 10 min. Both sample and equilibration solutions were adjusted to pH 6 using 0.1 M HCl. DNA samples were loaded onto the column using pressure injections for various injection times. A wash step was subsequently performed using 80% (v/v) 2-propanol in water to remove unbound DNA and proteins/contaminants, and then the DNA was eluted with TE buffer. Blank experiments were performed for each column formulation investigated, by replacing the DNA sample volume with water. All extraction experiments were performed at room temperature. To compare the DNA binding data obtained from the various monolith formulations investigated, the columns were systematically overloaded until reaching constant DNA elution peak areas. Excess DNA (not bound to the monolith), in the load and washing steps, and extracted DNA, in the elution step, were detected online and used to calculate the relative column capacity and extraction efficiency. For the purpose of this paper, the relative column capacity is defined as the maximum elution peak area obtained after overloading the monolithic column. The relative extraction efficiency is defined as the ratio of the elution peak area to the sum of load, wash, and elution peak areas when the column was not overloaded. DNA Extraction of Prepurified Human Genomic DNA and Whole Blood Samples. Extraction of prepurified DNA and whole blood were performed using the TMSPM/TMOS monolith and the QIAamp DNA minikit (Qiagen, Valencia, CA). To compare the efficiency of the two extraction methods, the same amount of DNA was loaded onto the columns. To do so, a syringe pump (KD Scientific) was used to deliver the DNA load solution at a constant flow rate (3 µL/min). Prepurified human genomic DNA was obtained from blood (60 µg/mL, A260/A280 ) 1.795), and 120 ng of DNA was loaded onto the monolith and Qiagen columns. Whole blood samples were chemically treated prior to extraction to release DNA from the white blood cells. For the extractions performed on the monolith, 2.5 µL of whole blood was incubated with 585 µL of load buffer (6 M Gu-HCl, 10 mM Tris-HCl, 1 mM EDTA, pH 5.8) and 15 µL of a 10 µg/µL proteinase K solution in a water bath at 56 °C for 10 min. Then 20 µL of the digested blood Analytical Chemistry, Vol. 78, No. 5, March 1, 2006

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sample was loaded onto the monolith, corresponding to 83 nL of whole blood and 4.9 ng of DNA. For the extractions performed using the Qiagen kit, whole blood was prepared according to the manufacturer’s guidelines. Due to the low volume of whole blood loaded onto the Qiagen column (83 nL), the blood sample was diluted in PBS buffer (2.5 µL of blood in 600 µL of PBS) prior to extraction. DNA was subsequently extracted from the monolith by washing away proteins and contaminants using 30 µL of a 80% (v/v) solution of 2-propanol in water and eluting DNA using 40 µL of TE buffer at a 3 µL/min flow rate. DNA extraction from the Qiagen column was performed as suggested by the manufacturer. For both extractions, the amount of DNA present in the elution fractions was subsequently quantified. DNA Quantification. Quantification was performed only for the extraction of purified human genomic DNA samples. Load, wash, and elution fractions were collected, and the amount of DNA in each fraction was subsequently quantified using the PicoGreen assay according to the manufacturer’s guidelines. RESULTS AND DISCUSSION DNA Extraction Using a TMSPM-Based Monolith. In the presence of a chaotropic solution, nucleic acids bind avidly to a hydrophilic silica surface. This has been described previously12 and represents the chemical basis for the most common form of DNA purification via interaction with a solid-phase surface.10 While silica beads represent the most common form of silica used for this purpose, it is feasible to utilize other forms. This was shown by the reported use of silica sol-gel structures for DNA capture under chaotropic conditions by both Wolfe et al.13 and Ferrance et al.41 The silica sol-gel monoliths were found to be functional for binding and extracting DNAshowever, Wolfe et al. found that the TEOS-based sol-gels did not yield extraction efficiencies comparable to silica beads, mainly because the sol-gel pore size was difficult to control, thereby inhibiting fluid flow through the matrix.13 The sol-gel reported by Ferrance et al.41 was produced using TMOS monomers and a porogen to provide the appropriate pore size, but these could not easily be localized within the microdevices. TMSPM is a silica monomer containing an acrylate group, which can also produce a sol-gel monolithic structure, but it relies on the acrylate functional groups for formation of the final monolith. Upon polymerization, this monomer forms a polyacrylate organic matrix around a silica backbone, while providing the ability to localize the monolith through UV-initiated polymerization. The stability of this monolith, the ability of the silica surface to bind DNA, and the tunable flow characteristics of these gels were first investigated to determine whether they could function as a suitable stationary phase for DNA extraction in microdevices. Initial investigations were performed using a 12-cm-long monolithic column fabricated in a fused-silica capillary. A fluorescently labeled PCR-amplified DNA fragment was used in these preliminary experiments. While we understand that a single fragment of low molecular weight cannot be viewed as representative of the heterogeneous character of human genomic DNA, it functions as a simple model to assess the potential of the monolith for DNA extraction. In addition, we utilized capillary electrophore(41) Ferrance, J. P.; Wu, Q.; Giordano, B.; Hernandez, C.; Kwok, Y.; Snow, K.; Thibodeau, S.; Landers, J. P. Anal. Chim. Acta 2003, 500, 223-236.

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Figure 1. Typical DNA extraction profile of a 380-bp fragment of the β-globin gene in human genomic DNA produced using a 10% TMSPM monolith: (1) TE buffer baseline (from elution step). (2) Loading step baseline. (3) Excess single- and double-stranded DNA (resulting from column overloading). (4) System peak (resulting from guanidine-2-propanol interaction). (5) Washing step baseline (2propanol). (6) Mixture of single- and double-stranded DNA in elution peak. (7) Scanning electron micrograph of the 10% TMSPM monolith internal microstructure.

Figure 2. Effect of sample loading time on DNA elution profiles, using a 10% TMSPM monolithic column. Column conditions: length 12 cm; back pressure 7 psi; running pressure 11 psi. The inset shows elution peak area versus sample loading time, displaying a stable elution peak area after reaching the column capacity.

sis (CE) instrumentation equipped with LIF detection as the platform to evaluate the chromatography (DNA load, wash, and elution steps) involved in the DNA extraction. The reasons for this are obvioussit provides an excellent platform for control of small-volume samples and reagents and has a sensitive, built-in LIF detection system. A typical extraction profile obtained from the CE using the 380-bp fragment of the β-globin gene amplified from human genomic DNA as the sample is presented in Figure 1. The baseline shifts observed in this profile (1, 2, 5) are due to the different buffers used in load, wash, and elution steps having different optical properties. The system peak (4), also present in blank profiles (as shown in subsequent Figure 2 with the trace at loading 0), is assumed to result from the interaction between guanidine and 2-propanol occurring when load and wash buffers come in contact. The extraction of a PCR primer sample showed that single-stranded DNA (ssDNA) could also be extracted using

the TMSPM monolith (data not shown). Both double-stranded DNA and ssDNA were therefore assumed to be present in the excess DNA eluted during the wash step (peak 3) and in the DNA detected during the elution step (peak 6). In the following experiments, only the elution portion of the extraction profile will be displayed as the efficiency of the monoliths will mainly be evaluated by their relative column capacity (obtained from the elution peak area). The effect of the TMSPM monomer concentration on the stability and back pressure was examined over a concentration range of 7-21%. Low TMSPM concentrations (