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HUMAN INTERPHASE CHROMOSOME (FISH probes (wcp (useless for idenfication…
HUMAN INTERPHASE CHROMOSOME
advantages
the ability to provide either an onchip
scan of the whole genome at extremely high resolution
or visualization of single peculiar genomic loci
able to analyze mixed DNA in large amount of cells
the capability to analyze genome organization,
structure and behavior in single cells at the DNA
(RNA) sequence level
by metaphase/ interphase analysis
FISH based-method
Denaturation/hybridization
Microscopic visual/digital analysis
Cells suspension/biopsies preparation for analysis
FISH probes
Site specific (40-70% hybridization efficiency)
wcp (useless for idenfication of no & structure of interphase chr.)
SKY
I-FISH (too problematic)
MFISH
(TTAGGG) repetitive-sequence DNA
classification
telomeric (I-FISH)
centromeric (I-FISH) : identification of numerical chr. abnormalities
Pooly applicable in diagnosis, Use in cancer and aging research
ICS-MCB
visualization of whole banded interphase chr. in their integrity
only one homologous pair chr. per analysis
Fiber FISH (Highest molecular cytogenetic resolution= ~2.3kb)
Immuno-FISH
applications
studies of chr. structure & organization
transplantation research
cancer research & diagnosis
identification of supramolecular disease mech.
Pod-FISH
suspension-FISH (S-FISH)
Golden standard
Cytogenetic banding analysis
Single-cell molecular cytogenetics
Metaphase plates
Different DNA probes sets
MFISH
SKY
Interphase nuclei (I-FISH)
I-FISH
Detect numerical chromosome imbalances
Aneuploidy
Polyploidy
Limitation
Signals not identical to chromosome no. (centromeric probes)
UNIQUE way to study variations & behaviour of genome in all cell types of human organisms, at all stages of cell and at molecular and supramolecular resolutions.
Problems
different hybridization efficiency complicate simultaneous application of different probes sets
signals site specific probes missed because of high brightness of wcp or centromeric probe signals @ hindered due to replicative signal appearance
solution
ICS-MCB
I-FISH applications
Somatic genomic variations
Interindividual genomic variations detected in interphase by parent-of-origin-determination FISH (pod-FISH)
Molecular cytogenetic diagnosis
limitations
Only use in research purpose, no diagnostic validity
No chromosomal mosaicism guidelines and criteria
No data or recommendations of correlation between metaphase and interphase diagnostic analysis of same individuals.
Genome organization in interphase
study of 3D preserved nuclei which requires specific conditions for cell cultivations using suspension-FISH (S-FISH) technique.