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Generation of Labeled Probes by Polymerase Chain Reaction

  • Y. M. Dennis Lo
  • Shu F. An
Protocol
  • 115 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Nucleic acid probes are an important tool in molecular diagnosis. To facilitate the detection of hybridized probes, they are labeled with a reporter molecule, which is usually a radioisotope. For diagnostic techniques carried out in a clinical laboratory, radioisotopes are hazardous and, thus, recently there is a move to use nonisotopic labels, such as biotin and digoxigenin. Nonisotopic probes also have the advantage of much better stability over time compared with isotopic probes that have limited half-lives.

Keywords

Polymerase Chain Reaction Sodium Dodecyl Sulfate Polymerase Chain Reaction Product Nitro Blue Tetrazolium Nitro Blue Tetrazolium 
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 deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase. J. Mol. Biol. 113, 237–247.PubMedCrossRefGoogle Scholar
  2. 2.
    Feinberg, A. P. and Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6–13.PubMedCrossRefGoogle Scholar
  3. 3.
    Lo, Y. M. D., Mehal, W. Z., and Fleming, K. A. (1988) Rapid production of vector-free biotinylated probes using the polymerase chain reaction. Nucleic Acids Res. 16, 8719.PubMedCrossRefGoogle Scholar
  4. 4.
    An, S. F., Franklin, D., and Fleming, K. A. (1992) Generation of digoxigenin-labeled double-stranded and single-stranded probes using the polymerase chain reaction. Mol Cell. Probes 6, 193–200.PubMedCrossRefGoogle Scholar
  5. 5.
    Chan, V. T. W., Fleming, K. A., and McGee, J. O. (1985) Detection of subpicogram quantities of specific DNA sequences on blot hybridisation with biotinylated probes. Nucleic Acids Res. 13, 8083–8091.PubMedCrossRefGoogle Scholar
  6. 6.
    Boehringer Mannheim. (1989) Biochemica-Applications Manual: DNA Labelling and Nonradioactive Detection. Boehringer Mannheim GmbH Biochemica, East Sussex, UK.Google Scholar
  7. 7.
    Barnes, W. M. (1994) PCR amplification of up to 35 kb DNA with high fidelity and high yield from Lambda bacteriophage templates. Proc. Natl. Acad. Sci. USA 91, 2216–2220.PubMedCrossRefGoogle Scholar
  8. 8.
    Weir, H. U. G., Segraves, R., Pinkel, D., and Gray, J. W. (1990) Synthesis of Y chromosome-specific labelled DNA probes by in vitro DNA amplification. J. Histochem. Cytochem. 38, 421–426.CrossRefGoogle Scholar
  9. 9.
    Duplaa, C., Couffinhal, T., Labat, L., Moreau, C., Lamaziere, J., and Bonnet, J. (1993) Quantitative analysis of polymerase chain reaction products using biotinylated dUTP incorporation. Anal. Biochem. 212, 229–236.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Y. M. Dennis Lo
    • 1
  • Shu F. An
    • 2
  1. 1.Department of Chemical Pathology, Prince of wales HospitalThe Chinese University of Hong KongHong KongPeople’s Republic of China
  2. 2.Institute of NeurologyUniversity of LondonLondonUK

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