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Journal of Microbiological Methods 54 (2003) 289 – 291 www.elsevier.com/locate/jmicmeth Note Improved recovery of DNA from polyacrylamide gels after in situ DNA footprinting Geertje van Keulena,* , Wim G. Meijer b a Department of Microbiology, Groningen Biotechnology and Biomolecular Science Institute (GBB), University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands b Department of Industrial Microbiology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland Received 16 January 2003; received in revised form 11 February 2003; accepted 11 February 2003 Abstract Methods used to date for the isolation of DNA from polyacrylamide gels are elution based, time-consuming and with low yield in DNA. This paper describes an improved system employing polyacrylamide gels made of a meltable matrix. The new system was successfully applied to in situ DNA footprinting following gel retardation assays. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Band shift; DNA footprinting; DNA isolation; In situ; Polyacrylamide; Recovery DNA footprinting (Galas and Schmitz, 1978) is widely used to map the precise DNA sequence to which transcription factors bind, leading to a detailed insight in the regulation of gene expression. When footprinting is performed in solution, the resulting footprint is often blurred because of the presence of unbound DNA and/or multiple DNA – protein complexes. For optimal footprinting results, unbound DNA should therefore be separated from DNA – protein complex(es) before loading the reaction mixture onto a denaturing acrylamide gel. Maximal footprinting information is thus obtained when the footprinting procedure is performed in situ, e.g. in * Corresponding author. Tel.: +31-5036-32150; fax: +31-503632154. E-mail address: [email protected] (G. van Keulen). nondenaturing acrylamide gels following band shift assays (Papavassiliou, 1994). If the complexes are sufficiently stable it is also possible to do the reverse: apply footprinted DNA(-complexes) to a nondenaturing acrylamide gel to separate the protein – DNA complexes. The critical step common to both procedures is the isolation of DNA from the acrylamide gel. Diffusion-driven gel elution and electro-elution are frequently used techniques for recovering footprinted DNA from acrylamide gels. However, these techniques are time consuming and are characterised by a poor yield and reproducibility. This paper describes a procedure for the isolation of DNA from acrylamide gels using a meltable acrylamide matrix to facilitate easy and fast (within 1 h) recovery of DNA after phenantroline footprinting in situ following a band shift assay. This technique was applied to analyse the interaction between the 0167-7012/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-7012(03)00045-9 290 G. van Keulen, W.G. Meijer / Journal of Microbiological Methods 54 (2003) 289–291 LysR-type transcriptional regulator CbbR and the cbb promoter of Xanthobacter flavus (Van Keulen et al., 1998). The cbb promoter has three, partly overlapping, CbbR-binding sites (IR1, IR2 and IR3; Fig. 1). Interaction of CbbR with these binding sites results in the formation of a complex of high electrophoretic mobility resulting from binding of one CbbR dimer to the DNA (binding site IR1 occupied) and a second complex of low electrophoretic mobility in which two CbbR dimers are bound to the DNA (binding site IR1 and IR2/3 occupied) (Shively et al., 1998). DNaseI footprinting in solution was used to characterise the interaction between CbbR and its cognate binding sites in greater detail (Van Keulen et al., 1998). However, the footprint of the low-mobility complex (two CbbR dimers bound to IR1 and IR2/3) was blurred due to the presence of unbound DNA and of the high-mobility complex (CbbR bound to only IR1) (Fig. 1, lanes 1 and 2). Even though a clear footprint was obtained for CbbR-binding site IR1, background bands were still visible; DNase I protection by CbbR of binding site IR2/3 is significantly less efficient than IR1. The extensive background is most likely due to unbound DNA and from the second high-mobility complex with one CbbR dimer bound. To improve the resolution of the footprint of the low-mobility complex, in situ copper-phenanthroline footprinting was carried out in a meltable nondenaturing acrylamide gel (Oligoprep, National Diagnostics). DNA was subsequently isolated from the gel in high yields with a large increase in resolution of the footprint of CbbR bound to binding site IR2/3 (Fig. 1). The procedure used was the following. CbbR protein was incubated with 32 P-labelled