DNAase I hypersensitive site mapping is frequently used to localize regulatory regions of eukaryotic genes. For reasons that are not yet understood, genes carry separate sequences that are sensitive to DNAase I cleavage, and these so-called hypersensitive site mappings vary with the activation state of the gene (Weintrauband Gmudine1976). This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
DNAase I hypersensitive site mapping
Materials and Instruments
Eukaryotic Cells Move makings For more product details, please visit Aladdin Scientific website.
Buffer A EDTA Ethanol Lysis buffer Phosphate buffer Tepan blue dye DNA enzyme I dilution buffer DNA enzyme I solution Protease K solution RNA enzyme solution Phenol: chloroform SDS buffer TE
Sorvall H1000B Turning head or equivalent Tubes with screw caps Oscillating water baths
Buffers and solutions
See Appendix 1 for the composition of storage solutions, buffers and reagents, and dilute the storage solution to the appropriate concentration.
Buffer A
50 mml/L Tris-Cl (pH 7.9)
100 mmol/LNaCl
3 mmol/LMgCl2
1 mmol/LDTT
0.2 mmol/L phenylmethylsulfonyl fluoride
Store Buffer A at 4°C. Add DTT and phenylmethylsulfonyl fluoride before use.
Add DTT and phenylmethylsulfonyl fluoride before use.
Ethanol
Lysis buffer
50 mmol/L Tris-Cl (pH 7.9)
100 mmol/L KCl
5 mmol/L MgCl2
0.05%(V/V) Saponin
50%(V/V) Glycerol
200 mmol/L β-liucoethanol
The lysis buffer is stored at 4°C. Add β-mercaptoethanol before use.
Phenol: chloroform
Phosphate buffer without calcium and magnesium salts
SDS buffer
20 mmol/L Tris-Cl (pH 7.9)
100 mmol/LNaCl
70 mmol/LEDTA (pH 8.0)
2% (m/V) SDS
SDS buffer was stored at room temperature.
TE
Taipan blue dye (0.4% m/V)
Appropriate amount of dye is dissolved in phosphate buffer free of calcium and magnesium salts and stored at room temperature.
Enzymes and buffers
DNA Enzyme I Dilution Buffer
10 mmol/LHEPES-KOH (pH 7.9)
30 mmol/L CaCl2
30 mmol/L MgCl2
50%(V/V) Glycerol
DNA enzyme I solution
Dilute the RNAase-free DNAase I product to a concentration of 10 units/ul with DNAase I Dilution Buffer. Store the solution at -20°C. The amount of DNAase I required to achieve a 0.001 change in A260 value per minute per milliliter of reactant is defined as 1 unit of DNAase I, using bovine thymus DNA as the substrate.
Proteinase K Solution
Dissolve Proteinase K in 50 mmol/L Tris-Cl (pH 7.9) and 100 mmol/L NaCl to a concentration of 0.2 mg/ml. Dispense the solution and store at -20°C.
RNAase Solution
RNAase product without DNAzyme is dissolved in TE (pH 8.0) to a concentration of 0.5 mg/ml.
Centrifuge and rotor head
Sorvall H1000B turntable or equivalent
Specialized equipment
Test tube with screw cap (50 ml)
Oscillating water baths preset to 50°C and 55°C
Additional reagents
The reagents listed in Chapter 6, Options 8 and 10 are required in Step 18 of this protocol.
Cells and Tissues
Eukaryotic Cells
Approximately 108 cells are used for each DNAase I hypersensitive site mapping assay.
Methods
1. Cells are spun or cultured in triangular flasks or petri dishes, then approximately 108 cells are harvested and washed twice with 25 ml of ice-cold phosphate buffer free of calcium and magnesium ions.
Alternatively, if by using fresh tissue, isolate nucleic acids as described in Step 1 of Scheme 1.
2. Resuspend the cell precipitate with 1.5 ml of ice-cold lysis buffer. Ice bath for 10 min to allow cell lysis.
3. 10 ul of cell lysate is mixed with an equal volume of 0.4% Taipan blue dye and observed under a microscope fitted with a 20x objective. Lysed cells and nucleic acids absorb the dye and appear blue, while non-lysed cells remain transparent because the dye is not permeable. The ice bath was continued until >80% of the cells were lysed.
4. Harvest nucleic acids from the lysed cell suspension by centrifugation at 1300 g (Sorvall H1000B turntable at 2500 r/min) at 4°C for 15 min.
5. Carefully remove the supernatant and resuspend the nucleic acid precipitate with 1.5 ml of ice-cold Buffer A. Collect nucleic acids by centrifugation as described in step 4.
6. Resuspend the nucleic acid precipitate with 4 ml of ice-cold Buffer A.
7. Make a series of dilutions of the standard DNAzyme I solution (1/40, 1/80, 1/160, 1/320, 1/640, 1/1280, 1/2560 with DNAzyme Dilution Buffer). Store the dilutions on ice.
8.-Series of tubes are labeled from 1 to 9, and 180ul of each of the nucleic acid suspensions from step 6 is added to each tube.
9. Tube 1 is filled with 20ul of DNAase dilution buffer without DNAase I and placed on ice until step 12 below. Tube 2 is filled with 20ul of DNAase dilution buffer without DNAase I and placed as described in step 11 below. Tube 1 and Tube 2 serve as controls.
10. From Tube 3 to Tube 9, add 20ul of increasing concentrations of diluent, i.e., 20ul of 1/2560 diluent to Tube 3, 20ul of 1/1280 diluent to Tube 4, etc. s
11. Place tubes 2 to 9 at 37°C for 20 min.
12. Terminate the reaction by adding 0.5 ml/L EDTA to each tube in 3 additions of 16.6ul each, shaking between additions. After all tubes have been processed, add 12ul of RNA May solution to each tube. Incubate the reaction at 37°C for 30 min to degrade the nuclear RNA.
13. Add 40ul of Proteinase K solution per tube to digest the nuclear proteins. Blow the mixture up and down to homogenize the solution. Add 100ul of SDS buffer to each tube and mix again. The tubes were rotated at 50°C for 16 h. The tubes were then incubated at 50°C for 16 h.
14. Add another 100ul of Proteinase K solution and continue to incubate at 50°C for 2 to 3 h.
15. Extract the mixture 3 times with phenol/chloroform. Extract gently. Add 3 times the volume of ice-cold ethanol and incubate for 30 min in an ice bath. Collect the DNA precipitate by centrifugation in a bench-top centrifuge at 1200 g (Sorvall H1000B head, 2400 r/min). Remove the supernatant and blot the remaining ethanol from the tube with a paper towel.
16. Add 200ul of TE to each tube and spin at 55°C overnight to dissolve the DNA.
IMPORTANT: For complete lysis of DNAase-treated DNA, prolonged incubation is required.
17. Determine the A260 value of the DNA resuspension and estimate its concentration.
18. Digest the DNA with a restriction enzyme, followed by Southern blotting and hybridization as described in Chapter 6, Protocols 8 and 10. Add 15-30ug of delayed restriction enzyme-digested genomic DNA to each lane of the agarose gel. 
