Principles of Fluorescence Spectroscopy

732 DNA TECHNOLOGY
ities and rearrangements, as well as monitoring bone marrow
cells following transplantation and cancer therapy.
166–172 The use of FISH relies on methods for labeling
DNA, computerized imaging, and high, sensitivity CCD
detection. FISH technology represents a combination of
modern optics, molecular biology, and fluorescence spectroscopy,
and promises to become a central tool in molecular
medicine.
21.9. DNA ARRAYS
Figure 21.56. Spectral karyotyping of 24 human chromosomes using 24 pointing probes. From [165].
DNA arrays provide a method for parallel high-throughput
analysis of gene expression. This capability has resulted in
a paradigm shift in biological research. Traditional experiments
in gene expression studied one or a few genes in an
organism. Presently it is possible to simultaneously study
the expression of thousands of genes in a single experiment.
173–180
Figure 21.57. Chromosome images of an ovarian carcinoma. Left,
inverted DAPI image. Middle, RGB image. Right, SKY classification
image. From [162].
DNA arrays consist of regular arrays of DNA fragments
or oligomers on a solid support, usually glass microscope
slides. These slides can contain more than 20,000 different
sequences or more in only a few square centimeters
of area. There are two general methods to prepare arrays, by
mechanical spotting of DNA solutions on slides or by lightgenerated
arrays. Spotted arrays are now being produced in
individual laboratories and in core facilities. Light-generated
arrays are more expensive to produce and are usually
manufactured commercially.
21.9.1. Spotted DNA Microarrays
Preparation of a DNA array is somewhat expensive and
complex (Figure 21.58). DNA clones are prepared by one
of several available methods. 180 Usually mRNA is isolated
from the desired sample and used to create cDNA using
reverse transcriptase. The use of mRNA or cDNA results in
DNA fragments that represent the expressed genes. The use
of mRNA or cDNA is generally preferable to using the
entire genome, which contains many regions that are repetitive
or not converted into gene products. The DNA clones
are then spotted onto microscope slides. Prior to spotting,
the slides are treated with polylysine or an aminosilane
reagent to cover the surface with positive charges, which
results in DNA binding to the surface. After drying, the
slides are illuminated with UV light, which probably
crosslinks the DNA to the surface. The surface is then treated
with succinic anhydride to remove the positive charges
on the surface, which would result in nonspecific binding.
Spotting of the slides is accomplished using automated
instruments designed for this purpose. 181 Spotting is usually
done using small capillaries that make contact with the