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Anal. Chem. 1999, 71, 2385-2389
High-Efficiency DNA Separation by Capillary
Electrophoresis in a Polymer Solution with
Ultralow Viscosity
Futian Han,†,‡ Bryan H. Huynh,† Yinfa Ma,*,† and Bingcheng Lin‡
Truman State University, 100 East Normal Street, Kirksville, Missouri 63501, and Dalian Institute of Chemical Physics,
Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, P.R. China
The viscosities of some polymer solutions for DNA separation in capillary electrophoresis are generally very high,
which makes them hard to pump into the capillaries. We
have developed a novel sieving buffer, based on lowmolecular-weight hydroxypropylmethylcellulose, to separate DNA fragments. The viscosity of this sieving matrix
was at least 1 order of magnitude lower than that of
traditional buffers with similar sieving effect. The influence
of additives such as urea and mannitol was investigated.
It was found that the double-stranded DNA (ds DNA)
fragments began to denature in 3.5 M urea, and 7 M urea
can denature the ds DNA completely. The presence of
mannitol will decrease the overlap threshold of the
polymer solution (the concentration at which the polymer
molecules begin to entangle with each other), which
makes it possible to separate DNA fragments in a polymer
solution of relatively low concentration. The influence of
the electrical field was also investigated, and it was found
that the mobility of DNA fragments up to 2000 bp in
length did not change greatly with different electric fields.
This phenomenon implies that the DNA fragments at this
range do not change their conformation with the increase
of electric field as was previously believed. The possible
mechanism for the separation of DNA fragments is also
discussed.
The charge densities of DNA fragments are independent of
molecular size. Therefore, the mobilities of DNA fragments of
different length are almost constant, which makes it impossible
to separate DNA fragments in free zone capillary electrophoresis.
Although some investigators have explored using free-solution
electrophoresis to separate molecule-tagged DNA fragments,1
molecular sieving must be applied to DNA separation by capillary
electrophoresis in most cases. Originally, a capillary electrophoresis system with various gels (capillary gel electrophoresis, CGE)
was used for the separation of biopolymers such as oligonucleotides, nucleic acids, and proteins. However, an easy-to-operate
and reproducible CGE system is difficult to obtain and the cross* Corresponding author: (phone) 660-785-4084; (fax) 660-785-4045; (e-mail)
[email protected].
† Truman State University.
‡ Dalian Institute of Chemical Physics.
(1) Heller, C.; Slater, G. W.; Mayer, P.; Dovichi N.; Pinto, D.; Viovy, J.; Drouin,
G. J. Chromatogr., A 1998, 806, 113-121.
10.1021/ac990160x CCC: $18.00
Published on Web 05/13/1999
© 1999 American Chemical Society
linked gel-filled capillaries do not have a long lifetime. In recent
years, entangled polymer solutions, such as poly(ethylene oxide),2
poly(ethylene glycol), poly(vinyl alcohol),3 glucomannan,4 uncross-linked polyacrylamide,5 and cellulose and its derivatives,6,7,8
were more and more frequently employed as media to achieve
molecular sieving of DNA fragments. Several models, such as the
Ogston model,9 the reptation model,10 and the biased reptation
model,11 were employed to explain the behavior of DNA fragments
in these kinds of sieving matrixes. But there still does not exist a
complete understanding of electrophoretic migration of DNA
fragments in polymer solutions. Therefore, it is hard to predict
the resolution of the migration and to design a sieving system
that satisfies one’s separation requirements. Generally, polymer
solutions with a wide range of concentrations were used for DNA
separation. The sieving ability varies greatly according to the
polymer concentration and chemical components. For sieving
matrixes prepared from cellulose and its derivatives, concentrated
solutions are generally required in order to get satisfactory
separation of smaller DNA fragments (<1000 bp) because of the
smaller mesh size of these solutions. The viscosities of such
solutions are high, and sometimes it is difficult to fill the capillary
with buffer for the commercially available instruments, which
generally do not have the ability to provide sufficient pressure.
