Electrophoretic mobility of DNA in gels. III ... - Wiley Online Library

Christoph Heller Jean Louis Viovy Ecole Superieure de Physique et de Chimie Industrielles de la Vile de Paris Laboratoire de Physico-Chimie Thhrique 10 rue Vauquelin 75231 Paris Cedex 05, France

Electrophoretic Mobility of DNA in Gels. 111. Experimental Study on Band Inversion

A series of experiments has been undertaken to study the phenomenon of band inversion that can occur during the separation of linear double-stranded DNA in agarose gels under constant electricfield. Wefound that there can be a considerableband inversion when the DNAfragments are moving as a single species. When separating mixtures offraments, as it is usually done in routine experiments, the band inversion effect is strongly reduced. Our data support the assumption that DNA-DNA interactions can play an important role in electrophoreticseparations. 0 I995 John Wiley & Sons, Inc.

INTRODUCTION Gel electrophoresis is routinely used to separate linear double-stranded DNA fragments of different size. Generally, the evaluation of the gels, i.e., the assignment of bands, is done under the assumption that there is a monotonous relationship between DNA size and electrophoretic mobility. However, this is not always the case. This was found out first with the introduction of field inversion gel electrophoresis, where a nonmonotonous behavior (band inversion) can be observed.1,2 Soon afterward, by using a computer simulation of the biased reptation model (BRM), Noolandi et al. found that even under constant electric field conditions a band inversion may occur, and they also observed this phenomenon e~perimentally.~-’Band inversion was also reported to take place in the separation of single-stranded DNA in denaturing polyacrylamide gels6 This behavior can be explained by “self-trapping” of DNA molecules. As a result of thermal fluctuations, during migration a DNA molecule Received May 2, 1994;accepted October 10, 1994. Biopolymers, Vol. 35,485-492 ( 1995) 0 1995 John Wiley & Sons, Inc.

can assume a compact U-shaped conformation, where both ends are close to each other in the direction of the electric field. In this case, the electric forces acting on the molecule cancel each other and the DNA will have a zero mobility. Detrapping can only be induced by thermal motion. Therefore both the probability of occurrence as well as the lifetime of those conformations are determined by fluctuation^.^^^ The lifetime of such U-shapes increases with the size of the DNA, whereas the probability of occurrence decreases. This means that the product of the lifetime and probability of the Ushapes will be maximum for medium-sized molecules and they will be trapped during a long time. Hence, they will be retarded and a mobility minimum will be observed. If the chain is stretched, U-shapes become very unlikely. In order to minimize band inversion, chain stretching should be maximized, e.g., by using lower agarose concentrations or higher electric fields. Beside this qualitative explanation, it was also possible to give a quantitative description for the

CCC 0006-3525/95/050485-08

485

486

Heller and Viovy

band inversion, using the biased reptation m ~ d e l . ~ - ~ RESULTS Recently, the BRM was modified by Duke et al. to take into account longitudinal fluctuations of the Constant Field Electrophoresis Followed chain.” The explanation for the band inversion reby FlGE mains valid in this “biased reptation with fluctuaWe have undertaken a series of electrophoretic septions” (BRF) model; however, the predicted dearations of DNA fragments in a size range of 2pendence of the mobility minimum on the electric 48.5 kilobase pairs (kbp) at three different agarose field E is different. The BRM predicts that the moconcentrations and at constant electric field lecular weight M with minimum mobility varies 0.25 and 5 V/cm (“separation strengths between proportional to E P 2 ,whereas the BRF gives MFmin step”). The separated fragments were then sub-E-1 1 1 jected to a second electrophoresisperpendicular to Interestingly, the phenomenon of band inverthe first one, using field inversion, in order to sepasion during DNA separation in agarose gels under rate the fragments in the compression zone constant field conditions has only been discovered (“analysis step”). An example for such a separaa few years ago and has never been described by tion is shown in Figure 1a. other groups since then. Recently, we undertook a The results (summarized in Figure 2a-c) show systematic study of DNA mobility in agarose gels I* the well-known behavior of the mobility in depenand we also observed some band inversion, but dence of molecular weight. In 1 and 1.5% gels, much less pronounced as described earlier.3-5 band inversion cannot be observed at all, whereas Therefore we camed out a series of experiments in in 2% gels there is a slight band inversion at electric order to find out the reason for this discrepancy field strengths below 1 V/cm. These results are esand to check the predictions of the modified reptasentially the same (within the range of experimention model. To distinguish DNA molecules of tal error) as published recently, l 2 but are in condifferent size but with the same mobility without trast to earlier observations,3-5 where band inverthe need of hybridization, two-dimensional gel sion already took place at gel concentrations as low electrophoresiswas used. as 1.2 and 1.4%.

