3 114 C.

Heller Electrophoresis 1998, 19, 3114-3127

Christoph Heller Finding a universal low viscosity polymer for DNA

separation (11)
Abteilung Lehrach,
Max-Planck-Institut fur When investigating the use of different polymers for capillary electrophoresis we
Molekulare Genetik, found that poly-N,N-dimethylacrylamide (pDMA) has a very low viscosity
Berlin-Dahlem, Germany compared to other polymers of comparable molecular mass and resolving power.
This makes it a potentially useful matrix for DNA separation in multi-capillary
electrophoresis, where short cycle times or low pressure for matrix replacement are
preferred. We have characterized this matrix by systematic studies on concen-
tration, chain length and field strength dependence. It is shown that pDMA
performs well for the separation of oligonucleotides and double-stranded DNA
fragments. Together with the application of DNA sequencing,pDMA is a universal
polymer for the separation of biological macromolecules.

1 Introduction solutions gave superior separations over dilute solutions

[ 151. Therefore, for many high resolution applications (e.g.,
During the last ten years, capillary electrophoresis (CE) has DNA sequencing), the properties of an entangled network
developed into a powerful analytical method and has also will most probably be needed. To date, a number of
shown great potential in the separation of biopolymers different polymers have been described for the separation of
currently being separated by slab-gel electrophoresis. CE, DNA in CE. Most of the polymers used are modified
however, has numerous advantages over slab-gel electro- polysaccharides: agarose [4, 161 and various cellulose
phoresis, which have already been described in detail [l]. derivatives such as methylcellulose (MC), hydroxyethyl-
CEs performance is comparable to HPLC, but has the cellulose (HEC), hydroxypropylcellulose (HPC) and
advantage that multiple capillaries can be run at the same hydroxypropylmethylcellulose (HPMC) [ 13-15, 17, 181.
time (see e.g. [2, 31). In the case of DNA separation, CE Synthetic polymers such as linear polyacrylamide and
offers full compatibility to existing biochemistry, which is alkyl-substituted derivatives [ 12, 19-23] as well as poly-
not always the case in alternative techniques, such as .ethyleneglycol (PEG) or polyethylene oxide (PEO) [2426]
MALDI-MS. Early attempts to apply CE to the size- have been used. This list shows that a great number of
separation of biomolecules were based on gel-filled polymers exists which could potentially be used as a
capillaries (e.g. agarose or cross-linked polyacrylamide) separation matrix for biological molecules, but not all of
[4, 51. However, gel-filled capillaries have several dis- them are suitable for standard CE devices. Especially in the
advantages: First, the filling of the capillary has to be done new multicapillary devices, a low viscosity to keep the
with great caution in order to avoid the introduction of air technical sophistication low is needed. Therefore, the ideal
bubbles. The shrinkage of the gel during polymerization can polymer should have at least the same separation properties
also be a source of bubbles [6]. Gels, and in particular as classical gels, combined with a low viscosity that would
polyacrylamide, can be degraded by hydrolysis, especially allow easy replacement.
at the alkaline pH normally used to separate biopolymers. It
has also been observed that during repeated use, bubbles
can form at the sample injection end of the capillary [7, 81. Recently, matrices have been described which can change
Gels can also dry out at the ends. their properties (notably the viscosity) if the temperature is
changed. Two mechanisms can be identified here: Either
the collapse of molecules at high temperature [27] or the
A solution to these problems is to use a polymer solution, formation of micelles with increasing temperature [28, 291.
which can be replaced after each run. Shortly after the first These ideas seem very promising, yet need to be better
DNA separations in gel-filled capillaries were performed [9, exploited. Also, they require a precise temperature control,
101, it was soon found that DNA can be separated in so- which puts higher demands on the instrumentation. Third,
called “semidilute” (i. e. “entangled”) polymer solutions the temperature requirements of the matrix have to be
(e.g. [ll-131). Later, it was proven that separation is adjusted to the temperature requirements of the analyte: For
possible even at concentrations below the overlap threshold example in DNA sequencing, a temperature of 50-60”C is
(dilute solutions) [14]. However, in a systematic study it needed to keep the molecules denatured. The rather low
was shown that for small dsDNA, the entangled polymer velocity in such matrices [28] might also be a concern.
Therefore, our approach is to develop a separation matrix
Correspondence: Dr. Christoph Heller, Max Planck-Institut fiir Mole- based on “classical” polymer entanglement. Recently, we
kulare Genetik, JhnestraBe 73, D-14195 Berlin, Germany, (Tel: +49- have described how such a polymer can be found and be
30-841 3-1710; Fax: 49-30-84 13-1380; E-mail: heller@mpimg-berlin- successfully used for the case of DNA separation [l]. The
dahlem.mpg.de)
present paper is a continuation of the earlier work and
Abbreviations: BRF, biased reptation with fluctuations; HPC, hydroxy- identifies a second polymer, poly-N,N-dimethylacrylamide
propylcellulose; PAA, polyacrylamide; pDMA, poly-N,N-dimethyl- (pDMA), as a good candidate for biomolecule separation.
acrylamide; TOPRO-1, quinolinium,4-[(3-methyl-2(3H)-benzothiazolyli- pDMA has been proposed before as a separation matrix for
dene)methyl]-l-[3-(trimethylammonio)propyl]-, diiodide CE [22, 301, but extensive, fundamental studies on this
Keywords. Separation matrix / Polymer solution I D N A separation I polymer have not been undertaken.
Viscosity

