Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics1-10 / 3

Figure 3.15

Renaturation kinetics actually seen for total

human genomic

DNA. The parameters plotted are the

same as in Figure 3.14.

phases to the reaction. About 10% of the DNA renatures almost immediately. It turns out

this is very simple sequence DNA, some of which is self-complementary, and can rena-

ture by folding back on itself to make a hairpin. Other single-stranded components of this

class form novel DNA ordered structures

like some triplet-repeating

sequences

(see

Chapter 13). About 40% of the DNA renatures 10

5 to

10

6 times as

fast as expected for

single-copy human DNA. This consists mostly of highly repeated sequences like the hu-

man

Alu

repeat. The

effective

C

0 t1/2

for

this DNA

is

about

1. The

remaining half

of the

genome does show the expected renaturation times for single-copy DNA.

The repeated sequences that renature fast, but intermolecularly, are mostly interspersed

with single-copy DNA. Thus the length of the DNA fragments used in a renaturation ex-

periment will have a profound effect on the resulting products that one might purify at

various times during the reaction. Repeats that come together fast will usually be flanked

by single-copy sequences that do not correspond. If one purifies double stranded material,

it will be a mixture of duplex and single strands, as shown in Figure 3.16. Upon further

renaturation

the dangling

single strands

will

find their

partners,

but

only

at

the

cost

of generating a complex crosslinked network of DNA (Fig. 3.16

b). Eventually this will

act

as such

a barrier to

diffusion that

some

of

the

strands will

never find

their

mates.

Figure

3.16

Effects of repeats on the structure of renatured DNA.

(a) Initial complex between

two DNAs

that

share partial sequence complementarity at a repeat.

(b)Subsequent complexes

formed at

C 0 t’s sufficiently large to allow duplex formation between single copy sequences.

86

ANALYSIS OF DNA SEQUENCES BY HYBRIDIZATION

where the sum is taken over every type of DNA species present;

m i is the number of times

a species of

size

N i is repeated. The complexity will determine the

hybridization

kinetics

of the slowest renaturing species in the sample.

HYBRIDIZATION ON FILTERS

In

contemporary

DNA work, hybridization of probes in solution to targets

immobilized

on filters is

much

more common than duplex formation in homogeneous solution. Two

basic

formats

are

most frequently used; these are summarized in Figure

3.18. In

a dot

The

exact mechanism of this immobilization is unknown,

but it is important,

especially

for

procedures in which the same filter is going to be subjected to many serial hybridiza-

tions. Generally, the DNA sample is denatured by alkali or heating to temperatures near

100°C just before immobilization so that what is

stuck to the filter

is largely single-

Figure 3.18

Hybridization on filters.

(a)A dot blot, in which DNA is spotted directly on a filter.

HYBRIDIZATION ON FILTERS

87

In the Southern blot format, a mixture of DNA species is size-separated by electrophore-

sis on polyacrylamide or agarose gels (see Chapter 5). A replica of the gel-separated material

is made by transfer of the DNA through the plane of the separation gel to a parallel filter, as

shown in Figure 3.18. In this way the targets of the subsequent hybridization reaction can be

characterized by their electrophoretic properties. Several methods exist for transfer of the gel

sample to the filter. These include bulk liquid flow through the capillary network of a stacked

set of paper sheets, vacuum transfer of the fluid phase of the gel, pressure transfer of the fluid

phase of the gel, or electrophoretic transfer of the DNA by using electrodes perpendicular to

the plane of the original gel separation. All of these methods work.

Several procedures exist where the transfer step can be avoided. One is prehybridiza-

tion of labeled probes to samples before electrophoresis. Another is drying the separation

gel and using it directly as a target for hybridization with radiolabeled probes. These pro-

cedures have never achieved the popularity of the Southern blot, mainly because they are

less amenable to successful serial probings.

There are two basic advantages of

filter hybridization

compared

with homogeneous

phase hybridization. The target samples are highly confined. Therefore many different sam-

ples can be reacted with the same probe simultaneously. The second advantage is that the

original target molecules are confined in separate microscopic domains on the filter surface.

