DMTase Mapping of DNAProtein Interactions

3.2.1. Growth of Strain(s) Under Experimental Conditions

The nature of the experiment will determine the details of this portion of the protocol. Variations may include growth of a single DMTase-expressing strain under several different conditions or growth of DMTase-expressing experimental and control strains under a single condition. Depending on the method of regulation, induction of the DMTase may be accomplished at high levels in a short pulse at the desired time, at lower levels over a longer period, or consti-tutively as in the case of a native DNA binding factor-DMTase fusion expressed from an endogenous constitutive promoter. At the completion of the experiment, total yeast DNA is rapidly isolated as in Subheading and subsequently subjected to selective deamination of unmethylated cytidines by bisulfite ion.

3.2.2. Bisulfite Deamination of Isolated Yeast DNA

1. Prepare the degassed water the evening prior to execution of subsequent steps.

2. Prepare the 3 N NaOH and 100 mM hydroquinone solutions. Prepare the sample denaturation buffer (SDB). Aliquot 10 ^L of SDB to a 0.65-mL microfuge tube for each sample. Add 20 ^L yeast DNA solution to the SDB and mix by pipeting. The samples should be left at RT while subsequent reagents are prepared.

3. Prepare the sodium metabisulfite solution (SMBS) and prewarm it to 50°C. It is not necessary to confirm the temperature prior to use.

4. While the SMBS is warming, denature the samples in a thermocycler for 5 min at 98°C.

a. At about the 4-min point, bring the SMBS to the thermocycler, uncap it, and stir briefly.

b. At 5 min, open the thermocycler while maintaining block temperature at 98°C and, working rapidly, open the first tube in the block, add 200 ^L SMBS, cap and remove the tube, vortex it immediately, and place it in a rack or float.

c. Proceed likewise for all the samples, using a new tip for each sample.

d. After all samples have been prepared, incubate them and the remaining SMBS at 50°C for 6 h in the dark (see Note 18).

5. During the incubation, label two sets of 1.7-mL microcentrifuge tubes. Attach one labeled Promega minicolumn to a 3-mL syringe barrel for each sample.

6. Near the conclusion of the incubation, attach the minicolumn assemblies to the vacuum manifold. Heat the 0.1X TE to 95°C. Prepare the desulfonation solution (DSS) and aliquot 8 ^L to each 1.7-mL microcentrifuge tube of the first labeled set from step 5. Read and record the final pH of the unused SMBS (see Note 19).

7. At the conclusion of the incubation, transfer the sample solutions to the second set of 1.7-mL microcentrifuge tubes from step 5.

a. Add 1 mL Promega Wizard® PCR Preps resin to each sample and vortex.

b. Transfer the resin to the syringe barrels on the vacuum manifold.

c. Apply the vacuum to draw the resin into the minicolumns.

d. Close individual stopcocks as the syringe barrels empty completely.

e. Reopen the stopcocks, add 2 mL 80% isopropanol to each syringe barrel, and apply the vacuum to draw the wash through the columns.

f. Repeat the wash with 1 mL 80% isopropanol.

g. After the washes have completely passed through all columns, reopen the stopcocks and apply the vacuum for 30 s to begin drying them.

h. Detach the minicolumns from the syringe barrels and press them into 1.7-mL microcentrifuge tubes (use a set of reusable tubes with lids removed that are designated for this purpose, not labeled tubes from step 5).

i. Centrifuge at 16,000g for 2 min to remove residual wash.

8. Transfer the minicolumns to the first set of labeled tubes. Add 52 ^L 95°C 0.1X TE to each minicolumn and let sit at RT for 5 min. Centrifuge for 20 s at 16,000g to elute the DNA. Mix the eluate and DSS by vortexing. Tap spin and incubate in a 37°C water bath for 15 min (see Note 20).

9. Add 18 ^L 10 M NH4OAc and mix by pipeting. Add 200 ^L 95% EtOH and vortex. Incubate overnight at -20°C.

10. Centrifuge the tubes at 16,000g for 20 min at RT to pellet the DNA. Draw off and discard the supernatant. Add 400 ^L 70% Et0H:30% 1X TE, pH 8.0, and vortex to wash. Centrifuge at 16,000g for 2 min, carefully draw off and discard the supernatant, centrifuge again, and carefully draw off and discard the remainder of the wash (see Note 21).

11. Dry the pellet briefly by centrifuging the open tube 1 min at approx 80g in an open microcentrifuge rotor. Before resuspending the pellet, check to be sure there is no residual EtOH wash left in the tube. Resuspend/dissolve the pellet in 25 ^L 0.1X TE. Incubate for 1 to 24 h at 4°C. Mix by pipeting and centrifuge prior to use. Store the deaminate at -20°C.

3.2.3. PCR Amplification of Deaminated DNA Template and Equalization of Product Concentration

1. Use 2 to 4 ^L of deaminate as template in the following 50-^L PCR reaction: dH 20 to volume, 1X Sigma JumpStart™ PCR buffer, 2.25 mM MgCl2, 0.2 mM dNTPs, 0.8 ^M b1 (or a1) primer, 0.8 ^M b2 (or a2) primer, 1.25 U Sigma JumpStart™ Taq DNA polymerase. Thermocycle as follows: 1 cycle of 3 min at 94°C, 30 cycles of 45 s at 94°C, 45 s at 5°C below the calculated Tm of the primers, and 1 min/kb at 72°C, and 1 cycle of 5 min at 72°C (see Note 22).