0 WILEY-VCH Verlag GmhH, 69451 Weinheim, 1998 0173-0835/98/1818-3114 $17.50+.50/0

Electrophoresis 1998, 19, 31 14-3127
2 Theory
2.1 Polymer solutions
The basics of polymer solutions have been described in
earlier publications (e.g. [31, 321). Therefore, we shall only
recall very briefly the major equations used in this study:
Polymer entanglement takes place above the overlap
threshold, or entanglement threshold, c*. Equation (1) is
the geometrical definition of the entanglement threshold,
i.e. c* is the concentration at which the polymer chains
(modeled as coils) touch each other [33, 341:
3 h!fJ4 3t N A R:
C* “N (1)
with R, and M, being the radius of gyration and the
molecular mass of the polymer, respectively, and NA being
the Avogadro number. For a given polymer, this threshold
can be experimentally determined by measuring the
viscosity of the polymer solution at different concentrations
and finding the point of departure from linearity on the
specific viscosity vs. concentration plot [18]. However, as
previously pointed out [34], this method depends essentially
on the accuracy of the experimental data and deviations
from linearity are difficult to estimate precisely.
Therefore, in order to determine the overlap threshold more
accurately, we need to know the radius of gyration of a
particular polymer. There are several ways to measure the
radius of gyration experimentally, but if M, is known, a
simple way to determine it, is to measure the intrinsic
viscosity in the dilute regime [35]:
[q] Z 6.2 R: N A / M ~ (2)
The value [q] (also called limiting viscosity number) is a
measure of the capacity of an isolated polymer to enhance
the viscosity of a solution and it increases with the
molecular mass. The viscosity q of a polymer solution
increases strongly with the concentration. However for very
dilute solutions, we can usually find the following relation:
with qs being the solvent viscosity and c being the polymer
concentration. Equation (3) describes the tangent to the
viscosity vs. concentration curve for c + 0. Usually, we
prefer to express the viscosity of a polymer solution relative
to the viscosity of the solvent: qrel = q/qs. For low
viscosities, this value approaches one. It is more useful to
take the difference between the viscosity and the solvent
viscosity and normalize it again to the viscosity of the pure
solvent. This is called the specific viscosity and approaches
zero for low viscosities: qspec= (q-qs)/qs= qrel- 1. In order
to estimate the influence of isolated polymers, we have to
divide this value by the concentration of the solution, which
gives us the reduced viscosity. Rearranging Eq. (3), we now
get:
which means that we can then determine the intrinsic
viscosity by extrapolating the reduced viscosity to zero
A low viscosity polymer for DNA separation 3 115
concentration. A second method to determine [q], is to
extrapolate the inherent viscosity qi,,h to zero:
[TI = In qrel/Cc -t 0 = qinh c + 0 (5)
The intrinsic viscosity has been measured for a number of
polymers and it was found that for many of them it fits the
following empirical expression (Mark-Houwink-Sakurada
equation) [36]:
[q] E K Mva (6)
where a and K are characteristic constants (“Mark-Houwink
constants”) for a given polymer-solvent system. For flexible
molecules, the value of a varies between 0.5 (polymer in an
ideal solvent) and 0.8 for so-called “swollen” chains. Mv is
the “viscosity average molecular mass”, which is different,
but often close to the weight average molecular mass, M,
WI.
If we neglect the difference between Mr and M , and
combine Eqs. (1), (2) and (6) we get a simple expression for
the overlap threshold:
c* z 1.5 [q]-l E (1.5/K) M,-“ (7)
For electrophoretic separations, we might want to know the
“pore size” of such a network. A chain segment between
two entanglement points can be regarded as an independent
subunit, and can itself undergo a random walk. The volume,
which this chain segment can take, is called a “blob” and is
described by the “blob size”, c b (analogous to the radius of
gyration). Viovy and Duke [34] argued that this “blob size”
should be used as a “pore size”, which can be calculated as
[33]:
kb = 1.43 R, (C/C*)-~’~ (c>c*) (8a)
(with c b in cm and concentration in g/mL). This means that
the “pore size” of an entangled polymer solution does not
depend on the degree of polymerization but only on its
concentration.
2.2 Electrophoresis theories
The mechanism of DNA movement in a porous matrix (gels
and entangled polymer solutions) under the influence of an
electric field has been the subject of intensive research and
reviewed in many articles (e.g., [31, 38, 391). Briefly, two
major regimes have been identified: The Ogston model [40]
- which assumes that the mobility of a particle is
proportional to the available volume fraction - describes
the mobility p of this particle as:
p = PO exp [-KRCl (9)
where is the mobility in pure solvent (free solution
mobility , KR is the retardation cuefficient (proportional to
1
(RD+ r) with r being the radius of the gel fibers and RD the
3 116 C. Heller
radius of gyration of the DNA molecule) and C is the gel
concentration. Recently, the Ogston model has come under
radical reconsideration [41], and an improved model is
under development. The Ogston model does not correctly
account for the mobility of large DNA molecules in gels, so
that a second type of model, the biased reptation model, was
proposed. In the original biased reptation model (BRM), the
DNA chain (which is much larger than a pore of the
separation matrix) is assumed to thread its way - without
changing its length - through a “tube” defined by the fibers
(for a rigid mesh) or the “blobs” (for a flexible network)
surrounding it. The BRM predicts (for low electric fields)
that the electrophoretic mobility in DNA gel electrophoresis
can be described by the following equations [39, 421:
cI/b= 1 / 3 N ~+ ~ ~ 1 9 1 < ND < ND” (10)
for small DNA (below a “critical length’, ND*) and:
plh sz ~ ~ -1113ND
9 ND > ND” (1 1)
for large DNA, with ND = MDIM, being the scaled
molecular size of DNA, i.e. the molecular size of DNA,
MD,related to the portion Ma of a DNA molecule that would
fit into one pore. E is the so-called “scaled electric field”,
proportional to the applied electric field strength. For small
molecular sizes, the BRM predicts that the mobility scales
as llsize; for large sizes it predicts a plateau mobility.
This theory was later improved by Duke et al. [43] to
include the fluctuations in the length of the DNA molecule.
This new model, called “Biased Reptation with Fluctua-
tions’’ (BRF), considers two different cases: first, when the
pore size a of the matrix is larger than the pore size bD of
DNA (a = albD > 1) and second, when the pore size is
smaller (so-called “tight gels”, a < 1). In both cases, we
have to again distinguish between two regimes, i.e. small
DNA (below a critical length ND*) and large DNA (above a
critical length). In the first case, the BRF predicts for the
mobility of small DNA:
and for large DNA:
As in the BRM, for small molecules, the mobility is
inversely proportional to the DNA size (“reptation without
orientation”), but for large DNA andlor high electric fields,
Eq. (13) reduces to: plpo M E, which means that the
mobility is independent of size (plateau mobility) and
separation fails (“reptation with orientation”).
In tight gels (a < l), reptation still occurs below a critical
size (Eq. 12), but for large DNA (ND > ND”), the existence
of three different regimes are predicted [43]:
plpo x E E < a7’2 (14a)
wo M 4 21s
) a > E > a7/2 (14b)
and: plh M E~ &>a (14c)
Electrophoresis 1998, 19, 3114-3127
In fact, the second regime (Eq. 14b) was observed by
Mitnik et al. [15] for DNA in entangled HPC solutions.
Polymer solutions differ from gels in that the physical
entanglements between the chains have a finite lifetime and
the dynamics of the matrix itself has to be taken into
account. Viovy and Duke [34] have adopted the physical
concept of “constraint release” (CR) to describe this
phenomenon. The net DNA mobility is then given by the
sum of the BRF and the CR mobility: ptot= hp +~CR.
Note, that the constraint release mobility is independent of
both, DNA size and applied electric field, but dependent on
the polymer concentration and the chainlength. Besides the
