Biology Reference
In-Depth Information
Table 6. Development of techniques employed for understanding sex differentiation genes
in fi shes
Technique
Reference
Principle/Advancement over the earlier one
in situ hybridization
(ISH)
Pardue and
Gall (1969)
ISH is a type of hybridization that uses a
labeled complementary DNA or RNA strand
(i.e., probe) to identify or localize a specifi c DNA
or RNA sequence in a section of tissue ( in situ )
or in the entire tissue (whole mount ISH). DNA
ISH determines the structure or integrity of
chromosomes. RNA ISH quantitates and localizes
mRNAs within tissue sections or whole mounts.
fl uorescence
in situ
hybridization
(FISH)
Lengauer et
al. (1990)
FISH, a cytogenetic technique, detects and localizes
the presence or absence of specifi c DNA sequences
on chromosomes. It uses fl uorescent probes that
bind to only those parts of the chromosome with
which they show a high degree of nucleotide
sequence homology. It is used to fi nd specifi c
features in DNA for use in genetic counselling,
medicine, and species identifi cation. It can also
quantitate and localize specifi c mRNAs within
tissue and also analyzes the spatial-temporal
patterns of gene expression within cells and tissues.
Immuno
staining/Immuno-
chemical staining/
Immunohisto-
chemistry
(IHC)
Coons et al.
(1941)
IHC staining of tissue sections or cells is one of the
most commonly used immunostaining techniques
and uses an antibody-based method to detect a
specifi c protein in a sample. It is extensively used
in basic research to understand the distribution/
localization of biomarkers or differentially expressed
proteins in different parts of a biological tissue.
Visualization of an antibody-antigen interaction
is accomplished in a number of ways. Otherwise,
the antibody can also be tagged to a fl uorochrome
such as fl uorescein. While the fi rst cases of IHC
staining used fl uorescent dyes, other non-fl uorescent
peroxidase and alkaline phosphatase are presently
used. These enzymes are capable of catalyzing
reactions that provide light microscopically
detectable colored products. Alternatively,
radioactive elements are used as labels, and the
immunoreaction is visualized by autoradiography.
PCR
Mullis and
Faloona
(1987); Mullis
et al. (1986),
see also Saiki
et al. (1988)
Used to amplify a single or a few copies of a
piece of DNA across several orders of magnitude,
generating thousands to millions of copies of a
particular DNA sequence. All PCR applications
employ a heat-stable DNA polymerase, such as Taq
polymerase, an enzyme originally isolated from the
bacterium Thermus aquaticus . This DNA polymerase
enzymatically assembles a new DNA strand from
single-stranded DNA as a template and DNA
oligonucleotides (also called DNA primers), which
are required for initiation of DNA synthesis. The
vast majority of PCR methods use thermal cycling,
i.e., alternately heating and cooling the PCR sample
to a defi ned series of temperature steps.
Table 6. contd....
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