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