Enzymatic Labeling of Nucleic Acids

Stanley Tabor1, Ann Boyle2

1 Harvard Medical School, Boston, Massachusetts, 2 Miles Research, West Haven, Connecticut
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.10
DOI:  10.1002/0471142735.im1010s02
Online Posting Date:  May, 2001
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Abstract

Extensive knowledge of the enzymology involved in biosynthesis and degradation of nucleic acids has permitted the development of simple methods for labeling RNA and DNA with radioisotopes or biotin. These labeled probes are used primarily for hybridization to nucleic acid fragments of interest in a variety of applications. A complete description of the methods available for such labeling is beyond the scope of this manual, but contained within this unit are protocols for oligonucleotideā€primed synthesis of radiolabeled and biotinylated DNA probes, in vitro synthesis of radiolabeled RNA, and end labeling of synthetic oligonucleotide probes.

     
 
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Table of Contents

  • Basic Protocol 1: 32P Labeling of DNA by Random Oligonucleotide–Primed Synthesis
  • Alternate Protocol 1: Labeling DNA with Biotin‐11‐dUTP Using Nick Translation
  • Support Protocol 1: Colorimetric Detection of Biotinylated Probes
  • Basic Protocol 2: In Vitro Synthesis of Radiolabeled RNA
  • Basic Protocol 3: End Labeling DNA by the Kinase Forward Reaction
  • Support Protocol 2: Removal of 5′ Phosphate Groups with Calf Intestine Phosphatase
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: 32P Labeling of DNA by Random Oligonucleotide–Primed Synthesis

  Materials
  • DNA sample
  • Restriction endonuclease (unit 10.8)
  • TE buffer ( appendix 2A)
  • 0.5 mM 3dNTP mix (minus dATP; unit 10.9)
  • Klenow fragment and 10× buffer (unit 10.9)
  • 10 µCi/µl [α‐32P]dATP (3000 Ci/mmol)
  • Random hexanucleotides (Pharmacia)
  • 0.5 M EDTA
  • 10 mg/ml tRNA
  • Additional reagents and equipment for restriction cleavage (unit 10.8), agarose gel electrophoresis (unit 10.5), ethanol precipitation and phenol extraction (unit 10.1), column chromatography of labeled nucleotides (unit 10.9), and acid precipitation (optional; unit 10.9)

Alternate Protocol 1: Labeling DNA with Biotin‐11‐dUTP Using Nick Translation

  Materials
  • E. coli DNA polymerase I and 10×buffer (unit 10.9)
  • 0.5 mM 3dNTP mix (minus dTTP; unit 10.9)
  • recipe0.5 mM biotin‐11‐dUTP stock
  • 100 mM 2‐mercaptoethanol
  • Sample DNA
  • 1 mg/ml DNase I stock (unit 10.9) prepared in 0.15 M NaCl/50% glycerol
  • 0.5 M EDTA
  • 10% (w/v) SDS
  • 100% ethanol
  • recipeSDS column buffer
  • Additional reagents and equipment for agarose gel electrophoresis (unit 10.4) and for preparation of spin column for separating labeled DNA from unincorporated precursors (unit 10.9)

Support Protocol 1: Colorimetric Detection of Biotinylated Probes

  Additional Materials
  • Biotinylated standard DNA ( protocol 2alternate protocol or GIBCO/BRL) and test DNA
  • recipeDNA dilution buffer
  • recipeAlkaline phosphatase pH 7.5 (AP 7.5) buffer
  • Blocking buffer: 3% (w/v) BSA Fraction V in recipeAP 7.5 buffer
  • 1 mg/ml streptavidin–alkaline phosphatase conjugate (GIBCO/BRL)
  • recipeAlkaline phosphatase pH 9.5 (AP 9.5) buffer
  • 75 mg/ml nitroblue tetrazolium (NBT)
  • 50 mg/ml 5‐bromo‐4‐chloro‐3‐indoyl phosphate (BCIP)
  • TE buffer ( appendix 2A)
  • Nitrocellulose or nylon filter
  • Sealable bags

Basic Protocol 2: In Vitro Synthesis of Radiolabeled RNA

  Materials
  • DNA template containing appropriate phage promoter
  • Restriction endonuclease (unit 10.8)
  • 5 mM 3NTP mix (minus UTP; unit 10.9)
  • [α‐32P]UTP (800 µCi/mmol)
  • 250 mM UTP
  • 10 mM spermidine (for SP6 reaction only)
  • T7, T3, or SP6 RNA polymerase and corresponding 10× buffer (unit 10.9)
  • 0.5 M EDTA (optional)

Basic Protocol 3: End Labeling DNA by the Kinase Forward Reaction

  Materials
  • 10× T4 polynucleotide kinase buffer (unit 10.9)
  • Dephosphorylated DNA ( protocol 3support protocol)
  • 10 µCi/µl [γ‐32P]ATP (specific activity >3000 Ci/mmol)
  • T4 polynucleotide kinase
  • 0.5 M EDTA (optional)
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Figures

Videos

Literature Cited

Literature Cited
   Carlson, D.D., Superko, C., Mackey, J., Gaskill, M.E., and Hansen, P. 1990. Chemiluminescent detection of nucleic acid hybridization. Focus 12:9‐12.
   Cherif, D., Bernard, O., and Berger, R. 1989. Detection of single copy genes by nonisotopic in situ hybridization on human chromosomes. Hum. Genet. 81:358‐362.
   Davanloo, P., Rosenberg, A.H., Dunn, J.J., and Studier, F.W. 1984. Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 81:2035‐2039.
   Gilman, M. 1989. Ribonuclease protection assay. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl, eds.) pp. 4.7.1‐4.7.8. Greene Publishing and Wiley‐Interscience, New York.
   Harrison, B. and Zimmerman, S.B. 1986. T4 polynucleotide kinase:Macromolecular crowding increases the efficiency of reaction at DNA termini. Anal. Biochem. 158:307‐315.
   Langer, P.R., Waldrop, A.A., and Ward, D.C. 1981. Enzymatic synthesis of biotinylated polynucleotides: Novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. U.S.A. 78:6633‐6637.
   Langer‐Safer, P.R., Levine, M., and Ward, D.C. 1982. Immunological method for mapping genes on Drosophilia polytene chromosomes. Proc. Natl.Acad. Sci. U.S.A. 79:4381‐4385.
   Leary, J.J., Brigati, D.J., and Ward, D.C. 1983. Rapid and sensitive colorimetric method for visualizing biotin‐labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio‐blots. Proc. Natl. Acad. Sci. U.S.A. 80:4045‐4049.
   Lichter, P., Cremer, T., Borden, J., Manuelidis, L., and Ward, D.C. 1988. Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 8:224‐234.
   Midgley, C.A. and Murray, N.E. 1985. T4 polynucleotide kinase; cloning of the gene (pseT) and amplification of its product. EMBO J. 4:2695‐2703.
   Moyzis, R.K., Buckingham, J.M., Cram, L.S., Dani, M., Deaven, L.L., Jones, M.D., Meyne, J., Ratliff, R.L., and Wu, J.R. 1988. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc. Natl. Acad. Sci. U.S.A. 85:6622‐6626.
   Richardson, C.C. 1981. Bacteriophage T4 polynucleotide kinase. In The Enzymes,Vol. 14A (P.D. Boyer, ed.) pp. 299‐314, Academic Press, New York.
   Tabor, S. and Richardson, C.C. 1985. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. U.S.A. 82:1074‐1078.
   Wilchek, M. and Bayer, E.A. 1988. The avidin‐biotin complex in bioanalytical applications. Anal. Biochem. 171:1‐32.
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