Biased Sinusoidal Field Gel Electrophoresis for Size-Dependent DNA Separation
TOSHlYUKl SHIKATA and T A D A O KOTAKA Depirtment of Macromolecular Science, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan
Although the basic mechanisms of separation have not yet been fully understood, the pulse field gel electrophoresis (PFGE)1-5is now a most routinely used technique for size-dependent separation of native DNAs of large size. An interesting feature of the PFGE method is a so-called antiresonance phenomenon6 in that an adequate choice of the pulse timing called the resonance time significantly reduces the electrophoretic mobility p of DNA fragment of a particular size, or virtually pins down the fragment but allows others to migrate with reasonable rates4a7The phenomenon was interpreted as s u c h a DNA, migrating through a gel, may assume two different states-the elongated state in which the DNA can migrate and the compact state in which it Field reversal with the resonance time presumably increases the chance of the particular DNA being trapped in the compact state and retard its migration. However, so far the timing was chosen arbitrarily, and thus the designing of optimal conditions was rather difficult. This is partly because a reversed pulse involves a set of higher order harmonics, and it is not certain which one is actually effective for trapping a particular DNA in the compact state. To resolve these problems and to achieve efficient size-dependent separation of DNAs of large size, we propose a new version that utilizes a sinusoidal field of strength E, and a given frequency f superposed on a steady bias field of strength Eb,for which the field is defined as E = Eb E, sin 27rft. We thus call this method a biased sinusoidal field ( B S F ) gel electrophoresis ( G E ) . The BSF method was tested on double-stranded DNAs with molecular weight (number of base pairs: b p ) ranging from 100 bp to megabase pair (Mbp) in agarose gel with gel concentration C,,, ranging from 0.25 to 1.5 wt %. The buffer was a standard 50 m M TBE, which consists of 50 m M Tris (hydroxymethyl) amino-methane (Tris base), 50 m M boric acid, and 1 m M EDTA. To apply a BSF we
+
~
Riopolymers, Vol 31, 253-254 (1991) 1991 J o h n Wiley & Sons, Inc.
(c
CCC 0006-3525/91/020253-02$0400
utilized a high-speed, high-voltage bipolar power amplifier and a conventional function generator. A submerged-type electrophoretic system was operated a t about 20°C. The details will be published elsewhere. In one series of experiments conducted in 0.5 wt % gel with Eb = 7.5 V cm-' > E, = 5.0 V cm and varying frequency f , we observed significant increase (as much as 25%) in the mobility p of all the DNAs at a constant frequency fM of' about 0.3 Hz that is independent of the DNA size but is dependent on the gel concentration C,,, in such a way f M a Cee17.This acceleration in p must reflect the nature of the gel but not that of the migrating DNAs. More striking findings are shown in the plots of fi us log f in Figure 1, in which three features of the BSFGE emerge. In 1.0 wt % gel with Eb = 2.5 V cm-' < E, = 7.5 V cm-' ( 1) the p at low f field is 100-30% larger than that at the high f field. ( 2 ) For each DNA the latter coincides with the p observed under the steady field. ( 3 ) For large DNAs with M more than 23.1 kilobase pair (kbp) the p exhibits a minimum a t a certain frequency f s (that we call the pin-down frequency) dependent on C,,, and on M of DNAs in such a way that f s cc Cge,-'M-'. The performance is essentially the same in the gels of different C,,, as long as it is not too high. In Figure 2, the set of the p data shown in Figure 1are replotted against the size of DNAs. We observe significant slow down or pinning down of a particular DNA a t a particular frequency. The minima in p of the large DNAs appearing a t fs are presumably due to the same mechanism reported in other conventional field inversion gel electrophoresis (FIGE)4,5.7 and crossed-field gel electrophoresis (CFGE) experiments. In our BSFGE method the size dependence of the pin-down frequency fs is so simple that the selection of the optimal conditions, say, to find a particular BSF frequency aiming at pinning down a particular, extremely large size DNA is quite easy and straightforward. We expect this method to be readily extended for size-dependent separation of DNAs of much larger size.
