Anel. Chem. 1992, 64, 2340-2351
1940
Enhanced Separation of DNA Restriction Fragments by Capillary Gel Electrophoresis Using Field Strength Gradients AndrPs Guttman,' Bart Wanders, and Nelson Cooke Beckman Instruments Inc., Fullerton, California 92634
T h e e f f ~ oef ~ d r k f h k l r t r ~ h g r a d k n t r o thewparatlon n of DNA reatrtction fragment8 waa Invostlgatd. A8 reported InourearWerwork, t h e m o b l M y o f d W f e r e n t r h e ~ m d . d DNA mokcuka k a function of the applkd ekctrlc field whkh wggertr that the uw of a nonuniform ( t h e varyhg) ekctrlc fkldmay Increasethe r.rdvlng power. We d o " t r a t e that In caplllary gd dectrophoreda enhamod wprrakn of DNA rostrlctlon fragments up to 1353 base p a h (bp) In dze can be achleved by mploylng the tkklatrength qadknt method. Tho ahape of the gradknt can be continuour or af1.pwW over the. Both method8 can k used to Increase wparatlon dfkkncy and redution In capllkry gd ehctrophoreda of doubb8trad.d DNA mokcukr.
INTRODUCTION Recently, there has been a great deal of activity in DNA analysis by capillary electrophoretic methods.'-3 Using this new and powerful technology,results have been published on the separation of single-stranded DNA molecules, such as in synthetic DNAanalysis4aswell as in the separation of doublestranded DNA molecules, particularly concerning PCR producta and restriction fragments.6 It was also demonstrated that high-efficiency, fast separations of DNA molecules can be achieved by the use of linear polyacrylamide gel-filled capillarycolumne.6~7Even fragments of the same chain length with different sequences were separated by this method due to differences in molecular conformation.8 Different field operation techniques have been described recently to achieve better separation of different size DNA molecules, mainly with slab gel electrophoresis. Dennison et al.9 employed conical or wedge-shaped slab gels to linearize the logarithmic distribution of bands (nonlinear voltage gradient method). Biggin et 81.10 studied the usefulness of a high ionic strength anode buffer where the resistance of the gel in the direction of the anode decreases and creates a negative field strength gradient along the DNA's migration path. They concluded that this particular method is not practical for ultrathin gels, as is also the case for capillary gel
* To whom correspondence and reprint requests should be addressed.
(1) Cohen, A. S.;Najarian, D. R.; Paulue, A,; Guttman, A.; Smith, J. A.; Karger, B. L. h o c . Natl. Acad. Sci. U.S.A. 1988,86,9660-9683. (2) Paulue, A.; Gaaamann, E.;Field, M. J. Electrophoresis 1990,11, 702-708. (3) Yin, H. F.; Lux, J. A,; Shomburg, G. J.HighResolut. Chromatogr. 1990,13,624-627. (4) Guttman, A.; Cohen, A. S.; Heiger, D. N.; Karger, B. L. Anal. Chem. 1990,62, 2038-2042. (5) Schwartz, H. E.; Welder, K.; Sunzeri, F.; Buech, M.; Brownlee, M. G. J. Chromatogr. 1991,559,267-283. ..(6) . - Heiger, D. N.; Cohen, A. 5.;Karger, B. L. J. Chromatogr. 1990,516, 33-48.
(7) Guttman, A.; Cooke, N. J. Chromatogr. 1991,559,285-294. (8) Guttman, A.; Nelson, R. J.; Cooke, N. J. Chromatogr. 1991,593, 297-303. (9) Dennison, C.; Linder, W. A.; Phillis, N. C. K. Anal. Biochem. 1982, 120,12-18. (10) Biggin, M. D.; Gibeon, T. J.; Hong, G. F. Proc. Natl. Acad. Sci. U.S.A. 1983,80,3963-3965. 0003-2700/92/0364-2348$03.00/0
electrophoresis.11 Ansorge et al. tried using an increasing cross-sectionalarea of the slab gels, producing a field gradient to achieve enhanced sharpening of bands, thereby increasing the number of resolvable bases per ge1.12 Cantor et del3 introduced the pulsed-field method (changing the direction and magnitude of the field in an oscillating manner), that takes advantage of the elongated and oriented configuration of large DNA (>SO kbp) molecules in gels. Heiger et ale6 described the capillary gel electrophoretic separation of double-stranded DNA molecules up to 23 kbp in size using the pulsed-field technique with very low gel concentrations. Although in slab gel operation there are some mechanical difficultiesinvolved in handling low-concentrationgels," this does not present a problem in capillary electrophoresis techniques. Demana and co-workers16 used an analyte velocity modulation method to increase separation power in capillary gel electrophoresis of DNA restriction fragments. In this paper, a simple field strength gradient method is described that provides enhanced resolution of doublestranded DNA molecules in capillary polyacrylamide gel electrophoresis.
