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

Hum Genet. 1997 Sep;100(3-4):472-6. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Affinity / methods*
  • DNA
  • Humans
  • In Situ Hybridization, Fluorescence / methods*
  • Male
  • Polymerase Chain Reaction / methods*
  • Repetitive Sequences, Nucleic Acid*

Substances

  • DNA