RNA SI Nuclease Mapping
Summary
SI nuclease is an endonuclease that was isolated from Aspergillus oryzae. It can degrade single-stranded nucleic acids but not double-stranded nucleic acids. In addition, it can degrade locally mismatched double-stranded molecules with high sensitivity, and even if there is only one base pair mismatch, it can be detected by cleavage by S1 nuclease. S1 nuclease is used to identify and cleave the mismatched or unduplicated regions, and the cleavage products are then analyzed by denaturing endocannabinoid gel electrophoresis.
Operation method
RNA SI Nuclease Mapping
Materials and Instruments
[γ-32P "ATP Suitable oligonucleotides DNA templatel 10XdNTP mixture KLenow fragments Suitable restriction endonucleases X-ray film Elution buffer tRNA Paraffin oil S1 Nuclease
10X kinase buffer PNK 10X replication buffer 6% polyacrylamide 8mol L urea solution (dissolved in 0.5XTBE) and formamide dye Sodium acetate 4X hybridization buffer Deionized formamide S1 buffer S1 termination buffer Isopropyl alcohol
Vertical electrophoresis unit Water bath Hybridization tubes Hybridization oven
Move
I. Materials and equipment
1) Vertical electrophoresis device
2) Water bath
3) Hybridization tube
4) Hybridization oven
5) Reagents for preparation of single-stranded DNA probes from single-stranded DNA templates. ①10X kinase buffer: 400 mmol/L Tris-HCl (pH7.8), 100 mmol/L MgCl2, 1OOmmol/L sparse ethanol, 250 ug/ml bovine blood albumin stored at -20 ℃ ②[ γ-32P ] ATP (specific activity 3000Ci/mmol) ③ Suitable Oligonucleotides: dissolved in distilled water at a concentration of l0 pmol/L, stored at -20 ℃ ④ PNK (polynuclemide). l0 pmol/L, stored at -20°C ④ PNK (polynuclemidekinase): polynucleotide kinase at 10 U/ul, stored at 20°C. ⑤ 10X replication buffer: 100 mmol/L Tris-HCl (ph7.5). ⑤10X replication buffer: 100 mmol/LTris-HCl (ph7.5), l00 mmol/L MgCl2, 500 mnnol/L NaCl, 10O mmol/L DTT ⑥DNA template: l mg/ml ⑦10XdNTP mixture: 5 mmol/LdATP, dCTP, dGTP, dTTP. ⑧KLenow fragments: large fragments of E. coli DNA polymerase I at a concentration of lOU/ul stored at -20°C,⑨ Suitable restriction endonuclease. ⑩6% polyacrylamide/8mol/L urea solution (dissolved in 0.5XTBE) and formamide dye. ⑪X-ray film: suitable sensitivity is required. ⑫ elution buffer: 50 mmol/L TrisHCl (pH7.5), 0.5 mmol/L LEDTA. ⑬ 3 mol/L sodium acetate (pH7.4) ⑭tRNA: 10 mg/ml storage solution, store at -20°C.
6) Hybridization and S1 analysis. ①4X hybridization buffer: 1.6 mmol/L NaCl, 40 mmol/L PIPES (pH6.4). ② Deionized formamide. ③ Paraffin oil. ④S1 buffer: 300 mmol/LNaCl30 mmol/L sodium acetate (pH4.5); 4.5 mmol/L zinc acetate, ⑤S1 nuclease: concentration of 10U/ul, stored at -20℃. ⑥S1 termination buffer: 2.5 mol/L ammonium acetate,50 mmol/L LEDTA ⑦Isopropanol
II. Methods of operation
(I) Preparation of single-stranded DNA probe
1. Preparation of single-stranded DNA probe from single-stranded DNA template
1) Labeling of oligonucleotide probe: Add the following components to 0.5 ml centrifuge tube plants.
Oligodeoxynucleotide 1ul
[ γ-32P ]ATP 10ul
l0x kinase buffer 2ul
dH2O 6ul
Polynucleotide kinase 1ul
Mix the above components, hold at 37℃ for 45 min to label the oligonucleotide probe, and finally hold at 95℃ for 2 min to inactivate the polynucleotide kinase.
2) Add 2ul of DNA template, 4ul of 10X buffer, 14ul of H2O, and hold at 65℃ for 10 min, 55℃ for lOmin, and 37℃ for lOmin.
3) Add 4ul NTP mixture, lul Klenow fragments, room temperature for 10 min, and then at 65 ℃ for 15 min, inactivate Klenow fragments.