253
254
BIOPOLYMERS, VOL. 31 (1991)
I
I
I
M = 600
I
I
I
bD
Of
0.4
-c >
N
E . V
5
f /Hz 0 =-lo00 ( s t e a d y )
0;
0
0 a V
D I
I
I
Figure 1. Frequency fdependence of the mobility p for DNAs with a variety of molecular weight M under biased sinusoidal field gel electrophoresis (BSFGE; see text). DNAs with M < 23.1 kbp were Hind I and I11 digests of 4X 174 and A-phage DNAs; those with 48.5 and 166 kbp were A-phage and Td4C-phage DNA, respectively; those with M between 1.9 Mbp and 230 kbp were chromosomal DNAs from a yeast (Sacchromyces ceruisinae YNN 295) commercialized as Yeast DNA-PFGE markers. Agarose gel of concentration C,,, of 1.0 w t % was used as a migration medium with 50 m M TBE buffer ( 5 0 m M Tris base, 50 m M boric acid, and 1 m M EDTA), which was kept at about 20°C through circulation. Ethidium bromide was added to the buffer with concentration of 0.5 mg L-' to visualize DNA bands with a uv illuminator. The bias field strength Eb was 2.5 V cm-' and the amplitude E, of the sinusoidal field, 7.5 V cm-' ( a n underbias condition). The data points on the right-hand side axis represent p under the ordinary steady field of E = 2.5 V cm-'. We thank our Ministry of Education (Monbusho) for financial support.
REFERENCES 1. Schwartz, D. C., Saffran, W., Welsh, J., Haas, R., Gldengerg, M. & Cantor, C. R. (1983) Quant. Biol. 47, 189-195.
0.1 0.03 0.01
0.003 O.PO1
I
I
3
L
I
5 l o g ( M I bp)
I
6
Figure 2. Replot of the mobilityp data (shown in Figure 1 ) under BSFGE against log (molecular weight/bp) at various frequencies.
2. Schwartz, D. C. & Cantor, C. R. (1984) Cell 3 7 , 6775. 3. Chu, G., Vollrath, D. & Davis, R. ( 1986) Science 2 3 4 , 1582-1585. 4. Carke, G. F., Frank, M. & Olsen, M. V. ( 1986) Science 2 3 2 , 65-68. 5. Lalande, M., Noolandi, J., Turmel, C., Rousseau, J. & Slater, G. W. (1987) Proc. Natl. Acad. Sci. USA 84,8011-8015. 6. Viovy, J. L. (1989) Electrophoresis 1 0 , 429-441. 7. Kobayashi, T., Doi, M., Makino, Y. & Ogawa, M. ( 1990) Macromolecules, 22, 4480-4481. 8. Lumpkin, 0. J. & Zimm, B. H. (1982) Biopolymers 2 1 , 2315-2316. 9. Slater, G. W. & Noodandi, J. (1986) Biopolymers 25, 431-454. 10. Slater, G. W. & Noolandi, J. (1989) Electrophoresis 10,413-428. 11. Akermann, B. & Jonnson, M. (1990) J. Phys. Chem. 9 4 , 3828-3838. Received August 7, I990 Accepted November 6, 1990
TOSHlYUKl SHIKATA and T A D A O KOTAKA Depirtment of Macromolecular Science, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan
Although the basic mechanisms of separation have not yet been fully understood, the pulse field gel electrophoresis (PFGE)1-5is now a most routinely used technique for size-dependent separation of native DNAs of large size. An interesting feature of the PFGE method is a so-called antiresonance phenomenon6 in that an adequate choice of the pulse timing called the resonance time significantly reduces the electrophoretic mobility p of DNA fragment of a particular size, or virtually pins down the fragment but allows others to migrate with reasonable rates4a7The phenomenon was interpreted as s u c h a DNA, migrating through a gel, may assume two different states-the elongated state in which the DNA can migrate and the compact state in which it Field reversal with the resonance time presumably increases the chance of the particular DNA being trapped in the compact state and retard its migration. However, so far the timing was chosen arbitrarily, and thus the designing of optimal conditions was rather difficult. This is partly because a reversed pulse involves a set of higher order harmonics, and it is not certain which one is actually effective for trapping a particular DNA in the compact state. To resolve these problems and to achieve efficient size-dependent separation of DNAs of large size, we propose a new version that utilizes a sinusoidal field of strength E, and a given frequency f superposed on a steady bias field of strength Eb,for which the field is defined as E = Eb E, sin 27rft. We thus call this method a biased sinusoidal field ( B S F ) gel electrophoresis ( G E ) . The BSF method was tested on double-stranded DNAs with molecular weight (number of base pairs: b p ) ranging from 100 bp to megabase pair (Mbp) in agarose gel with gel concentration C,,, ranging from 0.25 to 1.5 wt %. The buffer was a standard 50 m M TBE, which consists of 50 m M Tris (hydroxymethyl) amino-methane (Tris base), 50 m M boric acid, and 1 m M EDTA. To apply a BSF we
+
~
Riopolymers, Vol 31, 253-254 (1991) 1991 J o h n Wiley & Sons, Inc.