Thus they cannot react with each other. This largely avoids the formation of entangled ar-

rays, and it simplifies some of the problems caused by interspersed

repeated DNA se-

quences. Hybridization at surfaces does, however, produce some complications. The immo-

bilized target DNA is presumably attached to the surface at several places. This introduces a

number of obstacles to hybridization. The first of these is the potential for nonspecific attrac-

tion or repulsion of the probe DNA to the surface. More serious are the topological bound-

aries imposed by the points of surface-DNA attachment. These greatly reduce the effective

length of the target DNA. As measured by the

relative kinetics of

solution

and immobilized

HYBRIDIZATION ON FILTERS

89

BOX 3.4

(Continued)

way similar to the production of jumping probes described in Chapter 8). When the

probe

is hybridized to

the target, a substrate for DNA ligase is

created. After

ligation

the probe and target form a continuous stretch of double-stranded DNA. If this duplex

is greater than 10 bp in length, the probe and target strands are topologically

linked.

Thus, as described in Box 2.1, although strong denaturants

can melt the

double-

stranded region, the two resulting single strands cannot physically separate.

When padlock probes are used in a filter hybridization format, a single-stranded cir-

cular target is not required. Because the target is fixed

on the filter in multiple places,

as shown in Figure 3.19, each interior segment is topologically equivalent to circular

DNA. Thus padlock probes that are ligated after hybridization to such segments will

become topologically linked with them. This permits very stringent washing

condi-

tions to be used to remove any probe DNA that is hybridized but not ligated, or probe

DNA that is nonspecifically adsorbed onto the surface of the filter.

One variant on blot hybridization is the reverse dot blot. Here the probe is immobilized

on the surface while the labeled target DNA is introduced in homogeneous solution. This

really does not change the advantages and disadvantages of the blot approach very much.

Another variation on hybridization to blotted DNA samples is in-gel hybridization. Here

DNA samples are melted and reannealed in hydrated gels, sometimes in between two dis-

tinct electrophoresis fractionations. Such hybridization schemes have been developed to

specifically detect DNA fragments that have rearranged in the genome, by Michio Oishi,

and to isolate classes of DNA repeats, by Igor Roninsen.

The kinetics of hybridization of DNA on filters or in dried gels (Fig. 3.21) can be de-

scribed by a variation of the same basic approach used for solution hybridization kinetics.

The basic mechanism of the

reaction is still bimolecular. At

long distances

the

diffusion

of probe

DNA

in solution

toward the target

will

be more

efficient than

the

diffusion

of the immobilized target toward the probe, but these bulk

motions do not really matter

that much to the rate. What matters is diffusion once the two DNAs are in each others’

neighborhood, and here we can think of them as still

showing

equivalent behavior.

Usually, however, the relative concentrations of the probe and sample are very different.

Most often the initial probe concentration, [P]

0 , is much greater that the initial target con-

centration [

T] , and we say

that

the

probe

drives

the hybridization. The

amount of free

0

probe remains

essentially

constant

during

the reaction.

Starting

from

the

same basic

concentrations of [

P ] and [ T] , respectively.

0

0

Because [

P ] is constant,

the resulting

kinetics appear

as

pseudo–first

order

rather

than

0

second order. The rate equation can be integrated as

t

d [T] k

[P ]

dt

[T]

2

0

0

from the initial concentration of

target,

[

T] , at

time

zero,

to the

concentration

[

T], at

any later time

t, to give

0

ln

[T]

k

[P ] t

or

2

[T]0

0

[T]

exp ( k

[P

] t)

2

[T]0

0

The halftime of the reaction is defined as the time at which [

T]/[T]

1

. This can be

written as

0

2

t1/2

ln 2

ln 2

k

mC

/ 2N

k

2

[P

]

2

0

0

where

N

is the genome size of the probe,

C

0

is the probe concentration in nucleotides, and

m

reflects any repeated sequences or heterogeneity. Note that if the probe is single stranded, the

factor of two should be removed from the denominator. This allows us to write

C

0 t1/2

2 ln 2

N

mk

2

The equation is almost the same as the definition of

C

0 t1/2 for

ordinary

homogeneous so-

lution hybridization.

Occasionally the probe contains a trace radiolabel. The target is in great excess, and it

drives the hybridization reaction. The above equations are

exactly

the

same

except that

the role of target and probe are reversed, and

mC

0 /2N

refers to

target

DNA.

SENSITIVE

DETECTION

Hybridization frequently involves the detection of

very small

amounts

of

target

DNA.