two major mechanisms, Ogston sieving and reptation, more
regimes like “entropic trapping”, “reptation self trapping”
and “geometration” have been identified (see [38, 391 for
review).
The abovementioned mechanisms are true for matrices with
existing “pores” only. However, it was found, that DNA can
even be separated in very dilute polymer solutions, where
the concept of pores fails. For this case an “entanglement
coupling” mechanism was proposed [44, 451, where the
DNA molecules hook on to polymer molecules, dragging
them along during electrophoresis.
2.3 Resolution
To compare different separation techniques or protocols, we
need a measurement for the quality of the separation. In the
CE literature, different expressions, like “efficiency”,
“selectivity” and “plate height” have been used, resulting
in some confusion. Therefore we want to recall briefly the
main aspects: To measure the quality of a separation, the
resolution can be used, which is defined by the ratio of the
distance between two bands or peaks (distance between the
centers of gravity) and the average peak width. The peak
width can be measured at the baseline or at half height, wh:
Both, AX and wh are given in units of length, but may also
be regarded as the corresponding quantities in time units.
According to this definition the resolution depends on two
factors: The interband spacing - which is often called the
“selectivity” of a system - is given by the length of the
migration path, and the relative velocity difference between
the two species (which in turn is determined by the
migration mechanism):
AX = L Avlv.
The second factor is the band width, which can be
influenced by the migration mechanism [46, 471, but in
practice is a result of the independent dispersion effects. It
is equivalent to the so-called “efficiency”, described.by the
number of “theoretical plates”. However the efficiency
alone is not enough to describe the performance of a
separation. In other words, a capillary column with a higher
number of theoretical plates does not help if peak separation
fails due to the migration mechanism. On the other hand, if
two species differ greatly in their relative velocities, a low
plate number is acceptable. Landers [48] stated that ... it is
“
Electrophoresis 1998, 19, 31143121
important to note that it is, in fact, misleading to discuss
theoretical plates in electrophoresis. The concept is a carry-
over from chromatographic theory, where a true partition
equilibrium between two phases is the physical basis of
separation. In electrophoresis, separation of the components
of a mixture is determined by their relative mobilities in the
applied electrophoretic field, which is a function of their
mass, charge and shape...”. Also, as the number of
theoretical plates is usually given in plates per meter, this
can lead to a wrong assumption that the resolution would
scale linearly with the capillary length. For CE separations,
this is clearly not the case and we therefore recommend,
that resolution be used to describe the performance of a
system.
For the special case of DNA separation, it might be even
more convenient to take the reciprocal value of R,, and
normalize it to the DNA size [49, 501:
with Whl and Wh2 being the peak widths at half height and
AM the difference in size (in b or bp). This “separation
factor” S, which is equivalent to the “resolution per bp”
defined in [26] has the advantage of directly giving the
smallest difference in bp (or bases) that can be resolved.
3 Material and methods
3.1 Preparation of pDMA
pDMA was prepared according to a protocol by Chiari
et ul. [22]: N,N-dimethylacrylamide (DMA; Polysciences,
Warrington, PA, USA or Fluka, Buchs, Switzerland) was
dissolved in water to a final concentration of 10% w/w and
isopropanol was added in various amounts (0-5%). After
degassing (with a vacuum and ultrasound), 1 pL TEMED
per mL solution and 1 pL/mL of a 40% ammonium
persulfate solution was added. After careful mixture, the
solution was allowed to react for 1 h at 25°C or 50”C,
followed by incubation at 25°C overnight. The reaction
product was extensively dialyzed against water using
25 000 MWCO dialysis membranes (Roth, Karlsruhe,
Germany or Spectrum, Los Angeles, CA, USA) and
lyophilized. The recovery was 7 g of white solid per
100 mL of DMA solution.
3.2 Polymers and viscosity measurements
Dextran (average molecular mass 500 000 g/mol) was from
Sigma (Deisenhofen, Germany) and polyacrylamide (mo-
lecular mass 700 000 to 1 000000 g/mol) was from
Polysciences. No further information about the size
distribution (polydispersity) of these polymers was given
by the supplier. Kinematic viscosities were measured
manually with standard Ubbelohde viscometers (Schott,
Mainz, Germany) placed in a waterbath thermostated at
25°C or 40°C.
3.3 CE
For DNA separation the ABI 310 capillary electrophoresis
device (Perkin Elmer-ABI, Foster City, CA, USA) was
A low viscosity polymer for DNA separation 3 117
used, equipped with a 47 cm long (36 cm to the detection
window) 375/100 pm OD/ID fused-silica capillary (Poly-
micro Technologies, Phoenix, AZ, USA). As DNA samples,
we used @X174/HueIII, 10 bp ladder, 50 bp ladder, 1 kbp
ladder (all from Gibco BRL, Life Technologies, Eggenstein,
Germany), Marker V (Boehringer Mannheim, Germany), 20
bp ladder, pBR322/MspI and pUC19/Suu3AI (all from
Advanced Biotechnologies, Epson, UK). They were diluted
1000-fold in water to give final concentrations of 0.2-1 ng/
pL. As oligonucleotide standards, po1ydT25-30,
and polydA40-60 (all from Pharmacia, Uppsala, Sweden)
were dissolved in 1 mL TE (10 m~ EDTA, pH 8.0) and
diluted 1000-fold in water. For dsDNA and oligonucleotide
separations, 0.5 X TBE buffer (44.5 m~ Tris, 44.5 mM boric
acid, 1.25 m~ EDTA, pH 8.3; conductivity 625 pS) was
used as background electrolyte for the polymer (which was
only in the capillary) and as electrode buffer, supplemented
with TOPRO-I or Oligreen-I (Molecular Probes, Eugene,
OR, USA) added to a final concentration of 0.1 p ~All .
separations took place at 25OC. For the free mobility
measurements, the capillary was dynamically coated by
flushing a 1% pDMA solution (short chain) through the
capillary followed by rinsing with buffer (0.5 X TBE). This
coating proved to be stable for at least ten injections. The
mobility measurement was performed in 0.5 X TBE with
0.1 p~ TOPRO-1 at 25°C. For the experiments with
labelled DNA, Sizer 50-500 (Pharmacia) was used. For
denaturing conditions, 2 pL DNA were added to 8 pL
template suppression reagent (ABI), heated to 90°C for
2 min and chilled on ice before injection. As background
electrolyte, 0.5 X TBE with 7 M urea was used and 0.5 X
TBE as electrode buffer. Electrophoresis took place at
50°C. For nondenaturing conditions, the DNA was just
dissolved in water, urea was omitted and separation took
place at 25OC.
4 Results
4.1 Choice of polymer
The result of Eq. (8) means, in other words, that two
solutions of the same type of polymer and with the same
concentration but different molecular mass, will have the
same “pore size” as long as they are entangled. However,
their viscosity is dependent on the molecular mass, which
has important consequences for the choice of the appro-
priate polymer for CE. In an earlier work, by plotting the
intrinsic viscosity vs. mol. mass we could identify dextran
as a polymer with a potentially low viscosity [l]. Figure 1
shows the corresponding plot (calculated by Eq. 6) for
pDMA in comparison to dextran and linear polyacrylamide
(PAA) which is frequently used as a separation matrix. In
the literature, we find two rather different sets of Mark-
Houwink parameter values for pDMA, depending on the
temperature; at 25°C a = 0.81 and K = 0.0232 mL/g,
whereas at 4OoC, a = 0.65 and K = 0.020 mL/g [36]. From
Fig. 1 and Eq. (6) it is clear that the exponent a is the
predominant factor determining the intrinsic viscosity. The
difference in u at these two temperatures is rather
astonishing, as it would mean a roughly ten times lower
intrinsic viscosity at 40°C compared to 25°C. This indicates
a considerable change or collapse in polymer conformation.
From Fig. 1 we can see that pDMA (measured at 40°C) has
as low an intrinsic viscosity as dextran for all molecular
3 1 18 C. Heller
Electrophoresis 1998, 19, 31143127

