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Nick Translation and Random Hexamer Labeling of DNA

  • Jane Davenport-Jones
Protocol
  • 503 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Labeled nucleotides (radioactive or fluorescent) can be incorporated efficiently into double-stranded DNA by nick translation. Nick translation works by using DNase and DNA polymerase I enzymes. DNase cuts one strand of the DNA, exposing 5′-phosphoryl and 3′-hydroxyl (OH) termini DNA. Polymerase I adds deoxyribonucleoside triphosphate (dNTPs), including labeled dNTPs to the exposed 3′-OH strand and at the same time, the polymerase exonuclease activity digests from the exposed 5′ end. In this way, a new complementary strand, including labeled dNTPs is produced (1). It is also possible to incorporate radioactive nucleotides into DNA using a enzymatic primer extension technique (2). In this method, random hexanucleotides are annealed to the denatured DNA to be used as the probe. These are used as a primer for enzymatic extension in the presence of the four dNTPs, one of which is radiolabeled.

Keywords

Amersham Pharmacia Biotech Stop Solution Complementary Strand Nick Translation Unincorporated Nucleotide 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Rigby, P. W. J., Dieckmann, M., Rhodes, C., and Berg, P. (1977) Labelling deoxyribo-nucleic acid to a high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237–251.PubMedCrossRefGoogle Scholar
  2. 2.
    Feinberg, A. P. and Vogelstein, B. (1983) A technique for radiolabelling DNA restriction endonuclease fragments to a high specific activity. Anal. Biochem. 132, 6–13.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Jane Davenport-Jones
    • 1
  1. 1.Department of BiosciencesUniversity of HertfordshireHafieldUK

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