Human Genetics

, Volume 100, Issue 3, pp 472–476

Removal of repetitive sequences from FISH probes using PCR-assisted affinity chromatography

  • J. M. Craig
  • Jürgen Kraus
  • Thomas Cremer
Rapid communication

DOI: 10.1007/s004390050536

Cite this article as:
Craig, J., Kraus, J. & Cremer, T. Hum Genet (1997) 100: 472. doi:10.1007/s004390050536

Abstract

The vast majority of probes used in fluorescence in situ hybridization (FISH) contain repetitive DNA. This DNA is usually competed out of a hybridization reaction by the addition of an unlabeled blocking agent, Cot-1 DNA. We have successfully removed repetitive DNA from two complex FISH probe sets: a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) single human chromosome library and genomic DNA. The procedure involved hybridizing in solution a DOP-PCR-amplifiable probe set with a 50-fold excess of biotin-labeled Cot-1 DNA, and capturing the Cot-1 DNA-containing hybrids using streptavidin magnetic particles, followed by purification and reamplification of the unbound fraction. Probes were checked for depletion of repeats by hybridization to chromosomes without Cot-1 DNA. Results showed hybridization patterns comparable to those achieved with untreated probes hybridized with Cot-1 DNA.

Copyright information

© Springer-Verlag Berlin Heidelberg 1997

Authors and Affiliations

  • J. M. Craig
    • 1
  • Jürgen Kraus
    • 1
  • Thomas Cremer
    • 1
  1. 1.Institute of Anthropology and Human Genetics, University of Munich, Richard-Wagner-Strasse 10/1, D-80333 Munich, Germany Tel.: +49-89-5203-383; Fax: +49-89-5203-389DE

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