Macromolecules 1994,27, 6061-6067
6061
Periodic Motion of Large DNA Molecules during Steady Field Gel Electrophoresis Hidehiro Oana, Yuichi Masubuchi, Mitsuhiro Matsumoto, Masao Doi,' Yukiko Matsuzawa,' and Keuichi Yoshikawat School of Engineering and Graduate School of Human Informatics, Nagoya University, Nagoya 464-01, Japan Received April 26,1994; Revised Manuscript Received July 14,1994. ABSTRACT Direct observation of individual T4 DNA moleculesduring steady field gel electrophoresiswas carried out with fluorescencemicroscopy. Statistical analysesof the image data show that (i) DNA molecules change their conformation and velocity periodically even under a steady field, (ii) the characteristic time of theperiodicmotiondecreaseswithanincreaseoftbeelectricfieldand withadecreaseofthegelconcentration, and (iii) the rear end of DNA shows a periodic stickalip motion, while the front end moves with almost constant speed. A possible interpretation for the periodic behavior is presented. 1. Introduction
Ag-AgCI
Gelelectrophoresis is atechnique widely used toseparate DNA fragments of different lengths. Despite its wide usage, the detail of the underlying physics is not fully understood. The migration velocity of a DNA is a nonlinear function of the applied field, and its theoretical prediction has not been successful, especially when the applied field is time dependent.1.2 A large number of studies have been made to understand the nonlinear transport phenomena in the gel electrophoresis. A powerful technique is fluorescence microscopy? which enables a direct observation of the motion of DNA molecules during gel electrophoresis. By this technique, it has been found that even in the steady field, the dynamics of the DNA molecules is rather complex.~g As a DNA migrates through a gel network, it repeats a cycleof elongation and contraction. The DNA iselongated when it is hooked by an obstacle, while it contracts when it slides off the obstacle. This behavior has been described as a cyclic or inch-worm motion. Similar behavior of DNA has also been found by computer simulation^.'"^^ Deutsch et a1.10 solved a Langevin equation for a DNA chain in a field of obstacles and found that the velocity and the radius of gyration of DNA fluctuate violently around the mean value. Essentially the same phenomena have also been observed for other models."-14 Furthermore, it has been conjectured that the cyclic motion is related t o the phenomena of the minimum in the mobility in the field inversion gel electrophoresis (FIGE).'5 While the dynamics of DNA has been studied extensively by computer simulations, there have been few quantitative studies on the actual conformational change of DNA molecules. We have conducted a statistical analysis of the video images of DNA molecules obtained by fluorescence microscopy and reported preliminary results.9 In this paper, we describe the results in more detail and also give their gel concentration dependence. 2. Experimental Section 2.1. Materials. Bacteriophage T4 DNA (166kbp; contour length, 55pm) waspurcbasedfrom Nippon Gene. Agarose (FMC SeaKem LE) was dissolved in distilled water, using a microwave
*To whom correspondence should be addressed. 'Graduate School of Human Informatics. mAbstractpublished in Advance ACS Abstracts, September 1, 1994.
0024-9297/94/2227-6061$04.50/0
L-
electrodes
4-
microscope
Ag~electrodes 4m 0 m ~
Figure 1. Schematic cross-sectional new of the gel electrophoresis cell speciallydesigned for direct observation. The buffer wells,each withanAgelectrode.are filled with XO.5TBE buffer. oven. After the agarosesolution was cooled to about 60 OC, Trisborate (TBE, pH7.8) buffer, T4 DNA, ethidium bromide (EB intercalatingfluorescent dye), and 2-mercaptoethanol(%ME,as an antioxidant) were added to the solution. The final concentrations are as follows: T4 DNA, 0.15 pM (in nucleotide); EB, 0.15 p M , Tris-borate, 45 m M EDTA, 1.25 m M 2-ME, 4% (v/v); agarose, 0.75% (w/v), 0.9% (w/v). 1.25% (w/v), or 1.5% (w/v). Under these conditions, the intercalated EB has a negligibleeffect on the static properties of DNA.18J7 2.2. Gel Electrophoresis and Direct Observation. The observation of DNA molecules during gel electrophoresis was carried out in a specially-designedgel electrophoresiscell shown in Figure 1. The cell was prepared as follows. A DNAiagarose solution was placed on a 30 mm X 40 mm glass plate and covered by another glass plate of the samesize,and then the whole solution was cooled to be a gel. The thickness of the agarose layer was about 100 pm, which is sufficient to eliminate boundary effects of the glass plates.18 The electric field was applied by a potentiostat through apairofsilverwire electrodes40 mmapart. To compensate the polarization effect of the Ag electrodes, the cell had another pair of electrodes (Ag-AgCI) 32 mm apart, The Ag-AgCI electrodes monitor the actual field strength in the observation area, and the applied field strength was adjusted to keep the monitored field constant. The gel was excited by 520 nm UV, and individual DNA molecules were observed as fluorescenceimages with an inverted microscope (Nikon TMD) equipped with a X l o O oil-immersed objective lens. The images were recorded on S-VHS video tapes with a high sensitivity SIT camera and an image processor,Argus10 (HamamatsuPhotonics). Two series of experiments were done. First, the gel concentration C,I was fixed at 0.9 wt % and the strength of the electric field E was changed as 2,4,6, and 8 Vicm. Second, E was fixed at 8 V/cm and C,I was varied as 0.75,0.9,1.25, and 1.5 w t %. All experiments were done at room temperature (about 20 1 OC). 2.3. Image Analyses. The recorded images were processed by a programmable image analysis system PIAS3 (PIAS) which
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0 1994 American Chemical Society
