Advertisement

Internal Labeling of DNA Probes

  • Ralph Rapley
  • Bimal D. M. Theophilus
Part of the Methods in Molecular Biology book series (MIMB, volume 187)

Abstract

One of the most common precursors to undertaking a protocol for mutation detection is the production of a suitably labeled DNA probe (1). Labeled nucleotides (radioactive or fluorescent) can be incorporated efficiently into doublestranded DNA by a number of methods. One of the most common is by a process termed 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. Polymerase I adds 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 (2). It is also possible to incorporate radioactive nucleotides into a DNA using a enzymatic primer extension technique, usually termed random primer labeling (3). In this method, random hexanucleotides are annealed to denatured DNA to be used as the probe. These are used as a primer for enzymatic extension in the presence of the four deoxyribonucleotides, one of which is radiolabeled. Alternative probes may be prepared where the label occurs on one of the termini of the DNA, either the 3′ or the 5′ end. The protocol for this type of labeling is found in Chapter 3.

Keywords

Amersham Pharmacia Biotech Nick Translation Random Primer Label Alternative Probe Random Hexanucleotides 
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.
    Aquino de Muro M. (1998) Gene Probes, in The Molecular Biomethods Handbook (Rapley R. and Walker J. M., eds.) Humana Totowa, NJ.Google Scholar
  2. 2.
    Rigby P. W. J., Dieckmann M., Rhodes C., and Berg, P. (1977) Labelling deoxyribonucleic acid to a high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237–251.CrossRefGoogle Scholar
  3. 3.
    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 2002

Authors and Affiliations

  • Ralph Rapley
    • 1
  • Bimal D. M. Theophilus
    • 2
  1. 1.Department of BiosciencesUniversity of HertfordshireHatfieldUK
  2. 2.Department of HaematologyBirmingham Children’s Hospital NHS TrustBirminghamUK

Personalised recommendations