Localization and imaging of nucleic acids on nanoporous aluminum oxide membranes.

Abstract

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 Anopore™ (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 …