1 1
n
0, __ -pDMA 05% kop
> - pDMA 1% ISOP
E
- 1

0
.-
v)
c 1
.-
L

-c
4-0

1 1
1 o3 1 o4 1 o5 1 o6 1 o7 1o - ~ I o - ~I o - ~ I o-’ lo-’ I oo

Mol. Mass ( g h o l ) Concentration (g/mI)

Figure 1. Intrinsic viscosity (in d / g ) dependent on the molecular mass

for pDMA, dextran and PAA, calculated using Eq. ( 6 ) and data in [36].
1 o3
- - pDMA 0 5% lsop
sizes. This makes it a potentially good candidate for a low-
h
viscosity separation matrix. For efficient separation of DNA cn - pDMA 2% lsop
molecules, the “mesh size” of the polymer network is 1 - pDMA 3% lsop
important. Figure 2 (calculated using Eq. 8b) tells us that “E 1 o2
pDMA (at 4OOC) gives similar mesh sizes as dextran, which E - pOMA 5% lsop
v
is not surprising, as the Mark-Houwink values for dextran
and pDMA (4OOC) are close together [36]. +>r
.-
cn
4.2 Viscosity measurements ;10’
.-
pDMA is synthesized from the monomer, dimethylacryla- >
mide, by radical polymerization. The chain length can be

1 oo
10-5 1 0 - 4 1 0-3 1 o-2 l o - ’ 1 oo
Concentration (g/m I )
n Figure 3. Kinematic viscosity (in mm2/s or centiStokes) of different
E polymer solutions measured at 25OC dependent on the concentration.
t
W (A) pDMA solutions, prepared at 25°C in presence of 0%. 0.5, 1%,2% and
3% isopropanol; (B) pDMA solutions prepared at 5OoC in presence of 0%,
0.5%, I%, 2%, 3%, 4% and 5% isopropanol. For comparison, the
viscosities of a commercial PAA (M,700000-1 000 000) and a
commercial dextran preparation (M,500 OOO) are shown.

influenced in the following way; increasing the amount of

initiator (in this case ammonium persulphate ( A P S ) , in
combination with TEMED) or increasing the temperature
leading to a higher rate of nucleation and decrease in
polymer length. A more precise method to control the
polymer length is to stop the chain growth by using radical
1 o - ~ 1o - ~ 1 o - ~ 1 0-’ 1 o0 traps (“chain transfer reagents”), such as isopropanol.
Concentration (g/mI) Following the protocol described by Chiari et al. [22], we
produced pDMA solutions under different conditions and
Figure 2. “Mesh size” (in nm) for different polymer solutions dependent
determined their viscosities and used this as a measure the
on the concentration (only for c > c*), calculated using data from [36] and chain length. Note, that with the viscometers used here, we
Eq. (8b). determined the kinematic viscosity (measured in mm2/s or
Electrophoresis 1998, 19, 3114-3127 A low viscosity polymer for DNA separation 3 1 19

475 t 1
h
m
1 600
E t /

-
v

x 500
.-
v)
0
500
0
.-v) 3-F
> t / /
U
a,
0
3
-0
a,
I
I:

0 0,Ol 0,02 0,03 0,04

Concentration (g/ml)
Figure 4. Example for the estimation of the intrinsic viscosity by
extrapolating the reduced viscosity and the inherent viscosity of a polymer 0 1 2 3 4 5
solution (measured at 25°C) to zero polymer concentration. The plot shows
the values for a pDMA solution prepared at 25°C in presence of 2% lsopropanol Concentration (O/.)
isopropanol. The fitted lines (least square fits) intercept the y-axis at the
common value of 184 mUg.

centistokes), which differs from the dynamic viscosity

(measured in mPas or centiPoise) by the density of the
medium. However, for the dilute solutions, we can assume
that the density is close to one. Figure 3 shows the
viscosities (measured at 25°C) of 12 different pDMA
preparations synthesized at 25OC and 50°C respectively,
together with the viscosity of a dextran solution (mol. mass
500 kDa) and of linear PAA (700 000-1 000 000 Da), for
comparison.

During the last years, several groups have claimed to have

developed a “low viscosity polymer” (e.g., [25, 26,51,52]).
As this term can be misleading, we would like to give the
following arbitrary definition: Solutions in the viscosity
range from 1-10 centistokes should be called “very low
viscous”, whereas those from 10-100 centistokes should be
classified as having a “low viscosity”. Those matrices
having a viscosity of 100-1000 centistokes will be named
“medium viscous” and those above “highly viscous” (1000-
10 000 centistokes). Polymer solutions with a viscosity
beyond that limit can hardly be injected into capillaries with
commercially available equipment. From Fig. 3 we see that
pDMA solutions prepared in the presence of 1% isopropa-
no1 and more (at both temperatures) can be classified as
“low to medium viscous” in the useful concentration range
of 2-8%, whereas the viscosities of the preparations in
absence or at very low concentrations (0.5%) of isopropanol
extend into the “high viscous” range. All pDMA prepara-
tions are more viscous (at 25‘C) than M, 500 000 dextran
but have a lower viscosity than a commercially available
PAA preparation, which has a molecular mass in the same
range as the pDMA preparations made in presence of 0-1 %
isopropanol at 25°C and 50°C (see Table 1). From these
data, the intrinsic viscosity was determined by extrapolating
both the inherent viscosity and the reduced viscosity to zero
polymer concentration (see Eqs. 4 and 5). Both lines should
intercept the y-axis at the same point, as shown for example
0 1 2 3 4 5
lsopropanol Concentration (%)
Figure 5. Plot of the inverse of the molecular mass vs. the isopropanol
concentration during synthesis (0) at 25°C and (x) at 50°C. The molecular
mass data are based on the viscosity measurements (A) at 25°C and
(B) 40°C (see Table 1). The data can be fitted to the following relations
(straight lines): (A) 25°C: y = 0.72 + 0.53 X (3= 0.941) and 5OOC: y = 0.89
+ 0.64 X (2= 0.974); (B) 25°C: y = 0.56 + 0.55 X (3= 0.972) and 50°C: y
= 0.65 i0.75 X (3= 0.978).
As the Mark-Houwink parameters are published for differ-
ent temperatures ( i e . 25°C and 40°C [36]),the viscosities of
the same solutions were measured again at 40°C (not
shown) and the corresponding intrinsic viscosity was
determined. Table 1 shows the intrinsic viscosity values
for the different pDMA preparations at both temperatures,
together with the molecular mass and the overlaD threshold.
v

in Fig. 4. c*, calculated by Eq. (6) and (7). However, we <ant to point
3 120 C. Heller Electrophoresis 1998, 29, 3114-3127