EXPERIMENTAL SECTION Apparatus. In all of the experiments, the P/ACE System
2100capillaryelectrophoresis apparatus (BeckmanInstruments, Inc., Fullerton, CA) was used with the cathode on the injection side and the anode on the detection side. Therefore, the negatively charged DNA molecules migrate toward the anode in the gel (i.e., polymer network) fiied capillary column. The separations were monitored on column at 254 nm. The temperature of the capillary column was kept constant at 20 f 0.1 O C by the liquid cooling system of the P/ACE instrument. The electropherograms were acquired and stored on an Everex 386/ 33 computer. Chemicals. The 9x174 DNAHaeIII digest and the pBR322 DNA MspI digest restriction fragment mixtures (New England Biolabs, Beverly, MA) were diluted with deionized water to a concentration of 25 pg/mL before injection and were stored at -20 O C . Ultrapure grade acrylamide, Tris, boric acid, EDTA, ammonium persulfate, and tetramethylethylenediamine (TEMED)were used in the experiments (Schwarz/MannBiotech, Cambridge, MA). All buffer and acrylamide solutions were filtered through a 0.2-pm-pore-sizefilter (Schleicherand Schuell, Keene, NH) and carefully vacuum degassed. Procedum. Polymerizationof the linear polyacrylamidegel was initiatedby ammoniumpersulfate and catalyzedby TEMED in 100 mM Tris-borate, 2 mM EDTA buffer (pH 8.35) prior to inserting the reaction mixture into the 0.1-mm4.d. fuoed-silica capillary tubing (DB-226, J&W, Inc., Sacramento, CAI. The polymerization reaction mixture was injected into the capillary by means of a gastight syringe (Dynatech, Balton Rouge, LA). (11) Guttman, A. Beckman Instruments, Inc., Research and Development. Unpublished results, 1991. (12) Ansorge, W.; Labeit, 5.J. Biochem. Biophys. Methods 1984,10, 237-243. (13) Cantor, C. R.; Smith, C. L.; Mathew, M. K. Annu. Reu. Biophys. Biophys. Chem. 1988,17,287-304. (14) Fangman, W. L. Nucl. Acids Res. 1978,5,653-666. (15)Demana, T.; Lanan,M.; Morris, M. D. Anal. Chem. 1991, 63, 2795-2797. Q 1992 Amerlcen Chemical Socbty
ANALYTICAL CHEMISTRY, VOL. 04, NO. 20, OCTOBER 15, 1992
The use of low-viscositylinear polyacrylamide,not bound to the capillary wall, permits replacement of the gel-buffer system in the capillarycolumn by means of the rinse operation mode of the PIACE apparatus. The total length of the gel-filled capillary column was 470 and 670 mm (400 and 600 mm to the detection point), respectively. The samples were injected by the pressure injection mode of the PIACE system, typically 5 8, 0.5 psi (estimated injection amount: 0.1 ng of DNA). Field strength gradients were programmed in the P/ACE apparatus with continuously increasing or decreasing voltage separationmodes. In the stepwisefield gradientseparationmode, constant voltageswere used for different time periods as specified in the corresponding figure. The relative standard deviation (RSD) of the migration time was less than 2% (n = 12) when the temperature of the gel-filled capillary column was maintained within fO.l "C. The same capillary column could be used for approximately 100 runs with or without replacement of the separation gel-buffer system.
