Extraction and Amplification of Genomic DNA from Human Blood on Nanoporous Aluminum Oxide Membranes

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 localized to the AOM surface by filtration. We amplified target sequences within the localized gDNA in the presence of the membrane. To assess the impact of AOM on PCR (data not shown), we prepared β-globin calibration curves with Qiagen-purified gDNA, using half-log increments of gDNA quantities ranging from 100 pg to 320 ng, in the absence or presence of an 8.04-mm2 piece of 200-nm-pore-size AOM. DNA amplifications were performed with the iCycler iQ™ Real-Time Detection System (Bio-Rad) in a 50-μL PCR reaction containing 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 0.8 mM deoxynucleotide triphosphates (0.2 mM each), 25 U/mL iTaq DNA polymerase, 3 mM MgCl2, 0.5 μg/mL bovine serum albumin, 0.5 μM each β-globin primer [5′-ACACAACTGTGTTCACTAGC-3′ and 5′-CAACTTCATCCACGTTCACC-3′; GenBank accession no. GI:455025; positions 62150–62259R) (5)], and SYBR Green I dye. The reactions were cycled 45 times with a 20-s denaturation step at 95 °C and a 30-s annealing step at 55 °C, followed by a 30-s extension step at 72 °C. Fluorescence acquisition was done during a 10-s 80 °C hold after extension. Melting curves were obtained after cycling by denaturing samples for 30 s at 95 °C and then cooling to 70 °C for 10 s and subsequently increasing the temperature by 0.5 °C every 10 s, during which fluorescence was acquired. All data [crossing point (Cp) and melting curves] were analyzed with the iCycler iQ data analysis software. We lysed 1 μL of human whole blood that had been drawn into tubes containing dipotassium EDTA, sodium citrate, or sodium heparin as anticoagulant for 1 min at room temperature in a 100-μL final volume of 200 mmol/L NaCl, 10 mL/L Triton X-100, and 2.5 mg/mL Proteinase K (50 U/mg; Sigma). The lysate was then filtered through an AOM disc mounted on a specialized filtration card with 8.04-mm2 wells fabricated for this study. Filtration was performed under standard house vacuum, and 100 μL of lysate flowed completely through the AOM in <30 s. After filtration, the membrane was washed with 100 μL of 200 mmol/L NaCl and then separated from the card and transferred to a PCR reaction tube containing the aforementioned PCR master mixture. DNA amplifications, melting curves, and data analysis were carried out as described above with the exception that fluorescence acquisition during amplification was at 82 °C to eliminate artifacts attributable to nonspecific priming. Various lysis conditions were tested, and we determined that the conditions described above rendered a lysate that flowed rapidly through the AOM and allowed for robust localization of gDNA as determined by Cp values in real-time PCR. Amplification curves (Fig. 1A ) revealed that amplicons were produced in the lysates from blood collected in all anticoagulants filtered on AOM, with Cp values indicating the presence of 3–12 ng of gDNA (26–29 cycles; Fig. 1B ). Furthermore, melting analysis (Fig. 1C ) demonstrated that the amplification products were specific, representing the 110-bp β-globin amplicon, with a melting temperature of ∼85 °C. The fact that equal volumes of citrate-, EDTA-, or heparin-anticoagulated blood yielded amplicons with equivalent Cp values indicated that the PCR inhibition usually associated with citrate and heparin by other preparative nucleic acid methods (11)(12) was not an apparent factor after localization of the gDNA to the membrane and subsequent amplification. Although the theoretical estimate of the gDNA content in 1 μL of whole blood [mean leukocyte count ∼7000 cells/μL, measured on a Coulter AC.T (Beckman Coulter, Inc.)] suggested that ∼50 ng should have been present on the membrane, the AOM capacity for a monolayer of DNA is ∼1.6 ng/mm2 (based on the surface area of a double-stranded DNA molecule >1000 bp); thus, at capacity, an 8.04-mm2 AOM would saturate at ∼13 ng. Another factor, the increase in temperature for fluorescence acquisition, led to an apparent decrease in PCR efficiency of 25%, based on the calibration curve as illustrated in Fig. 1B , as a result of a decrease in the amount of double-stranded amplicon, and subsequently the intercalated SYBR Green I, present during fluorescence acquisition. Considering the apparent AOM capacity and the apparent decrease in exponential amplification, the mean gDNA yield based on Cp values was comparable to that of silica-based purification methods such as the QIAamp DNA Blood Kit (Qiagen), which reports 15–60 ng of total nucleic acid per 1 μL of whole blood. Notably, the Qiagen purification protocol requires several centrifugation or vacuum steps and a final elution, with the silica matrix being inhibitory to PCR amplification. Localization of gDNA from whole blood to the AOM and subsequent amplification. (A), amplification curves of AOM-localized gDNA from blood drawn into citrate (solid black line), EDTA (dot-dashed black line), or heparin (dashed black line). A no-template control (dashed gray line), 3.3 ng of gDNA (light gray line), and 33 ng of gDNA (dark gray line) are shown for comparison. (B), determination of gDNA recovery from whole blood in citrate (▪), EDTA (▴), or heparin (•) compared with log(gDNA) from calibration curves derived from Cp values from real-time PCR amplification. Equation for the line: y = −3.98x + 31.31 (R2 = 0.9976). (C), melting curve analysis for the samples above (lines are the same as for amplification curves in A). Note the single peak in the no-template control at ∼75 °C, which represents primer-dimer, and the β-globin product peak at ∼85 °C in all gDNA samples as well as in the blood samples. We conclude that localization, amplification, and detection of nucleic acid targets from biological samples such as whole blood is feasible on and in the presence of the AOM. Furthermore, the process, as we have described it here, is rapid and avoids several of the drawbacks of current nucleic acid purification methods, such as accumulation of inhibitory factors, inhibition of enzymatic processes by the matrix itself, the need for many manual or robotic steps, and ultimately the consumption of time. We have also demonstrated (data not shown) that this process can allow for genomic analysis of severely hemolyzed blood, analysis of HIV RNA directly from plasma, and genotyping from bacterial cultures. We are currently investigating the nucleic acid capacity of the AOM and have developed and fabricated a self-contained reaction tube consisting of the AOM on a scaffold for filtration, which is then sealed for PCR, thus eliminating the need for separation of the AOM from a filtration device. Experiments are underway to develop this technology into a high-speed, stand-alone molecular diagnostic device capable of rapid isolation and analysis of nucleic acids for a variety of clinical diagnostic analyses.