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

INADEQUACY OF REPTATION MODELS FOR PFG

151

where

A z is the absorbance with

polarizers set parallel to the field, and

A y is the ab-

sorbance with perpendicular polarizers. This means that the net LD of oriented DNA will

be negative, as shown in Figure 5.19. The magnitude of the LD is a measure of the net lo-

cal orientation of the DNA helix axis.

The LD of DNA in the absence of an electrical field is zero because there is no net orien-

tation. When the LD of DNA in a gel is monitored during the application of a square wave

electrical field pulse, very surprising results are seen. What was expected is shown in Figure

5.20a :

a monotonic increase in

LD until the orientation saturates; then

a monotonic

Figure 5.20 Linear dichroism of DNA in a gel, as a result of a single applied electrical field pulse

of sufficient intensity to cause a saturating level of orientation.

(a) Expected result.

(b) Observed re-

sult (from Nordén et al., 1991).

modeled as a set of rigid obstacles. Most early simulations were done in

two dimensions

to limit the amount of computer time required. This is unlikely to allow a realistic picture

of either the gel or the DNA. However, more recent three-dimensional simulations are at

least in

qualitative

agreement

with the earlier two-dimensional results. Undoubtedly

the

model

of the gel used in

these

simulations

is unrealistically

crude;

the potential

used to describe the interaction

of

the

DNA

with

the gel is hopelessly oversimplified.

The

DNA model itself

is not terribly

accurate.

Despite all these reservations, the

picture

DNA

TRAPPING ELECTROPHORESIS

155

Such

increases

in

computation

speed are needed because two additional

variations

of

PFG show considerable promise for enhanced size resolution, but

each

of

these intro-

duces additional complications into both the experiments and any attempts

to simulate

them.

DNA TRAPPING ELECTROPHORESIS

The technique of DNA trapping

electrophoresis

was

devised by

Levi

Ulanovsky

and

Walter

Gilbert

as

an approach

to improving

the

resolution

of

DNA

sequencing

gels.

of the pore in order for

net motion to occur. Above a certain DNA size, thermal

energies

may be insufficient to

allow

such

backtracking,

and once

trapped,

the

tethered DNA

might remain so indefinitely.

A

typical experimental

result

is shown

in

Figure 5.25.

Figure 5.26

Effect of a bulky end label on

DNA mobility

in gels.

(a)

Comparison of DNA with

DNA terminally labeled with streptavidin.

(b)

Trapping

of DNA in a pore because of the

bulkiness

of the streptavidin.

in Figure 5.23.

If the picture just described is

accurate, one can make the further prediction that

FIGE will assist the escape of the

trapped DNA. These results are shown

in Figure

5.27. The three curves in this figure

show the size dependence of DNA mobility under

three different FIGE conditions with

DNAs tethered to streptavidin. Very slow FIGE

leads to a reappearance of the limiting mobility. Apparently under these conditions all

trapped molecules can be rescued. Very rapid FIGE fails to remove any trapping

effects.

SECONDARY PULSED FIELD GEL ELECTROPHORESIS (SPFG)

157

Apparently

these pulses are too short

to allow the trapped molecules to escape.

Intermediate FIGE conditions produce a very impressive increase in the useful separation

range of the gel. Overall, DNA trapping electrophoresis appears to be a very interesting

idea

worth

further elaboration and pursuit. It

underscores

the

fact

that

trapping,

and

not sieving, appears to be the dominant effect underlying DNA electrophoretic size sepa-

rations.

SECONDARY PULSED

FIELD

GEL

ELECTROPHORESIS

(SPFG)

The

history

of

SPFG

is

amusing.

Tai

Yong

Zhang,

a technician then working with

us

in the

early 1990s, was instructed to perform the experiment shown schematically

in

Figure

5.28. The

notion

was

to

use

a series

of

rapid field

alternations to

perturb

the

motion

of the head of an oriented DNA chain moving

in

response

to

a

slowly

varying field. The result should be to

fold

the chain into a zigzag pattern, which ought

to

affect

its subsequent orientation kinetics markedly

when

the

primary

field

direc-

tion is switched. Zhang, whose English

was quite imperfect at the time, misunderstood

the

original

instructions and

did

the

experiment

shown

schematically in

Figure 5.29

a .

He

applied

periodic, short

intense

pulses,

along the direction of net

DNA

motion. At

about

the

same time Jan Noolandi’s group

did a similar experiment, shown in Figure

5.29 b .

They

applied short, intense pulses opposite

to

the

direction of net DNA motion.

The

results

of both experiments, now called SPFG, are quite similar. The overall rate of

DNA motion is dramatically increased. In addition, under some SPFG conditions, greatly

improved resolution is seen, and larger molecules can be handled than is possible with

conventional PFG alone under comparable field strengths and primary pulse times. Some

examples are shown in Figure 5.30.

While the actual mechanism for the success of SPFG is unknown, one suggested

mechanism shown in Figure 5.31 seems quite reasonable. DNA in PFG spends most of its

time

trapped

on

obstacles. As long

as

the

field

direction

remains constant,

the

applied

Figure 5.30 Examples of the effect of secondary pulses on PFG behavior. Separation of chromo-

somal DNA molecules in the size range between 50 kb and 5.8 Mb by SPFG. Samples are (lane 1)

S. pombe,

(lane 2) Pichia 1A which consists of a mixture of

P. scolyti

and P. mississipiensis,

(lane

3)P. scolyti,

(lane 4)P. mississippiensis,

(lane 5)S. cerevisiae,

and (lane 6) lambda concatemers.

Separation on a 1% agarose gel using

(a) a pulse program as follows: (i) 4800 s, 2.2 volts/cm pri-

mary pulses with 1 : 15 s (1 second pulse every 15 seconds), 6 volts/cm secondary pulses for 12 h,

(ii) 2400 s, 2.8 volts/cm primary pulses with 1 : 15 s, 6 volts, cm secondary pulses for 12 h, (iii) 240

s, 6 volts/cm primary pulses with 1 : 15 s; 10 volts/cm secondary pulses for 21 h, and (iv) 120 s, 6

volts/cm primary pulses with 1 : 15 s, 10 volts/cm secondary pulses for 10 h.

(b) The same primary

pulse program shown in

(a) but without secondary pulses. (From Zhang et al., 1991.)

Primary field strength

Agarose gel concentration

Primary pulse time

Particular type of agarose (low melting, high

Angle of alternation of primary fields

endoosmosis, etc.)

Secondary field strength

Ionic strength

Direction of secondary field

Temperature

Secondary pulse time

Specific ions present (e.g., acetate versus borate)

Phase between secondary and primary fields

Homogeneity of primary field

Homogeneity of secondary field

One

possible

consequence of the way in which

large

DNA is

made and

the

obstacles it must overcome to enter a gel

is shown

schematically

in

Figure

5.33.

Vaughan Jones,

a

mathematical topologist at

the University of California, Berkeley,

in

thinking

about large

DNA

molecules,

suggested

in

1989

that

above

a critical

DNA

size

the

conformation

of

these chains

would

always

contain

at

least

one

mation

poses

no problems inside cells where topoisomerases exist in abundance that

can tie

and

untie knots at will. Similarly it is no problem for an isolated DNA mole-