In addition, a viscous solution may stick to the electrode and
contaminate the sample during the injection. Great care must be
taken when these kinds of buffers are used. On the basis of a
theory of entangled polymer solutions, one can easily predict that
shorter polymers can form a sieving net in the solution giving it
a relatively low viscosity and a smaller mesh size.12 But the shorter
polymers were not often used. One reason is that the sieving
(2) Zhang, N.; Yeung, E. S. J. Chromatogr., A 1997, 768, 135-141.
(3) Dolnik, V.; Novotny, M. V. J. Microcolumn Sep. 1992, 4, 515-519.
(4) Izumi, T.; Yamaguchi, M.; Yoneda, K.; Isobe, T.; Okuyama T.; Shinoda, T.
J. Chromatogr., A 1993, 652, 41-46.
(5) Ruiz-Martinez, M. C.; Salas-Solano, O.; Carrilho, E.; Kotler, L.; Karger, B.
L. Anal. Chem. 1998, 70, 1516-1527.
(6) MacCrehan, W. A.; Rasmussen, H. T.; Northrop, D. M. J. Liq. Chromatogr.
1992, 15 (6, 7), 1063-1080.
(7) Kim, Y.; Morris, M. D. Anal. Chem. 1994, 66, 1168-1174.
(8) Barron, A. E.; Soane, D. S.; Blanch H. W. J. Chromatogr., A 1993, 652,
3-16.
(9) Ogston, A. G. Trans. Faraday Soc. 1958, 54, 1754-1757.
(10) Slater, G. W.; Rousseau, J.; Noolandi, J.; Turmel, C.; Lalande, M. Bioploymers
1988, 27, 509-524.
(11) Slater, G. W.; Noolandi J. Bioploymers 1989, 28, 1781-1791.
(12) Grossman, P. D.; Soane, D. S. Biopolymers 1991, 31, 1221-1228.
Analytical Chemistry, Vol. 71, No. 13, July 1, 1999 2385
ability of these shorter polymers is not competitive enough.13 We
have not seen any reports regarding the use of cellulose derivatives with very low molecular weight (as low as 10 000) as sieving
buffers. In this paper, we have developed a sieving matrix for DNA
separation. The matrix is based on low-molecular-weight hydroxypropylmethylcellulose (HPMC) and has a remarkably low viscosity. The influence of urea and mannitol on the separation of DNA
fragments was investigated, and the effect of electric field on
separation was also evaluated.
EXPERIMENTAL SECTION
Chemicals and Reagents. The HPMC with low molecular
weight (HPMC-5, the viscosity of which in 2% aqueous solution
is 5 cP and the molecular weight is ∼10 000) is from Aldrich
(Milwaukee, WI). Other HPMCs (HPMC-50, HPMC-100, and
HPMC-4000, the viscosities of which in 2% aqueous solutions at
25 °C are 50, 100, and 4000 cP, respectively, and the molecular
weights are about 11 500, 12 000, and 86 000, respectively) are
from Sigma (St. Louis, MO). Tris(hydroxymethyl)aminomethane,
mannitol, boric acid, urea, ethylendiaminetetraacetic acid disodium
salt (EDTA), and the PBR322/HaeIII DNA standard were also
purchased from Sigma. The DNA marker contains 22 fragments,
8, 11, 18, 21, 51, 57, 64, 80, 89, 104, 123, 124, 184, 192, 213, 234,
367, 434, 458, 504, 540, and 587 bp long, respectively. A DNA lowrange marker was obtained from Life Technologies Inc. (Gaithersburg, MD), which contains six DNA fragments ranging from
100 to 2000 bp. The samples were diluted with deionized water
to a concentration of ∼40 µg/mL and kept at -20 °C until use.
Deionized water (resistance g18 MΩ) was prepared with a Milli-Q
System (Millipore, Bedford, MA).