2949
0
0
9 9
E O
0
9
0
0
9
N
m 0
5
0
0
9
9
In
u) 0
0
8
RESULTS AND DISCUSSION By the use of a low concentration gel (less than 5 % linear polyacrylamide) in capillary electrophoresis, it is possible to achievegood separationof a wide size range of double-stranded DNA molecules. Separations are comparable to or better than those achieved with agarose in slab gel operation.5 Using a relatively high electricfield,DNA molecules can be separated with high resolution in a relatively short time. However, at high field strengths, the elecrophoretic mobility of DNA molecules becomes field-dependent.16 Furthermore, it is known that chain entanglementplays a significantrole in the separation of DNA molecules in a gel of a given pore size;" this entanglement is a function of the molecular size and the applied electric field.15 The main challenge is to find the appropriate field for the optimal separation of a mixture of DNA molecules with different chain lengths in a given gel matrix. At low field strengths a sieving effect applies and an inversely proportional relationship between mobility and molecular size is observed.l8 With higher field strengths, a different phenomenon appears. The DNA chain becomes more oriented because the field biases the direction of the leading end of the molecule.'8 This lea& to an increase in mobility with increasing field strength, particularly for the larger size molecules. In other words, by application of a high electric field, the longer chain length DNA molecules might be partially or completely stretched alongthe alignment of the field. Thus the electrophoretic mobilities of these big molecules become size-independentcausing poor separation at high field strengths.19 It should be noted that in capillary polyacrylamide gel electrophoresisthis effect appears to occur with fragment lengths longer than loo0 bp. Interestingly, for very short chain length fragments (50 min). However, some of the smaller fragmentsare not fully resolved. As Figure 1A shows, (16) Flint,D. H.; Harrington, R. E. Biochemietry 1972,11,4868-4884. (17) Smizek, D. L.; Hoegland, D. A. Science 1990,248, 1221-1223.
(18)De Gennee, P.G.Scaling Concepts in Polymer Physics; Comell University Pram: Ithaca, NY, 1979; Chapter 3. (19)Lumpkin, 0.J.; Dejardin, P.; Zimm, B. H. Biopolymer8 1985,24, 1573-1593.
e
9 9
.I m c
F
0
9 2
0
0
9
s
0
9
0
0
9
m
N
N
N
9
C
1
I
c' -
0
0
0
9 v
S n c
e
0 0
0
y1
Lo
9
0
9
h
0
0
min rd
Fl~uro1. Separation of the 4X774DNA restrlctbnfragment mlxtve by capillary gel electrophoresis using constant applied electric fleld @"static): (A) lOO,(B)2OO,(C)5OOV/cm. PeakswereidmUfbd by their increasing area, whlch correlates to the chain lengths: 1 = 72,2 = 118,3 = 194, 4 = 234, 5 = 271,6 = 281, 7 = 310,8 = 003, 9 = 872, 10 = 1078, 11 = 1353 bp. Conditions: replaceable polyacrylamidegel column, effective length to detector 40 cm, total len@ 47 cm; buffer, 0.1 M Trls-borate, 2 mM EDTA (pH 8.35).
there is an incomplete separation between peak 6 and peak 7 (271- and 281-bp fragments). Similar results have been attributed by Karger and co-workers6 to diffusional band broadening due to the long separation time in very diluted gel-filled capillary columns. Increasing the applied electric field to 200 V/cm completes separation of all 11fragments in 27 min (Figure 2B). The larger DNA molecules probably start to align with the electric field, and therefore resolution in the large-size range is not as complete as in Figure 1A. A further increase in field strength to 500 V/cm (Figure lC), causes stronger alignment20 of the larger fragments with a concomitant loss of resolution, since the sieving matrix can no longer separate some of the aligned molecules (peaks 9 and 10). However, the separation time decreases to 7 min and the lower molecular weight fragments (peaks6 and 7) are separated completely due to the high applied electric field.19 On the basis of the results shown above we reasoned that enhancedseparation of DNA molecules could be achieved by applyinga nonuniform electric field (field Strength gradient) at time. In this way all the different molecular weight range DNA molecules can be exposed to the electric field strength that is optimal for their separation. In capillary polyacrylamide gel electrophoresis of DNA, when a uniform electricfield (E)is applied to a charged polyion under steady-state conditions,? the electrophoretic velocity (v) can be expressed by the product of the electric field and ~
~~
~
~~
~
(20) Slater, G.W.; Noolandi, J. Biopolymers 1986,25, 431-454.
