Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics1-10 / 5

Figure 5.1

Schematic illustration of separation

(from top to bottom of a tube)

of DNAs of differ-

ent base composition by density gradient equilibrium ultracentrifugation;

is density, Absorbance

(A 260) is proportional to DNA concentration.

gation is topological properties. Velocity sedimentation

can be

used to

discriminate

among linear DNAs, relaxed circles, and supercoiled circles. Equilibrium density gradient

separations are also very powerful for this discrimination because, under the right circum-

stances, supercoils will bind very different amounts of intercalating dyes, like ethidium

bromide, than bound by relaxed circles or linear duplexes (see Cantor and Schimmel,

1980, ch. 24). This leads to large density shifts. Thus such fractionations have played a

major role in early studies of DNA topology, and they have served as major tools for the

separation of plasmid DNAs key in many recombinant DNA experiments. Once again,

however, as the need for dealing

with very large numbers of samples in parallel grows,

these centrifugation methods have to be replaced by others. The capacities of existing

centrifuges simply cannot cope with large numbers of DNA samples, and loading and un-

loading centrifuges is a procedure that is very difficult to automate efficiently. In addition

even the best protocols for DNA size or topology separation

by ultracentrifugation have

far lower resolution than typical electrophoretic methods.

ELECTROPHORETIC

SIZE

SEPARATIONS

OF DNA

In contemporary methods of DNA analysis, electrophoresis dominates size fractionations

for all species from oligonucleotides with a few bases up

to intact

chromosomal DNAs

with sizes in excess of 10

7 bp. Rarely does a single method

prove to be so powerful

across such a broad size range. The size resolution for optimized separations is a single

base up to more than 1000 bases for single-stranded DNAs, a few percent in size for dou-

ble strands up to about 10 kb, and progressively lower resolution as DNA sizes increase

until, for species in excess of 5 Mb, only about 10% size resolution is available.

Size

is

the

overwhelming

factor that determines the separation power of double-

stranded DNA electrophoresis. In general, double-stranded DNA fractionations are car-

ried out in

dilute aqueous buffer

near neutral pH. Base composition

and base

sequence

ELECTROPHORESIS WITHOUT GELS

133

Figure 5.2

Behavior of highly charged

molecules in electrophoresis.

(a)

Structure of single-

stranded DNA without denaturants, or in the presence of strong denaturants where charge and fric-

tion are both proportional to length.

(b) Structure of short double-stranded DNA, where charge and

friction are both proportional to length.

(c) Proteins, denatured by SDS, form micelles where charge

and friction are both proportional to length.

sequence in the special case of sequences that

lead to pronounced DNA bending. The

most common example are AA’s or TT’s spaced at intervals comparable to the helical re-

peat distance. When this occurs, the slight bends at each AA are in phase, and the overall

structure can be markedly curved.

Electrophoretic fractionations of single-stranded DNA are mostly carried out in the

presence of strong denaturants such as high temperature or high concentrations of urea or

formamide. Under these conditions most of the potential secondary structure of the DNA

strands is eliminated, and size dominates the

electrophoretic separations (Fig.

5.2

a ).

Sometimes, however, single-stranded DNA electrophoresis is carried out under conditions

that allow substantial secondary structure formation. In this case it is the extent of folded

structure that tends to dominate the separation

pattern. We will discuss these cases

in

MOTIONS OF DNA MOLECULES IN GELS

135

Figure 5.3

Convective instabilities in electrophoresis.

(a) Vertical electrophoresis.

(b) Horizontal

electrophoresis.

little

to do

with the above considerations. Almost all electrophoresis is done with gels or

other

support

matrices, even when the molecules involved do not behave as free draining.

Figure 5.4

Motion

(shown by arrows)

of molecules in gels.

(a) Gel filtration (molecular

sieve

chromatography) where large molecules elute more rapidly than small molecules.

(b)

Gel elec-

trophoresis where small molecules migrate more rapidly than large molecules.

volume as they

move through the gel, since they can meander through both the gel matrix

and the spaces

between the particles. As a result their net motion is considerably retarded.

Thus large species elute more rapidly than small species in gel filtration chromatography.

COMPLEX EFFECTS OF GEL STRUCTURE

AND BEHAVIOR

137

teraction with the solute are not considered at all. However, for DNA electrophoresis we

now know that the details of the gel structure, its behavior, and its interactions with the

solute matter considerably. Unfortunately, our current knowledge about the structure of

gels, and the ways macromolecules interact with them, is very slight. For example, exper-

iments have shown that the gels used for typical DNA separations can respond directly to

electrical fields. The result is to change the orientation of gel fibers in order to minimize

their interaction energy with the applied field. In general, macroscopic measures of gel

fiber orientation show that these direct gel-field effects occur at field strengths higher than

those typically employed is most electrophoresis.