Table 1. Intrinsic viscosities, calculated molecular masses, and overlap thresholds for different pDMA solutions, prepared at 25°C and 50°C in the
presence of various amounts of isopropanol
Polymerization Isopropanol Intrinsic Viscosity Molecular mass (Da) c* (%) at 25OC Intrinsic Viscosity Molecular mass (Da) c* (%) at 40°C
temperature conc. (%) ( W g ) at 25°C (a = 0.81, K = 0.023) (mL/g) at 40°C (a = 0.65, K = 0.02)

25OC 0 345 141 OOO 0.43 210 1536OOO 0.7

25'C 0.5 239 90 000 0.65 n.d. n.d. n.d.
25°C 1 .o 214 78 000 0.7 152 934 OOO 0.95
25°C 2.0 184 65 OOO 0.8 120 649 000 0.125
25°C 3.0 125 40 OOO 1.2 91 424 OOO 0.165
50°C 0 332 135 OOO 0.45 187 1 285 OOO 0.8
50°C 0.5 217 79 OOO 0.69 n.d. n.d. n.d.
50°C 1 .O 168.5 58 OOO 0.89 135 778 OOO 1.1
50°C 2.0 148.5 50 000 1 .o 100 490 OOO 1.5
5OoC 3.0 110 34 OOO 1.4 77 328 OM)* 1.95
50°C 4.0 97.5 29 OOO 1.5 n.d. n.d. n.d.
50°C 5.0 83.5 24 OOO 1.8 n.d. n.d. n.d.
* The molecular mass of pDMA prepared under similar conditions was determined as 225 000 Da [22].
n.d., not determined

out here, that this estimation is strictly valid for mono-
disperse polymers (when Mr = M,). Therefore, the
molecular mass values and the c* values should only be
used with caution when working with poorly fractionated
polymers.
The degree of polymerization in reactions with chain
transfer can be described by the so-called Mayo-equation
[37]:
IlX, = (l/Xn)o + k [S]/[M]
with X, being the degree of polymerization, and [S] and [MI
being the concentration of the chain transfer reagent and the
monomer, respectively. As in our case, the monomer
concentration was the same in all reactions, a plot of the
inverse molecular mass vs. isopropanol concentration
should yield a straight line. Figure 5 shows that, within
experimental error, our data can be fitted to a straight line
and satisfy in fact Eq. (17).
The most striking result from Table 1 is that the molecular
mass, obtained from the intrinsic viscosities of the same
solutions but measured at different temperatures, differ
quite considerably (up to tenfold or more). We have no
independent estimation of the molecular mass, but Chiari et
al. [22] measured the size of pDMA prepared with 3%
isopropanol at 5OoC. Their value of 225 kDa is relatively
close to the value of 328 kDa estimated for our preparation
at 40°C (Table 1). Given the fact, that the preparations have
been done in different labs and with monomer from
(17) different suppliers and probably different degrees of
degassing, they are still in good agreement. However, it
indicates that the published Mark-Houwink values for 25°C
[36], are not applicable here. Therefore, in the following,
we will use the molecular mass values obtained with
viscosity measurements at 40°C. Taking these values, we
can claim from Fig. 3 that a solution of pDMA with a
molecular mass of up to 1.5 million has a lower viscosity (at
the same concentration) than a solution of PAA with a
molecular mass of about 700 000-1 000 000 Da.

Time (sec)

500 600 700 800 900 1000

I I I I I I

Figure 6. Electrophoretic separation of @X174/HueIII DNA (0.5 Fg/mL) in a pDMA matrix prepared at 50°C in presence
of 2% isopropanol (490 kDa preparation). Conditions: electrokinetic injection at 1 kV for 5 s; separation in 0.5 X TBE
containing 2.5% w/w pDMA and 100 n~ TOPRO-1 at 10 kV (210 V/cm) in a 47 crn long (36 cm to the detector) fused-silica
capillary (100 pm ID).Peaks: 1,72 bp; 2,118 bp; 3, 195 bp; 4,234 bp; 5,271 bp; 6,281 bp; 7,310 bp; 8,603 bp; 9,872 bp;
10, 1078 bp and 11, 1353 bp.
Electrophoresis 1998, 19, 31 14-3127 A low viscosity polymer for DNA separation 3 121

I I I Ill IV IV Ill II

4
E 3
0
\

>
-.
v
h
2
v)

10 100 1000 10000 0 0,002 0,004 0,006 0,008

Size (bp) 1/ s i z e
I II Ill IV

n
\
v)
\

E 0
=I
v

X Figure 7. (A) Dependence of the electrophoretic mobility of linear dsDNA

fragments on the size, separated in pDMA (490 kDa) at different
0 concentrations and at 210 Wcm. 0.5 X TBE containing 100 nM TOPRO-
1 was used as electrode buffer and background electrolyte. (B) Semi-
I I 00
1 9 1 1 I l l I l l I logarithmic plot of the electrophoretic mobility vs. size for 7.5% and 10%
I I I pDMA (490 kDa) at 210 V/cm (conditions as in A) and for 1% HPC (1
mio) at 270 Vkm. ( C ) Plot of mobility vs. the inverse of DNA size for
0 200 400 600 800 10001200 separation in 2.5%, 5%, 7.5% and 10%pDMA at 210 v/cm (conditions as
in A). The zones I-IV correspond to the different regimes explained in the
Size (bp) text .

