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Technical Briefs |
1 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT;
2 Department of Pathology, University of Utah, Salt Lake City, UT;
aaddress correspondence to this author at: ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108; fax 801-584-5114, e-mail eralimc{at}aruplab.com
The technologies currently available for nucleic acid analysis in the clinical laboratory rely on target amplification methods, such as PCR or transcription-mediated amplification, or signal amplification methods, such as branched-chain DNA, hybrid capture, Invader technology, and fluorescent in situ hybridization. In current nucleic acid analyses, a multistep process consisting of sample preparation and purification followed by amplification and detection is required. A comprehensive method for direct localization and visualization of individual molecules would provide an additional method for qualitative or quantitative nucleic acid analysis. We evaluated a nanoporous aluminum oxide membrane (AOM) as a support for direct localization and visualization of individual nucleic acid molecules. The process described here allows nucleic acids to be localized by filtration onto AOM with direct imaging using nucleic acid-binding stains. Additionally, we present a method for modifying the membranes to improve the optical properties.
The membranes used in this study are commercially available under the trade name AnoporeTM (Whatman International) and are composed of aluminum oxide with a mean pore size of 200 nm and a nominal thickness of 60 µm (1). The mean width between the pores is 50 nm, and the membranes have a pore density of 
108 pores/cm2. The pores are evenly distributed in a honeycomb pattern across the surface, with a porosity of 50%. Because the length of one DNA base pair is 0.34 nm (2)(3), a linear double-stranded DNA molecule will extend 600 bp across a single 200 nm pore, and 150 bp of the molecule will cover the area between the pores.
The uniform pore structure, the relatively rigid and flat surface, and the high porosity and flow rate make AOMs an attractive material for sample filtration and nucleic acid visualization. The surface properties of aluminum oxide allow for chemical modifications to the membrane that can enhance their use as a substrate for nucleic acid analysis.
Because the membranes would be used as a surface for nucleic acid filtration as well as visualization, we evaluated the background autofluorescence of an Anopore membrane compared with a polyvinylidene fluoride (PVDF) membrane filter, and for reference purposes, to a clear low-fluorescence glass cover slip and fused silica. Autofluorescence was determined by measuring relative fluorescence generated during excitation at 488 nm. The autofluorescences of the PVDF, Anopore, glass, and silica were 104, 122, 18, and 0.4 relative fluorescence units (RFU), respectively. The autofluoresecence of Anopore was much lower than that of the PVDF membrane but not as low as for the glass or silica. These results indicated that the Anopore membranes would provide a better membrane than the PVDF for nucleic acid analysis but that improvements to further lower the autofluorescence of the Anopore would be advantageous.
Black AOMs with improved optical properties were prepared by electroless nickel deposition (4). ACS-grade or better reagents and HPLC-grade water were used for this process and throughout the study. The black membranes were prepared by briefly soaking Anopore membranes in a solution of 50 mg/L palladium(II) chloride in acetone and allowing them to air dry. The membranes were then placed in a solution of 25 g/L nickel(II) sulfate hexahydrate (NiO4S · 6 H2O), 15 g/L sodium acetate (C2H3NaO2), 4 g/L borane-dimethylamine complex (C2H10BN), and 2 mg/L lead(II) acetate trihydrate (C4H6O4Pb · 3 H2O) adjusted to pH 5.9. The membranes remained in this solution for 5 min until they were optically black; they then were washed with deionized water and dried at 45 °C. When analyzed for background autofluorescence as described previously, the black membranes had background autofluorescence of 3.0 RFU, lower than glass (18 RFU) and more comparable to fused silica (0.4 RFU). The black membranes serve as a convenient, single platform for concentrating and localizing nucleic acids and additionally provide a low background autofluorescence surface for visualization.
For nucleic acid localization and imaging, the black AOMs were attached to custom filtration units that provided support for the filtration of solutions through the membranes. The detection and imaging system consisted of an argon ion laser with an emission wavelength of 488 nm (American Laser Corporation); a x10, 0.3 NA objective (Olympus America); and a D535/40M bandpass filter (Omega Optical). Images were acquired with a charge-coupled device camera (MaxCam with back-thinned Marconi Chip; Finger Lakes Instrumentation) and MaxIm DL CCD Imaging Software, Ver. 3.10 (Diffraction Limited). Image analysis was accomplished with IMAQ Vision Builder, Ver. 6.1 (National Instruments Corporation). Imaging could also be performed on a standard fluorescence microscope with the appropriate filters.
