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

A number of variants of PFG exist where multiple field directions are used. These are

DNA size on FIGE mobility is complex, as we will demonstrate, and it is also rather sensitive to overloading by too high sample concentrations.

Figure 5.11 Schematic of a contemporary PFG apparatus in which computer-controlled individual electrodes can be used to generate a variety of field shapes in a single apparatus.

146 PRINCIPLES OF DNA ELECTROPHORESIS

MACROSCOPIC BEHAVIOR OF DNA IN PFG

The results of a large number of systematic studies on DNA mobilities in PFG are summarized in Figures 5.12 through 5.14. The major variables explored have been DNA size, the field strength, the angle between the fields, and the pulse time. Other parameters are also known to be important, such as the gel concentration (and the details of the type of agarose used), the buffer type, the ionic strength, and the temperature. These will not be

considered further. The effect of angle is relatively slight in the range around 120°. However, regular oscillation of homogeneous electrical fields between two sets of parallel

electrodes 90° apart does not produce PFG fractionations. More complex sets of 90° pulses, with varying durations or field strengths, or multiple directions, have been shown

to be effective under some circumstances.

The PFG behavior of DNAs up to about a Mb in size with 120° alternating fields is shown in Figure 5.12 as a function of pulse time. A very simple picture suffices to explain these results, but it begs the question of the details of how DNA moves in a gel. At very long pulse times, the process of reorientation should require an insignificant fraction of each pulse period.

As a result the DNA moves by essentially ordinary electrophoresis, in a zigzag pattern centered along the average of the two field directions. Because ordinary electrophoresis is dominant, there is no net effect of DNA size on mobility; the DNAs are essentially fully oriented almost all the time. At very short pulse times, the field changes direction much more rapidly than the DNA molecules can reorient. They experience a constant net field which is just the vector sum of the two distinct applied fields. They move in response to this net average field by ordinary electrophoresis. Since they become oriented and elon-

gated, their net mobility is size independent and in fact is the same as their mobility at very long pulse times.

At intermediate pulse times, DNA reorientation processes occupy a significant fraction of each pulse period. This leads to a marked decrease in overall electrophoretic mobility. The pulse time at which mobility is a minimum increases roughly linearly with DNA size. Larger DNAs have a progressively broader response to pulse time. The result is that one can find pulse times that afford very good resolution of particular DNA size classes.

Figure 5.12 PFG mobility as a function of pulse time for DNAs of different size, observed using 120° alternate field directions.

148 PRINCIPLES OF DNA ELECTROPHORESIS

Figure 5.15 The ratchet model for PFG, proposed by Edwin Southern of Oxford University.

effectively explained why 90° angles might be ineffective, and why mobility should decrease linearly with DNA size. This ratchet model of DNA motion in gels is shown in Figure 5.15. In the ratchet model the head of the moving DNA is oriented along the applied field direction. When the field direction is switched, the molecule attempts to reorient by a reptation motion. If this were led by the head, it would require that the chain bend through an acute angle. It would seem easier, instead, for the tail to lead the reorientation, since that would require bending the chain through a much less sharp obtuse angle. This leads to retrograde motion until the entire chain has changed orientation. The ratchet model leads directly to a linear dependence of mobility on DNA size because the mobil-

ity decreases with the length of the retrograde motion. It also justifies the poor perform-

INADEQUACY OF REPTATION MODELS FOR PFG

Three specific observations of macroscopic DNA behavior are very difficult to reconcile

Figure 5.16 Behavior of DNA in ordinary electrophoresis at high field strengths in dense gels: Mobility, , as a function of molecular weight, M.

The second perplexing observation is DNA trapping. For DNAs with sizes above a particular threshold, at high enough fields no PFG motion could be observed. The threshold size decreased with increasing field strength. This DNA trapping implies more complex DNA-gel interactions than contained in simple reptation models. A final, devastating observation was made in ordinary DNA electrophoresis in high concentration gels at very high field strengths. Here it was observed that, under some conditions, the mobility of DNA was no longer a monotonically decreasing function of DNA size. Instead, at a particular DNA size, a minimum

in mobility is observed, just as in FIGE, even though in these experiments a constant, uniform applied electrical field was employed (Figure 5.16).

Three different approaches have been used to examine in detail the nature of DNA motions in gels under the influence of electrical fields. All of these produced unexpected results, totally inconsistent with simple reptation pictures. This was true, even in ordinary

electrophoresis with constant fields, or pulsed fields in a single direction. Several different groups reported, almost simultaneously, the detection, by UV fluorescence microscopy, of single DNA molecules undergoing gel electrophoresis. In order to do these experiments, the

DNAs were prestained with an intercalating dye like acridine orange or ethidium bromide.

dyes, except for the predictable consequences of this increase in length.

When observed in the microscope at constant field strength, the motion of DNA molecules was very irregular. In the absence of a field, the molecules were coiled and moderately compact (Figure 5.17). This is expected from random walk models of polymer chain statistics. On application of an electrical field, the molecules elongate, orient, and move parallel to the field. However, the head soon collides with some obstacle in the gel. It stops moving, but the rest of the molecule does not. As a result the elongated chain collapses; the tail may even overtake the head. A very condensed configuration is formed

may pull free, and the result is a very elongated, tethered structure. Finally the motion of one end dominates; the DNA slips free of the obstacle and starts to run as an elongated aligned structure until it impacts on the next obstacle. Thus the DNA spends most of its

150 PRINCIPLES OF DNA ELECTROPHORESIS

Figure 5.17 DNA behavior in conventional gel electrophoresis as visualized by fluorescence microscopy of individual stained DNA molecules.

There may be DNA sizes where unhooking from obstacles is especially difficult at particular field strengths. This would explain the minimum in ordinary electrophoretic mobility seen macroscopically for certain DNA size ranges at high field strengths in dense gels. When DNA molecules undergoing PFG are viewed by fluorescence microscopy, addi-

tional unexpected behavior is seen (Fig. 5.18). When the field is rotated by 90°, DNA molecules respond to the new direction not by motions of their ends but by herniation at

several internal sites. This produces a series of kinks which start to move in the direction of the new field. These kinks grow and compete. Eventually one dominates, and this becomes the leading edge of the moving DNA. At some subsequent point the hairpin struc-

ture presumably unravels, and the DNA attains full elongation again. Note that this picture is quite at odds with the reptation model where the DNA is supposed to remain in its

tube, except for motions at the ends. It suggests that a tube model is not at all appropriate for DNA in a gel.

The second experimental approach that revealed unexpected complexities of DNA behavior in gels was measurement of bulk electrophoretic orientation by linear dichroism

(LD). Here the absorbance of polarized UV light by DNA in gels in the presence of an electrical field was measured. The base pairs of DNA are the dominant absorber of near-

UV light (wavelengths around 260 nm). As shown in Figure 5.19, the base pairs preferentially absorb light polarized in the plane of the bases. DNA tends to orient in an electrical

field with the helix axis parallel to the field. The LD is defined as

LD A z A y

Figure 5.18 Fluorescence microscopic images of DNA in a gel after a 90° rotation of the electrical field direction. Shown from left to right are successive time points.