Tish in pathology
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third-strand in situ hybridization TISH pathology
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https://www.scribd.com/presentation/670820037/FISH-TISH-Copy
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TISH in Pathology - Third-Strand In Situ Hybridization
TISH stands for Third-Strand In Situ Hybridization. It is a molecular cytogenetic technique related to, but distinct from, the more common FISH (Fluorescence In Situ Hybridization).
What is TISH?
TISH is a method in which a synthetic oligodeoxyribonucleotide "third strand" is used to bind to intact, non-denatured duplex DNA in fixed metaphase chromosome spreads or interphase nuclei. Unlike FISH, which requires prior denaturation of the target DNA to allow probe binding, TISH exploits triple-helix (triplex) DNA formation - the probe binds as a third strand to the major groove of the native double helix.
The key technique was described by Johnson & Fresco (1999) at Princeton University (PMID: 10398847).
How TISH Works (Key Steps)
- Cells/chromosomes are fixed on a microscope slide (standard fixation)
- No denaturation step - the DNA remains in its native double-stranded form
- A homopyrimidine third-strand oligonucleotide probe (modified with psoralen and biotin) is applied at pH 6.0 (slightly acidic conditions favor Hoogsteen/reverse-Hoogsteen hydrogen bonding needed for triplex formation)
- The probe hybridizes to a specific purine-rich target sequence in the major groove
- UVA photofixation cross-links the psoralen-modified probe to the target, stabilizing the triplex
- Detection is via fluorescein isothiocyanate (FITC)-labeled avidin, rendering the signal fluorescent
How TISH Differs from FISH
| Feature | FISH | TISH |
|---|---|---|
| DNA state | Denatured (single-stranded) | Native (double-stranded) |
| Probe binding mechanism | Watson-Crick base pairing | Hoogsteen/triplex formation |
| Denaturation step required? | Yes | No |
| Probe type | Any complementary sequence | Homopyrimidine oligonucleotides targeting purine-rich regions |
| Target sequences | Broad | Specific purine-rich duplex sequences |
| Chromatin state | Disrupted by denaturation | Native chromatin preserved |
Why Is TISH Significant?
- It allows interrogation of native chromatin without disrupting DNA-protein interactions (unlike FISH, which denatures the DNA and destroys associated proteins)
- Has potential for flow cytometric applications to non-denatured chromosomes
- Demonstrated ability to identify chromosome 17-specific D17Z1 haplotypes (centromeric alpha-satellite region) with reproducible centromere specificity
- Potential applications in diagnostic pathology, biochemistry, and cytogenetics
Context: ISH Techniques in Pathology
For comparison, the main ISH-based techniques used in modern diagnostic pathology include (per the Tietz Textbook of Laboratory Medicine, 7th Ed.):
- FISH - fluorescent probes, requires denaturation; gold standard for detecting gene amplifications, deletions, and translocations (e.g., BCR-ABL in CML, HER2 in breast cancer)
- CISH (Chromogenic ISH) - similar to FISH but uses brightfield microscopy instead of fluorescence
- RNA-ISH - detects mRNA expression in tissue (e.g., HPV RNA detection in head and neck pathology)
- TISH - specialized technique using triplex probe binding to non-denatured DNA; primarily a research/specialized tool
TISH remains more of a research tool than a routine clinical assay, but its ability to preserve native chromatin architecture gives it unique advantages in certain biochemical and cytogenetic investigations.
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