Bacteriophage 
DNA (New England BioLabs) was localized on the black AOM and visualized as a linear form after staining in a 1:10 000 dilution of SYBR Green I (Molecular Probes) in 10 mmol/L Tris, 1 mmol/L EDTA, pH 8.0 (Tris-EDTA). The phage genome is 48 500 bp, with a contour length of 16.5 µm. In our optical system, this corresponds to 13 pixels in length. The images of DNA detected in this study were consistent with the expected molecular dimensions. Unprocessed and processed images of DNA are presented in panels A and B of Fig. 1
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Wash studies were performed on DNA localized to the black AOM to evaluate characteristics such as rinse off, photobleaching, and destaining of the nucleic acids on the membranes. Wash solutions were prepared in Tris-EDTA, pH 7.5, containing 50 mmol/L phosphate, sodium chloride, sodium carbonate/bicarbonate, or sodium borate. These solutions and water were used as washes after localization of DNA stained with a 1:100 000 dilution of SYBR Gold (Molecular Probes) in Tris-EDTA. No movement of the DNA in the images was noted with any of the solutions tested. In some images, DNA was not visible after washes with the borate and phosphate solutions; however, repeat exposure of the samples to the SYBR Gold solution revealed that the nucleic acid remained. The borate and phosphate solutions likely removed the SYBR Gold stain from the nucleic acid but did not move the DNA.
Dilutions of DNA from 0 to 5000 molecules per field of view (FOV) were prepared in a 1:10 000 dilution of SYBR Green I in Tris-EDTA, localized on the black membrane, and imaged. The images were processed and counted in the IMAQ software to determine the observed number of molecules per FOV. A plot of the observed molecule count vs the expected count of molecules is shown in Fig. 1C
. The dilution series is linear to 100 DNA molecules per FOV.
The apparent 100-count background limit observed when we used nucleic-acid-binding dyes in this system may have been attributable to contamination from fluorescent particulates and/or exogenous nucleic acid contamination potentially present on the base membrane or associated with reagents used to produce the black AOM. Even with a background of 100 counts, these data support the potential of this system to provide a means for direct quantification of nucleic acids or organisms.
To further assess the potential usefulness of the black AOM for localizing and imaging larger nucleic acid molecules, we studied human genomic DNA. Leukocytes were prepared from whole blood by Ficoll gradient centrifugation. We lysed the purified leukocytes by adding 190 µL of 5 g/L sodium dodecyl sulfate in Tris-EDTA to 10 µL of leukocytes and incubating at 56 °C for 20 min. The DNA was stained with a 1:10 000 dilution of SYBR Gold prepared in Tris-EDTA, and the solution was filtered through a black AOM. Pipetting of the leukocyte-sodium dodecyl sulfate-SYBR Gold solution was minimized to reduce the amount of DNA strand breakage.
Images of human genomic DNA isolated from leukocytes are shown in Fig. 1D
. The lengths of most of the DNA fragments ranged from 40 to 70 pixels, which corresponds to DNA strands of 150 000–260 000 bp. The linear strand (1) indicated in Fig. 1D
is 50 pixels, or 190 000 bp, and the curved strand (2) in Fig. 1D
is 65 pixels, or 250 000 bp. Longer strands of genomic DNA could be imaged if the solutions were handled without any pipetting steps (data not shown).
We have presented a method for the modification of commercially available Anopore membranes that improves optical properties and allows for the localization and direct visualization of nucleic acids by filtration and imaging with nucleic-acid-binding stains. The ability to filter solutions through an AOM, thereby localizing nucleic acids or other targets, and to subsequently perform direct detection without the need for transfer, is a simple and straightforward process. The possibility of quantitative analysis of nucleic acids is supported by the good linearity observed for the dilution series of DNA molecules. Additional studies are underway to develop methods for hybridization of labeled probes that will allow specific identification of localized nucleic acids.
Anopore membranes have also been used for the collection, staining, and enumeration of microorganisms with nucleic-acid-binding stains (5)(6). The membranes have additionally been used as supports for cell growth (7)(8). It is expected that the modified membranes may be useful for these types of applications and that the improved optical properties could enhance visualization of stained organisms or cells.
We continue to evaluate the black AOM as a surface for localization and imaging of nucleic acids as well as bacterial and viral targets, and we are examining the use of clinical specimens for applications that could improve sensitivity and processing time for clinical testing.
References
The following articles in journals at HighWire Press have cited this article:
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  S. Dames, L. K. Bromley, M. Herrmann, M. Elgort, M. Erali, R. Smith, and K. V. Voelkerding A Single-Tube Nucleic Acid Extraction, Amplification, and Detection Method Using Aluminum Oxide J. Mol. Diagn., February 1, 2006; 8(1): 16 - 21. [Abstract] [Full Text] [PDF]
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