FlGE Followed by Constant Field Electrophoresis MATERIAL AND METHODS Electrophoresis was performed using low electroendosmosis agarose (Seakem LE, lot nos. 603393 and 63492, FMC) at concentrations of I , 1.5, and 2%in 0.5X TBE (pH 8.3), essentially as described.’’ All separations under constant electric field conditions were performed at 10°C with active temperature control and buffer recirculation, whereas field inversion gel electrophoresis (conditions: 0.5 s at 5 V/cm forward pulse, 0.5 s at 2.5 V/cm backward pulse, 1% agarose in 0% TBE) was done at room temperature. For two-dimensional gels, unstained slices of the first dimension were trimmed with a scalpel to about 5 X 0.5 X 0.5 cm, turned by 90” around their longitudinal axis, and cast into a new agarose gel.’ Separation at constant field was followed by field inversion gel electrophoresis (FIGE) or vice versa. As size standards, a mixture of X-DNA (Appligene) and a X-Hind111 digest (New England Biolabs-NEB) at a concentration ratio of 1.5:2.5, and a mixture of XDNA, X-XhoI digest (NEB), and X-Hind111 digest (NEB) at a concentration ratio o f 2 . 3 5 5 , were used. Restriction enzyme digestions were performed according to the instructionsof the suppliers.

In a second kind of experiment (example in figure l b ) , the DNA samples were first separated by FIGE ( “preseparation step”) and then subjected to constant field electrophoresis in a second dimension (“separation step”). In some cases, this separation step was done in the same gels as those used in the experiments described above, ensuring identical conditions (e.g., Figure 1 ). But even when different gels were used, the mobilities of the smaller (2-6 kbp) and larger (48.5 kbp) molecules, which are not retarded, were the same at the respective electric field strengths, indicating a good reproducibility. This kind of experimental procedure corresponds to the one employed by Noolandi and coworkers, 3-5 who used isolated DNA fragments rather than a mixture. In fact, as can be seen in figure 2d-f, electrophoresis of isolated DNA fragments results in a nonmonotonous relationship between mobility and molecular weight, in agreement with their results. The onset of the band inversion effect is a function of agarose concentration and electric field

Electrophoretic Mobility of DNA in Gels. 111

487

FIGURE 1 Two-dimensional electrophoresis of a mixture of DNA fragments (A, A-XhoI and A-Hind111) . (a) Constant field electrophoresis followed by field inversion electrophoresis. (b) Field inversion electrophoresis followed by constant field electrophoresis. Conditions: Constant field 2 V/cm, 2% agarose in 0.5X TBE at 10°Cfor 24 h. Field inversion: 0.5 s at 5 V/cm forward pulse, 0.5 sat 2.5 V/cm reverse pulse, 1% agarose in 0.5X TBE at room temperature for 8 h.

strength. In 1% gels it can only be observed at electric fields I0.5 V/cm; in 1.5% gels this limit increases to 2 V/cm, whereas at 2% a field of 3 V/cm is sufficiently low to induce band inversion.

Dependence on Electric Field Strength Both parameters, agarose concentration and electric field strength, do not only determine the onset of band inversion, but also the position of the mobility minimum on the molecular weight scale. Figure 3 shows a plot of the molecular weight with minimum mobility vs the electric field strength. In 1% gels, the position of the mobility minimum remains constant, whereas at higher concentrations the minimum shifts to lower molecular weight with increasingfield strength. The function is sigmoidal, i.e., the minimum mobility remains constant in the high field and low field limit, with a field depenin between. dence of about MflminE-' to

-

Influence of High Molecular Weight DNA From the experiments described above, it became clear that the effect of band inversion is dependent on the sample used. When isolated single species of DNA fragments are subjected to electrophoresis, band inversion occurs at low electric field and high agarose concentration. However, if a mixture of fragments is electrophoresed, the effect of band inversion can be reduced or canceled. Apparently, interactions between the molecules of different species play a role. We assumed that the presence of high molecular weight or even low molecular weight DNA is responsible for the cancellation of band inversion. For practical reasons, we used one-dimensional electrophoresisat a constant field of 2 V/cm and a concentration of 2% agarose for the following experiments. Under these conditions, it is the second largest fragment of the A-Hind111 digest (9.4 kbp),

488

Heller and Viovy

which has a minimum mobility, when electrophoresed as a single species. In a beginning experiment, A-DNA (48.5 kbp) was omitted from the original mixture containing A, A-XhoI, and A-Hind111 DNA, but band inversion was not observed (not shown). Only when the A-XhoI digest (33.5 and 15 kbp) was also left out (i.e., A-Hind111 alone as sample), band inversion occurred (not shown).

