KEY TECHNIQUE

FISHing for Specific Sequences

PROBLEM: Stains can identify some differences in chro- 1 Label

Probe Target
mosome structure or various types of nucleic acids, but can- probe with
not identify specific DNA sequences. How can specific DNA fluorescent tag
sequences be identified along chromosomes?

SOLUTION: Fluorescence in situ hybridization (FISH)

allows detection of specific nucleic acid sequences in an intact
cellular sample.
3 Denature
2 Denature nucleic acid
Key Tools: Fluorescently labeled nucleic acid probe; pre- probe in sample
served tissue sample; an incubator or oven for heating samples;
fluorescence microscope.

Details: Nucleic acids have several key properties that allow

them to be used as probes for detecting specific sequences in
4 Cool slightly; allow
DNA or RNA. First, they can undergo hybridization: hydrogen
probe to hybridize
bonding with complementary bases in a sequence-specific with sample
fashion. Second, hybridization is reversible: it depends on
pH and temperature. FISH capitalizes on these properties by
creating fluorescent DNA using molecular biology techniques
(Figure 16A-1, ● 1 ). The labeled probe is then denatured into
single strands by heating to near boiling (● 2 ). After the tissue
sample itself has been treated and heated to allow access to 5 Wash unbound probe;
view in microscope
its nucleic acids (●
3 ), the probe is applied to the tissue sample,
which is cooled somewhat to allow for optimal hybridization
(●4 ). After washing away unbound probe, the bound fluorescent
probe can then be detected with a sensitive camera attached to
a fluorescence microscope (● 5 ).
The example in Figure 16A-1 involves direct labeling of the
probe. In some FISH applications, the probe DNA is labeled with
a tag that allows a second fluorescent molecule to bind to the
probe. This adds extra steps, but can amplify weak signals in a
manner very similar to indirect immunostaining (see Chapter 1).
In some cases, FISH is used to detect very specific DNA
sequences within chromosomes. Two examples you will
­encounter in this chapter are centromeres and telomeres
(see Figure 16-21). In the cytogenetics laboratory, FISH is
often performed on metaphase-arrested cells to detect whole

Figure 16A-1  Fluorescent in Situ Hybridization (FISH). See

text for description of steps.

The bacterial DNA residing in this region forms a threadlike
mass of fibers packed together in a way that keeps the nucle-
oid distinct from the rest of the cell. The DNA of the bacterial
chromosome is negatively supercoiled and folded into an ex-
tensive series of loops averaging about 20,000 bp in length.
Because the two ends of each loop are anchored to structural
components that lie within the nucleoid, the supercoiling of
any individual loop can be altered without influencing the su-
percoiling of adjacent loops.
The loops are thought to be held in place by RNAs and
proteins. Evidence for a structural role for RNA in the bacterial
chromosome has come from studies showing that treatment
468
with ribonuclease, an enzyme that degrades RNA, releases
some of the loops, although it does not relax the supercoil-
ing. Nicking the DNA with a topoisomerase, on the other
hand, relaxes the supercoiling but does not disrupt the loops.
The supercoiled DNA that forms each loop is organized into
beadlike packets containing small, basic proteins, analogous
to the histones of eukaryotic cells (discussed below). Current
evidence suggests that the DNA molecule is wrapped around
particles of the basic protein. Thus, from what we know so far,
the bacterial chromosome consists of supercoiled DNA that
is bound to small, basic proteins and then folded into looped
domains.

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 Figure 16A-2  Painting
Chromosomes Using FISH.
(a) The 46 chromosomes of
a metaphase-arrested cell
from a human male have been
“painted” using multicolor
FISH and a combination of
specific probes. After pro-
cessing using a computer,
chromosome pairs (1-22) and
sex chromosomes (X and Y)
11 X 12 15 6
6 10 can be arranged for clarity, as
8 Y 13 12 in the bottom image. (b) The
5 chromosomal territories of
21 the chromosomes of a human
17 male interphase cell can be
1 2 3 visualized using similar tech-
4 5
niques. All different types of
decondensed chromosomes
6 7 8 9 10 11 12 1 can be identified in the bottom

Chapter 16  | The Structural Basis of Cellular Information: DNA, Chromosomes, and the Nucleus
20 8
3 14 1 4 19 image.
13 14 15 16 17 18 7 18
18 7 2 16 9
19 20 21 22 X Y

(a) Metaphase chromosomes (b) Interphase chromosomes

chromosomes. In cells treated with microtubule inhibitors, chro-
mosomes remain condensed, making them easy to visualize by
FISH. When the probes are selected so that they can hybridize
over large portions of a specific chromosome, the result is a
karyotype in which each chromosome can be “painted” using
a ­different color (Figure 16A-2a, top). Sophisticated analy-
sis of the resulting fluorescence images can be performed to
­distinguish many different probes applied to single tissue sam-
ple. Each ­chromosome can then be identified and the images
of the painted chromosomes can be arranged using a computer
(Figure 16A-2a, bottom). Such data provide even more specific
information about the karyotype of a patient than through the
use of stains (see Figure 16-22) and can be useful in diagnosing
chromosomal rearrangements in cancer cells or in human genetic
disorders.
FISH has also proven useful for identifying the positions of
interphase chromosomes, i.e., the positions of chromosomes
in the nucleus when cells are not dividing (Figure 16A-2b, top).
FISH has revealed that interphase chromosomes occupy dis-
tinct chromosomal territories (Figure 16A-2b, bottom). Although
the positions vary from cell to cell, FISH has been essential in
showing that the nucleus is a much more orderly place than
might have been suspected based on general staining tech-
niques. This realization is spurring current research on how this
spatial organization affects the function of specific genes.
QUESTION: Suppose you are trying FISH for the first
time, and you forget to heat your probe before applying it to
your tissue sample. What do you expect to happen, and why?

Bacterial Plasmids. In addition to its chromosome, a bac-
terial cell may contain one or more plasmids. Plasmids are
relatively small, usually circular molecules of DNA that carry
genes both for their own replication and, often, for one or
more cellular functions (usually nonessential ones). Most
plasmids are supercoiled, giving them a condensed, compact
form. Although plasmids replicate autonomously, the replica-
tion is usually sufficiently synchronized with the replication
of the bacterial chromosome to ensure a roughly comparable
number of plasmids from one cell generation to the next.
In E. coli cells, several classes of plasmids are recognized: F
(fertility) factors are involved in the process of conjugation,
a sexual process we will discuss in Chapter 25; R (resistance)
factors carry genes that impart drug resistance to the bacte-
rial cell; col (colicinogenic) factors allow the bacterium to se-
crete colicins, compounds that kill other bacteria lacking the
col factor; virulence factors enhance the ability to cause dis-
ease by producing toxic proteins that cause tissue damage or
enzymes that allow the bacteria to enter host cells; and meta-
bolic plasmids produce enzymes required for certain metabolic
reactions. Some strains of E. coli also possess cryptic plasmids,
which have no known function and possess no genes other
than those needed for the plasmid to replicate and spread to
other cells.
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