Thus it is essential that the probe be equipped with a very sensitive detection system. The

most conventional approach is to

use

a

radiolabeled

probe.

32 P

is

the

most

common

choice because its short half-life and high energy create a very intense radiodecay signal.

The key to the use of

32 P is to incorporate the

label

at very

high specific activity at many

places in the probe. High specific activity means that many of the phosphorous atoms

in

the probe

are

actually

32 P

and not

31

P. The most

common

way

to

do this is random prim-

ing of DNA polymerase by short oligonucleotides to incorporate [alpha

32 P]-labeled de-

oxynucleoside triphosphates ([

32 P]dpppN’s) at many places in the chain. There is a trade-

SENSITIVE DETECTION

91

off between priming at too

many places, which gives probes too short to

hybridize well,

and priming too infrequently, which runs the risk of not sampling the probe uniformly. In

principle,

the highest

specific

activity

probes would

be

made

by

using

all

four

32

32

P]dpppN’s are usually employed.

[ P]dpppN’s. In practice, however, only one or two [

Attempts to make even hotter probes are usually plagued by technical difficulties.

The short half-life

of

32 P

is

responsible

for

its

intense signal, but it also leads the

major disadvantage of this detection system: The probes, once made, are rapidly decay-

ing. A second disadvantage of very high energy radioisotopes like

32 P is that the decay

produces a track in a photographic film or other spatially resolved detectors that can be

significantly longer than the spatial resolution of the target sample. This blurs the pattern

seen in techniques

like

autoradiographic detection of high-resolution DNA sequencing

gels or in situ hybridization analysis (Chapter 7) of gene location on chromosomes. To

circumvent this problem, lower-energy radioisotopes like

35 S-labeled thio-derivatives of

dpppN’s can be incorporated into DNA. However, to avoid the disadvantages of radioiso-

topes altogether, many alternative detection systems have been explored. A few will be

discussed here. The principal concern with each is the ratio of the target signal to the

background signal. The advantage of radioactive detection is that there is no intrinsic

background from the sample (just a counting background due to cosmic

rays and

the

like). This advantage disappears when chemical or spectroscopic detection methods are

used.

After

32 P,

fluorescence

is currently

the

most popular method

for

DNA detection,

with

SENSITIVE DETECTION

93

There are a number of different ligands and polyvalent reagents that have been widely

used for nucleic acid analysis. Several of the reagents are illustrated in Figure 3.24.

Potentially the most powerful, in several respects, is the combination of biotin and strepta-

vidin or avidin. Biotin can be attached to the 5

- or 3 -ends of nucleic

acids, as

discussed

earlier, or it can be incorporated internally by various biotinylated dpppN analogues.

Streptavidin and avidin bind biotin with a binding constant

K

a

approaching 10

15 under phys-

iological conditions. The three-dimensional structure of streptavidin is known from two X-

ray crystallographic studies (Figure 3.25). Since

avidin

and

streptavidin are

tetravalent,

there is a natural amplification route. After attaching single streptavidins to biotinylated

probes, the three remaining biotin-binding sites on each protein molecule can be coupled to

a polyvalent biotinylated molecule such as a protein with several biotins. Then more strepta-

vidin can be added, in excess; the network is grown by repeats of such cycles (Fig. 3.26

a ).

In practice, for most nucleic acid applications, streptavidin, the protein product from the

bacterium

Streptomyces avidinii,

is preferred

over

egg

white avidin

because

it has

an iso-

electric point near pH 7, which reduces nonspecific binding to DNA, and because it has no

carbohydrate, which reduces nonspecific binding to a variety of components and materials

in typical assay configurations.

Most

other

stoichiometric

amplification systems

use monoclonal

antibodies

with

high affinity for haptens that can easily be coupled to DNA. Digoxigenin and fluorescein

are the most frequent choices. After the antibody has been coupled to the hapten-labeled

DNA, amplification can be achieved by sandwich techniques in which a haptenated sec-

ond antibody, specific for the first antibody, is allowed

to bind, and then additional

cy-

cles of monoclonal antibody and haptenated second antibody are used to build up a net-

work. An example in the case of fluorescein is shown in Figure 3.26

b. Other haptens that

have proved useful for such amplification systems are coumarin, rhodamine, and dinitro-

phenyl. In

all of

these cases a

critical concern

is the

stability of the network. While anti-