Capillary Electrophoresis Separation. All the experiments
were carried out on the Beckman P/ACE 5500 capillary electrophoresis system (Fullerton, CA). The fused-silica capillary (37 cm
in length with 30 cm from inlet to detection window, 50 µm i.d.)
was purchased from PolyMicro Technologies, Inc. (Phoenix, AZ).
The capillary was precoated with polyacrylamide as described
elsewhere14 to prevent adsorption and to eliminate electroosmotic
flow. The background electrolyte (BGE) was 0.1 M Tris-0.1 M
boric acid-2 mM EDTA with or without urea. Solutions with
concentrations of urea ranging from 1 to 10 M were investigated
in this study. A 0-12% (w/v) mannitol additive was added to the
buffer to investigate the influence of mannitol and evaluate the
optimum concentration of mannitol for the best sieving effect. The
sieving solutions were prepared by adding the polymers to the
BGE and stirring the mixture until all the polymer was dissolved
and the solution became homogeneous. The solutions were not
filtered or degassed before use.
The capillary was rinsed first with water and then with buffer
before injection. The rinsing time varies from a range of 3 to 20
min for each buffer according to its viscosity. The outside wall of
the capillary at the inlet end and the electrode were cleaned to
prevent the sample from being contaminated by viscous buffers.
The sample was introduced into the capillary by electromigration
at 10 kV for 5 s. The electrophoresis was carried out under
negative polarity at 25 °C. An ultraviolet detector was used with
the filter setting at 254 nm, and the data were collected and
(13) Barron, A. E.; Blanch H. W.; Soane D. S. Electrophoresis 1994, 15 (5), 597615.
(14) Han, F.; Xue, J.; Lin, B. Talanta 1998, 46, 735-742.
2386 Analytical Chemistry, Vol. 71, No. 13, July 1, 1999
Figure 1. Electropherogram of the PBR322/HaeIII in different
sieving matrixes. Conditions: capillary, 37 cm × 50 µm i.d. with 30
cm effective length; injection, 10 kV for 10 s; running voltage, 6 kV
from negative to positive; detection, UV at 254 nm; sieving buffers,
(A) 4% HPMC-5, (B) 2.0% HPMC-50, (C) 1.8% HPMC-100, and (D)
1.2% HPMC-4000. The sieving buffers were prepared in 0.1 M Tris0.1 M boric acid-2 mM EDTA solution.
processed by the Beckman PACE Station software. The viscosity
of each buffer was measured with an Ostwald viscometer at a
constant temperature of 25 °C.
RESULTS AND DISCUSSION
Four HPMCs with different molecular sizes, HPMC-5, HPMC50, HPMC-100 and HPMC-4000, were investigated to compare the
sieving abilities of those polymers. The PBR322/HaeIII DNA
marker was employed as a sample to test the sieving matrixes. It
was found that the larger fragments can be easily separated with
relatively low polymer concentration while the smaller fragments
can only be separated with high polymer concentration. For
HPMC-50, HPMC-100, and HPMC-4000, the baseline separation
of the PBR322 marker can be achieved (except for the 123/124
pair, which migrate together) only when the concentrations
reached as high as 2.0, 1.8, and 1.2%, respectively. The concentrations of the four smallest DNA fragments were not high enough
for successful detection. It was found that, for HPMC-5, there was
no satisfactory separation even when the concentration reached
as high as 4% (Figure 1A). In another independent study, it was
found that mannitol can enhance the separation ability of cellulosebased polymer solutions because mannitol and boric acid may
form a chain bridge between HPMC molecules through the
tetraborate structure.14 Experiments with HPMC-5 also show
dramatically enhanced resolution when mannitol is present. With
the addition of mannitol, even 2% HPMC-5 gave good resolution
of the DNA standards (Figure 2). The influence of the mannitol
concentration was investigated, and it was found that 6% (w/v)
mannitol was the optimum concentration for the 2% HPMC-5
solution. It was also found that more mannitol was needed for
the more concentrated HPMC-5 solutions because 12% mannitol
would result in an optimized separation in the 4% HPMC-5 buffer
Figure 4. Plot of [Vn)0/VDNA - 1] as a function of HPMC-5
concentration.
