In situ hybridization and Chromosome painting FISH & Zoo-FISH

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

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

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?

FISH Mapping

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

For improved resolution Interphase FISH For improved resolution Chromatin fibers extended - act as template Order loci & estimate distances Best in resolution limits of ~ 25kb - 300 kb 250 kb 400 kb

Fiber FISH Chromatin extracted from cells Stretched mechanically on glass slides used as template for hyb. Best in resolution limits of ~ 25kb - 300 kb

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?

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.

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 !

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

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

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

Across species Species ‘A’ Species ‘B’

Use of human chromosome 17 probe on cattle chromosomes

Zoo-FISH HSA17 HSA3 HSA9 HSA21

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

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

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

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

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

Any limitations ? Size of detected segment How much evolutionarily diverged are the species ? Closely related species Species that diverged 60-80 Myrs ago 100-150 Myrs ago over 300 Myrs ago

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

Human-mouse chromosome similarity a range