4.3 Separations mobility vs. size plot: medium-sized molecules (250-700

4.3.1 Influence of polymer concentration
The use of pDMA for DNA separation in CE has been
described before [22, 27, 301, but for a detailed character-
ization of this new matrix, we need to undertake more
systematic studies. To study the influence of the polymer
concentration, we have picked the 490 kDa preparation
(polymerized in the presence of 2% isopropanol at 50°C),
which showed a very good performance at low viscosity in
pilot experiments (see Fig. 6 as an example). Figure 7a
shows the dependence of the electrophoretic mobility of
DNA on polymer concentration at an electric field strength
of 210 Vlcm. Above a size of about 70 bp, we can observe
the usual sigmoidal curve on the double logarithmic
bp) are well resolved (regime 111), whereas larger ones (>
700 bp, regime IV) and smaller ones (70-250 bp, regime 11)
show less difference in mobility. This behavior is identical
to that observed in other polymer solutions such as HPC,as
described before [15]. Looking closer at the large molecules
we observe, however, that the mobility does not fully level-
off as it does in gels (see, e.g. [53]), but that some
separation is still possible. Again, this effect has been
observed before and we attribute it to the flexible nature of
the network (see below) [15].
However, we focused our attention on the small DNA
range, as in earlier works, we found some indication for
a new separation regime [I]. Separation of such small
3122 C. Heller
\
0 0
2 - 0
0 mobility vs. concentration plot should yield a straight line as
0
W
0
0
0
0
0 From this, we can conclude, that only the smallest
0
0 2 4 6 8 1 0 1 2 [15, 54, 551, but in full agreement to the recently measured
pDMA Concentration (%)
Figure 8. Semilogarithmic plot of the electrophoretic mobility vs. pDMA solution (0% pDMA) confirming these data.
concentration (“Ferguson plot”) for four different DNa fragments, each
selected from one of the four regimes of Fig. 7a. Conditions as in Fig. 7.
The straight line represents a fit of the data for 20 bp to the following
relation: Y = 4.45 exp[-0.10], (3 = 0.975).
molecules has hardly been studied in detail to date, as in
usual agarose gels, they are not separated and also only
recently, intercalators with strong fluorescence and
strong binding became commercially available, making
it possible to visualize those short molecules without the
need to overload the gel or the capillary. Figure 7a also
shows that for the small molecules (8-70 bp, regime I)
the mobility does not continue to level-off as it is
described in numerous publications (e.g., [38, 53-58] for
separation in gels and [15, 281 for separation in polymer
solutions), but that it increases with decreasing DNA
size. This effect, which has been verified with different
DNA standards (to exclude effects of repeating sequen-
ces) is most pronounced in 10% pDMA. In more dilute
solutions, the intermediate plateau, named regime 11, is
barely visible and there is a direct crossover from
regime I (with the shallower slope) to regime I11 (steep
slope).
In order to check for Ogston type separation, the data from
the 5 % and 10 % pDMA experiments have been replotted
in a semilogarithmic plot, together with the data obtained in
1 % HPC (Fig. 7b), which was published previously [l].
Again, we can identify the different regimes (from left to
right): a steep slope (regime I), followed by a plateau (only
for 10 % pDMA, regime 11) which then leads to another
regime (regime 111) with a steep slope, but different from
regime I. With increasing size, the curve turns into a regime
with a very shallow slope again (regime IV), indicating the
onset of the reptation with orientation mechanism. As the
Ogston model is in principle applicable to rigid fibre-like
obstacles, it is not obvious what role the “fiber radius” r
(Eq. 9) in a polymer solution plays here. However, if we
take the molecular radius of a polymer in the order of 1-
1.5 nm, it becomes negligible compared to the radius of
Electrophoresis 1998, 19, 31 14-3127
gyration of DNA. In this case, Eq. (9) predicts that the
Ogston-type separation should lead to a straight line with
negative slope on a semilogarithmic mobility vs. size plot.
However, as we observe two regimes with strong negative
slopes (Fig. 7b), it is not obvious, which one corresponds to
the Ogston regime.
However, Eq. (9) also tells us that a semilogarithmic
well. We picked one representative DNA fragment, from
each of the four regimes, and drew a plot (so-called
Ferguson plot [59], Fig. 8). The only fragment showing a
straight line in the Ferguson plot is the shortest one (20 bp).
fragments (regime I) are in fact separated by an Ogston-
like separation mechanism. The straight line intercepts the
y-axis at a value of 4.45 x cm2Ns (free solution
mobility), which is slightly higher than previously evaluated
free mobility in TBE of 4.4 f 0.1 X lo4 cm2/Vs [60]. In
an independent experiment, we obtained a value of 4.37 X
lo4 cm2Ns, when directly measuring the mobility in free
Having identified regime I as the Ogston regime, we now
can attribute the regime I11 to the reptation regime, whereas
the border to regime IV indicates the onset of the reptation
with orientation mechanism, in analogy to earlier results
[15]. In regime I11 we do in fact obtain the steepest slope,
but we do not reach a slope of -1 as required by the
reptation model. However, considering the dynamic
nature of the network and the rather strong electric field
(210 V/cm) used here, we cannot expect to observe “true”,
i.e. unbiased reptation. In order to check for a possible
underlying reptation, we replotted a subset of the data of
Fig. 7a as a linear mobility vs. inverse of size plot (Fig. 7c).
If constraint release (or other processes) added to the overall
mobility, we would not find a slope of -1 in the double
logarithmic mobility vs. size plot. However, the linear
mobility vs. l/size plot still yields a straight line. Indeed, in
Fig. 7c, we found a linear relationship within the domain of
regime 111, indicating a mobility inversely proportional to
the size in agreement to Eqs. (10) and (12), respectively.
4.3.2 Influence of electric field
Besides the polymer concentration, the electric field
strength is one of the major factors influencing the mobility
of DNA in a porous matrix. As the abovementioned
electrophoresis models are strictly valid for zero or low
electric field only it is important to measure the field
dependence of a separation method in order to test the
applicability of these theories. Figure 9a shows again a
double logarithmic plot of mobility vs. size, obtained in the
same matrix (5% pDMA 490 kDa), but at different electric
fields. Again, the result is similar to those obtained in other
matrices [15]. In the example shown here, a slope of -1
could again not be reached, not even at a higher con-
centration (Fig. 9b). This and the fact that the mobility does
not fully level-off for long DNA indicates that the
entanglement between the chains is not strong enough for
the chain length here to demonstrate an “ideal” reptation
regime. A subset of the data from Fig. 9a was replotted in
Electrophoresis 1998, 19, 31 143127 A low viscosity polymer for DNA separation 3123

IA
A

6
Fi 0
150
150
210
Vlcm
Vlcm
Vlcm
-c
506 bp
1018 bp
+ 300 Vlcm 2036 bp
12219 bp
X X x u x

n n n
..0
- -

xXX

10 100 1000 10000 10 100 1000

Size (bp) El. Field (V/cm)

€ 2

Figure 9. (A) Dependence of the electrophoretic mobility on the size of

linear dsDNA fragments separated in 5% pDMA (490 kDa) at different
electric fields. Other conditions as in Fig. 7a. The straight line is for
illustration purposes only and has a slope of -1. (B) Dependence of the
electrophoretic mobility on the size of linear dsDNA fragments separated
in 10%pDMA (490 kDa) at different electric fields. Other conditions as in
Fig. 7a. The straight line illustrates a slope of -1. (C) Dependence of the
10 100 1000 10000 electrophoretic mobility of linear dsDNA fragments on the electric field in
5% pDMA (490 m a ) . Data are from Fig. 9A. The lines are for illustration
Size (bp) purposes only with the nonhorizontal line having a slope of 0.4.

order to demonstrate the dependence of the mobility on the
electric field (Fig. 9c). Again, the results are in agreement to
those obtained in HPC [15]. At low electric field and/or
small size, the mobility remains constant (but depending on
the size), whereas larger molecules show an electric field
dependence. The slope of 0.4 for long DNA and high
electric field indicates that the BRF in the regime of “tight
gels” (Eq. 14b) is applicable here.
4.3.3 Influence of chain length
From theoretical considerations and from earlier works, it is
known that the chain length of the polymer also has an
influence on the separation. This is not only true for
nonentangled solutions [14], but also above the entangle-
ment threshold: If the purely topological entanglement is
not strong enough ( i e . if there are not enough entanglement
points between the chains), then the polymer chains can
move themselves away from the entanglement points
(“constraint release”). From microscopic observations
(see, e.g. [61] for review), we also know that individual
DNA molecules can hook around a polymer chain and drag
it along before disengaging from it [ 151. This dragging of
polymers is similar to the “entanglement coupling mech-
anism’’ in dilute solutions [14]. This dynamic nature of the
network can not only change the mobility of the DNA (e.g.
the recovery of separation in the oriented regime), it can
also influence the peak width and therefore the resolution.
This effect can be very important for DNA sequencing,
where single base pair resolution is required. This means
that highly entangled polymer solutions are needed which
can only be achieved by rather long polymer chains (if
cross-linked polymers are to be avoided). On the other
hand, we would like to keep the viscosity low. Therefore we
need to find a compromise between these two contrary
requirements. Another compromise has to be found between
speed (high electric field) and resolution (low electric field).
 C. Heller Electrophoresis 1998, 19, 31143127

