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Improved method of isolation of DNA from celloidin‐embedded tissue

1999; Wiley; Volume: 109; Issue: 5 Linguagem: Inglês

10.1097/00005537-199905000-00028

1531-4995

P. Suzanne Hart, A. Charla Vlaservich, Jai H. Ryu, Robert I. Kohut,

RNA and protein synthesis mechanisms

Isolation of DNA from celloidin-embedded temporal bones has recently been reported.1 Analysis of DNA from such tissues by the polymerase chain reaction (PCR) is now feasible and has been recently reviewed.2 Such studies have demonstrated the presence of inherited mutations resulting in disorders of the inner ear, deletions associated with aging, and the presence of viral sequences.3-6 Most isolation techniques use phenol/chloroform extraction, the presence of which can inhibit amplification if not completely removed.1, 4, 7 As the amount of DNA in a 20- to 25-μm temporal section is very small, every effort should be made to maximize DNA yield and remove all organic solvents in order to achieve efficient amplification. We now report an improved method of DNA isolation from celloidin-embedded tissues that eliminates the use of organic solvents. The DNA isolated via this method is free of organic inhibitors and results in consistent PCR products compared with DNA isolated following conventional methods. After removal of the celloidin from the 20-μm temporal bone section as described by Simpson and Smith,4 180 μL of digestion buffer (100 mmol/L NaCl, 10 mmol/L Tris-Cl, pH 8.0, 0.5% SDS, 25 mmol/L EDTA) and 20 μl of proteinase K (20 mg/mL) were added, the samples vortexed and incubated at 55°C for 48 hours. After the addition of 210 μL of 100% ethanol to the tube, the samples were vortexed and applied to a QIAamp spin column (Qiagen, Chatsworth, CA). The samples were centrifuged at 12,000g for 1 minute at room temperature. The spin column was transferred to a clean 2 mL collection tube and 500 μL of Buffer AW added. The samples were centrifuged, the column transferred to a clean collection tube, and another 500 μL of Buffer AW added. Samples were centrifuged at 12,000g for 3 minutes. After transferring the column to a sterile 2-mL microfuge tube, the DNA was eluted with 100 μL of water that had been preheated to 70°C. After the addition of the water to the column, the samples were incubated at room temperature for 1 minute and then centrifuged at 12,000g for 1 minute. To increase the yield, a second elution with 100 μL of 70°C water was performed. DNA was also isolated following the method of Wackym.1, 8 As a positive control for amplification, DNA was isolated from peripheral blood using the QIAamp kit according to the manufacturer's instructions. Samples were stored at 4°C until ready to use in PCR. A 110-bp fragment of the β-globin gene was amplified using oligonucleotides BGL 101, (5′-ACACAACTGTGTTCACTAGC-3′) and BGL 102, (5′-CAACTTCATCCACGTTCACC-3′) (Operon Technologies) in a 20-μL reaction mixture containing approximately 50 ng of temporal bone lysate, 1X buffer D [60 mmol/L Tris-HCL, pH 8.5, 15 mmol/L (NH4)2SO4, 3.5 mmol/L MgCl2)] (Invitrogen, San Diego CA), 125 μmol/L of each dNTP (Promega, Madison WI), 1 μmol/L each of primers, and 0.625 units of Taq polymerase (Promega). Using a GeneAmp PCR System 2400 (Perkin-Elmer, Foster City, CA) the reaction mixtures were incubated for 5 minutes at 94°C before undergoing 35 cycles of PCR, consisting of a 15-second denaturing at 94°C, a 30-second annealing at 60°C, and a 45-second extension at 72°C, followed by a final extension at 72°C for 5 minutes. Ten microliters of the PCR products were electrophoresed through a 2.0% agarose gel containing 0.25 ng/mL ethidium bromide. As controls for amplification, 100 ng of DNA isolated from peripheral blood (the positive control) and a water blank (the contamination control) were amplified as described above. Polymerase chain reaction amplification of the β-globin gene. A 110-bp product of the β-globin gene was amplified in a final volume of 20 μL containing 15 mmol/L NH4SO4, 60 mmol/L Tris pH 8.3, 3.5 mmol/L