Primer Extension

Steven J. Triezenberg1

1 Michigan State University, East Lansing, Michigan
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.8
DOI:  10.1002/0471142727.mb0408s20
Online Posting Date:  May, 2001
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Abstract

This protocol can be used to map the 5' terminus of an RNA and to quantitate the amount of a given RNA by extending a primer using reverse transcriptase. The primer is an oligonucleotide (or restriction fragment) that is complementary to a portion of the RNA of interest. The primer is end‐labeled, hybridized to the RNA, and extended by reverse transcriptase using unlabeled deoxynucleotides to form a single‐stranded DNA complementary to the template RNA. The resultant DNA is analyzed on a sequencing gel. The length of the extended primer maps the position of the 5' end of the RNA, and the yield of primer extension product reflects the abundance of the RNA.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1:

  Materials
  • Diethylpyrocarbonate (DEPC; unit 4.1)
  • 10× T4 polynucleotide kinase buffer (unit 3.4)
  • 0.1 M and 1 M dithiothreitol (DTT; appendix 22)
  • 1 mM spermidine
  • 50 to 100 ng/µl oligonucleotide primer (5 to 10 µM; unit 2.11)
  • 10 µCi/µl [γ‐32P]ATP (3000 Ci/mmol)
  • 20 to 30 U/µl T4 polynucleotide kinase (unit 3.10)
  • 0.5 M EDTA, pH 8.0 ( appendix 22)
  • TE buffer, pH 8.0 ( appendix 22)
  • Cation‐exchange resin (e.g., Bio‐Rad AG 50W‐X8), equilibrated in 0.1 M Tris˙Cl (pH 7.5)/ 0.5 M NaCl
  • Anion‐exchange resin (e.g., Whatman DE‐52), equilibrated in TEN 100
  • TEN 100 buffer: 100 mM NaCl in TE buffer, pH 7.5 ( appendix 22)
  • TEN 300 buffer: 300 mM NaCl in TE buffer, pH 7.5 ( appendix 22)
  • TEN 600 buffer: 600 mM NaCl in TE buffer, pH 7.5 ( appendix 22)
  • Total cellular RNA (units 4.1 4.3)
  • recipe10× hybridization buffer
  • 0.1 M Tris˙Cl, pH 8.3 ( appendix 22)
  • 0.5 M MgCl 2
  • 1 mg/ml actinomycin D (store at 4°C protected from light; unit 1.4)
  • 10 mM 4dNTP mix (unit 3.4)
  • 25 U/µl AMV reverse transcriptase (unit 3.7)
  • recipeRNase reaction mix
  • 3 M sodium acetate ( appendix 22)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (unit 2.1)
  • 100% and 70% ethanol
  • Stop/loading dye (unit 7.4)
  • 9% acrylamide/7 M urea gel (unit 2.12)
  • Silanized glass wool and 1000‐µl pipet tip ( appendix 3B)
  • 65°C water bath
Additional reagents and equipment for denaturing gel electrophoresis (units 2.12 & 7.6), phenol extraction and ethanol precipitation of DNA (unit 2.1), and autoradiography ( 3.NaN)NOTE: Water 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.
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Figures

Videos

Literature Cited

Literature Cited
   Graves, B.J., Eisenberg, S.P., Coen, D.M., and McKnight, S.L. 1985. Alternate utilization of two regulatory domains within the Moloney murine sarcoma virus long terminal repeat. Mol. Cell. Biol. 5:1959‐1968.
   Jones, K.A., Yamamoto, K.R., and Tjian, R. 1985. Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro. Cell 42:559‐572.
   McKnight, S.L. and Kingsbury, R. 1982. Transcription control signals of a eukaryotic protein‐encoding gene. Science 217:316‐324.
   Mierendorf, R.C. and Pfeffer, D. 1987. Sequencing of RNA transcripts synthesized in vitro from plasmids containing bacteriophage promoters. Methods Enzymol. 152:563‐566.
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed., p. 11.39. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
   Sasse‐Dwight, S. and Gralla, J.D. 1991. Footprinting protein‐DNA complexes in vivo. Methods Enzymol. 208:146‐168.
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