IN SITU HYBRIDIZATION
Veronica Descotte
Research in Molecular Genetics
Macalester College
December 2002


Defiition of the Technique
Applications
Different types of probes for ISH
Probe Design
Three main ways to do ISH
Whole mount ISH in C.elegans
References
Gallery


I. Definition of the Technique

   i. Method to visualize nucleid acid sequences by hibridization of complimentary probe sequences
  ii. It enables to precisely localize and identify individual cells that contain a specific sequence we are interested.

 

II. Applications


    A. Clinical: Prenatal diagnoses diagnoses (inherited  chromosome aberrations); postnatal diagnoses of inherited                 genetic diseases; viral and bacterial  disease diagnoses.
    B. Research: Mapping of chromosomal genes; nuclear organization; tumor biology; developmental biology                       (temporal/spatial expression of genes)


III. Different types of probes for ISH

   A. PCR amplification of repeat sequences of tDNA
   B. Oligos specific to the repeat elements
   C. cDNA/genomic DNA cloned in vectors
   D. In vitro transcripts of DNA


IV. Probe Design

   Efficiency of detecting a hybridized probe depends on the target sequence


V. Three main ways to do ISH

   A. Whole Mount
       a. Determination of the spatial distributions of RNAs
       b. Method of choice to determine the expression patterns of gene products
       c. Simultaneaous detection of different transcripts
   B. Sections
       a. Frozen sections; paraffin-embedded sections
   C. Chromosomal
       a. DNA is visualized by ISH; other cellular components are aften visualized by indirect immunofluorescence


VI. Whole mount ISH in C.elegans

   A. Collection of embryos
   B. Permeabilization and fixation of embryos
   C. Probe Synthesis
   D. Prehybridization
   E. Hybridization
   F. Post-hybridization washes
   G. Probe Detection
   H. Appropriate Controls
       1. Positive: prove with known distribution
       2. Negative: ommision of probe and/or DIG ab; incubation with sense strand

VII. References

Epstein H.F., and Shakes D.C. (1995) Canenorhabditis elegans,  modern biological analyses of an organism. Academic Press, California

Patel, N.H., and Goodman, C.S (1992). Preparation od digoxigenin-labeled   single strabded DNA probes. In 'Non-radioactive labeling and detection of biomolecules' (C.Kessler, ed). Springer-Verlag, Berlin

Seydoux, G., and Fire, A. (1998). Whole-mount in situ hybridization  for the detection of RNA in Caenorhabditis elegans embryos. In 'Caenorhabditis elegans, modern biological analyses  of an organism' (H.Epstein, ed.) Academic Press, California

Seydoux, G., and FIre, A. (1994) Soma-germline assymetry in the  distributions of embryonic RNAs in C.elegans. Development, 2823-2834

Spector, A.L., et.al., (1998) Cell laboratory manual vol.3; Cold Spring Harbor Laboratory Press, Cold  Spring Harbor.


Gallery


cey-2 in situ
cey-2insitu