Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

I. Introduction
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FIGURE 2-2 Fluorescent in situ hybridization. Two color FISH on
canine metaphase chromosomes. Two loci labeled red and green are
assigned to canine chromosome 9 (CFA9).
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FIGURE 2-3 Restriction site for the restriction enzyme Eco RI. The
double-stranded DNA is cleaved along the line.
a bright fluorescent signal. With the development of fluorescent
labels that have specific emission spectra, multiple
DNA probes can be hybridized simultaneously to a single
chromosome preparation, allowing their ordering on a chromosome
( Fig. 2-2 ). Another useful application of multicolor
FISH is called chromosome painting : multiple probes
distributed throughout the length of one chromosome
are labeled with the same color dye at a density such
that the entire chromosome is covered by fluorescence.
As chromosome-specific probe sets are hybridized with different
colors, each chromosome reveals its unique color,
which is particularly useful to examine chromosomal abnormalities
like deletions, duplications, and translocations of
chromosomal segments. Because FISH allows only lowresolution
mapping (probes 1Mb apart), other techniques
need to be applied for finer, high-resolution mapping.
b . Restriction Enzyme Mapping
Restriction endonucleases are enzymes isolated from various
strains of bacteria that recognize and cleave specific
double-stranded DNA sequences, called restriction sites,
with the majority of sites consisting of only four to seven
nucleotides (see the example in Fig. 2-3 ). A DNA segment,
digested by a specific restriction enzyme, is cut into
smaller DNA fragments of different sizes depending on the
number and location of the recognition sites present within
the DNA sequence. The differently sized fragments can be
separated by agarose or polyacrylamide gel electrophoresis.
A simple way to create a restriction map of a smaller
genome is to first cut the DNA using two separate reactions,
each with a different restriction enzyme, and then in
an additional reaction simultaneously with both enzymes
to compare the resulting fragment size patterns. This will
allow one to assess the number of restriction sites for each
enzyme by single digests and then the relative positions to
each other by the double digest ( Fig. 2-4 ). However, with
an increasing size of the DNA segment to be mapped, the
number, sizes, and order of resulting fragments become too
complex. Then analysis requires cloning smaller fragments
or other mapping techniques.
c . Sequence Tagged Site (STS) Mapping
STSs are short nonrepetitive DNA segments that are located
at unique sites in the genome and can be easily amplified
by the polymerase chain reaction (PCR). Common sources
to obtain STSs represent expressed sequence tags (ESTs),
microsatellites (discussed later), and known genomic
sequences that have been deposited in databanks. ESTs are
short sequences obtained by converting mRNA into complementary
DNA (cDNA). They are unique and valuable
sequences, because they represent parts of expressed genes
of the cells or tissue used for the mRNA extraction. To construct
a genome map using STSs, different DNA resources,
sometimes called a mapping reagent, can be used. The most
common resources are radiation hybrid panels or clone
libraries, both of which can be constructed using either
whole genome sequences or a single chromosome.
i . Radiation Hybrid (RH) Mapping Radiation cell
hybrids are typically constructed using cells from two
different species. Cells from the organism whose genome
is to be mapped (donor) are irradiated with a lethal dose
and then usually fused with rodent (recipient) cells. The
irradiated chromosomes break at random sites and, after
cell fusion with the recipient cells, the donor chromosome
fragments are incorporated into the recipient chromosomes.
Consequently each hybrid cell line derived from a single cell
contains different parts of the donor’s chromosomes, which
were incorporated at random.
Radiation hybrid mapping is based on this artificially
induced random breaking of the genomic DNA into smaller
fragments. The original order of these fragments to each
other is determined by ascertaining that specific DNA
sequences are found to be in the same clones, which means
that they segregate together because of their close physical
proximity in the genome. For detailed mapping, fewer
than 100 hybrid cell lines are necessary. For example, irradiated
canine cells were fused with recipient hamster cells,
and 88 cell lines were selected ( Hitte et al. , 2005 ). To map
the canine genome, DNA from each cell line is being tested
for the presence or absence of unique canine markers, like
STSs. If two markers are originally located closely on a
chromosome, a break between the markers is unlikely, and,
therefore, they will mostly be found together in the same
cell line. In contrast, if they are farther apart or even on