1. In situ hybridization and Chromosome painting
FISH & Zoo-FISH

2. I. In situ hybridization
PROBE
TARGET
modify or label
Hybridization
Q: where does the probe ‘bind’ on the target?
use approaches to detect the bound probe
use approaches to detect the chromosome

3. In situ hybridization PROBE TARGET DNA (RNA)
small-medium sized DNA clones
nuclear DNA
cDNA
plasmid
lambda
cosmids
chromosomes
Mechanically
Stretched
DNA
large sized DNA clones
BACs
PACs
YACs

4. Important considerations
In situ hybridization
Important considerations
PROBE
TARGET
small/medium/ large
sized DNA clones
nuclear DNA
1. How can DNA hybridize to DNA?
2. What needs to be done?
3. What precautions to take while using
medium/large DNA probes?
4. How to minimize non-specific hybridization?

7. FISH Mapping

8. Multicolor FISH
Using more than one probe at a time
Detecting using different fluorophores
Best for closely located loci - deriving order
Can be used for chromosome identification

9. For improved resolution
Interphase FISH
For improved resolution
Chromatin fibers extended - act as template
Order loci & estimate distances
Best in resolution limits of ~ 25kb kb
250 kb
400 kb

10. Fiber FISH
Chromatin extracted from cells
Stretched mechanically on glass slides
used as template for hyb.
Best in resolution limits of ~ 25kb kb

12. II. Chromosome painting
Resources
flow sorted chromosomes
chromosome specific libraries
microdissected chromosomes
Concept
Hybridize to
metaphase
chromosomes
Obtain DNA specific
to a chromosome
Label
Species specific
visualize
How is it different from routine in situ hybridization?

14. 7 colour chromosome paints from a palette of 3 fluorochromes
and a DAPI counterstain for a variety of applications such as
Leukaemia / lymphoma monitoring or early indicative diagnosis.

16. Chromosome painting Major uses
Studying chromosomal abnormalities: routine cytogenetics
Detecting minute chr. rearrangements
Cancer Genetics: multiple ‘paints’
SKY: spectral karyotyping
one-step solution to
studying entire karyotype !

17. Chromosome microdissection
Chromosomal DNA scraped
Whole or defined segments
Several copies needed for efficient amplification
Resultant DNA
purified
amplified
labeled
used as hyb. probe
Major contribution
chromosome analysis
genome analysis

19. Cross species chromosome painting
Probe & target same species
What if probe & target from different species?
Can it work? How/Why?
Evolutionarily
closely related species
distantly related species
Major contribution
comparative genomics
karyotype evolution

20. Across species (Zoo-FISH) Comparative chromosome painting
Across evol. closely related species
primates
bovids
equids
Across evol. distantly related species
human & cattle/pig
birds and mammals

21. Across species
Species ‘A’
Species ‘B’

22. Use of human chromosome 17 probe on cattle chromosomes

23. Zoo-FISH
HSA17
HSA3
HSA9
HSA21

24. The essence of comparative painting work
Whole chromosome conservation
Conservation of whole arms or large chromosomal segments
Neighboring segment combinations
Relative size of homologous segments maintained
Conserved gene order + rearrangements

26. Bi-directional painting
Refinement of comparative data
Bi-directional painting
Species A
Species B

27. Unidirectional painting Bi-idirectional painting
Pig
Cattle
Sheep
Buffalo
Horse
Dog
Cat
Rabbit
Mink
Donkey
Goat
Chicken
Pig
Dog
Cat
Rabbit
Giant Panda
Red fox
Indian muntjac

28. Comparative animal genomics
Develop gene rich maps
Generate high resolution comparative maps with human/mouse
Increase the accuracy of alignment
Maximize the ‘candidate approach’ output

29. GENOMES From anatomy to physiology comprehensive mol. genetics
Human genomics - entering a new era !
Animal genomics - trying to catch-up !
New vision of
comprehensive mol. genetics
Functional Genomics
Searching the role/function of individual gene
searching the network within which they work
Comparing the network in different species
Comparative Functional Genomics

30. Any limitations ? Size of detected segment
How much evolutionarily diverged are the species ?
Closely related species
Species that diverged
60-80 Myrs ago
Myrs ago
over 300 Myrs ago

32. A HUMAN-CHICKEN COMPARATIVE OVERVIEW (X AND Z)
HSA18
HSA8
GGA1
GGA4
HSA9
HSA5
Human X
Chicken Z

34. Human-mouse chromosome similarity
a range