Extraction and amplification of genomic DNA from human blood on nanoporous aluminum oxide membranes.

Abstract

Purification of genomic DNA from biological samples is a requisite initial step in the performance of many molecular diagnostic assays. Traditional purification methods based on phenol–chloroform extraction and ethanol precipitation have been largely supplanted by adsorption and elution procedures based on silica and coated magnetic bead matrices. In most applications, the purified genomic DNA must be physically separated from the adsorption matrix before downstream processes such as amplification by PCR. Separation is done to reduce inhibitory effects of adsorption matrices on amplification processes, which may result from matrix interactions with enzymes and other reaction components (1). The requirement for separation necessitates the addition of a transfer step performed by either manual or robotic means. To further streamline molecular diagnostic analyses, new purification matrices are needed. Specifically, a matrix capable of purification that does not inhibit amplification chemistries would open the path to development of instrumentation that performed purification, amplification, and detection in an integrated format. We have been investigating the properties of a commercially available porous aluminum oxide membrane (AOM) (2) for nucleic acid purification. Commercially available AOM, distributed under the trade names Anopore™ and Anodisc™ by Whatman, Inc., are manufactured with pore size options of 20, 100, and 200 nm and a membrane thickness of ∼60 μm. The membranes are rigid, with low autofluorescence and a porosity of 50%, allowing for high liquid flow rates. The membrane core component is aluminum oxide, which is relatively biologically inert but can be derivatized to display functional amine groups by silanization. In addition to standard filtration applications, AOM have been used as a support matrix for cell growth (3) and as a substrate for enzyme immobilization in biosensor applications (4). Here we describe conditions for AOM-based purification of genomic DNA (gDNA) from human blood in which blood is lysed and gDNA is …