RNA mapping with S1 nuclease
RNA mapping with S1 nuclease
Three different nucleases, S1 nuclease, RNAase, and exonuclease VII, are used to quantify RNA, to identify intron positions, and to characterize the positions of the 5' and 3' ends of mRNA on cloned DNA templates. Nuclease S1 is used to analyze protection assays when detecting RNA hybridized to a DNA template, and RNAase is used when detecting RNA hybridized to RNA from a DNA template. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Mapping of RNA with S1 nuclease
Principle
Three different nucleases, S1 nuclease, RNAase, and exonuclease VII, are used to quantify RNA, to identify intron positions, and to characterize the positions of the 5' and 3' ends of mRNA on cloned DNA templates. Nuclease S1 is used for protection assay analysis when detecting RNA hybridized to a DNA template, and RNAase is used when detecting RNA hybridized to RNA from a DNA template.
Materials and Instruments
T4 Phage polyribonucleotide kinase Klenow fragment of E. coli DNA polymerase S1 nuclease S1 nuclease digestion buffer Restriction endonuclease Carrier RNA Standard RNA Single-stranded RNA DNA probe Move I. Materials For more product details, please visit Aladdin Scientific website.
Ammonium persulfate Reproduction buffer Ethanol Gel elution buffer RNA hybridization buffer S1 Nuclease termination buffer Phenol Chloroform RNA sampling buffer Sodium acetate TE Tetramethylethylenediamine Trichloroacetic acid
Denaturing polyacrylamide gel Water bath Whatman 3 MM filter paper
1. Buffers and solutions
Ammonium persulfate (10%)
10X compounding buffer (100 mmol/L Tris-Cl (pH 7.5), 100 mmol/L MgCl2, 0.5 mol/L NaCl, 100 mmol/L dithiothreitol (DTT))
Ethanol
Gel elution buffer (0.5 mol/L ammonium acetate, 1 mmol/L EDTA ( pH 8.0), 0.1% (m/V) SDS)
RNA hybridization buffer
S1 Nuclease termination buffer (4 mol/L ammonium acetate, 50 mmol/L EDTA (pH 8.0), 50 ug/ml carrier RNA)
Phenol: chloroform (1:1, V/V)
RNA Sampling Buffer
Sodium acetate (3 mol/L, pH 5.2)
TE ( pH 7.6)
Tetramethylethylenediamine (TEMED)
Trichloroacetic acid (TCA) (1% and 10%)
2. Enzymes and buffers
T4 phage polyribonucleotide kinase
Klenow fragment of E. coli DNA polymerase (10 units/μl)
S1 Nuclease (used with S1 Nuclease Digestion Buffer)
S1 Nuclease Digestion Buffer (0.28 mol/L NaCl, 0.05 mol/L sodium acetate (pH 4.5), 4.5 mmol/L ZnSO4-7H2O )
Restriction endonuclease
3. gel
Denaturing polyacrylamide gel with 8 mol/L urea 
4. nucleic acids and oligonucleotides
Vector RNA (yeast tRNA)
Solution containing four dNTPs (20 mmol/L) (Dissolve dNTP with 25 mmol/L Tris-Cl (pH 8.0) and store in small portions at -20°C.)
Standard RNA
Synthetic oligonucleotides dissolved in distilled water (10 pmol/μl )
Template DNA ( 1 μg/μl), single stranded
Single-stranded RNA
5. Probes
Uniformly labeled and single-stranded DNA probes
6. radioactive compounds
[ γ-32P ] ATP (10 mCi/ml, 3000 Ci/mmol)
7. Specialized equipment
Water bath, preset at 65°C, 85°C, and 95°C, respectively, to obtain suitable digestion temperature as well as ideal hybridization temperature.