Figure 2. Electropherogram of the PBR322/HaeIII in 2.0% HPMC-5
+ 6% mannitol. Peak identification: (1) 51, (2) 57, (3) 64, (4) 80, (5)
89, (6) 109, (7) 123/124, (8) 184, (9) 192, (10) 213, (11) 234, (12)
267, (13) 434, (14) 458, (15) 504, (16) 540, and (17) 587 bp. Other
conditions are the same as in Figure 1.
ment coupling mechanism” to explain the migration behavior of
DNA fragments in ultradilute polymer solution.13 On the basis of
the DNA/polymer interaction theory, Hubert and co-authors also
developed a theoretical model to interpret the separation of DNA
fragments in very dilute (HEC) hydroxyethylcellulose solutions.15
The prediction from their theory was in good agreement with the
experimental data from Barron’s work. According to Hubert’s
theory, a DNA fragment drags along polymer molecules it
encounters during migration. Taking account of several factors
that influence migration, asymptotic relative velocity (VDNA/Vn)0)
can be described as
(
(
VDNA
L
) 1/ 1 + γC 1 + β
Vn)0
MDNAb
Figure 3. Viscosity as a function of the concentration of HPMC-5
with and without urea and mannitol additives.
(data not shown). According to a theory of the role of an entangled
polymer solution, only when the concentration of the solution is
higher than the overlap threshold concentration can a network
be formed in the solution.12 The threshold concentration of
HPMC-5 (both with and without 6% mannitol) can be determined
by measuring the viscosities of the polymer solutions with different
concentrations and plotting the viscosity as a function of the
polymer concentration (Figure 3). From Figure 3 we can see that
the threshold for the HPMC-5 itself is ∼3.5%, and mannitol can
decrease the threshold of this polymer to 2.5%, thus making it
possible for to separate DNA fragments in a relatively low
concentration.
It was found that good separation can be achieved at concentrations under the threshold concentration. Considerable separation can even be achieved in the 1.5% HPMC-5 solution with 6%
mannitol. In this case, neither the Ogston model nor the reptation
model will be suitable for explaining the mechanism of separation.
Barron and co-authors developed a so-called “transient entangle-
))
-1
(1)
where VDNA is the mean velocity of DNA in a polymer solution,
Vn)0 is the DNA velocity in a solution without polymer, C is the
polymer concentration, L is the contour length of the polymer,
MDNA is the DNA fragment length in base pairs, b is the contour
length of one DNA base pair, and β and γ are constants related
to the polymer and buffer. Since we were unable to find the L
value of HPMC-5, we were not able to use the fully functional
form of eq 1. But since L, MDNA, b, β, and γ are all constants, a
plot of [Vn)0/VDNA - 1] as a function of polymer concentration
will still result in a straight line. We tested our data for the 2000bp fragment using this method (Figure 4), and it was found that
our data were in good agreement with this theory. It was worth
noting that the concentration of our polymer solution was not very
low. From Figure 4, we can see that a good linear relationship
was obtained within a wide range of polymer solutions (0-4%).
This result indicated that Hubert’s theory may not only be applied
to interpret the DNA migration in ultradilute polymer solution
but also be applied to interpret the sieving mechanism of DNA
fragments in a low-molecular-weight polymer solution.
DNA conformation may have great influence on the mobility
of the DNA fragments in nongel sieving electrophoresis. Therefore, denaturant may be necessary in the buffers to eliminate the
conformation influence in some occasions.16 Urea is often employed as a denaturant in biological laboratories, and in this study,
(15) Hubert, S. J.; Slater, G. W.; Viovy J. Macromolecules 1996, 29, 1006-1009.