MgCl2, 1 μmol/L each of primers (forward 5′-ACACAACTGTGTTCACTAGC-3′; reverse 5′-CAACTTCATCCAGCTTCACC-3′), 125 μmol/L each of dNTPs, and 0.625 U of Taq DNA polymerase. Thirty-five cycles of denaturation at 94°C for 15 seconds, annealing at 60°C for 30 seconds and extension at 72°C for 45 seconds, followed by a final extension of 72°C for 5 minutes were conducted. Samples (10 μL) were separated by electrophoresis through 2% agarose gels containing ethidium bromide and visualized with ultraviolet light. A. Mass ladder. B. Negative control (no DNA). C. Genomic DNA isolated from peripheral blood. D. Temporal bone lysate isolated according to the modified procedure. E. Temporal bone lysate isolated according to the method of Wackym et al.1, 8 F. 1-kb ladder. The lower band visible in Lanes D and E represents primer dimer. The PCR products were prepared for sequencing by excising the bands from the agarose gel and extracting the fragments using a Qiagen Gel Clean-up Kit. The sense and antisense strands of each PCR product were directly sequenced on an ABI Prism 310 Genetic Analyzer (Perkin-Elmer) using four dye terminator chemistry. Approximately 1 to 3 ng of purified product and 3.2 pmol primer were added to premised reagents from the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit, FS (Perkin-Elmer) and underwent a cycle sequencing reaction in a GeneAmp PCR System 9700 (Perkin Elmer). The linear amplification started with a 10-second denaturation at 96°C, 5-second annealing at 50°C, and 4-minute extension at 60°C. The fluorescently labeled sequencing products were separated from residual reaction reagents using a Centri-Sep spin column (Princeton Separations, Aldelphia, NJ) and electrophoresed on POP6 capillary at 1500 V for 30 minutes. Sequencing data were automatically collected and analyzed by the ABI Prism 310 software. As shown in Figure 1, DNA obtained by the method described in this report resulted in an amplification product of the correct size. However, when DNA was isolated with phenol/chloroform and purified by ethanol precipitation as described by Wackym et al.,1, 8 DNA amplification was not consistent, which sometimes resulted in little or no product. The amount of amplified product obtained from DNA isolated by the modified method was at least four times that obtained by the organic method. The PCR products were directly sequenced to verify the specificity of amplification. As shown in Figure 2, sequence of the 110-bp product matched with the published sequence of the β-globin gene. The 60-bp product observed in some reactions, predominantly in the organic protocol, was confirmed by sequencing to be primer dimers (data not shown). DNA sequence analysis of the 110-bp β-globin fragment shown in Figure 1. A. Peripheral blood product. B. Temporal bone product. The modified procedure described in this report is a more reliable method for obtaining quality DNA from celloidin-embedded temporal bones than the method described by Wackym.1 The results from this study confirm the findings of Simpson and Smith4 that PCR amplification is unreliable using DNA isolated according to the Wackym method. Although DNA is obtainable via the Wackym method, residual organics from the extraction and isolation procedure can inhibit subsequent amplification, resulting in little or no product. Simpson and Smith4 described an alternate method of DNA purification using a Centricon microconcentrator to remove residual phenol from the extraction process. However, ether (used in dissolving celloidin) is not chemically compatible with the microconcentrators. As a result, ether may leach through the membrane, contaminate the DNA, and inhibit PCR amplification. The advantage of the modified procedure described here is that no organics are used for DNA extraction or isolation; thus, the isolated DNA is free of organic solvents that may interfere with the subsequent amplification process. Also, the method is simple, quick, and straightforward and does not require specialized equipment that would prohibit its use in other laboratories.