6062 Oana et al.
Macromolecules. Vol. 27,No. 21, I994
converts the analog image data into digital data of 512 X 512 pixels with 256 degrees of brightness. After eliminationof the backgroundnoisewitbfiiltersofPIASS, the DNA image function I(x,y,t),which takes 1 if the pixel ( x , y ) is brighter than a certain threshold or 0 if it is not, is obtained as a function oft. Here the x-axis is taken along the direction of the applied field. Then, the position vector of the center of mass of a DNA molecule r ( t ) = (rAt),r J t ) ) was calculated by
I I1 111 I V
v VI
10
3.0 2.5
8
2.0 R,
vi 6 (msi 4
1.5 Ivmi 1.o
2
0.5 0.0
0 0
1
2
3
4 S I (Si
f
i
7
8
Figure 2. Example of the time development of u,, RI, and R. of T4 DNAduringgelelectrophoresis. The fieldstrengthis8V/cm, and the gel concentration is 1.5 wt %. I
where
TI
111 IV
v
VI
(3)
From the series of data r ( t ) ,the center of mass velocity uJt) along the applied field was computed by r,t+ UJt) =
(
At
a
Figure 3. Digitized images of T4 DNA at the stages I-VI in Figure 2. The scale bar is 20 pm long.
r,t--At
; I At- (
(4)
Although the video image was obtained every 8, At was taken to be 0.4 8 , since small At gives large statistical fluctuations of um. To characterize the size of a DNA molecule, the radii RI and
R. (RI > R.) which are along the two principal axes repsectively
were computed as Rl(t) = [;(M,.
+ M,) + z[(M.. 1 - M,)'+
4Mz~11/2]1/2 (5)
where Mx=,Mn, and MZyare the components of the twodimensional radius of gyration tensor defined as
3
We use RI and R. separately here instead of Rz = lZ (R? + R,2)1'/* as in ref 9 to characterize the size of DNA, because thus defined R, overestimates the radius of gyration when the DNA is extremely elongated in one direction. Tracing the image of each DNA molecule was possible for about 25 s at most in the usual measurement, due to the effect that DNA molecules go out ofthe observation area (about 80pm X60pm)byelectrophoresisorgooutoffocusbyverticaldiffusion. The sampling interval was 0.1 8. From the observed data, ahout 10 sequences of images (i.e., 10molecules) at each condition were chosen for further analyses.
3. Results 3.1. Image Data. Figure 2 is an example of the time development of u,(t), Rdt), and R&). It is seen that Rdt) and u,(t) show large fluctuations from the average value, while fluctuations of R,(t) remain rather small. As has been reported already,Pg the large fluctuations of Rl(t) and u,(t) are related to the characteristic motion of DNA. Figure 3 shows the actual conformations of DNA at each stage indicated by I-VI in Figure 2
Figure 4. Ensemble averages of ur of T4 DNA under various field strengthsE. The gel concentrationis 0.9 wt 7%. Error bars are the standard deviation calculated for the set of molecules at each condition. A functionu, = E'.=is shown by the solid curve.
Stage I. A DNA molecule in the gel has a coiled conformation. A t this stage, R1 and R. are about the same size as those in buffer solutions."JS Stage 11. The DNA begins to be stretched since ita middle part is caught by a gel fiber. StageIII. The A-conformationofthe DNAgrows with its apex fixed at the gel fiber. A t this stage, the migration velocity u,(t) becomes the smallest. Stage IV. The longer arm in the A-conformation becomes dominant and begins to pull the other arm. Consequently, the dominant arm becomes longer and the shorter one becomes even shorter. Stages V and VI. The DNA is released from the gel fiber and shrinks rapidly, retrieving the coiled conformation. During this process, the velocity of migration becomes a maximum. Before going to the detailed discussion on such timedependent conformational change, we first describe the results of the time-averaged quantities. 3.2. Average Velocity a n d Average Size. Figures 4 and 5 show the average velocity as a function of the field strength - E and the gel concentration C,l. The velocity u, increases in proportion to Emwith n 1.5 and decreases in proportion to C& with @ -0.9. These results are consistent with the standard macroscale gel e l e c t r o p h ~ r e s i s . ' ~The ~ ~ values of are also in reasonable agreement with reported values obtained by the usual gel electrophoresis; for example, Shikata and KotakaZ3 reported that is ahout 0.6 pm/s under E = 2.5 V/cm and
