S1 Analysis of Messenger RNA Using Single‐Stranded DNA Probes

John M. Greene1, Kevin Struhl2

1 Massachusetts General Hospital, Boston, Massachusetts, 2 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.6
DOI:  10.1002/0471142727.mb0406s45
Online Posting Date:  May, 2001
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Abstract

This method takes advantage of the ability of oligonucleotides to be efficiently labeled to a high specific activity at the 5' end through the use of kinase. The oligonucleotide is hybridized to a specific single‐stranded template containing the complementary sequence to the oligonucleotide, and this hybrid is extended through the use of the Klenow fragment of E. coli DNA polymerase I. The mixture is cut with a restriction enzyme to give the probe a defined 3' end, and the probe is isolated on an alkaline agarose gel. Before using this protocol it is first helpful to have an M13 clone. If this is unavailable, a double‐stranded plasmid clone of the region to be studied may be used, as described in an alternate protocol. Another alternate protocol describes the use of long oligonucleotides as probes for S1 analysis (useful for rapid and easy quantitation of the level of mRNA produced from a characterized promoter). For the mapping of the 5' end of an RNA species, hybridization of the probe to RNA is then carried out. S1 nuclease is added to digest all of the unhybridized portion of the probe. Electrophoresis of the hybrid on a denaturing polyacrylamide gel allows a determination of the length of the remaining DNA fragment. This length equals the distance between the 5' end of the probe to the 5' end of the RNA, defining the transcriptional start site to the nucleotide. By performing the hybridization reaction in vast probe excess, quantitation of the relative amounts of RNA can be estimated between samples.

     
 
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Table of Contents

  • Section II: Analysis of RNA Structure and Synthesis
  • Basic Protocol 1: S1 Analysis of mRNA Using M13 Template
  • Alternate Protocol 1: Synthesis of Single‐Stranded Probe from Double‐Stranded Plasmid Template
  • Alternate Protocol 2: Quantitative S1 Analysis of mRNA Using Oligonucleotide Probes
  • Support Protocol 1: Controls for Quantitative S1 Analysis of mRNA
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: S1 Analysis of mRNA Using M13 Template

  Materials
  • Diethylpyrocarbonate (DEPC)
  • Low gelling/melting temperature agarose (unit 2.6)
  • recipeAlkaline pour buffer
  • recipeAlkaline running buffer
  • [γ‐32P]ATP (10 mCi/ml, 6000 Ci/mmol)
  • 100 µg/ml oligonucleotide primer (unit 2.11)
  • 10× polynucleotide kinase buffer
  • T4 polynucleotide kinase (unit 3.10)
  • 18 µg M13mp template DNA containing sequence of interest
  • 10× TM buffer ( appendix 22)
  • 4 mM dNTP mix (unit 3.4)
  • Klenow fragment of E. coli DNA polymerase I (unit 3.5)
  • 10× restriction buffer (unit 3.1)
  • 40 U restriction endonuclease (Table 97.80.4711)
  • 5 M ammonium acetate
  • 100% ethanol
  • recipeAlkaline loading buffer
  • TE buffer ( appendix 22)
  • recipe10 mg/ml tRNA
  • Buffered phenol (unit 2.1)
  • 3 M and 0.3 M sodium acetate, pH 5.2
  • 70% ethanol/30% DEPC‐treated H 2O
  • recipeS1 hybridization solution
  • recipe2× S1 nuclease buffer (unit 3.12)
  • 2 mg/ml single‐stranded calf thymus DNA
  • S1 nuclease (unit 3.12)
  • recipeS1 stop buffer
  • Formamide loading buffer (unit 2.12)
  • Additional reagents and equipment for ethanol precipitation (unit 2.1) and agarose and denaturing polyacrylamide gel electrophoresis (units 2.5 and 7.6, respectively)
Water and sodium acetate should be treated with DEPC to inhibit RNase activity. See unit 4.1, reagents and solutions, for instructions.CAUTION: DEPC is a suspected carcinogen and should be handled carefully.

Basic Protocol 2:

  Additional Materials
  • recipe10× NaOH/EDTA solution
  • 1.5 M ammonium acetate, pH 4.5

Alternate Protocol 1: Synthesis of Single‐Stranded Probe from Double‐Stranded Plasmid Template

  Additional Materials
  • 2 pmol each oligonucleotide probe
  • 4 M ammonium acetate
  • BioGel P‐2 (or equivalent resin; optional)
  • recipe3× aqueous hybridization solution (optional)
  • 0.5 M EDTA
  • 0.1 M NaOH
  • Additional reagents and equipment for acid precipitation (unit 3.4) and denaturing polyacrylamide gel electrophoresis (unit 7.6)
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Figures

Videos

Literature Cited

Literature Cited
   Berk, A.J. and Sharp, P.A. 1977. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease‐digested hybrids. Cell 12:721‐732.
   Casey, J. and Davidson, N. 1977. Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucl. Acids Res. 4:1539‐1552.
   Favaloro, J., Treisman, R. and Kamen, R. 1980. Transcription maps of polyoma virus‐specific RNA: Analysis by two‐dimensional nuclease S1 gel mapping. Meth. Enzymol. 65:718‐749.
   Weaver, R.F. and Weissman, C. 1979. Mapping of RNA by a modification of the Berk‐Sharp procedure: The 5′ termini of 15S β‐globin mRNA precursor and mature 10S β‐globin mRNA have identical map coordinates. Nucl. Acids Res. 7:1175‐1193.
Key Reference
   Sharp, P.A., Berk, A.J. and Berget, S.M. 1980. Transcription maps of adenovirus. Meth. Enzymol. 65:750‐768.
  Contains a fairly detailed discussion of S1 mapping procedures using double‐stranded probes as well as S1 endonuclease and its optimal digestion conditions.
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