In Situ Hybridization (ISH)

Introduction

In Situ Hybridization (ISH) is a technique that allows for precise localization of a specific segment of nucleic acid within a histologic section. The underlying basis of ISH is that nucleic acids, if preserved adequately within a histologic specimen, can be detected through the application of a complementary strand of nucleic acid to which a reporter molecule is attached.

Visualization of the reporter molecule allows to localize DNA or RNA sequences in a heterogeneous cell populations including tissue samples and environmental samples. Riboprobes also allow to localize and assess degree of gene expression . The technique is particularly useful in neuroscience.

In situ hybridization probes

• double-stranded DNA (dsDNA) probes
• single-stranded DNA (ssDNA) probes
• RNA probes (riboprobes)
• synthetic oligonucleotides (PNA, LNA)

Labeling techniques

• radioactive isotopes
  • ³²P
  • ³⁵S
  • ³H
• non-radioactive labels
  • biotin
  • digoxigenin
  • fluorescent dye (FISH)

Applications of In Situ Hybridization

• microbiology (classic target - 16S rRNA) - morphology and population structure of microorganisms
• pathology (pathogen profiling, abnormal gene expression)
• developmental biology (gene expression profiling in embryonic tissues)
• karyotyping and phylogenetic analysis (unique FISH patterns on individual chromosomes, chromosomal abberations)
• physical mapping (mapping clones on chromosomes and direct assignment of mapped clones to chromosomal regions associated with heterochromatin or euchromatin)

Probe

References

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