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In Situ Hybridization

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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
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Double-stranded DNA (dsDNA) probes
Single-stranded DNA (ssDNA) probes
RNA probes (riboprobes)
Synthetic oligonucleotides (PNA, LNA)
Labeling techniques
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Radioactive isotopes
o 32P
o 35S
o 3H
Non-radioactive labels
o biotin
o digoxigenin
o fluorescent dye (FISH)
Applications of In Situ Hybridization
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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 aberrations)
Physical mapping (mapping clones on chromosomes and direct assignment of mapped
clones to chromosomal regions associated with heterochromatin or euchromatin)
Generation of riboprobes