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DNA Footprinting Using Embryonic Extracts 191The first protocol described is that of the chemical nuclease copperorthophenanthroline (OP–Cu). Cleavage occurs at the phosphodiester backboneof nucleic acids at physiological pHs and temperatures (6). Protectionfrom (OP) 2 CU + -mediated cleavage implies that at least a portion of the proteinoccupies the minor groove (see Note 1). This OP–Cu footprinting is novel, asthe scission reaction can take place within the polyacrylamide matrix followinggel retardation. The second protocol is that of dimethyl sulfate (DMS) protectionor methylation footprinting. This technique requires that theprotein–DNA complex is briefly treated with DMS immediately prior to gelretardation. The manner of this chemical modification arises from a mild exposureof the DNA to the oxidizing agent DMS. Principally, methylation occurson exposed guanine residues at the N7 position (major groove) and, to a lesserextent, adenine residues at the N3 position (minor groove). Methylated DNA islater cleaved by piperidine treatment. Although this technique does not providethe same resolution as the above in situ approach, DMS demonstrates areduced sensitivity to conformational variability (see Note 1). The final techniqueis that of methylation interference using dimethyl sulfate. This assayidentifies the extent of protein contacts to specified bases within the recognitionsequence. Methylation interference is the most widely used of thefootprinting techniques and is often used for comparison with other characterizedfactors (see Note 2) (7).2. Materials2.1. Maxam and Gilbert Sequencing Reagents1. DNase- and RNase-free water (Sigma, Poole, UK).2. 90% Formic acid (Merck, Lutterworth, UK).3. 10 M Piperidine (Merck).4. 10 mg/mL Salmon sperm DNA (Boehringer Mannheim, Lewes, UK).5. Parafilm.6. Water-saturated butan-1-ol (Merck).7. Heating block.8. Variable-speed bench-top centrifuge.9. 3 M Sodium acetate (pH 4.5–5.5) (Merck).10. 100% and 70% Molecular-biology-grade ethanol (EtOH) (Sigma).2.2. Copper Phenanthroline Reagents1. 40 mM 1,10 Phenanthroline (0.079 g dissolved in 10 mL EtOH, prepared fresh).2. 9 mM Cupric sulphate (0.022 g dissolved in 10 mL H 2 O, prepared fresh).3. 28 mM 2,9-Dimethyl-1,10-phenanthroline (0.104 g dissolved in 10 mL EtOH,prepared fresh).4. 50 mM Tris–HCl, pH 8.0 (Sigma).5. β-Mercaptoproprionic acid (Sigma).
192 Orford, Mernagh, and Guille2.3. DNA Extraction and Preparation.1. 3MM Paper (Whatman, Maidstone, UK).2. DE-81 Paper (Whatman).3. Western-blotting tank (Bio-Rad, Hemel Hemstead, UK).4. 3L1XTris-borate EDTA (TBE) Ultrapure: 0.089 M Tris-borate pH 8.3, 2 mMEDTA (National Diagnostics, Hull, UK).5. Elution buffer: 1 M NaCl, 20 mM Tris pH 8, 1 mM EDTA.6. Biophenol–chloroform–iso-amyl-alcohol (Camlab).7. Chloroform (Merck).8. 1 µg/µL tRNA (Boehringer Mannheim).9. Spin-X tubes (Sigma).2.4. Footprinting Gel Materials1. Sequencing gel plates.2. Sequencing gel tank. Acrylamide sequencing grade 40% (29:1)acrylamide:bisacrylamide solution (gas stabilized) (National Diagnostics).3. Silane (Sigma).4. Yellow sealing tape.6. 10% (w/v) Ammonium persulfate (APS) (store solution at 4°C and use within1 wk) (Sigma).7. N,N,N'N'-Tetramethylethylenediamine (TEMED) (Sigma).8. Urea (Sigma).9. Formamide loading dye: 10 mL formamide, 10 mg xylene cyanol, 10 mg bromophenolblue, 200 µL 0.5 M EDTA pH 8.2.5. Dimethyl Sulfate Footprinting Reagents1. 10M Dimethyl sulfate (Sigma).2. 1X TE (10 mM Tris pH 7.5, 1 mM EDTA).3. Dimethyl-sulfate (DMS) buffer: 60 mM NaCl, 10 mM Tris-HCl, pH 8.0, 10 mMMgCl 2 , 1 mM EDTA.4. 250 mM DTT stored at –20°C (Sigma).3. Methods3.1. Preparation of G + A Ladder: Maxam and GilbertThe preparation of a suitable marker is essential to orient and analyzefootprinting gels. Both strands of the DNA fragment to be used in thefootprinting assays should be sequenced. This protocol involves extensive use ofradioactivity, always wear gloves, glasses, work behind protective screens andmonitor for contamination.1. Take 15–20 fmol of 5' end-labeled dsDNA in Eppendorf tubes (+/– labeledstrands separately) (see Chapter 15 and Notes 2 and 3), to each tube add 1.5 µL10 mg/mL Salmon sperm DNA and 10 µL Sigma water.
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Analysis of mRNA Levels by RT-PCR 4
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70 Bertwistleparative techniques. F
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Synthetic mRNA for Microinjection 9
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112 Guilleantibodies (16), and anti
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118 Guille4. The next morning, the
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120 Guille10. Incubate the oocytes
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122 Guilleencoding a protein (4F2hc
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