Time ( s )

200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400

2400
A 2

1600

800

B 2
4000

3000

2000

1000

Figure 10. Separation of a single-stranded linear DNA standard labelled with fluorescein in (A) short chain pDMA
(490 kDa) and (B) long chain pDMA (1536 m a ) . Common conditions: electrokinetic injection at 5 kV for 10 s; separation
at 5OoCin 0.5 X TBE containing 5% w/w pDMA and 8 M urea at 10 kV (210 V/cm) in a 47 cm long (36 cm to the detector)
fused-silica capillary (100 pn ID). Electrode buffer was 0.5 X TBE. Peaks: 1, 50 b; 2, 100 b; 3, 150 b; 4, 200 b; 5,250 b;
6, 300 b; 7, 350 b; 8,400 b; 9, 450 b and 10, 500 b. The peaks to the left of the 50 b fragments represent unknown shorter
products which are present in the sample, according to the supplier.

Table 2. Resolution of ssDNA and dsDNA fragments in pDMA of different chain lengthsa)
Peak pair S (ssDNA) S (ssDNA) S (dsDNA) S (dsDNA)
(b or bp) (short chain pDMA) (long chain pDMA) (short chain pDMA) (long chain pDMA)
100/150 1.06 0.644 3.81 4.01
400/450 5.89 2.623 5.37 5.41
a) The separation factor S is calculated according to Eq. (16).

To avoid any effects from the intercalator and to simulate

the conditions of a sequencing run, fluorescein-labelled
DNA was used. Figure 10 shows the separation of a ssDNA
standard under the same conditions (210 Vkm, 5% pDMA),
however a polymer of different chain length was used. As a
result, the larger DNA fragments travel faster in pDMA of
490 kDa molecular mass than in pDMA of 1536 kDa
molecular mass, which can be explained by the less rigid
network in the first case. Also, in 490 kDa pDNA the peaks
appear to be broader. The same experiment was performed
with the same DNA standard, but under nondenaturing
conditions (not shown). In this case, the difference in
mobility and band broadening between the separations in
low molecular mass polymer and high molecular mass
polymer were rather small.
900 950 1000 1050 1100
Using the definition in Eq. (16) for the resolution, we obtain
Time (sec) lower separation factors with increasing polymer size for
ssDNA, but hardly any effect for dsDNA (Table 2). For the
Figure 11. Electrophoreticseparation of poly dT25-30in a pDMA solution.
Conditions:electrokineticinjection at 3 kV for 5 s; separation in 0.5 X TBE short DNA fragments, the separation factor of 1 and 0.644
containing 10%w/w pDMA (490 kDa) and 0.1 m Oligreen I at 10 kV in a respectively, is certainly sufficient for DNA sequencing in
47 cm long (36 cm to the detector) fused-silica capillary (100 pm ID). both cases. However for longer fragments, the observed
Electrophoresis 1998, 19, 31 14-3 127
separation factor of > 5 (for the separation in short chain
pDMA) is hardly adequate for sequencing. A separation
factor of 2.6 (400 bp fragment separated in long chain
pDMA) should still be sufficient for correct base-calling in
DNA sequencing using four different colors for the four
different bases and in fact, successful sequencing up to 600
bases with a pDMA matrix was demonstrated [30]. The
peak width in the ssDNA separations and dsDNA
separations did not differ substantially. The reason for the
difference in separation factor under denaturing and non-
denaturing conditions (i.e. 0.644 vs. 4.61 for the 100/150
pair in long chain pDMA) was mainly due to the difference
in L?Ix: The dsDNA peaks eluted closer together than the
corresponding ssDNA peaks.
A similar effect was observed by Chu’s group (Figs. 6 and 7
in [62]): 8% linear PAA (M, 350000) separates small
DNAs as well as 8% linear PAA ( M,1 000 Om),but fails to
separate the larger fragments. This leads to the conclusion
that although two entangled polymer solutions of the same
type but different size give the same mesh size at the same
concentration, for some applications, like sequencing, we
still need rather long polymers (i.e., strong entanglement) in
order to avoid network rupture. If this is not possible,
stronger interactions between the chains (i.e., micelles or
cross-links) will be necessary for long reads. This, in turn,
will be at the cost of a higher viscosity.
4.3.4 Oligonucleotides
For oligonucleotide separation, we again chose the pDMA
preparation of size 490 kDa and a concentration of 10 % w/
w (viscosity: about 300 mm2/s).Figure 11 shows a capillary
electrophoretic separation of a poly-T standard. As can be
seen, we obtained very high resolution of oligonucleotides
in the size range of 25 to 30 bases. Baseline resolution was
achieved up to a size of 60 bases (not shown). This is
comparable in separation performance and migration time
to previously reported separations with gel-filled capillaries
[63-651, and high viscosity linear polymer matrices [21,
66-68].
5 Discussion
We used theoretical considerations as a tool to find a low or
medium viscosity matrix for capillary electrophoretic
separations. As in the case of dextran [l], this method
was also successful for identifying pDMA as a second
candidate. However, the discrepancies between the Mark-
Houwink values at different temperatures make it clear that
this method relies greatly on the accuracy of the published
data. The use of linear pDMA as a matrix for DNA
separations in CE has been described before [22, 301, and it
has also been used in its cross-linked form in slab-gel
electrophoresis [69]. We have extended these previous
studies to prove its use for routine applications such as
oligonucleotide separations and dsDNA separations. Along
with dextran [l], pDMA shows a lower viscosity than
previously used polymers, which makes it an ideal
separation matrix in combination with a number of
commercially available electrophoresis devices. Compared
to dextran, it gives faster separation and sharper peaks (for
comparison, see Fig. 6 here and Fig. 7 in [l]) under the
A low viscosity polymer for DNA separation 3 125
otherwise same conditions. However, one major advantage
is its self-coating ability: Due to its hydrophobic nature,
pDMA has a high segmental adsorption energy and coats
the inner capillary wall, providing an effective suppression
of osmotic flow [30]. Both properties, low to medium
viscosity and self-coating, make this polymer valuable
for the use in multi-CE (Heller et al., in preparation):
The possibility of using low pressure (up to 5 bar) and
noncoated capillaries makes such an instrument much
cheaper and easier to maintain. Noncoated capillaries can
be washed under harsh conditions, making it possible to
recover them in case of clogging and prolonging their
lifetime considerably.
pDMA has a higher hydrolytic stability than PAA [70]. This
again makes it a useful matrix for biopolymer separation,
where basic conditions are used. Further investigations to
test the use of this matrix for separation of other polymers
such as PNA and proteins are underway. Double-logarith-
mic mobility vs. size plots of dsDNA in pDMA and in other
polymer solutions show that separation is recovered for
small DNA molecules. More detailed studies in high
concentration pDMA reveal that in fact four different
regimes can be distinguished. The new regime (called
“regime I” in Fig. 7) has - to our knowledge - not been
described before, presumably, because most separations
where done with samples larger than about 100 bp.
In an earlier publication [l], it was speculated that the
influence of the sequence on the mobility in free solution
might play a role: The four different bases have different
mobilities in free solution, but in long DNA molecules this
effect is leveled out. Here, we used different DNA standards
having rather different sequences. Therefore we can rule out
that a possible sequence dependence of DNA mobility may
override the effective mobility due to sieving. Separation by
an “entropic trapping” mechanism is rather unlikely. As in
entangled polymer solutions, we expect more uniform pores
than in gels and we also expect a much steeper slope in the
double logarithmic mobility vs. size plot [41, 531.
In this new, more detailed study, we proved that it is this
new regime (regime I) which can in fact be attributed to
Ogston-type sieving, even at the strong electric fields used
here. This means that in a series of earlier publications,
where only three regimes (i.e. regimes 11-IV, in our
notation) were distinguished, the plateau regime on the left
hand side of a double logarithmic mobility vs. size plot
(here: regime II) was wrongly attributed to the Ogston-
regime. In fact, it is doubtful, that a real Ogston-type
separation was truly observed before for DNA separation in
polymer solutions. Even in agarose gels, Ogston-type
sieving could only be proven when the mobilities were
extrapolated down to zero electric field [54, 551. The fact
that in earlier studies, small molecules (< 100 bp) were not
considered, might have lead to a wrong assignment of
regimes.
Viovy and Heller [31] estsimated the radius of gyration of
DNA by the so-called Kratky-Porod formula. Short dsDNA
molecules (< 70 bp) had a radius of gyration of less than
8 nm. From Fig. 2 we see that a 10% pDMA solution has a
“pore size” of about 7 nm. It is therefore clear, that only
short molecules can actually be sieved in an Ogston-like
3 126 C. Heller
manner, in agreement to the observations made above.
Molecules larger than about 70 bp are larger than the
average pore size and can no longer be sieved. However,
they are still smaller than the persistence length of dsDNA
(about half the Kuhn length), which is about 450 A,
corresponding to about 150 bp [71]. Therefore, the shorter
molecules cannot be regarded as globular structures, or even
as an “equivalent sphere”, but as rods. Such rods will move
in a different manner than globular particles, for which the
Ogston model was originally developed. The lack of
flexibility also hinders them from moving in a reptation-
like manner. This might explain the existence of regime II
in high concentration solutions: The molecules are too big
to be sieved, but too small to effectively reptate.
Under our conditions, reptation-like movement only takes
place above a size of about 250 bp (regime 111), i.e. larger
than the persistence length. However, the electric field
strengths used here are still too high to observe the true
reptation mode. Also, the reptative motion seems to be
overridden by the movement of the obstacles themselves.
The same is true for regime IV, where reptation with
orientation occurs. Whereas in rigid gels, this orientation
effect hinders the separation of large molecules, we still get
some separation when using polymer solutions. Again, we
can attribute that to the flexible nature of the matrix.
However, the fact, that this recovery of separation does not
considerably change with increasing polymer concentration
nor with decreasing electric field, makes it difficult to judge
which process (constraint release or network rupture by
dragging) is the dominant one.
From the theory of polymer physics, it can be assumed that
the “pore size” of a network does not change with the chain
length of the polymers as long as they are entangled (Eq. 8).
With this knowledge, we can design a matrix, which has
long enough chains to be entangled (for high resolutive
separation), but at the same time has a low enough
molecular mass to give a low viscosity. This approach is
very successful for the separation of dsDNA and short
oligonucleotides, but fails for longer ssDNA fragments,
such as sequencing products. Probably due to the flexible
nature of the network, the peak width increases and the
resolution decreases with DNA size. To counterbalance this
effect, we have to increase the chain length again, however
at the cost of increased viscosity. We have shown that
indeed for ssDNA, this approach is successful in narrowing
the band width. In a more empirical manner, this route was
also chosen by Karger’s group [72]: By developing matrices
with very long PAA chains, the read length could be
increased, however at the cost of an extremely high
viscosity (around 30000 cP) and the danger of shearing.
As the PAA concentration can hardly be decreased any
further (otherwise the pore size would become too large),
there is probably a limit to this approach. With pDMA,
however, having a lower viscosity than PAA of comparable
size (see Fig. 3), we are confident that the situation can be
improved.
Using dsDNA of the same length, we did not observe this
effect (Table 2). This is due to the higher stiffness of
dsDNA compared to ssDNA (which has a persistence length
of only about 4 nm [73]). Double-stranded DNA molecules
of up to 500 bp are less than four persistence lengths long.
Electrophoresis 1998, 19, 3114-3127
These molecules can hardly hook onto polymer fibers,
dragging them along.
We therefore conclude that pDMA with its low to medium
viscosity, high stability and self-coating ability is an ideal
polymer for CE separation of DNA and most probably for
other biomolecules as well. Its separation properties are
very similar to those of other polymers such as HPC and
dextran. We obtained even sharper peaks and therefore
better resolution than with other matrices.
DNA separation in entangled pDMA (and other polymer)
solutions can be explained by the same mechanisms as
separation in gels. However, the situation is slightly more
complex due to the dynamic nature of the network: On one
hand, the polymer chains themselves can move, leading to a
renewal of the obstacles. The constraint release mobility
should be independent of DNA size and electric field.
However, the polymer chains can also be actively dragged
by the moving DNA (“network rupture”), a process which
should in fact be dependent on the DNA size and the
electric field. Important consequences of such processes -
which are difficult to distinguish - are: (i) The separation in
the reptation regime is not as good (a steep slope is not
obtained), (ii) separation is recovered in the orientation
regime (regime IV) and (iii) peak width increases with
increasing DNA size (for single stranded DNA). Longer
polymer chains can help to reduce these effects, however at
the cost of higher viscosity.
Note added in proof: After preparation of the manuscript,
the molecular mass of some pDMA preparations was
determined by gel permeation chromatography. These
experiments gave a weight average molecular mass of
216 kDa (polydispersity:4.4) for the short chain preparation
and 1150 kDa (polydispersity: 5.3) for the long chain
preparation. We thank M. Millequant (Lab0 PCM ESPCI,
Paris, France) for performing these measurements.
I woud like to thank R. Reinhard for providing the capillary
electrophoresis instrument and I. Reese, R. Steinkamp and
V. Egelhofer f o r their help with the characterization of the
polymer solutions and some DNA separations. M. Chiari
was kind enough to provide the polymerization protocol
prior to publication. I also thank J.-L. Viovy for introducing
me to the subject of polymer solutions and for helpful
discussions and very helpful comments. This work was
supported by a grant from the German Ministry of Research
(BMBF}, FKZ 0311040 and by grants from the Commission
of the European Union, CT 96-1158 and CT 97-2627.
Received May 13, 1998
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