Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics1-10 / 7

unique PCR amplification can be used. Thus this genetic map cannot be used to

locate

genes on the basis of their phenotype. Not only must DNA be available, but enough of it

must be sequenced to allow for the production of appropriate PCR primers. The

pheno-

IN SITU HYBRIDIZATION

219

be captured on the same film, eliminating the need for a separate autoradiographic devel-

opment step. Short-lived radioisotopes like

32P have decay tracks that are too long for mi-

croscopic images, and considerable resolution is lost by imprecision in the location of the

origin of a track. Longer-lived isotopes like

3H or

14 C would have shorter tracks, but these

would be less efficient to detect, and more seriously, one would have to wait unrealisti-

cally long periods of time before an image could be detected. The

technique in

wide-

spread use today depends on several cycles of stoichiometric amplification such as strep-

tavidin biotin amplification (see Chapter 3) to increase the sensitivity of fluorescent

detection. It is called FISH, for fluorescent in situ hybridization.

Metaphase chromosomes, until recently, were the predominant samples used for DNA

mapping by in situ hybridization. A schematic illustration is given in Figure 7.10. In typi-

cal protocols, cell division is stopped, and cells are arrested in metaphase by adding drugs

such as colchicine; chromosomes are isolated, dropped from a specified height onto a mi-

croscope slide, fixed (partially denatured and covalently

crosslinked), and aged. If this

seems like quite a bit of magic, it is. The goal is to strike a proper balance between main-

taining sufficient chromosome morphology to allow each chromosome to

be

recognized

and measured, but disrupting enough chromosome structure

to expose

DNA

sequences

that can hybridize with the labeled DNA probe. Under such circumstances the hybridiza-

tion reactions are usually very inefficient. As a result a probe with relatively high DNA

complexity must be used to provide sufficient illumination of the target site. Typically one

starts with probes containing a total of 10

4 to 10

5 base pairs of DNA from the site of inter-

est, and these probes are broken into small pieces prior to annealing to facilitate the hy-

bridization.

Recently improved protocols and better fluorescence

microscopes

have allowed

the

great excess of

C

0 t 1 DNA (see Chapter 3). A typical metaphase FISH result with a sin-

gle-copy DNA probe is shown schematically in Figure 7.11. In an actual color image a

bright pair of yellow fluorescent spots would be seen on a single red chromosome. The

yellow comes from fluorescein conjugated to the DNA probe via streptavidin and biotin.

The red comes from a DNA stain like DAPI used to mark the

entire chromosome. The

pair of yellow dots result from hybridization with each of the paired sister chromatids. In

the simplest case one can determine the approximate chromosomal location by measuring

the relative distance of the pair of spots from the ends of the chromosome. The identity of

the particular chromosome is revealed in most cases by its size and the position of its cen-

tromeric constriction.

The interpretation of FISH results requires quantitative analysis of the image in the

fluorescent microscope. It is not efficient to do this by photography and then

processing

of the image. Instead, direct on-line imaging methods are used. One possibility is to equip

a standard fluorescence microscope with a charge couple device array (CCD) camera that

can acquire and process the image as discrete pixels of information. The

alternate

ap-

proach is to use a scanning microscope like a confocal laser microscope that records the

image’s intensity as a function of position. In either case it is important to realize that the

number of bits of information in a single microscope image is considerable; only a small

amount of it actually finds its way into the final analyzed probe position. Either a large

amount of mass storage must be devoted to archiving FISH images,

or a procedure

must

be developed to allow some arbitration or reanalysis of any discrepancies in the data after

the original raw images have been discarded.

Many enhancements have been described that allow FISH to provide more accurate

chromosomal locations than the simple straightforward approach

illustrated

in

Figure

7.11. Several examples are given in Figure 7.12. Chromosome banding provides a much

more accurate way of identifying individual chromosomes and subchromosomal regions

than simple measurements of size and centromere position. Each chromosome in the mi-

croscope is an individual—the amount of stretching and the nature

of any distortion can

vary considerably. Clearly, by superimposing banding on the emission from single-copy

labeled probes, one not only provides a unique chromosome identifier but also local mark-

ers at the band

locations

that allow more accurate positioning of

the single-copy

probe.

IN SITU HYBRIDIZATION

221

Figure 7.12

Some of the refinements possible in metaphase FISH. (

a ) Simultaneous use of a sin-

gle-copy probe and a repeated DNA probe like the human

Alu

sequence which replicates the band-

ing pattern seen in ordinary Giemsa staining. (

b ) Use of three different single-copy DNA probes.

quences in

the

human have preferential locations within the traditional Giemsa bands.

Thus

Alu

repeats prefer light G bands, while L1 repeats prefer dark G bands. One can use

two different colored fluorescent probes simultaneously, one with a single-copy sequence,

the

other with

a cloned repeat, and the results, like those shown in Figure 7.12, represent

a major improvement.

