Biology Reference
In-Depth Information
testing when livestock or germplasm from them are exported to regions with-
out the disease.
12.4.2 Molecular Cytology
Three breakthroughs in cytogenetic techniques revived this approach to sys-
tematic and evolutionary studies. The first was the discovery that hypotonic
treatment spreads metaphase chromosomes, allowing more accurate counts of
chromosome numbers and details of chromosome morphology. The second was
the development of chromosome-banding techniques that allow the identifica-
tion of specific types of DNA within homologous chromosomes. The third was
the development of
in situ
hybridization
techniques that allow specific DNA
sequences to be localized to particular segments of the chromosomes.
In situ
hybridization involves annealing single-stranded probe molecules to
target DNA to form DNA duplexes.
In situ
hybridization is effective in locat-
ing satellite DNA, ribosomal gene clusters, or duplicated genes of polytene
chromosomes and can even locate single-copy DNA on mitotic chromosomes.
Chromosomal DNA is denatured in such a way that it will anneal with high
efficiency to complementary ss nucleic-acid probes to form hybrid duplexes.
Because chromosomal DNA is complexed with proteins and RNA, the efficiency
of
in situ
hybridization is determined by how well the chromosomal DNA can be
denatured, how much DNA is lost during fixation and treatment, and whether
chromosomal proteins are present in the region of interest (
Sessions 1996
).
Autoradiography is used to detect where hybridization between a radioactive
probe and its target DNA occur. Nonradiographic labeling techniques such as
biotinylation or fluorescence can be used, as well.
Chromosome morphology may be used as a taxonomic character. In many
cases, chromosomes can be identified by their relative size, centromere position,
and secondary constrictions. Many chromosomes, particularly polytene chromo-
somes, have complex patterns of bands or other markers that can be used to
identify specific populations or to discriminate between closely related species.
Q, G, or C banding produces distinctive patterns that identify chromosomes in
most species.
Q banding
is the simplest technique, and involves treating chromosome prep-
arations with quinacrine mustard or quinacrine dihydrochloride, which produces
fluorescent bands that are brightest in AT-rich regions of the chromosomes.
Q banding is visible only with UV optics and the bands fade rapidly.
G banding
involves treating chromosome preparations with trypsin or NaOH and staining
with Giemsa in a phosphate buffer, a process that yields alternating light and