2960
ANALYTICAL CHEMISTRY, VOL. 64, NO. 20, OCTOBER 15, 1992
the electrophoreticmobility of the DNA molecule at the given field strength (p): (la) However, this basic equation should be modified when a nonuniform field is applied [E(t)l since then the applied electric field is changed with respect to time (t), causing a change in the electrophoretic velocity of the polyion. We also should consider that the electrophoretic mobility of the DNA molecule is a function of the electric field [p(E)],as we reported earlier?, so we can write U
=
IO
d
u(t) = r(E)E(t) (1b) Equation l b states that the actual velocity, u(t), of a DNA molecule is influenced by the field strength in use at a given time and by the mobility, which is also a function of the field strength. Thus, when a nonuniform electric field is applied, the electrophoretic acceleration (a) can be expressed as the change in electrophoretic velocity, i.e. the product of the electrophoretic mobilityand the field strength at a given time: a = dv/dt = d ( d ) / d t (2) where du and dt are the electrophoretic velocity and the time incrementa, respectively. On the basis of our earlier as a first approximationwe can consider that the mobility of the double-strandedDNA molecules has a field-independent (h)and a field-dependent component: (3) cc = cco + S'E where is an extrapolated value of the electric field vs the mobility plot to zero field strength for a given chain length DNA molecule and SIis the slope value of the same plot (r2 = 0.987). Since this slope values show a linear relationship with the chain length of the DNA molecules in the range we examined,,' the following equation will hold
S, = A + S,n
(4)
where A is a constant for a given gel-buffer system, Sz is the slope of the SIversus n plot (r2 = 0.9921, and n is the chain length (base pair number) of the DNA molecule. Combining eqs 1-4 we obtain a = dvldt = d[ro + (A + S,n)ElE/dt
(5)
a = podE/dt + (A + S n dE2/dt
(6)
thus I
where term I is the field strength only and term I1 is the fieldand chain-length-dependent component. As an example, when the electric field strength is a linear function of time E=B+Ct (7) where B and C are constanta, then the electrophoretic acceleration of the polyion can be simply expressed as in eq 6 a
11
= const, + tconst,
(8)
Peak efficiency (N) and resolution (R,) are also affected by the momentaryfield 8trength.a Thus, we concludethat after the proper substitutions, the change in the theoretical plate number is a linear function of the acceleration
where 1 is the effective length of the capillary and D is the diffusion coefficient of the solute. The change in resolution (21) Guttman, A.; Cooke, N. Anal. Chem. 1991, 61, 2038-2042.
''1. 9
'1 .
2
i iiij
I
+!;
0
0 0
2
'0
0
9
nun2
Flgwo 2. Separatlon of the 4x174 DNA restrictkn fragment mlxtve by -Wry polyacrvlrr~@ w wino an Involtage gradlent. The dotted line represents the current output. Condltlons: contkluous Reldstrength gradlent, 0-400 Vlcm In 20 mkr; other c "as In Figure 1.
-
is proportional to the aquare root22 of the acceleration, dRJ dt d ( a W d t , when a linear field strength gradient is used. Since different applied electric fields are optimal for the separation of different size DNA fragmenta,13-16J+21 the use of a field strength gradient gives the opportunity to increase significantly the resolving power of the technique. Field strength gradienta can be used in increasing, decreasing, or stepwise modes or in any combinationthereof, if necessary. Figure 2 shows a separation of the 4x174DNA restriction fragmentmixture using increasing electric field strength over time (0-400 V/cm in 20 min). Full separation of all the test mixture components was achieved in lese than 19 min. As Figure 2 shows,the apparent efficiency (i.e., theoretical plate number' of the last several peaks (9-11) is greater compared to Figure lB, where full separation of all the sample componenta was also attained. In this example, these fragmentsmigrate faster past the detector window due to the higher field strength applied at the last part of the separation. Thus, consistent with the above, the apparent theoretical plate value N (eq 9, Table I), seem to be higher. (It is important to note here that the effect of the same phenomenon should be considered in peak area quantitation). Separation time was decreased by one-third by using the increasing field strength gradient method (compare to Figure 1B). Conversely, the applied electric field can be decreased over time, as shown in Figure 3. Here, a decreasing field strength gradient of 4OO-lOo V/cm in 20 min was applied to the linear polyacrylamide-filledcapillary column. Baseline separation of all 11 componenta of the 4x174 restriction fragment mixturewas achieved in lesa than 10min, which is comparable to the separation time shown in Figure 1C. In the case of Figure 3, the larger fragments migrated past the detector window slower due to the lower field strength at the end of the separation. This causes an apparent loea in efficiency (eq 9, Table I) and resolution, particularly for the last threepeaks. In some instances the continuous ramping does not give sufficientseparation between very closely related compounds (e.g., same chain length but different sequence DNA fiagmenta). During continuous ramping, the time the DNA molecules are exposed to the field that is optimal for their separation may be too short. In this case a stepwiae field (22) Karger, B. L.; Cohen, A. 5.; Guttman, A. J. Chromotogr. 