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© 2004 American Association for Clinical Chemistry, Inc.
Technical Briefs |
Extraction and Amplification of Genomic DNA from Human Blood on Nanoporous Aluminum Oxide Membranes
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 marc.elgort{at}aruplab.com
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 AnoporeTM and AnodiscTM 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 ß-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 iQTM 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 ß-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.
ß-Globin amplification curves in the absence and presence of the membrane demonstrated increasing Cp values with decreasing quantities of gDNA, shifting 1.6 cycles with each half-log dilution, with no amplification detected in the no-template control. A single melting peak at 85 °C, representing the melting temperature of the 110-bp ß-globin amplicon, was observed in reactions containing gDNA, with or without AOM, consistent with previously reported results (6). An additional peak at 75 °C was observed that decreased with increasing gDNA and was the sole peak observed in the no-template control, thus likely corresponding to primer-dimer. Because fluorescence acquisition was done at 80 °C, the primer-dimer amplification did not influence the actual amplification curves (7). A plot of the Cp values vs the log of the amount of gDNA in the absence or presence of AOM demonstrated that the membrane had no discernable impact on PCR. The regression lines for each calibration curve overlapped with nearly equivalent slopes. Using a method for determining PCR efficiency based on the relationship of the slope of the calibration curve and exponential amplification of the amplicon (8):
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3.3-cycle change between log changes in DNA). Although this method did not take into account the absolute PCR efficiency within each reaction (9)(10), it was suitable for these comparisons and demonstrated that the AOM had no inhibitory effect on PCR. 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 ß-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.
 View larger version (18K): [in a new window] |
Figure 1. 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 (
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), 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
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