Download Improved recovery of DNA from polyacrylamide gels after in situ

Journal of Microbiological Methods 54 (2003) 289 – 291
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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.