(16) Han, F,; Lin, B.; Xu, Q.; Shen, Y.; Wu, G. Chromatographia 1999, 49, 179184.
Analytical Chemistry, Vol. 71, No. 13, July 1, 1999
2387
Figure 5. Urea influence on the migration of DNA fragments.
Table 1. Comparison of Viscosity and Efficiency of the
Buffers Based on HPMCs with Different Molecular
Sizes
2% HPMC-5
+ 7 M urea
2%
1.8%
1.2%
+ 6% mannitol HPMC-50 HPMC-100 HPMC-4000
viscosity (cP)
efficiency
(plates/m)
5.7
1 800 000
30.8
600 000
46.0
840 000
335.3
530 000
urea also served to denature the double-stranded (ds) DNA
fragments. The influence of urea was investigated by adding
different amounts of urea to the mannitol-modified 2% HPMC-5
sieving buffer and plotting the migration time as a function of urea
concentration (Figure 5). Figure 5 shows some unique sigmoidal
curves for the DNA fragments ranging from 51 to 587 bp. Since
single-stranded DNA (ss DNA) has lower mobility than ds DNA,
the mobility of a ds DNA fragment will decrease when part of the
double-stranded molecule denatures to single strand. From Figure
5, one can easily see that ds DNA began to denature when the
urea concentration reached ∼3.5 M and continued to denature
with the increase in urea concentration. When the urea concentration reaches ∼7 M, the ds DNAs are completely denatured and
higher urea concentration will not lead to a large increase in
migration time; therefore, 7 M urea is necessary to completely
eliminate the influence of conformation.
One of the main advantages of this sieving medium is the low
viscosity, which makes it very easy and convenient to use. The
viscosities of the buffers with different molecular sizes were
measured with an Ostwald viscometer under the same conditions,
The results are listed in Table 1. It was found that the viscosities
of the sieving buffers containing larger HPMC molecules were
much higher than that of the HPMC-5 solution. Therefore it took
a much longer time (e.g., 20 min) for the instrument to fill the
capillary with a very viscous buffer. Furthermore, the viscous
buffers are more likely to stick on the electrode and/or the
capillary and may contaminate the sample if there is no cleaning
procedure before injection. On the contrary, the mannitol-modified
HPMC-5 buffer has a viscosity equivalent to water, thus making
it very easy to operate. Table 1 also shows the efficiency of the
different sieving buffers in terms of plate numbers. We can see
that the mannitol-modified low-viscosity buffer also give the
highest efficiency up to 1 800 000 plates/m, which makes it an
2388 Analytical Chemistry, Vol. 71, No. 13, July 1, 1999
Figure 6. Electrical field influence on the migration of DNA
fragments (51, 234, 587, and 2000 bp). Sieving buffer: 2%HPMC-5
+ 7 M urea + 6% mannitol. Inset: migration time vs electric field for
the 2000-bp fragment in the undenatured 2% HPMC-50 buffer.
ideal sieving matrix for DNA analysis.The influence of electric
field was also investigated in this study. On the basis of the biased
reptation model, the coil of the chainlike molecules tends to be
elongated under high fields.12 This conclusion does not agree with
what we observed in our experiments. Figure 6 shows the
variation of the migration time of the 51-, 234-, 587-, and 2000-bp
fragments with variation of the electrical field in the urea- and
mannitol-modified 2% HPMC-5 buffer. The curve indicates that
the migration times of the fragments are inversely proportional
to the electric field. When we draw the plot of reciprocal migration
time as a function of electric field, we obtain some linear
relationships (R > 0.9990). The relationship can be described by
1/t ) K1E + K2
(2)
where t is the migration time of the DNA fragments, K1 is a
constant related to the size of a DNA fragment, K2 is another
constant related to the system (temperature, capillary inner
diameter, sieving buffer, etc.), and E is the electric field. The
mobility of a DNA fragment is defined by
µ ) L/(tE)
(3)
where µ is the mobility and L is the effective length of the capillary.
