In situ Hybridization (ISH). Method of localizing, either mRNA within the cytoplasm or DNA within the chromosomes, by hybridizing the sequence of interest to a complimentary strand of a nucleotide probe .
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Method of localizing, either mRNA within the cytoplasm or DNA within the chromosomes, by hybridizing the sequence of interest to a complimentary strand of a nucleotide probe.
Nucleic acid hybridization is a fundamental tool in molecular genetics. It takes advantage of the complementary nature of double stranded DNA or RNA to the DNA or even RNA to RNA.
0.05% 0.02% 0.005% 0.002%
Spinal Cord; probe PLP mRNA
BM: Non-radioactive in situ hybridization, 1996
RNA-RNA > RNA-DNA > DNA-DNA
- Chemical coupling of a modified reporter molecule. The reporter molecule can bind with high affinity to another ligand (Biotin, Digoxigenin).
Disadvantage: hazard, long exposure times
Cut with EcoRI
Use T-3 polymerase
Cut with BamHI
Use T-7 polymerase
Ref: Anne Ephrussi, Daniel St Johnston, 2004, Cell, 116 (2), pages 143-152, 23 Jan
Characterization of chromosomal translocations
2. Aneuploidy analysis
3. Cancer specific chromosome deletions
FISH + + + + + + +
acute promyelocytic leukemia
Green + RED = YELLOW
of chromosome 13
in pancreatic carcinoma
Two green, two reds
on different chromosomes
– no deletion
Two green, one red –
One red is deleted.
Interphase FISH, relaxed chromatin
GREEN SIGNAL SERVE AS A CONTROL PROBE
ON A SAME CHROMOSOME.