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Technical Briefs |
a address correspondence to this author at: Regions Hospital, Department of Pathology, 640 Jackson St., St. Paul, MN 55101
Differential display analysis (DDA) has become a useful and popular technique to identify differentially expressed genes in many tissues and cell types. Recovery of DNA fragments, however, from silver-stained nondenaturing polyacrylamide gel is difficult. Numerous techniques for extracting DNA from the polyacrylamide gel have been reported, including using untreated dried polyacrylamide flakes (1) , leeching with water ((2)), excision and boiling in 1x PCR buffer (3) , and heating overnight in 0.5 mol/L ammonium acetate (4) . The potential problems with these techniques are that inhibitors can be carried over into the PCR reaction and that inconsistent preparation can lead to poor re-amplification. In addition, most of these techniques require excessive thermal cycling to re-amplify the fragment. We developed a technique in which the DNA is electroeluted out of the acrylamide onto a DEAE membrane. With this technique, multiple samples can be processed and purified simultaneously, leading to reproducible and consistent re-amplification reactions.
Cytogenetically normal amniocytes were grown in duplicate in Chang's in situ media (Irvine Scientific). Messenger RNA was isolated with an oligo dT/streptavidin magnetic bead capture system (Promega Corp.) (5) and converted to cDNA using MMLV reverse transcriptase and a degenerative oligo dT primer (5'-TTTTTTTTTTTTGC). For each DDA analysis, the following 20-µL reaction was set up: 1x PCR buffer with 1.5 mmol/L MgCl2, 20 µmol/L each dNTP, 0.5 µmol/L oligo dT primer, 0.25 µmol/L upstream primer (5'-CTGCTTGATG, 5'-GATCCAGTC, 5'-GATCGCATTG, and 5'-AAACTCCGTC), 30 ng of cDNA, and 0.625 unit of Taq DNA polymerase (Perkin-Elmer) (6) . Forty thermal cycles of 95 °C, 30 s; 40 °C, 1 min; and 72 °C, 1 min were used. The DDA products were ethanol-precipitated, rehydrated in water, size-separated through a 5% nondenaturing polyacrylamide gel, and visualized by silver staining (7) .
The DDA amplification products were excised from the dried polyacrylamide with sterile water and a clean scalpel. The excised acrylamide fragments were placed into the wells of a 2% agarose gel (Life Technologies) and flattened against the edge nearest the cathode. The wells were carefully filled with cooled 2% molten agarose. Once the agarose was set, DEAE membranes (Schleicher & Schuell), cut into 0.5 x 0.75-cm pieces, were inserted into the gel ~0.5 cm below each well. A narrow weighing spatula was used to facilitate the insertion of the dry DEAE membrane. The DNA was electrophoresed at 10 V/cm for 90 min. The DEAE membranes were removed and placed into microfuge tubes containing 500 µL of low-salt NET buffer (0.15 mol/L NaCl, 0.1 mmol/L EDTA, and 20 mmol/L Tris, pH 8.0). The low-salt NET buffer was replaced with 50 µL of the high-salt NET buffer (1 mol/L NaCl, 0.1 mmol/L EDTA, and 20 mmol/L Tris, pH 8.0) and incubated at 65 °C for 30 min to elute the DNA. The DNA was then precipitated with 2.5 volumes of ethanol and rehydrated in 15 µL of water. Five microliters of the eluted DNA was amplified using the same conditions as described above. Amplified fragments were ligated into a T-vector (Promega) and sequenced.
This technique was used to purify DDA fragments excised from dried
nondenaturing polyacrylamide gels. Fig. 1
shows the amplification of four purified DDA fragments. A
single band is present in each lane. With this technique, DNA ranging
in size from 100 to 700 bp was precipitated in sufficient quantity for
several re-amplification reactions. In addition, because a 2% agarose
gel in Tris-borate-EDTA was used, multiple samples could be processed
without the use of Tris-acetate-EDTA, low-melt agarose, specialized
pipet tips, or electrophoresis chambers (8) . Numerous
authors have shown that DNA can be purified by elution onto DEAE
membranes through agarose (9)(10) , and Dretzen
et al. (11) showed that DNA could be eluted onto DEAE
membranes directly from polyacrylamide gels. Electroeluting DNA
directly out of large polyacrylamide gels, however, is not practical
because these gels are very thin and dried.
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We have shown here that DNA can be efficiently eluted out of acrylamide and through agarose onto a DEAE membrane, using standard laboratory protocols and equipment. With this technique, numerous samples can be consistently and reliably processed, producing inhibitor-free DNA. This technique should aid in the cloning and sequencing of DDA products.
Acknowledgments
Ramsey Foundation Grant N590 supported this work.
Footnotes
Regions Hospital, St. Paul, MN 55101
fax (612) 221-2741, e-mail David.J.Lakatua{at}Healthpartners.com
References
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