Advanced polymers for DNA separation
Valessa Barbier
Ã
and Jean-Louis Viovy
Recent research to improve matrices for DNA separation has
resulted in the development of advanced polymers for use in
capillary electrophoresis and, more generally, for electrophoresis
in microchannels. To date, the most commonly used matrix is
linear polyacrylamide (LPA). Unfortunately, the high-molecular
weight LPA solutions required for achieving good resolution lead
to very viscous solutions. Moreover, the coating ability of LPA is
very poor. For these reasons, many research groups have
developed low-viscosity matrices, which make microchannel
®lling easier, and self-coating matrices, which are able to reduce
ef®ciently the electro-osmotic ¯ow and the interaction of DNA
with the capillary wall. To this purpose, thermo-adjustable
viscosity polymers represent a very clever and interesting class
of matrices.
Addresses
Institut Curie, UMR168, 11 rue Pierre et Marie Curie, 75005 Paris, France
Ã
e-mail: valessa.barbier@curie.fr
Current Opinion in Biotechnology 2003, 14:51±57
This review comes from a themed issue on
Analytical biotechnology
Edited by Norm Dovichi and Dan Pinkel
0958-1669/03/$ ± see front matter
ß 2003 Elsevier Science Ltd. All rights reserved.
DOI 10.1016/S0958-1669(02)00007-1
Abbreviations
AM acrylamide
B polyoxybutylene
DEA diethylacrylamide
ds double-stranded
E polyoxyethylene
IPN transient interpenetrating network
LCST lower critical solution temperature
LPA linear polyacrylamide
P polyoxypropylene
PAM polyacrylamide
PDMA N,N-polydimethylacrylamide
PNIPAM poly(N-isopropylacrylamide)
PVP polyvinylpyrrolidone
PHEA poly-N-hydroxyacrylamide
ss single-stranded
Introduction
Capillary electrophoresis is rapidly becoming the refer-
ence method for DNA analysis. The most remarkable
achievement of this technique has been the sequenc-
ing of the human genome, which was performed twice
as fast as anticipated thanks to the development of
fully automated capillary array electrophoresis sequen-
cers [1].
The choice of the separation matrix dramatically in¯u-
ences separation performance and, therefore, its possible
applications. Concerning DNA sequencing, LPA (linear
polyacrylamide) is currently the best performing matrix in
terms of read length and separation time [2±8]. At this
time, optimal results are obtained with a 2.5% w/v mix-
ture (2% w/v LPA 1:7 Â 10
7
Da plus 0.5% w/v LPA
2:7 Â 10
5
Da) and 1300 bases have been sequenced
in 2 h with 98.5% accuracy for a single-stranded (ss)
DNA M13 template [6]. These performances, however,
have required several years of effort and optimisation. In
particular, such results can only be obtained with poly-
mers of extremely high molecular weight (typically
higher than 10
7
Da), which are obtained by emulsion
polymerisation [9]. Commercial solutions based on this
polymer, such as CEQ separation gel I
TM
(Beckman
Coulter [10])orLongRead
1
matrix (Molecular Dynamics
[11]), lead in routine applications to a read length in the
order of 700 bases in less than 2 h: this is much less than
obtained with the ultra-high molecular weight polymers,
but these are the best commercial matrices in this respect.
LPA has two major disadvantages, however: it has very
high viscosity owing to its high molecular weight and
no coating ability. Even if LPA is a shear-responsive
polymer, viscosity still remains very high. For example,
the zero-shear viscosity of a 2% w/v LPA solution
(9 Â 10
6
Da) is equal to 260 000 mPas compared with
27 000 mPas at a shear rate of 1.32 s
À1
. Therefore, capil-
lary ®lling becomes dif®cult and requires a high loading
pressure (7 Â 10
6
Pa or more). Because LPA has a poor
coating ability, it has very poor electro-osmotic ¯ow
regulation [12] and capillaries need to be coated before
use. (Electro-osmotic ¯ow is a motion of the ¯uid induced
by the counterions present in the Debye layer close to the
charged capillary wall. In practice, a dispersion is
observed that probably arises from unavoidable non-uni-
formity of the capillary surface potential.) This is why a
lot of work has been devoted to developing low-viscosity
and/or self-coating matrices [13±15,16
].
In this review, we focus on the most recent advances
made in the development of matrices for DNA separation
in capillaries and more generally in microchannels. First
we discuss self-coating matrices, and then thermo-adjus-
table-viscosity polymers.
Self-coating matrices
Permanently coated capillaries are rather expensive to
produce. Because the coating polymer can be hydrolysed
or fouled with time, such capillaries have a moderate
lifetime. Moreover, they cannot be regenerated when
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