Labeling and Colorimetric Detection of Nonisotopic Probes

Ann Boyle1, Heather Perry‐O'Keefe2

1 Miles Research Center, West Haven, Connecticut, 2 Millipore Corporation, Burlington, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 3.18
DOI:  10.1002/0471142727.mb0318s20
Online Posting Date:  May, 2001
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Abstract

Although a number of different nonisotopic labels have been described in the literature, biotin and digoxigenin are used most frequently and are commercially available. Either label can be easily incorporated into DNA probes and be detected colorimetrically; a number of fluorochromes, as well as alkaline phosphatase and horseradish peroxidase (which produce colored precipitates) are available directly conjugated to anti‐digoxigenin antibodies and to avidin. Chemiluminescent detection methods and indirect immunofluorescent techniques also provide sensitive alternatives for many molecular biology applications. This unit presents two protocols for incorporating biotinylated nucleotides into DNA probes by nick translation and random‐primed synthesis. A describes colorimetric detection of the probes, which also serves to check the extent of nucleotide incorporation. An describes adaptations of the basic protocols for incorporation of digoxigenin‐modified nucleotides.

     
 
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Table of Contents

  • Specialized Applications
  • Basic Protocol 1: Preparation of Biotinylated Probes by Nick Translation
  • Basic Protocol 2: Preparation of Biotinylated Probes by Random Oligonucleotide–Primed Synthesis
  • Support Protocol 1: Colorimetric Detection of Biotinylated Probes
  • Alternate Protocol 1: Preparation and Detection of Digoxigenin‐Labeled DNA Probes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Preparation of Biotinylated Probes by Nick Translation

  Materials
  • E. coli DNA polymerase I (unit 3.5) and 10× buffer (unit 3.4)
  • 0.5 mM 3dNTP mix (minus dTTP; unit 3.4)
  • recipe0.5 mM biotin‐11‐dUTP stock
  • 100 mM 2‐mercaptoethanol (2‐ME)
  • Test DNA
  • 1 mg/ml DNase I stock (unit 3.12) prepared in 0.15 M NaCl/50% glycerol
  • DNA molecular weight markers (unit 2.5)
  • 0.5 M EDTA, pH 8.0 ( appendix 22)
  • 10% (w/v) SDS
  • 100% ethanol
  • recipeSDS column buffer
  • 1‐ml syringe
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5) and removal of unincorporated nucleotides by Sephadex G‐50 spin columns (unit 3.4)

Basic Protocol 2: Preparation of Biotinylated Probes by Random Oligonucleotide–Primed Synthesis

  Materials
  • Linear template DNA
  • recipeBiotinylated random octamers
  • recipedNTP/biotin mix
  • 5 U/µl Klenow fragment (unit 3.5)
  • TE buffer, pH 7.5 ( appendix 22)
  • 0.5 M EDTA, pH 8.0 ( appendix 22)
  • 4 M LiCl
  • 100% and 70% ethanol, ice‐cold

Support Protocol 1: Colorimetric Detection of Biotinylated Probes

  Additional Materials
  • Biotinylated standard DNA (basic protocols 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 (AP) 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, pH 8.0 ( appendix 22)
  • Small piece of membrane (e.g., 5 × 3 cm2): nitrocellulose or uncharged nylon
  • Sealable bags
NOTE: To avoid nonspecific background, wear powder‐free gloves when handling the membranes.
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Figures

Videos

Literature Cited

Literature Cited
   Beck, S. and Koster, H. 1990. Applications of dioxetane chemiluminescent probes to molecular biology. Anal. Chem. 62:2258‐2270.
   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.
   Feinberg, A.P. and Vogelstein, B. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6‐13.
   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 Drosophila polytene chromosomes. Proc. Natl. Acad. Sci. U.S.A. 79:4381‐4385.
   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.
   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.
   Wilchek, M. and Bayer, E.A. 1988. The avidin‐biotin complex in bioanalytical applications. Anal. Biochem. 171:1‐32.
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