1989, 492,585-614.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 20, OCTOBER 15, l9Q2 2SSl
Table I. Theoretical Plate Number Values (RSD< S%, n = 9) of the Peaks on Figures 1-4, Calculated by Using the System Gold Software Package (Beeban Instruments, Inc., Fullerton, CA) 100 V/cm 200 V/cm 500 V/cm 0-400 V/cm 400-100 V/cm peak no. no. of base pairs (Figure 1A) (Figure 1B) (Figure 1C) (Figure 2) (Figure 3) 1 2 3 4 5 6 7 8 9 10 11
72 118 194 234 271 281 310 603 872 1078 1353
267 315 314 173 295 187 266 028 234 530 244 754 143 187 147 648 110 304 86 257 61 032
285 570 296 041 288 121 296 556 286 551 294 239 191 084 194 253 149 061 100 610 88 550
231 663 225 221. 233 902 226 452 201 401 255 325 146 538 164 707 147 018
2 238 505 1659 932 1585 342 1555 559 1274 003 1 380 669 1483 281 1411 394 1325 371 1 162 954 1 131 967
233 525 207 590 215 614 207 910 189 650 197 177 204 222 145 678 101 351 113 796 116 819
0
11
/H
0
8 5 c
0
9
Q
f
min
J9 CD 9 0,
Flguro 5. Separation of the 4x174 DNA restrlctbnfragment mlxtwe byapHlarypolvaclytam@electrophoresisu gradient. The dotted llne represents current output. Condltions: contlnuous field strength gradient, 400-100 V/cm In 20 mln; other condltbns as In Figure 1.
strength gradient is better because closely related molecules are exposed to their optimal separation field strength for a longer period of time, resulting in better resolution. Figure 4 show the separation of a pBR322 DNA restriction fragment mixture employing a stepwise voltage gradient method in a longer capillary column (effective column length 60 cm). The method consisted of three consecutive steps, 100 Vtcm from 0 to 30 min, 200 V/cm from 30 to 70 min, and 400 V/cm from 70 to 100 min. In this way, full separation of almost all the componentswas attained. It is worth noting the baseline separation of peaks 8 and 9 (147-mers)and peaka 10 and 11 (160-mers1, since these components have the same chain length but different sequences. These fragments could have been separated previously only by means of capillary affinity gel electrophoresisusingethidium bromide as an intercalating affinity ligand.21
CONCLUSION A simple field strength gradient method was introduced in order to increase the resolving power in capillary polyacrylamide gel electrophoresis separation of DNA restriction fragment mixtures. The use of increasing, decreasing, or stepwisevoltage gradienttechniquesshowedthat the resolving power can be optimized for a given DNA chain length range, and separationtime can be significantlyreduced. In our study on the separation of the 9x174 DNA restriction fragments by capillary polyacrylamide gel electrophoresis, the best separation with minimum time requirement was achieved by
0
9
.%?
;m
0
9 0
0
9 YI 0
0 0
P
0
9
min
g
Flgurr 4. Separatlonof the pBR322 DNA restrlctlonfragment mixtwo by caplllary polyactylamide gel electrophoresis using an lncreaslng stepwlse gradient fleld. Channel B represents cwrent output. Condltbna: fieM strengths, 100 V/cm for 0-40 mln, 200 V/cm for 40-70 mln, 400 V/cm for 70-100 mln; effecthm column length,60 cm; other condltbns as In Flgure 1. Peaks: 1 = 26, 2 = 34,3 = 67,4 = 76,5 = 90,6 = 110,7 = 123,8 = 147,Q = 147, 10 = 160, 11 = 160, 12 = 180, 13 = 190, 14 = 201, 15 = 217, 16 = 238, 17 = 242, 18 = 309, 19 = 404, 20 = 527, 21 = 622 bp.
using a continuously decreasing applied electric field. It is important to note that with the use of field strength gradient methods, the apparent peak efficiencyand resolution may be midleading since the different size components migrate past the detector windowwith a velocity that is determined by the voltage in use at that point in time. Other types of gradients may be employed, such as current, power, and temperature, and the combination of those can also be used to optimize capillary gel electrophoretic separations of a given sample mixture.
ACKNOWLEDGMENT We gratefully acknowledge Barry L. Karger for his stimulating discussions. We further thank Judy Nolan,Herb E. Schwartz,and Tom van de Goor for reviewingthe manuscript before submission. The help of Phyllis Browning in the preparation of the manuscript is also highly appreciated.
RECEIVED for review April 16, 1992. Accepted July 14, 1992.