(c
CCC 0006-3525/91/020253-02$0400
utilized a high-speed, high-voltage bipolar power amplifier and a conventional function generator. A submerged-type electrophoretic system was operated a t about 20°C. The details will be published elsewhere. In one series of experiments conducted in 0.5 wt % gel with Eb = 7.5 V cm-' > E, = 5.0 V cm and varying frequency f , we observed significant increase (as much as 25%) in the mobility p of all the DNAs at a constant frequency fM of' about 0.3 Hz that is independent of the DNA size but is dependent on the gel concentration C,,, in such a way f M a Cee17.This acceleration in p must reflect the nature of the gel but not that of the migrating DNAs. More striking findings are shown in the plots of fi us log f in Figure 1, in which three features of the BSFGE emerge. In 1.0 wt % gel with Eb = 2.5 V cm-' < E, = 7.5 V cm-' ( 1) the p at low f field is 100-30% larger than that at the high f field. ( 2 ) For each DNA the latter coincides with the p observed under the steady field. ( 3 ) For large DNAs with M more than 23.1 kilobase pair (kbp) the p exhibits a minimum a t a certain frequency f s (that we call the pin-down frequency) dependent on C,,, and on M of DNAs in such a way that f s cc Cge,-'M-'. The performance is essentially the same in the gels of different C,,, as long as it is not too high. In Figure 2, the set of the p data shown in Figure 1are replotted against the size of DNAs. We observe significant slow down or pinning down of a particular DNA a t a particular frequency. The minima in p of the large DNAs appearing a t fs are presumably due to the same mechanism reported in other conventional field inversion gel electrophoresis (FIGE)4,5.7 and crossed-field gel electrophoresis (CFGE) experiments. In our BSFGE method the size dependence of the pin-down frequency fs is so simple that the selection of the optimal conditions, say, to find a particular BSF frequency aiming at pinning down a particular, extremely large size DNA is quite easy and straightforward. We expect this method to be readily extended for size-dependent separation of DNAs of much larger size.
253
254
BIOPOLYMERS, VOL. 31 (1991)
I
I
I
M = 600
I
I
I
bD
Of
0.4
-c >
N
E . V
5
f /Hz 0 =-lo00 ( s t e a d y )
0;
0
0 a V
D I
I
I
Figure 1. Frequency fdependence of the mobility p for DNAs with a variety of molecular weight M under biased sinusoidal field gel electrophoresis (BSFGE; see text). DNAs with M < 23.1 kbp were Hind I and I11 digests of 4X 174 and A-phage DNAs; those with 48.5 and 166 kbp were A-phage and Td4C-phage DNA, respectively; those with M between 1.9 Mbp and 230 kbp were chromosomal DNAs from a yeast (Sacchromyces ceruisinae YNN 295) commercialized as Yeast DNA-PFGE markers. Agarose gel of concentration C,,, of 1.0 w t % was used as a migration medium with 50 m M TBE buffer ( 5 0 m M Tris base, 50 m M boric acid, and 1 m M EDTA), which was kept at about 20°C through circulation. Ethidium bromide was added to the buffer with concentration of 0.5 mg L-' to visualize DNA bands with a uv illuminator. The bias field strength Eb was 2.5 V cm-' and the amplitude E, of the sinusoidal field, 7.5 V cm-' ( a n underbias condition). The data points on the right-hand side axis represent p under the ordinary steady field of E = 2.5 V cm-'. We thank our Ministry of Education (Monbusho) for financial support.
REFERENCES 1. Schwartz, D. C., Saffran, W., Welsh, J., Haas, R., Gldengerg, M. & Cantor, C. R. (1983) Quant. Biol. 47, 189-195.
0.1 0.03 0.01
0.003 O.PO1
I
I
3
L
I
5 l o g ( M I bp)
I
6
Figure 2. Replot of the mobilityp data (shown in Figure 1 ) under BSFGE against log (molecular weight/bp) at various frequencies.
2. Schwartz, D. C. & Cantor, C. R. (1984) Cell 3 7 , 6775. 3. Chu, G., Vollrath, D. & Davis, R. ( 1986) Science 2 3 4 , 1582-1585. 4. Carke, G. F., Frank, M. & Olsen, M. V. ( 1986) Science 2 3 2 , 65-68. 5. Lalande, M., Noolandi, J., Turmel, C., Rousseau, J. & Slater, G. W. (1987) Proc. Natl. Acad. Sci. USA 84,8011-8015. 6. Viovy, J. L. (1989) Electrophoresis 1 0 , 429-441. 7. Kobayashi, T., Doi, M., Makino, Y. & Ogawa, M. ( 1990) Macromolecules, 22, 4480-4481. 8. Lumpkin, 0. J. & Zimm, B. H. (1982) Biopolymers 2 1 , 2315-2316. 9. Slater, G. W. & Noodandi, J. (1986) Biopolymers 25, 431-454. 10. Slater, G. W. & Noolandi, J. (1989) Electrophoresis 10,413-428. 11. Akermann, B. & Jonnson, M. (1990) J. Phys. Chem. 9 4 , 3828-3838. Received August 7, I990 Accepted November 6, 1990
