Construction of Repeat-Free Fluorescence in Situ Hybridization Probes

Nucleic Acids Res. 2012 Feb;40(3):e20. doi: 10.1093/nar/gkr1123. Epub 2011 Nov 28.

Abstract

FISH probes are generally made out of BAC clones with genomic DNA containing a variable amount of repetitive DNA that will need to be removed or blocked for FISH analysis. To generate repeat free (RF) Probes without loss in genomic coverage, a random library is made from BAC clones by whole-genome amplification (WGA). Libraries are denatured in the presence of excess C(0)t-1 DNA and allowed to re-anneal followed by digestion of all double-stranded elements by duplex-specific nuclease (DSN). Selective amplification of all elements not containing repetitive sequences is realized by a sequential amplification. The final RF products can be re-amplified and used as a stock for future probe production. The RF probes have a lower background, the signal intensity build up is faster and there is no need for blocking DNA. The signal to background ratio of the RF was higher as compared to repeat containing probes.

MeSH terms

  • Cell Line, Tumor
  • Chromosomes, Artificial, Bacterial
  • DNA / chemistry
  • Female
  • Fluorescent Dyes / chemistry*
  • Gene Library
  • Humans
  • In Situ Hybridization, Fluorescence*
  • Metaphase
  • Polymerase Chain Reaction
  • Repetitive Sequences, Nucleic Acid

Substances

  • Fluorescent Dyes
  • DNA