Molecular markers
Fluorescent in situ hybridization (FISH)
FISH probes

Topic Completed: 1 April 2012

Minor changes: 15 February 2019

Copyright: 2003-2018,, Inc.

PubMed Search: FISH probes[TI]

Rodney E. Shackelford, D.O., Ph.D.
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Cite this page: Shackelford R. FISH probes. website. Accessed May 28th, 2020.
Definition / general
  • Like most assays involving nucleic acid binding and detection, the specificity of FISH depends on the nucleotide sequence of the probe and the stringency of the annealing and washing conditions used
Nucleic acid types
  • Double stranded DNA probes: more common; require denaturation prior to application and often reanneal to each other, lowering their effectiveness
  • Single stranded DNA probes
  • Oligodeoxyribonucleotide probes: usually short, up to 100 base pairs
  • RNA probes: usually single stranded complementary to target nucleic acid sequence; can be very specific but are easily degraded by mild alkaline conditions or Rnases, as are RNA target hybridization sequences
Detection types
  • Fluorescent probes: most common, includes rhodamine, Texas Red and fluorescein; used in most current assays; useful because require no secondary detection reagents, can be stored for relatively long periods, have strong signals with fluorescent excitation
  • Enzymatic probes: includes digoxygenin / antidigoxygenin, biotin / streptavidin; works well but often has high background signals; analysis is limited by short half lives of enzymatic activities
  • Radioactive probes: used in early studies, includes H3 and P32; cumbersome to use, take days - weeks to complete, require extra safety precautions and are limited by radioactive decay of probes
Target types
  • Bind specific sequences (or locus specific FISH): bind one nucleotide sequence found at one chromosomal region or specific to a few diseases; also used to identify deletions (with another probe used as a positive control) or amplifications of specific gene sequences; examples - BCR / ABL fusion transcript due to t(9;22)(q43;q11.2) in CML
  • Bind unique sequences: bind unique sequences found on different chromosomes, such as centromeric and subtelomeric regions; used to identify chromosomal changes in malignancy, birth defects or developmental delay
  • Whole chromosome probes: collections of many probes, each specific for different regions of each chromosome and each labeled with a different fluorophore, which together gives each chromosome a unique color; used to locate translocations difficult to otherwise identify
  • Probe length: ranges from short oligonucleotides of 100 base pairs to nucleic acid sequences over 1 million base pairs; probe must be large enough to bind specifically to target but not so large that its size interferes with efficient hybridization; optimal probe size depends on its target sequence - specific unique target sequences require shorter probe sequences but large target sequences (chromosomal deletions over large areas) often need larger probes
  • Probe sequence and target binding: based on sequence from human genome project, grown in bacteria containing plasmids with specific sequences
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