When DNA is present, applied electrical fields lead to changes in the gel not seen in the

absence of DNA. These changes presumably reflect distortion and orientation

of the gel

caused by motion of the DNA and direct gel-DNA interactions. It is not known if DNA has

any indirect effect on the interaction between the gel and the electrical field. What is even

less clear is whether the effects of DNA electrophoresis through the gel matrix lead to any

irreversible changes in the gel

structure. Certainly the overall forces of interaction between

a highly charged macromolecule and an obstructive stationary phase could be considerable.

We know that gels

can usually not be reused more than a few times for

electrophoresis

without a serious degradation in their performance. This is attributed to some breakdown in

necessary gel properties. How much of this is due to electrochemical attack on the gel ma-

trix, and how much to DNA-mediated damage, remains unknown.

The buffer system used to cast the gel, and used in the actual gel electrophoresis itself,

matters a great deal. For example, agarose gels cast or run in Tris (trihydroxymethyl-

aminomethane) acetate and Tris borate behave very differently. This is believed to reflect

the direct interaction of borate ion with cis hydroxyls on the agarose fibers. Finally the

specific monomers and crosslinkers used in gels, like polyacrylamide,

can have a pro-

found effect on gel running speed, resolution, and the ability of the gel to discriminate un-

usual DNA structures. For simplicity, we will ignore all of these complications in the dis-

cussion that follows.

In general, polyacrylamide-like matrices are used for small, single-stranded DNAs in

the size range of 1 to 2 kb. They are also used for small double-stranded DNAs. Agaroses,

with much larger pores, are used for larger double-stranded DNAs, ranging from 1 kb to

more than 10 Mb in size. These gel systems were not specifically designed to handle nu-

cleic acids, and there is no particular reason to think that they are anywhere optimal for

these materials. However, some alternative materials are clearly undesirable for DNA sep-

arations because they cannot be made sufficiently free of nucleases or

because they

demonstrate nonspecific adsorption of DNA molecules.

The ultimate solution for DNA separations may be to use micro-fabricated separation

matrices. Here one uses techniques like microlithography to manufacture a custom-

designed surface or

volume containing specific

obstacles to modulate DNA

movement

and prevent gross convection. Robert Austin at Princeton University has recently demon-

strated the electrophoresis of DNA on a microlithographic array. He constructed a regular

array of posts spaced

at 1-

intervals, sticking up from

a planer surface. In the presence

of an electrical field, DNAs in solution move through these posts (Fig. 5.5). The much

larger DNA molecules tend to get hooked on the posts; they eventually are pulled off and

net motion through

the array

continues. This

carefully controlled situation appears

to

BIASED REPTATION MODEL

OF DNA BEHAVIOR IN

GELS

139

This redefines the tube. The model has the DNA behaving much in the same way as a

snake in a burrow; hence the term reptation from the word reptile.

In polymer diffusion, when the head of the chain emerges from

its

tube, it makes

a

random choice from among the pores available to it (Fig. 5.6

b ). The elegant

simplifica-

tion offered by the reptation model is that to explain DNA motion,

one need only con-

sider what is happening at the head and the tail. The remainder of the complex network of

pores, and the detailed configuration of the DNA (except for the overall length of

the

tube) can be ignored. Indeed this model works very well to explain polymer diffusion in

condensed phases. To adapt the reptation model to electrophoresis, it

was

proposed that

the influence of the electrical

field biases the choice of pores made by

the head of the

DNA molecule (Fig. 5.6

c ). In this biased reptation model, one only needs to consider the

knowledge of the detailed configuration of the DNA or the gel pores.

The biased reptation model makes the

following predictions:

For

relatively

small

DNAs (small for a particular combination

of field strength and

gel

matrix),

diffusion

tends to balance out most of the effects of the applied field. The DNA molecule retains a

chain configuration that is highly coiled, and the ends have a wide

choice of

pores. For

much larger DNAs, the electrical field dominates over random diffusion. The molecule

becomes highly elongated and highly oriented. The ends have a relatively small choice of

pores, and there is more frictional interaction between the DNA and the pores. The pre-

dicted result of this on electrophoretic behavior is shown in Figure 5.7. Most experiments

conform very well to this prediction. At a given field strength, up to some critical DNA

length, the mobility drops progressively with DNA size. However, once a point is reached

where the DNAs are fully oriented, they all move at the same speed. For typical agarose

gel electrophoresis conditions, this plateau occurs at around 10 kb. Fully oriented mole-

cules in gels display a constant charge and friction per unit

length;

thus

their elec-

trophoretic mobility is size independent.