4) Add appropriate amount of NaCl or correct the concentration of the mixture by dilution, add 20U of restriction endonuclease, 37 ℃ holding time 60 min
5) Add 2ul of tRNA stock solution A1 times the volume of 3mol/L sodium acetate, 3 times the volume of ethanol 5 in dry ice for 10 min, centrifuge at 12000 g for 15 min to precipitate the sample.
6) Add 20ul of formamide dye to resuspend the precipitate, and take the sample on a 6% polyacrylamide/urea gel at 95℃ for h, and end the electrophoresis when the bromophenol blue reaches the bottom of the gel.
7) Expose the gel to X-ray film for 5 min to determine the position of the single-stranded DNA fragments. Cut off the corresponding bands and place in 500ul elution buffer overnight.
8) Add 50ul of sodium acetate T20ugtRNA, lml ethanol. Place at 20℃ for 2 h. Centrifuge at 12000 g for 20 min.
9) Resuspend the precipitate in dry formamide (lO5 cmp/ul) after radioactivity counting and store at 20°C. This probe is used for hybridization and S1 analysis.
2. Preparation of single-stranded DNA probes from double-stranded DNA templates
Except for the following steps, the remaining steps are 1 "Preparation of single-stranded DNA probes from single-stranded DNA templates".
1) First cut the double-stranded plasmid (20ug) with the selected restriction endonuclease, and resuspend the precipitate in water at a concentration of lmg/ml.
2) Step 2) of the replication step 1 "Preparation of a single-stranded DNA probe from a single-stranded DNA template" needs to be carried out quickly. After heat inactivation of polynucleotide kinase, add 2~5ug of digested plasmid, 4ul of 10X Recovery Buffer, and 14ul of water. Hold at 95°C for 5 min, place the plants on ice immediately, and then proceed to step 3) in the "Preparation of a single-stranded DNA probe from a single-stranded DNA template" procedure.
3) Omit restriction enzyme digestion, i.e., step 4) in 1 "Preparation of single-stranded DNA probe from single-stranded DNA template".
(ii) Hybridization and S1 nuclease analysis
1) Add the following components to a 0.5 ml Eppendorf tube:
Probe (10000cpm total) 10ul
4X Hybridization Buffer 5ul
RNA (total poly(A)+RNA or synthesized in vitro) 5ul
Paraffin oil (to prevent evaporation) 20ul
Mix well and hold at 37℃ for 4 to 16 h.
2) Add 200 ul of S1 buffer (containing 100U of S1 nuclease), mix well, and place at 37°C for 5~30 min. Add 50 ul of S1 termination buffer to terminate the reaction, and add 40 ug of tRNA, 300ul of isopropanol. Plants on dry ice for 15 min, centrifuge at 12000 g for 15 min.
3) Wash the precipitate with 80% ethanol, air dry and resuspend in 3ul of formamide dye, heat at 95°C for 5 min, sample on 6% polyacrylamide/urea gel, electrophoresis until bromophenol blue reaches the bottom of the gel, and then expose the gel to the light for 8 to 48 h.
Caveat
1) The length of the oligonucleotide is 20~25 nucleotides, and it is complementary to the analyzed DNA.2) Single-stranded or double-stranded DNA templates should be prepared according to standard methods. The advantage of using single-stranded DNA templates to prepare probes over double-stranded DNA templates is that the yield of probes is much higher, with specific activities of up to 1X106-1.5×106cpm when prepared with single-stranded templates.3) To increase the yield of fragments, restriction endonucleases should be used to cleave at the 3' end of the oligonucleotide hybridization at 200-600 nucleotides, as longer fragments are difficult to recover from acrylamide gels. If the restriction endonuclease used cuts more than one site on the DNA template, there is no obstacle as long as they are located outside the probe sequence.4) Hybridization times vary depending on the type of RMA being analyzed. For cytoplasmic total RNA or poly(A)+RNA samples, hybridization should be performed at 37°C for at least 12 h. For in vitro transcribed RNA, hybridization should be performed for 2 to 4 h.5) When analyzing in vitro transcribed RNA, the S1 nuclease action time should be 15 min, 30 min, or 60 min, so as to determine the conditions required for complete digestion of all single-stranded nucleic acids, including the free probes, and for analyzing cytoplasmic total RNA or poly(A)+RNA, the optimal digestion time of S1 nuclease is 30 min, which is often longer than that required for in vitro transcribed RNA. The optimal digestion time for S1 nuclease when analyzing cytoplasmic total RNA or Poly(AVRNA) is 30 min, which is often longer than the time required to digest isolated RNA (15 min).
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