2. Check for successful amplification of PCR product by electrophoresis of 1 ^L of the unpurified reaction on a 1% agarose-TAE-EtBr mini-gel for 30 min at 100 V. Visualize by UV illumination. PCR product is purified with Microcon PCR filters according to the manufacturer's directions. Dissolve product with and elute off filter in 30 yL st.dH2O. Store at -20°C (see Note 23).

3. In preparation for sequencing primer extensions, quantify the relative product concentration by electrophoresing 2 yL of each product to be sequenced on a 1% agarose-TAE-EtBr mini-gel for 30 min at 100 V and UV imaging. Obtain a linear digital image of the gel (i.e., not saturated owing to overexposure) under UV transillumination. Using image analysis software, calculate the relative intensities of the product bands. Equalize product concentrations by diluting products to the level of the least concentrated of the panel with st.dH2O. Store all dilutions at -20°C (see Note 24).

3.2.4. 32P End-Labeling of Primer To Be Used for Sequencing Extension

1. 1.0 to 1.2 pmol of primer (1.0-1.2 yL at 1 y M) will be used for each extension. Set up a labeling reaction in the following ratio with some excess in a 1.7-mL microcentrifuge tube: 10.0 yL dH2O, 2.0 yL 10X PNK buffer (supplied by manufacturer), 1.0 yL 20 y M b1 (or a1) primer, 1.0 yL 10 U/yL T4 PNK, and 6.0 yL 1.7 y M 6000 Ci/mmol (10 mCi/mL) [y32P]ATP, for a total of 20.0 yL.

3. During the incubation, pack two 1-mL syringe barrels with silanized glass wool to approx 0.1 mL graduation. Fill the barrels to the top with Sephadex G-25-80 equilibrated in dH2O. Remove the caps from two 1.7-mL screw-cap tubes and place them into two 15-mL centrifuge tubes. Place the syringe barrels into the tube assemblies such that the slip-tip ends sit in the 1.7-mL tubes and the shoulders rest on the 15-mL tubes. Pack the columns by centrifuging the assemblies 5 min at 1800g at RT. Remove the assemblies, decant the water from the collection tubes, replace them, fill the columns again to the top with Sephadex, and centrifuge again under the exact same conditions. Decant the water as before and add 100 yL dH2O to the top of each column. Reassemble the spin column apparatus and centrifuge again under the exact same conditions.

4. At the conclusion of the incubation, centrifuge the reaction microcentrifuge tube for 5 min at 16,000g.

5. Using new 1.7-mL screw-cap tubes for collection, assemble the spin column apparatus again. Pipet the entire volume of the primer-labeling reaction onto the top of the first column. Centrifuge exactly as in step 3, with a separate balance. Upon completion, pipet the entire volume of the eluate onto the top of the second column and repeat the centrifugation. Note the volume of the eluate from each purification for estimation of primer concentration.

3.2.5. Primer Extension Sequencing

1. Use 2 to 4 yL of equalized PCR product (see Subheading 3.2.3.) as template in the following 8-yL primer extension reaction: dH2O to volume, 1X Epicentre SequiTherm™ buffer, 150 yM ddGTP, 50 yM d[A,C,T]TP, 1.0 to 1.2 pmol primer (see Subheading 3.2.4.), 1.25 U Epicentre SequiTherm™ thermostable DNA polymerase. Thermocycle as follows: 1 cycle of 2 min at 94°C, 15 cycles of 30 s at 94°C, 30 s at 5°C below the calculated Tm of the primers, and 1 min/kb at 70°C, and 1 cycle of 2 min at 70°C. At the conclusion of the cycling, put the tubes on ice, tap spin, add 4 pL stop dye to each, and mix by pipeting. Store at -20°C (see Note 25).

3.2.6. Electrophoresis and Imaging of Primer Extension Products

1. Cast a 35 o 43 o 0.04 cm polyacrylamide denaturing sequencing gel with the following composition: dH2O to volume, 4 to 6% 19:1 acrylamide/bisacrylamide, 1X TBE, 50% urea, polymerized with APS and TEMED. Insert a 40-well straight-tooth comb, and allow to polymerize overnight (see Note 26).

2. Carefully remove the comb and assemble the electrophoretic apparatus. Buffer compositions should be determined by the desired separation pattern (see Note 4).

3. Using a syringe, flush out air bubbles and urea from the wells with buffer. Flush out any bubbles from the bottom edge of the gel with buffer. Prior to loading, pre-electrophorese the gel for approx 30 min at 60 W to warm it.

4. Near the end of the preelectrophoresis, denature the primer extension reactions for 5 min at 70°C in a thermocycler. Quick chill the samples on ice at the conclusion of the incubation; do not allow them to cool gradually. Flush the wells with buffer prior to loading. Load 3 to 4 pL of each sample per well. If empty lanes are to be left between samples, load an equal volume of dummy dye in the corresponding wells. Load two to three wells with dummy dye on both sides of the loaded primer extension reactions (see Note 27).

5. Electrophorese at 60 W. Under gradient buffer conditions, electrophoresis times vary from 2.5 to 4 h at 60 W and should be determined by the number, lengths, and relative positions of fragments to be resolved.

6. Dry the gel for 2 h under heat and vacuum. Expose the dried gel to a phosphor screen. Image using a Molecular Dynamics Storm860 phosphorimager. A representative image is shown in Fig. 2.

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