The accuracy gained by FISH over conventional radioactively labeled in situ hy-

bridization

is

illustrated in Figure 7.13. The further increase in accuracy when FISH is

of FISH

mapping even further by the simultaneous use of multiple single-copy DNA

probes. For example, as shown in Figure 7.12, when metaphase chromosomes are hy-

bridized

with three nearby DNA segments, each labeled with a

different color fluo-

rophore, a

distinct pattern of six

ordered dots is seen. Each color

is a pair of dots on the

two sister chromatids. The order of

the colors gives the order of the probes down to a res-

Figure 7.13

Examples of the accuracy of various in situ

hybridization mapping techniques. In each case the probe is

the

blast1 gene.

Dots show the apparent position

of the

probe

on individual

chromosomes. (

a ) 32P-labeled probe

on Giemsa-banded chromosomes. (

b ) Fluorescein-la-

beled probe on fluorescently banded chromosomes. (

c )

Fluorescein-labeled probe on unbanded chromosomes.

(Adapted from Lawrence et al., 1990.)

IN SITU HYBRIDIZATION

223

Today metaphase FISH provides an extremely effective way

to assign a large number

of DNA probes into bins along a chromosome of interest. An example from work on

chromosome 11 is shown in Figure 7.14. Note, in this

case, that the

mapped, cloned

probes are not distributed evenly along the chromosome; they

tend to cluster

very much

224

CYTOGENETICS AND PSEUDOGENETICS

HIGH-RESOLUTION

FISH

To increase the resolution of FISH over what is achievable with metaphase chromosomes,

it is necessary to use more extended chromosome preparations. In most cases this results

in sufficient disruption of chromosome morphology that no apriori assignment of a single

probes to particular chromosomes or regions is possible. Instead, what is done is to deter-

mine the relative order and distances among a set of closely spaced DNA probes by si-

multaneous multicolor FISH, as we have already described for metaphase chromosomes.

The different approaches reflect mostly variations in

the particular

preparation

of

ex-

tended chromosomes used.

Several different methods exist for systematic preparation of partially decondensed

chromosomes. One approach is to catch cells at the pro-metaphase stage, before chromo-

somes have become completely condensed. A second approach to

fertilize

a

hamster

oocyte with a human sperm. The result of this attempted interspecies cross is called a

humster. It does not develop, but within the

hamster nucleus the human chromosomes,

which are highly condensed in the sperm head,

become partially decondensed; in this

state they are very convenient targets for hybridization. A third approach is to use condi-

tions that lead to premature chromosome condensation.

The most extreme versions of FISH use highly extended DNA samples. One way to

accomplish this is to look at interphase nuclei. Under these conditions the chromatin is

mostly in the form of 30 nm fibers (Chapter 2). One can make a crude estimate of the

length of such fiber expected for a given length of DNA as follows. The volume of 10 bp

of DNA double helix is given by

r 2 d

where

r

is the radius and

d

is the pitch. Evaluating

these as roughly 10 Å and 34 Å, respectively, yields 10

4 Å per 10 bp or 10

3 Å per bp. The

volume of a micron of 30-nm filament is

r 2 d,

where

r

is 150 Å and

d

is 1

10 4 Å.

This is 8

10 8 Å3, and roughly half of it is DNA. Thus one predicts that a micron of 30-

nm filament will contain on average about 0.4 Mb of DNA. This estimate is not in bad

agreement with what is observed experimentally (Box 7.1). Since the resolution of the

fluorescence microscope is about 0.25

, the ultimate resolution of FISH based on inter-

phase chromatin should be around 0.1 Mb.

Even higher resolution is possible if the DNA is extended further

in

methods

called

fiber FISH.

One way to do this is the technique known as a Weigant halo. Here, as shown

schematically in Figure 7.15, nuclei are prepared and then treated so that the DNA is de-

proteinized and exploded from the nucleus. Since naked DNA is about 3 Å per base pair,

such samples should show an extension of 3

10 3 Å

per kb

, w

hic

h is

0.3

per kb.

Thus the ultimate resolution of FISH under

these circumstances could approach 1000

base pairs. To take advantage of the high resolution afforded by extended DNA samples,

one must use multicolored probes to

distinguish their order, and then estimate the

dis-

tance between the different colors. The probes themselves will occupy a significant dis-

tance along the length of the DNA, as shown in Figure 17.16.

Interphase

chromatin,

or

naked DNA, in

contrast to metaphase chromosomes, is not

a unique structure, and

it

is

not

rigid

(Fig.

7.17

a ).

To

estimate

the

true

distance

between markers from the apparent separation of two probes in the microscope, one must

correct for the fact that the DNA between the markers is not

straight. The problem be-

comes ever more severe as the distance between

the markers increases (Fig. 7.17

b ). No

single molecule measurement

will

suffice

because

there is

no

way

of knowing

what the