Whatman 3 MM filter paper (or equivalent) 
II. Methods
Preparation of randomly labeled single-stranded DNA probes
1. Prepare a polyacrylamide microgel (13 cm X 15 cm X 0.75 cm ) containing 8 mol/L urea ( e.g. Bio-Rad Mini-Protean).
(1) Mix the following reagents
7.2 g urea
1.5 ml 10X TBE
Prepare a polyacrylamide gel containing the desired concentration by adding an appropriate amount of 40% acrylamide (acrylamide: methacrylamide 19:1).
(2) Add water to a final volume of 15 ml.
(3) Stir at room temperature on a magnetic stirrer until the urea is dissolved. Add again:
120 μl 10% ammonium persulfate
16 μl TEMED
Quickly mix the solution and pour the gel into the mold of the microgel.
2. mix the following reagents while the gel sets:
10 pmol ( 1 μl) unlabeled oligonucleotide
10 μl [ γ-32P ] ATP (10 mCi/ml, 3000 Ci/mmol)
2 μl 10X polynucleotide kinase buffer
6 μl H2O
10 units (1 μl) of polynucleotide kinase
The reaction mixture was incubated at 37℃ for 45 min, and then incubated at 95℃ for 3 min to inactivate the polynucleotide kinase.
3. Add to the kinase reaction:
2 μl (2 μg) single-stranded DNA template
4 μl of 10X Recovery Buffer
14 μl H2O
The reaction mixture was incubated at 65°C for 10 min and then cooled to room temperature.
4. Add to the reaction mixture from step 3:
4 μl dNTP mixture
1 μl (10 units) of Klenow fragment of E. coli DNA polymerase I
The reaction mixture was incubated at room temperature for 15 min and then at 65℃ for 3 min to inactivate the DNA polymerase.
5. Adjust the ionic composition and pH of the reaction mixture to suit the restriction enzyme. Add 20 units of restriction enzyme and digest for 2 h at the appropriate temperature.
6. Add to the restriction endonuclease digestion reaction:
2 μl carrier RNA
5 μl 3 mol/L sodium acetate (pH 5.2)
The DNA probe was recovered by standard ethanol precipitation method.
7. Dissolve the DNA with 20 μl of Gel Sampling Buffer, incubate at 95℃ for 5 min to denature, and then quickly cool to 0℃.
Purify the probe by gel electrophoresis.
8. While the DNA is incubating at 95°C, wash the sample wells of the gel to remove the urea and add the probe immediately.
9. Electrophoresis is performed until the bromophenol blue reaches the bottom of the gel (about 30 min at 200 mA).
10. Remove the perfusion device and allow the gel to adhere to the bottom glass plate. Wrap the gel and glass plate with plastic film (e.g. Saran film). Make sure there are no air bubbles between the gel and the plastic film.
NOTE: Wear eye protection when prying off the glass plate.
11. Expose the gel to x-ray film. Make permanent marks on the film at the corners and edges of the slide. Also make marks for the position of bromophenol blue and xylene cyanide blue.
An exposure of 2 to 10 minutes is usually sufficient to obtain an image of the radiolabeled probe.
12. Separate the glass plate from the film and remove the radiolabeled strips with a scalpel. Expose the cut gel on a new piece of film to assure that the area of the gel containing the correct molecular size bands has indeed been cut down.
13. Transfer the cut gel fragments into a sterilized microcentrifuge tube and add just enough volume of gel elution buffer to cover the gel (250 to 500 μl). Leave the capped centrifuge tube on a shaker overnight at room temperature.
14. Centrifuge at maximum speed for 5 min.
15. Transfer the supernatant into a new centrifuge tube using an automated pipetting device, taking care not to aspirate polyacrylamide. The labeled probe should be approximately 10,000 cpm/μl by liquid flash spectroscopy.
16. Store the probe at -70°C. 
Hybridization between the RNA to be tested and the radiolabeled DNA probe
17. Divide 0.5-150 μg of RNA (test and standard) equally into each sterilized microcentrifuge tube. Add an excess of uniformly labeled single-stranded DNA probe to each tube.
