Labeling DNA and Preparing Probes

Karl A. Haushalter1

1 Harvey Mudd College, Claremont, California
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 8.4
DOI:  10.1002/9780470089941.et0804s00
Online Posting Date:  October, 2008
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Abstract

Labeling nucleic acids with radioisotopes, fluorophores, biotin, or digoxigenin enables their detection and analysis. When designing a labeling strategy, consider the intended application, the source of nucleic acid, and the type of label to incorporate. DNA oligonucleotides can be 5′ end‐labeled with radioisotopes in a reaction catalyzed by T4 polynucleotide kinase, or nonisotopic labels can be incorporated into oligonucleotides during DNA synthesis. Larger DNA substrates can be labeled by 5′ end labeling (radioisotopes) or labeled uniformly along the length of the DNA by nick translation or random primed synthesis (using radioisotope or nonisotopic labels). The labeled DNA can be used for a variety of applications, including probing Southern blots, probing northern blots, in situ hybridization, quantifying real‐time PCR results, and gel shift assays.

Keywords: nucleic acid probe; end‐labeling; random primed synthesis; nick translation; radioisotope; fluorophore; biotin; digoxigenin; polynucleotide kinase; terminal transferase

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Safety Considerations
  • Protocols
  • Basic Protocol 1: 5′ End‐Labeling of DNA with T4 Polynucleotide Kinase
  • Basic Protocol 2: Labeling DNA by Nick Translation
  • Basic Protocol 3: Labeling DNA by Random Primed Synthesis
  • Support Protocol 1: Purification of Labeled Probes Using Gel‐Filtration Spin Columns
  • Reagents and Solutions
  • Understanding Results
  • Troubleshooting
  • Variations
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: 5′ End‐Labeling of DNA with T4 Polynucleotide Kinase

  Materials
  • 10 mCi/ml [γ‐32P]adenosine‐5′‐triphosphate ([γ‐32P]ATP; sp. act., 6000 Ci/mmol; lower‐specific‐activity ATP can be used, depending on the sensitivity needs)
  • 10× PNK buffer (see recipe or use buffer supplied with the enzyme)
  • DNA substrate with free 5′‐hydroxyl end(s)
  • 10 U/µl T4 polynucleotide kinase
  • 0.5 M EDTA, pH 8.0 (unit 3.3)
  • 65° and 75°C water baths or heat blocks
  • Shielded radiation containers (unit 2.3)
  • Additional reagents and equipment for removing unincorporated nucleotides ( protocol 4) and radiation safety and measurement, including quantifying specific activity of radiolabeled DNA (unit 2.3)

Basic Protocol 2: Labeling DNA by Nick Translation

  Materials
  • dNTP with label (e.g., 10 µM [α‐32P]dNTP; see Table 8.4.3)
  • 0.5 mM 3dNTP mix (omitting the labeled dNTP; see Table 8.4.4)
  • 10× E. coli DNA polymerase I buffer (see recipe)
  • Deoxyribonuclease I (DNase I), diluted ∼10,000‐fold from 1 mg/ml stock in enzyme diluent (see recipe)
  • 5 to 15 U/µl E. coli DNA polymerase I
  • DNA to be labeled
  • 0.5 M EDTA, pH 8.0 (unit 3.3)
  • TE buffer, pH 8.0 (unit 3.3)
  • 15°C water bath
  • Shielded radiation containers (unit 2.3)
  • Additional reagents and equipment for removing unincorporated nucleotides ( protocol 4), radiation safety and measurement, including quantifying specific activity of radiolabeled DNA (unit 2.3), nucleic acid blotting (unit 8.2), agarose gel electrophoresis (unit 7.2), and digital image analysis (unit 7.5)
    Table 8.4.4   Materials3dNTP Nucleotide Mixes for Nick Translation and Random Primed Synthesis

    Modified nucleotide with label Contents of 3dNTP mix (0.5 mM each)
    dATP (e.g., biotin‐11‐dATP, [α‐32P]dATP) dCTP, dGTP, dTTP
    dCTP dATP, dGTP, dTTP
    dGTP dATP, dCTP, dTTP
    dTTP (dUTP) dATP, dCTP, dGTP

Basic Protocol 3: Labeling DNA by Random Primed Synthesis

  Materials
  • dNTP with label (e.g., 10 µM [α‐32P]dNTP; see Table 8.4.3)
  • 0.5 mM 3dNTP mix (omitting the labeled dNTP; see Table 8.4.4)
  • 10× E. coli DNA polymerase I buffer
  • 3 to 8 U/µl E. coli DNA polymerase I large fragment (Klenow fragment)
  • DNA to be labeled
  • Random hexanucleotide primers (Sigma cat no. H‐0268)
  • 0.5 M EDTA, pH 8.0 (unit 3.3)
  • TE buffer, pH 8.0 (unit 3.3)
  • Boiling water bath
  • Additional reagents and equipment for removing unincorporated nucleotides ( protocol 4), radiation safety and measurement, including quantifying specific activity of radiolabeled DNA (unit 2.3), nucleic acid blotting (unit 8.2), agarose gel electrophoresis (unit 7.2), and digital image analysis (unit 7.5)
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Figures

Videos

Literature Cited

Literature Cited
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