0.1

0.01

--

Dependence on DNA Concentration I

J

I

1.5 X

I I

1 o3

1 o4

U

1

0.1

0.01 L

I 1

5

From this experiment it became evident that it is the presence of high molecular weight DNA that is responsible for the cancellation of band inversion. In order to study this effect in more detail, we “isolated” the 9.4 kbp fragment, by further digesting the A-Hind111 fragments with PvuII and SacII. Keeping the total amount of DNA constant, this double digest cuts down the large 23.1 kbp fragment and the medium-sized 6.5 kbp fragment to pieces smaller than 4.3 kbp but leaves the 9.4 kbp fragment intact. Again band inversion occurred, as in the experiment with A-Hind111 DNA (see lane b of Figure 4 ) . In a second step, we now added A-DNA in increasing amounts to the A-HindIII-PvuII-Sac11 digest and separated the samples. The addition of high molecular weight DNA has indeed an effect on the mobility of the fragment with minimum mobility: With increasing amounts of A-DNA, the 9.4 kbp fragment is “taken along” by the high molecular weight DNA and comigrates, i.e., the band inversion effect is cancelled. The mobility of the shorter DNA molecules is not affected by the addition of A-DNA (Figure 4 ) . In a third experiment, a fixed amount of A-DNA was added to the digest and the mixture was serially diluted as a whole (i.e., the ratio of high molecular weight DNA to medium-sized DNA remains constant). When separating these dilutions at 2 V/ cm in 2% agarose, a cancellation of band inversion takes place at high sample concentration,whereas inversion is recovered with a dilute sample ( Figure 5 ). Therefore, it is the amount of high molecular weight DNA, i.e., its local concentration in the gel,

L

FIGURE 2 Dependence of the electrophoretic mobility of linear double-stranded DNA fragments on the molecular weight in 1, 1.5, and 2% agarose gels at different

electric field strengths (constant field) in 0.5X TBE at 1O’C. ( a-c) Constant field electrophoresis followed by field inversion electrophoresis. (d-f) Constant field electrophoresis with preceding field inversion electrophoresis. Conditions as in Figure 1.

Electrophoretic Mobility of DNA in Gels. III

;E \%

0.1

I

0.01

105

I

1o4

10'

1.5 % n

5

0.1

2 3. f

5

w

-E= CI

0

0.01

1 o3

tt

0.01 O .

10'

10'

2 %

i

103

104 size (bp)

FIGURE 2 (Continued)

10'

-

5Vlm 3V1m -c 2 V l m Vlcm 0.5 Vlcm

-t- 1 --)t

489

Heller and Viovy

mum mobility, are “dragged along” by the faster ones and band inversion is canceled.

DISCUSSION

11

FIGURE 3 Dependence of the molecular weight with minimum mobility on the electric field strength for three different agarose concentrations. Same conditions as in Figure 1.

that determines the occurrence of band inversion. In other words, if sufficient high molecular weight DNA with a higher mobility is present, the medium-sized molecules, which would have mini-

The series of experiments described in this paper supplements our recent study about the mobility of double-stranded DNA fragments in agarose gels.’’ We could show, that if single species of DNA fragments are subject to electrophoresis, a band inversion occurs at low electric field strength and at high agarose concentration, in agreement to earlier result~.~ This - ~ kind of experiment is the one that should be used to test theoretical predictions of DNA mobility, as these normally do not take into account DNA-DNA interactions. These new results ( Figure 2d-f) should therefore replace those published recently (Fig. lc, e, and f of Ref. 12). However, the conclusions drawn there, concerning the validity of the biased reptation with fluctuation model, are not affected by the band inversion effect: As the mobility of DNA with low molecular weight (i.e., smaller than the molecular weight with minimum mobility) is not changed, the reptation regime is still present (slope of - 1 in a log p vs log M , plot ) .

FIGURE 4 Influence of high molecular weight DNA on band inversion. Lanes a and i: ADNA 100 ng; lanes b-h X-HindIII-PvuII-Sac11DNA ca. 100 ng mixed with 0 (b), 10 (c), 20 ( d ), 5 0 ( e) , 100 ( f ) ,200 (g) , and 300 ng; ( h ) of A-DNA. The fragment sizes of the A-HindIIIPvuII-Sac11 digest are 9416, 4361, 4268, 4194, 3916, 3755, 3638, 2802, and 2255, and 18 smaller fragments, not seen on the gel.

