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DNase I FOOTPRINTING
23 DNase I FOOTPRINT!NG Barbara Krummel
PCR can be used to prepare microgram quantities of double-stranded DNA fragments for use in experiments such as gel shift assays, DNase I footprinting, and in vitro transcription (Higuchi et al. 1988; Krummel and Chamberlin, in press). T h e use of P C R obviates the need for convenient restriction sites, large amounts of plasmid D N A as starting material, and gel isolation of products. T h e P C R product can be radioactively labeled at only one or both of the 5' ends. A simple procedure for the preparation of a uniquely end-labeled D N A fragment for use in DNase I footprinting is described (Figure 1).
Protocols
DNA To Be Amplified The DNA starting material can be supercoiled or linearized plasmid DNA prepared by a CsCI banding procedure or from a small-scale
184
PCR Protocols: A Guide to Methods and Applications Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
23. DNase I Footprinting
185
I. C i r c u l a r o r l i n e a r D N A starting
material
II. E n d - l a b e l o n e P C R p r i m e r 1-2 h r .
III. P C R r e a c t i o n 2-4
hr.
Fragment of
endpoints
restriction
site
independent availability
* IV.
Extraction
and concentration
1 hr.
Figure 1
Steps r e q u i r e d t o o b t a i n a u n i q u e l y 5 ' - e n d - l a b e l e d D N A f r a g m e n t . T h e
t i m e s i n d i c a t e d are a p p r o x i m a t e a n d d e p e n d o n t h e exact p r o c e d u r e u s e d .
lysis procedure (Maniatis et al. 1982); DNA fragments are also suitable. End-Labeled Primer Choosing the primer: Typically primers 20 to 25 nucleotides in size with at least a 5 0 % G + C content and no obvious self-complemen tarity are used (see Chapter 1). Size of fragment to be amplified: If o n e fragment will be used to obtain a footprint from each strand, the region of interest should
186
Part Two. Research Applications
ideally be close to the center of the fragment and not more than ap proximately 100 bp f r o m either e n d . The distance between the foot print region and the fragment end is limited by the resolution avail 3 2 gel system. able on the denaturing 3 2 nec Incorporation of P at the 5' e n d : The amount of primer "A" 32 essary for the PCR is determined (see following) and then P-labeled at the 5' end using [y- P]ATP (NEN; 6000 Ci/mmol) and T4 polynu 32 cleotide kinase (Maniatis etal. 1982, p. 22). The completed reaction is extracted once w i t h phenol. The unincorporated [y- P]ATP can be removed by centrifugal gel filtration using Sephadex G-10 (Phar macia) or ethanol precipitation w i t h the addition of 10 m M MgCI 2 (final concentration) to the standard conditions of 0.3 M sodium acetate and 2.5 volumes of 100% ethanol.
PCR Reactions (final volume 200 /d) are performed in a 0.5-ml mi crofuge tube. 250 ng of template DNA (e.g., 4-kb plasmid) 20 /xl of 10x PCR buffer (1 x concentrations: 50 m M KCI, 10 m M Tris-HCI (pH 8.3), 2.5 m M MgCI 2, 0.01% gelatin w/v) 50 pmol end-labeled primer "A" (from labeling protocol)
50 pmol primer " B " 3 /xl of 10 m M dNTPs (a mixture of all four dNTPs at 10 m M each) 5 units of Taq DNA polymerase (Perkin-Elmer Cetus) Cycling reactions are performed in a DNA thermal cycler. Temperature Time 94°C (denaturation) 60 seconds 37°C (annealing) 40 seconds 72°C (extension) 60 seconds The total number of cycles is 20 to 25, and a last cycle w i t h a 10minute extension time is included. This increased extension is in tended to allow complete reannealing and/or extension so that little single-stranded DNA remains. A higher annealing temperature (55°C instead of 37°C) may disrupt potential secondary structure in the primers and can result in more specific amplification (Saiki et al. 1988). Larger fragments may require longer extension times. The 6 f r o m a 200-/xl reaction is 2 to 3 /xg typical yield of a 300-bp fragment with a specific activity of 5 x 10 c p m / p m o l .
23. DNase I Footprinting
187
Extraction and Precipitation of PCR Before extraction, 1% of the total reaction volume is electrophoresed on an agarose minigel to evaluate the reaction. If the plasmid template and unused primer will not interfere significantly with binding experiments, the reaction is extracted o n c e with phenol and o n c e with chloroform, then ethanol-precipitated. A Centricon 100 microconcentrator (Amicon) can be used to remove unused primer, while removal of both primer and plasmid starting material is ac complished by nondenaturing PAGE. To quantitate the DNA, an ali quot is electrophoresed on an agarose minigel with size standards of known concentration. If the unused primer has not been re moved, the cpm/ng of the fragment product cannot b e determined by simply counting an aliquot. The band of interest can b e excised from the agarose gel and dried onto filter paper; the total cpm can be determined by scintillation counting.