18. Precipitate the RNA and DNA by adding 10% by volume of 3 mol/L sodium acetate (pH 5.2) and 2.5 times the volume of ice-cold ethanol. place at 0°C for 30 min and centrifuge at 4°C for 15 min at maximum speed to recover nucleic acids. Remove the supernatant from the ethanol, wash with 70% ethanol and centrifuge again. Carefully remove all ethanol and leave the precipitate containing RNA and DNA at room temperature until the ethanol residue has evaporated. 
19. Dissolve the nucleic acid precipitate with 30 μl of hybridization buffer. Blow several times to ensure that the precipitate is thoroughly dissolved.
20. Place the cap on the centrifuge tube tightly and incubate the hybridization reaction in a water bath at 85°C for 10 min to denature the nucleic acids.
21. Quickly transfer the tube to a water bath set to the hybridization temperature (typically 65°C). Do not allow the tube to cool below the hybridization temperature during the transfer. Hybridize DNA and RNA for 12-16 h at the selected temperature.
S1 Nuclease Digestion of DNA-RNA Heterodimers
22. Take care to keep the centrifuge tube submerged in water and open the cap. Quickly add 300 μl of ice-cold S1 Nuclease Digestion Buffer and immediately remove the tube from the water bath. Gently shake to mix the contents of the tube and transfer the tube to a water bath set to the S1 Nuclease Digestion temperature. Allow to incubate for 1-2 h, depending on the degree of digestion required. 
23. Cool the reaction system to 0°C. Add 80 μl of S1 Ribonuclease to the reaction system. Add 80 μl of S1 Nuclease Termination Buffer and mix the solution by shaking.
24. Extract the reaction system once with phenol: chloroform. Centrifuge the reaction system in a microcentrifuge tube at maximum speed for 2 min at room temperature and transfer the upper part to another centrifuge tube. Add twice the volume of ethanol, mix well, and allow to stand at -20°C for 1 h. The solution is extracted once with phenol: chloroform.
25. Recover the nucleic acids by centrifugation at 4°C for 15 min at maximum speed. Carefully remove all supernatant and leave the open centrifuge tube at room temperature until the visible ethanol residue evaporates.
Analyze S1 nuclease products by gel electrophoresis
26. Dissolve the nucleic acid precipitate with 4 μl TE buffer (pH 7.6). Add 6 μl of Sampling Buffer and mix well.
27. Incubate the nucleic acids at 95°C for 5 min and immediately transfer to ice. Slightly centrifuge the sample so that it collects at the bottom of the centrifuge tube.
28. Examine the radiolabeled DNA by gel electrophoresis with polyacrylamide 8 mol/L urea.
29. When the tracer dye has moved the appropriate distance across the gel, turn off the power to disassemble the electrophoresis unit. Gently skid the corner of the large glass plate and remove the large glass plate from the gel. For orientation, cut off a corner of the gel.
30. Transfer the glass plate containing the gel to a tray containing an excess of 10% trichloroacetic acid. Slightly shake the tray for 10 min at room temperature.
31. Pour off the 10% TCA solution and replace with an excess of 1% TCA. Shake for 5 min at room temperature.
32. Pour off the 1% TCA solution and lightly rinse the immobilized adhesive with deionized water. Take the glass plate out of the tray with the adhesive and place it on a flat surface. Remove excess water with a paper towel.
33. Cut a piece of Whatman 3 MM filter paper (or equivalent) that is 1 cm longer than the glue on each side. Place the paper on top of the adhesive and turn the glass plate upside down to transfer the adhesive to the filter paper.
34. Remove the glass plate and place the gel in a gel dryer at 60°C for 1 to 1.5 hours.
35. Obtain a radiographic autoradiogram of the dried gel. Scan the image with optical densitometry or phosphorimaging, or cut off the gel containing the fragments and measure them with a liquid flash spectrophotometer.