ElectrophoreticMobility of DNA in Gels. 111

491

FIGURE 5 Influence of sample concentration on band inversion. Lanes a and n: A-DNA 100 ng; lane b: A-HindIII-PvuII-Sac11 DNA ca. 100 ng mixed with 250 ng of A-DNA; lane c: AHindIII-PvuII-Sac11 DNA ca. 100 ng only; lanes d, f, h, j, 1: same sample as in lane b, diluted by l : l S ( d ) , 1:2.25(f), 1:3.37(h), 1:5(j),and 1:7.6(1).Lanese,g,i,k,m:samesampleasin laneqdilutedby 1:1.5 (e), 1:2.25(g), 1:3.37(i), I:5(k),and 1:7.6(m).Fragmentsizesasin Figure 4.

The mobility of DNA fragments with high molecular weight (48.5 kbp, i.e., larger than the molecular weight with minimum mobility), did not change either, regardless of the method used, which means that they regained the “reptation with stretching” regime. This is expected, as the stretching reduces the probability for the formation of Ushapes, which are responsible for the band inversion effect. Therefore, the linear mobility vs electric field dependence of long chains remains valid, in agreement with the predictions of the BRF model. The dependence of the “position” of the mobility minimum on the molecular weight scale is more complicated than predicted, but an E-’ dependence could not be detected. The E-’ dependence that can be observed at medium field strength is closer to the predictions of the new BRF model; however, the amount of data is too small to allow a final conclusion. The second kind of experiment used, i.e., the separation of a mixture of DNA species, is of much more practical importance. Depending on the composition of the sample, band inversion can occur, making the identification of bands in one-dimensional gels difficult. In conclusion, the criteria for the occurrence of band inversion during the separation of double-

stranded DNA in agarose gels at constant electric field are the following: 1. The electric field must be low (< 3 V/cm). 2. The agarose concentration must be high ( 2 1%). 3. The sample should not contain large amounts of high molecular weight DNA.

Erroneous band assignment due to band inversion could have important consequences in some applications, such as gene mapping, forensic testing, etc. As band inversion affects medium-sized DNA, it takes place in the upper part of the gel (“compression zone”). Therefore, we generally recommend avoiding any attempt of band identification in this part of the gel, especially for unknown samples, as the mobility of these molecules can vary with the presence or the absence of high molecular weight DNA. However, as the high molecular weight DNA does not affect the mobility of the smaller molecules, it is safe to use the shorter fragments (i.e., those in the part of the gel below the compression zone) for band assignment. We thank F. Caron for his help and L. Oury and C. Serre

492

Heller and Viovy

for their assistance. This work was supported by a fellowship from the European Community to CH.

5. 6.

REFERENCES 1. Carle, G. F., Frank, M. & Olson, M. V. ( 1986) Science 232,65-68. 2. Heller, C. & Pohl, F. M. ( 1989) Nucleic Acids Res. 17,5989-6003. 3. Noolandi, J., Rousseau, J., Slater, G. W., Turmel, C. & Lalande, M. (1987) Phys. Rev. Lett. 58, 2428243 1 . 4. Lalande, M., Noolandi, J., Tunnel, C., Brousseau,

7. 8. 9. 10. 1 1.

12.

R., Rousseau, J. & Slater, G. W. (1988) Nucleic Acids Res. 16,5427-5437. Slater, G. W., Turmel, C., Lalande, M. & Noolandi, J. ( 1989) Biopolymers 28, 1793-1799. Brassard, E., Tunnel, C. & Noolandi, J. ( 1992)Electrophoresis 13,529-535. Doi, M., Kobayashi, T., Makino, Y., Ogawa, M., Slater, G. W. & Noolandi, J. ( 1988)Phys. Rev.Lett. 61,1893- 1896. Viovy, J . L. ( 1988)Europhys. Lett. 7,657-661. Dhjardin, P. ( 1989)Phys. Rev. 40,4752-4755. Duke, T. A. J., Semenov, A. N. & Viovy, J. L. ( 1992) Phys. Rev. Lett. 69,3260-3263. Duke, T., Viovy, J. L. & Semenov, A. N. ( 1994)Biopolymers 34,239-247. Heller, C., Duke, T. & Viovy, J. L. ( 1994) Biopolymers34,249-259.