COBRA: Combined Binary Ratio Labeling of Nucleic-Acid Probes for Multi-Color Fluorescence in Situ Hybridization Karyotyping

Nat Protoc. 2006;1(1):264-75. doi: 10.1038/nprot.2006.41.

Abstract

Combined binary ratio labeling (COBRA) is designed to increase the multiplicity of fluorescence in situ hybridization (FISH)--i.e., the number of targets that can be distinguished simultaneously. In principle, chemical (ULS), enzymatic (nick translation or random priming) or PCR-based labeling procedures of probes can be used. The method was originally designed to label chromosome-painting probes, but has also been used for probe sets specific for subtelomeric regions. COBRA imaging requires a digital fluorescence microscope equipped for sequential excitation and recording of color images. Staining of all 24 human chromosomes is accomplished with only four fluorochromes, compared with five for methods based on combinatorial labeling. The COBRA procedure takes approximately 6 h laboratory work, 2-3 d incubation and a few hours imaging. The method is routinely applied in research (cultured cells from human or mouse origin) or to support clinical diagnosis, such as postnatal and perinatal genetic testing and in solid tumors.

MeSH terms

  • Animals
  • Chromosome Painting / methods*
  • Chromosomes, Human
  • Fluorescent Dyes / chemistry*
  • Humans
  • Karyotyping / methods*
  • Microscopy, Fluorescence
  • Nucleic Acid Probes / chemistry*
  • Polymerase Chain Reaction

Substances

  • Fluorescent Dyes
  • Nucleic Acid Probes