From eqs 2 and 3, we can get
µ ) K1L + K2L/E ) µ0 + K2L/E
(4)
where µ0 was defined as the optimized mobility of a DNA fragment
in this buffer, which is the maximum mobility that a DNA fragment
can reach because K2 is a negative value of all cases. µ0 can be
determined experimentally by plotting the reciprocal migration
time as a function of electric field and multiplying the slope by
the effective length of the capillary. Differentiation of the above
leads to
dµ/dE ) -K2L/E2
(5)
Equation 5 indicates that the mobility variation with the change
of electric field is inversely proportional to the square of the
electric field. From the experimental data, we can see that dµ/
dE is a very tiny (almost zero) value, which means that the
influence of the electric field on mobility is so slight that it can
be neglected. As a matter of fact, the second term of eq 4 is at
least 2 orders of magnitude less than the first term of eq 4 in all
of this study. The above description states that the mobility of
the DNA fragments does not change greatly under different
electric fields ranging from 80 to 480 V/cm. We also tested a larger
fragment from the low-range DNA sample and our calculations
show that the mobilities of the DNA fragments up to 2000 bp in
length also do not change greatly while the electric field changes.
We even obtained the same results from the 2% HPMC-50
undenatured buffer (inset of Figure 6). Therefore, this phenomenon is not the result of using the denaturing buffer, which can
eliminate the influence of the secondary or higher order structure
of the DNA fragments. This result is not consistent with the biased
reptation model,11 which suggests that the DNA molecule may
change its conformation when the electrical field increases,
because the change in the conformation will lead to great change
in the mobility.
On the basis of this phenomenon, we suggest that the electric
field does not influence the resolution of the DNA fragments as
much as what is thought. Figure 7 shows the resolution of the
80/89,184/192, and 540/587 fragment pairs under different electric
fields. Resolution was calculated using a traditional chromatographic method,17 and the result shows that there is no significant
change in the resolution of 80/89, 184/192, and 540/587 fragment
pairs when the electric fields changed from 80 to 320 V/cm. The
resolution of these fragment pairs decreased slightly when the
electric fields changed from 320 to 480 V/cm, probably due to
band-broadening resulted from the Joule heating effect. Therefore,
a higher electric field should be used to get faster separation until
Joule heating becomes obvious.
CONCLUSION
The mannitol-modified low-molecular-size HPMC buffer has
very low viscosity, which makes it easy for capillary filling,
flushing, and refilling. This is a great advantage over those buffers
(17) Snyder, L. R.; Kirkland, J. J. Introducation to Modern Liquid Chromatography;
John Wiley & Sons: New York, 1979; Chapter 2.
Figure 7. Resolutions of the 80/89, 184/192, and184/192 fragment
pairs under different electrical fields. The line was drawn through the
data to guide the eye.
with high viscosities. Mannitol can enhance the separation by
interacting with HPMC molecules and decreasing the threshold
of the polymer solution. Satisfactory resolution of DNA fragments
can be achieved in the solution with a concentration even lower
than the overlap threshold concentration. A theory used to
interpret the DNA separation mechanism in a ultradilute polymer
solution is found to fit the data presented in this paper for the
low-molecular-weight solution. A concentration of 7 M urea is
necessary for the complete denaturing of the ds DNA fragments.
The electric field does not have much influence on the mobility
of the DNA fragments, which implies that the DNA fragments up
to 2000 bp do not change their conformation with a change of
electric field.
ACKNOWLEDGMENT
The authors thank Dr. Kenneth Martin and Dr. Kenneth
Fountain of Truman State University for their help on the
manuscript. This work was supported by an Internal Faculty
Research Grant from Truman State University awarded to Y.M..
We also appreciate the support from the National Natural Science
Foundation of China.
Received for review February 10, 1999. Accepted April 6,
1999.
AC990160X
Analytical Chemistry, Vol. 71, No. 13, July 1, 1999
2389