Enzymatic Labeling of DNA

Stanley Tabor1, Kevin Struhl1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Appendix 3E
DOI:  10.1002/0471142905.hga03es00
Online Posting Date:  May, 2001
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Abstract

This appendix describes the preparation of uniformly labeled radioactive probes by nick translation and random oligonucleotide‐primed synthesis, as well as the end‐labeling of oligonucleotides by T4 polynucleotide kinase. Accompanying these protocols are methods for removing unincorporated dNTP precursors with spin columns and for measuring the specific activity of a probe by acid precipitation.

     
 
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Table of Contents

  • Basic Protocol 1: Uniform Labeling of DNA by Nick Translation
  • Alternate Protocol 1: Labeling DNA by Random Oligonucleotide–Primed Synthesis
  • Basic Protocol 2: 5′‐End‐Labeling Oligonucleotides Using T4 Polynucleotide Kinase
  • Support Protocol 1: Spin‐Column Procedure for Separating Radioactively Labeled DNA from Unincorporated dNTP Precursors
  • Support Protocol 2: Measuring Radioactivity in DNA and RNA by Trichloroacetic Acid (TCA) Precipitation
  • Reagents and Solutions
     
 
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Materials

Basic Protocol 1: Uniform Labeling of DNA by Nick Translation

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2; for suppliers, see suppliers appendix.
  • 0.5 mM 3dNTP mix (minus dATP; appendix 2A)
  • 5 to 15 U E. coli DNA polymerase I and recipe10× buffer (see recipe)
  • 100 µCi [α‐32P]dATP (3000 Ci/mmol)
  • 1 mg/ml DNase I stock solution, diluted 1/10,000 in recipestandard enzyme diluent (see recipe) just before use
  • DNA to be labeled
  • 0.5 M EDTA
  • 10 mg/ml tRNA
  • TE buffer, pH 8.0
  • Additional reagents and equipment for phenol extraction ( appendix 3C)
CAUTION:32P is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Alternate Protocol 1: Labeling DNA by Random Oligonucleotide–Primed Synthesis

  Additional MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2; for suppliers, see suppliers appendix.
  • recipe10× E. coli polymerase I buffer (see recipe)
  • 3 to 8 U/µl Klenow fragment
  • Random hexanucleotides
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7) and ethanol precipitation ( appendix 3C)
CAUTION:32P is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Basic Protocol 2: 5′‐End‐Labeling Oligonucleotides Using T4 Polynucleotide Kinase

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2; for suppliers, see suppliers appendix.
  • 50 mM Tris⋅Cl, pH 7.5
  • 10 mM MgCl 2
  • 5 mM DTT
  • 1 to 50 pmol dephosphorylated DNA, 5′ ends
  • 50 pmol (150 µCi) [γ‐32P]ATP (specific activity >3000 Ci/mmol)
  • 50 µg/ml BSA
  • 20 U T4 polynucleotide kinase
  • 0.5 M EDTA
  • 75°C water bath or heating block
  • Additional reagents and equipment for phenol/chloroform extraction ( appendix 3C)
CAUTION:32P is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Support Protocol 1: Spin‐Column Procedure for Separating Radioactively Labeled DNA from Unincorporated dNTP Precursors

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2; for suppliers, see suppliers appendix.
  • Resin: e.g., Sephadex G‐50 (Pharmacia) or Bio‐Gel P‐60 (Bio‐Rad)
  • TE buffer, pH 8.0
  • Radioactive sample: reaction mixture containing radioactive precursors ( protocol 1 and protocol 2)
  • 500‐ml screw‐cap bottle
  • IEC Clinical centrifuge
  • 5‐ml disposable syringe
  • Silanized glass wool
  • 50‐ml polypropylene tube
CAUTION:32P is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Support Protocol 2: Measuring Radioactivity in DNA and RNA by Trichloroacetic Acid (TCA) Precipitation

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2; for suppliers, see suppliers appendix.
  • Radioactive sample: reaction mixture containing radioactive precursors ( protocol 1 and protocol 2)
  • 500 µg/ml sonicated salmon sperm DNA (unit 4.3) in TE buffer, pH 8.0
  • 10% (w/v) trichloroacetic acid (TCA) or protocol 1acid precipitation solution (see recipe), ice‐cold
  • 100% ethanol
  • Glass microfiber filters (2.4‐cm diameter, Whatman GF/A)
  • Filtration device
  • Scintillation fluid (toluene‐based)
CAUTION:32P and toluene‐based scintillation fluid are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.
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