Discussion PCR provides a rapid and simple method for the preparation of double-stranded D N A fragments. 3T2h e procedure outlined here is for the preparation of a uniquely P-end-labeled fragment for use in DNase I footprinting. T h e same P C R protocol is followed for prepar ing unlabeled D N A fragment or D N A labeled at both ends. Clearly this procedure could also be used to incorporate nonradioactive molecules such as biotin, specifically at the 5 ' end (see Chapter 13). No problems have been encountered because of misincorporation by Taq D N A polymerase that would lead to a population of fragments with a mixed D N A sequence. However, the product of the PCRs de 32 scribed here is not always a homogeneous preparation of full-length double-stranded D N A molecules. When the P-end-labeled product is electrophoresed on a denaturing polyacrylamide gel, the result may be a "ladder" of fragments ranging in size from full length to that of the original primer. Every position appears to be represented, but the full-length material predominates (90 to 9 5 % ) . We believe that these shorter fragments are due to the presence of incompletely replicated molecules. Nondenaturing PAGE does not provide separa tion of the two populations. Reducing the total number of cycles or
188
Part Two. Research Applications
increasing the annealing temperature to 55°C (closer to the optimum temperature of the enzyme) results in no significant change in the product. This phenomenon has not interfered with the use of PCR-generated D N A in DNase I footprinting experiments. However, P C R has not reproducibly provided a suitable substrate for the hydroxyl radi cal cleavage reaction, which requires a D N A template with a very low background of cleaved or nicked molecules (Tullius et al. 1987).
Literature Cited Higuchi, R., B. Krummel, and R. K. Saiki. 1988. A general method of in vitro prepara tion and specific mutagenesis of D N A fragments: study of protein and D N A in teractions. Nucleic Acids Res. 1 6 : 7 3 5 1 - 7 3 6 7 . Krummel, B., and R. Chamberlin. 1989. R N A chain initiation by Escherichia coli R N A polymerase. Structural transitions of the enzyme in early ternary com plexes. Biochemistry. In press. Maniatis, T., E. Fritsch, and J. Sambrook. 1982. In Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primer-directed enzymatic amplification of D N A with a thermostable D N A polymerase. Science 2 3 9 : 4 8 7 - 4 9 1 . Tullius, T. D., B. A. Dombroski, M. E. A. Churchill, and L. Kam. 1987. Hydroxyl radi cal footprinting: a high-resolution method for mapping protein-DNA contacts. Methods Enzymol. 1 5 5 : 5 3 7 - 5 5 8 .
PCR can be used to prepare microgram quantities of double-stranded DNA fragments for use in experiments such as gel shift assays, DNase I footprinting, and in vitro transcription (Higuchi et al. 1988; Krummel and Chamberlin, in press). T h e use of P C R obviates the need for convenient restriction sites, large amounts of plasmid D N A as starting material, and gel isolation of products. T h e P C R product can be radioactively labeled at only one or both of the 5' ends. A simple procedure for the preparation of a uniquely end-labeled D N A fragment for use in DNase I footprinting is described (Figure 1).
Protocols
DNA To Be Amplified The DNA starting material can be supercoiled or linearized plasmid DNA prepared by a CsCI banding procedure or from a small-scale
184
PCR Protocols: A Guide to Methods and Applications Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
23. DNase I Footprinting
185
I. C i r c u l a r o r l i n e a r D N A starting
material
II. E n d - l a b e l o n e P C R p r i m e r 1-2 h r .
III. P C R r e a c t i o n 2-4
hr.
Fragment of
endpoints
restriction
site
independent availability
* IV.
Extraction
and concentration
1 hr.
Figure 1
Steps r e q u i r e d t o o b t a i n a u n i q u e l y 5 ' - e n d - l a b e l e d D N A f r a g m e n t . T h e
t i m e s i n d i c a t e d are a p p r o x i m a t e a n d d e p e n d o n t h e exact p r o c e d u r e u s e d .
lysis procedure (Maniatis et al. 1982); DNA fragments are also suitable. End-Labeled Primer Choosing the primer: Typically primers 20 to 25 nucleotides in size with at least a 5 0 % G + C content and no obvious self-complemen tarity are used (see Chapter 1). Size of fragment to be amplified: If o n e fragment will be used to obtain a footprint from each strand, the region of interest should
186
Part Two. Research Applications
ideally be close to the center of the fragment and not more than ap proximately 100 bp f r o m either e n d . The distance between the foot print region and the fragment end is limited by the resolution avail 3 2 gel system. able on the denaturing 3 2 nec Incorporation of P at the 5' e n d : The amount of primer "A" 32 essary for the PCR is determined (see following) and then P-labeled at the 5' end using [y- P]ATP (NEN; 6000 Ci/mmol) and T4 polynu 32 cleotide kinase (Maniatis etal. 1982, p. 22). The completed reaction is extracted once w i t h phenol. The unincorporated [y- P]ATP can be removed by centrifugal gel filtration using Sephadex G-10 (Phar macia) or ethanol precipitation w i t h the addition of 10 m M MgCI 2 (final concentration) to the standard conditions of 0.3 M sodium acetate and 2.5 volumes of 100% ethanol.
PCR Reactions (final volume 200 /d) are performed in a 0.5-ml mi crofuge tube. 250 ng of template DNA (e.g., 4-kb plasmid) 20 /xl of 10x PCR buffer (1 x concentrations: 50 m M KCI, 10 m M Tris-HCI (pH 8.3), 2.5 m M MgCI 2, 0.01% gelatin w/v) 50 pmol end-labeled primer "A" (from labeling protocol)
50 pmol primer " B " 3 /xl of 10 m M dNTPs (a mixture of all four dNTPs at 10 m M each) 5 units of Taq DNA polymerase (Perkin-Elmer Cetus) Cycling reactions are performed in a DNA thermal cycler. Temperature Time 94°C (denaturation) 60 seconds 37°C (annealing) 40 seconds 72°C (extension) 60 seconds The total number of cycles is 20 to 25, and a last cycle w i t h a 10minute extension time is included. This increased extension is in tended to allow complete reannealing and/or extension so that little single-stranded DNA remains. A higher annealing temperature (55°C instead of 37°C) may disrupt potential secondary structure in the primers and can result in more specific amplification (Saiki et al. 1988). Larger fragments may require longer extension times. The 6 f r o m a 200-/xl reaction is 2 to 3 /xg typical yield of a 300-bp fragment with a specific activity of 5 x 10 c p m / p m o l .
23. DNase I Footprinting
187
Extraction and Precipitation of PCR Before extraction, 1% of the total reaction volume is electrophoresed on an agarose minigel to evaluate the reaction. If the plasmid template and unused primer will not interfere significantly with binding experiments, the reaction is extracted o n c e with phenol and o n c e with chloroform, then ethanol-precipitated. A Centricon 100 microconcentrator (Amicon) can be used to remove unused primer, while removal of both primer and plasmid starting material is ac complished by nondenaturing PAGE. To quantitate the DNA, an ali quot is electrophoresed on an agarose minigel with size standards of known concentration. If the unused primer has not been re moved, the cpm/ng of the fragment product cannot b e determined by simply counting an aliquot. The band of interest can b e excised from the agarose gel and dried onto filter paper; the total cpm can be determined by scintillation counting.
Discussion PCR provides a rapid and simple method for the preparation of double-stranded D N A fragments. 3T2h e procedure outlined here is for the preparation of a uniquely P-end-labeled fragment for use in DNase I footprinting. T h e same P C R protocol is followed for prepar ing unlabeled D N A fragment or D N A labeled at both ends. Clearly this procedure could also be used to incorporate nonradioactive molecules such as biotin, specifically at the 5 ' end (see Chapter 13). No problems have been encountered because of misincorporation by Taq D N A polymerase that would lead to a population of fragments with a mixed D N A sequence. However, the product of the PCRs de 32 scribed here is not always a homogeneous preparation of full-length double-stranded D N A molecules. When the P-end-labeled product is electrophoresed on a denaturing polyacrylamide gel, the result may be a "ladder" of fragments ranging in size from full length to that of the original primer. Every position appears to be represented, but the full-length material predominates (90 to 9 5 % ) . We believe that these shorter fragments are due to the presence of incompletely replicated molecules. Nondenaturing PAGE does not provide separa tion of the two populations. Reducing the total number of cycles or
188
Part Two. Research Applications
increasing the annealing temperature to 55°C (closer to the optimum temperature of the enzyme) results in no significant change in the product. This phenomenon has not interfered with the use of PCR-generated D N A in DNase I footprinting experiments. However, P C R has not reproducibly provided a suitable substrate for the hydroxyl radi cal cleavage reaction, which requires a D N A template with a very low background of cleaved or nicked molecules (Tullius et al. 1987).
Literature Cited Higuchi, R., B. Krummel, and R. K. Saiki. 1988. A general method of in vitro prepara tion and specific mutagenesis of D N A fragments: study of protein and D N A in teractions. Nucleic Acids Res. 1 6 : 7 3 5 1 - 7 3 6 7 . Krummel, B., and R. Chamberlin. 1989. R N A chain initiation by Escherichia coli R N A polymerase. Structural transitions of the enzyme in early ternary com plexes. Biochemistry. In press. Maniatis, T., E. Fritsch, and J. Sambrook. 1982. In Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primer-directed enzymatic amplification of D N A with a thermostable D N A polymerase. Science 2 3 9 : 4 8 7 - 4 9 1 . Tullius, T. D., B. A. Dombroski, M. E. A. Churchill, and L. Kam. 1987. Hydroxyl radi cal footprinting: a high-resolution method for mapping protein-DNA contacts. Methods Enzymol. 1 5 5 : 5 3 7 - 5 5 8 .