cbb promoter DNA (100,000 cpm/reaction) at 30 jC as described earlier (Van Keulen et al., 1998). The reaction mixture was subjected to nondenaturing gel electrophoresis at 4 jC using a prerun Oligoprep gel in 1 Oligoprep buffer. The gel was prepared according to manufacturer’s instructions by adding 17.5 ml of Oligoprep concentrate to 1.25 ml of Oligoprep buffer and 6.25 ml deionized water which was polymerized by adding 10% APS and TEMED. Upon completion of electrophoresis, the Oligoprep gel was subjected to copperphenanthroline footprinting basically as described (Papavassiliou, 1994). The bands with specific DNAcomplexes and free DNA were cut from the gel and macerated by forcing it through a 20-G needle punc- Fig. 1. Improved recovery of DNA from a meltable acrylamide matrix resulting in high-quality copper-phenanthroline footprints in situ. Lane 1: Control DNaseI footprinting reaction without protein. Lane 2: DNaseI footprinting of CbbR in solution. Lane 3: Unbound DNA footprinted with copper-phenanthroline and isolated from an Oligoprep gel. Lane 4: Low-mobility complex DNA with two CbbR dimers bound footprinted with copper-phenanthroline and isolated from an Oligoprep gel. GA indicates GA chemical sequencing reaction. Boxes on the right of footprints represent the sequences protected by CbbR. IR1 and IR2/3 indicate binding sites for CbbR. G. van Keulen, W.G. Meijer / Journal of Microbiological Methods 54 (2003) 289–291 ture hole in a 0.5-ml vial into a 1.5-ml tube by centrifugation. DNA was isolated by dissolving the gel paste with 5 volumes of OligoPrep Dissolution Reagent at 60 jC for 30 min. The volume of the DNA solution was decreased from 600 to 20 Al using glassmilk elution according to the manufacturer’s instruction (Geneclean II, Bio101). DNA concentration by ethanol precipitation or using commercially available kits which use chaotropic agents such as guanidine compounds was not suitable due to the coprecipitation of the OligoPrep gel matrix with the DNA. DNA samples were denatured and equal amounts of radioactivity was loaded onto a 6% sequencing gel together with a G + A sequencing ladder as described previously (Van Keulen et al., 1998). The results show that the meltable matrix did not interfere with the chemical nuclease reaction. The method described here is clearly superior to conventional techniques (diffusion-driven and electro-elution) to isolate footprinted DNA. The yield of the latter was often too low to enable identification of footprint ladders (data not shown). In situ techniques clearly improve the quality of footprinting results, and meltable acrylamide matrices such as Oligoprep shorten the protocol significantly. Using the procedure described here, a high-quality footprinting experiment can be completed within 1 day. The advantages of the procedure are summarized in Table 1. The isolation of DNA from meltable matrices is highly efficient, making this a suitable procedure for other applications requiring acrylamide separations (e.g. purification of oligonucleotides). As the matrix is free of nuclease activity, it can be employed to isolate RNA as well. Furthermore, proteins can be 291 Table 1 Advantages of using a meltable polyacrylamide gel over conventional techniques for the isolation of nucleic acids from polyacrylamide gels Meltable polyacrylamide gel Conventional methods (e.g. electro-elution, diffusion-driven elution) Time Yield 30 min good Equipment/ chemicals standard laboratory equipment and chemicals standard procedures, nuclease free good 2 – 16 h poor due to DNA adhering to the dialysis membrane and/or diffusion-driven process expensive dialysis membranes, expensive electro-elution equipment advanced skills required, handling increases risk of nuclease contamination poor Handling Reproducibility isolated from these gels if SDS is added before the polymerization of the gel. References Galas, D.J., Schmitz, A., 1978. DNase footprinting: a simple method for the detection of protein – DNA binding specificity. Nucleic Acids Res. 5, 3157 – 3170. Papavassiliou, A.G., 1994. 1,10-Phenantroline-copper ion nuclease footprinting of DNA – protein complexes in situ following mobility-shift electrophoresis assays. Methods Mol. Biol. 30, 43 – 78. Shively, J., van Keulen, G., Meijer, W.G., 1998. Something from almost nothing: carbon dioxide fixation in chemoautotrophs. Annu. Rev. Microbiol. 52, 191 – 230. Van Keulen, G., Girbal, L., van den Bergh, E.R.E., Dijkhuizen, L., Meijer, W.G., 1998. The LysR-type transcriptional activator CbbR controlling autotrophic CO2 fixation by Xanthobacter flavus is an NADPH sensor. J. Bacteriol. 180, 1411 – 1417.