Labeling DNA and Preparing Probes

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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‐³² P]adenosine‐5′‐triphosphate ([γ‐³² P]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‐³² P]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‐³² P]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‐³² P]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

Figure 8.4.1 Representative fluorophores that can be used to label nucleic acids. (A ) Cy5: maximum absorbance, 649 nm, maximum emission, 670 nm; (B ) fluorescein: maximum absorbance, 494 nm, maximum emission, 518 nm.

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Figure 8.4.2 Biotin and digoxigenin.

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Figure 8.4.3 T4 polynucleotide kinase catalyzes the transfer of the γ phosphate from ATP to the 5′ hydroxyl of the DNA substrate.

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Figure 8.4.4 Structure of [γ‐³² P]ATP.

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Figure 8.4.5 The nature of the ends of DNA affect the efficiency of labeling by T4 polynucleotide kinase.

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Figure 8.4.6 The nicking activity of deoxyribonuclease I (DNase I).

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Figure 8.4.7 The 5′→3′ exonuclease activity of E. coli DNA polymerase I.

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Figure 8.4.8 The 5′→3′ polymerase activity of E. coli DNA polymerase I.

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Figure 8.4.9 DNA labeling by nick translation.

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Figure 8.4.10 Structure of [α‐³² P]deoxynucleotide used for radiolabeling DNA in nick translation or random primed synthesis.

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Figure 8.4.11 Biotin‐11‐dUTP: an example of a modified nucleotide than can be incorporated by E. coli DNA polymerase I.

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Figure 8.4.12 DNA labeling by random primed synthesis.

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Videos

Literature Cited

Literature Cited
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Labeling DNA and Preparing Probes

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited