U.S. patent application number 12/960630 was filed with the patent office on 2011-03-31 for matrix and dynamic polymer systems and compositions for microchannel separation.
Invention is credited to Annelise E. Barron, Christopher P. Fredlake, Cheuk Wai Kan.
Application Number | 20110073477 12/960630 |
Document ID | / |
Family ID | 38006522 |
Filed Date | 2011-03-31 |
United States Patent
Application |
20110073477 |
Kind Code |
A1 |
Barron; Annelise E. ; et
al. |
March 31, 2011 |
Matrix and Dynamic Polymer Systems and Compositions for
Microchannel Separation
Abstract
Matrix polymers and dynamic coating polymers, compositions
thereof and related methods, systems and apparatus for microchannel
separation.
Inventors: |
Barron; Annelise E.;
(Evanston, IL) ; Kan; Cheuk Wai; (Medford, MA)
; Fredlake; Christopher P.; (Chicago, IL) |
Family ID: |
38006522 |
Appl. No.: |
12/960630 |
Filed: |
December 6, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11591915 |
Nov 1, 2006 |
7862699 |
|
|
12960630 |
|
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60732398 |
Nov 1, 2005 |
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Current U.S.
Class: |
204/451 ;
204/601 |
Current CPC
Class: |
G01N 27/44747 20130101;
G01N 27/44752 20130101 |
Class at
Publication: |
204/451 ;
204/601 |
International
Class: |
C25B 9/00 20060101
C25B009/00; G01N 27/00 20060101 G01N027/00 |
Goverment Interests
[0002] This invention was made with government support under Grant
No. 1 R01 HG019770-01 awarded by the National Institutes of Health
and Grant No. DMR-0076097 awarded by the National Science
Foundation. The government has certain rights in the invention.
Claims
1. A DNA sequencing or genotyping composition for microchannel
electrophoresis comprising a hydrophobic separation matrix
component and a hydrophilic wall coating component wherein said
microchannel electrophoresis sequences of from between 500 to 800
bases in 12 minutes or less.
2. The composition of claim 1 wherein said hydrophobic separation
matrix component comprises a polyacrylamide of a formula
CH.sub.2C(R)C(O)NR'R''.sub.n, wherein R is selected from H and
methyl, and R' and R'' are independently selected from C.sub.1 to
about C.sub.8 linear alkyl moieties C.sub.1 to about C.sub.8
alkoxy-substituted linear alkyl moieties, C.sub.1 to about C.sub.8
branched alkyl moieties and C.sub.1 to about C.sub.8
alkoxy-substituted branched alkyl moieties, and copolymers thereof,
said polyacrylamide at least partially water soluble; and said
hydrophilic wall coating component comprising a
poly(N-hydroxyethylacrylamide).
3. The composition of claim 2, wherein said matrix component is
selected from poly(N,N-dimethylacrylamide) and a
poly(N,N-dimethylacrylamide) copolymer.
4. The composition of claim 3, wherein said matrix component
comprises about 3% (w/v) to about 5% (w/v)
poly(N,N-dimethylacrylamide) in an aqueous medium.
5. The composition of claim 4, wherein said matrix component
comprises about 3% (w/v) poly(N,N-dimethylacrylamide), with a
weight average molar mass ranging from about 3 to about 5 MDa.
6. The composition of claim 4, wherein said matrix component
comprises about 1% (w/v) to about 2% (w/v)
poly(N,N-dimethylacrylamide), with a weight average molar mass
ranging from about 200 to about 300 kDa.
7. The composition of claim 1 wherein said microchannel
electrophoresis sequences of from between 500 to 600 bases in less
than 7 minutes.
8. The composition of claim 1 wherein said microchannel
electrophoresis sequences up to 630 bases in about 9 minutes.
9. The composition of claim 1 wherein said microchannel
electrophoresis sequences about 800 bases in about 12 minutes.
10. A microchannel electrophoresis system for DNA and RNA
separations, said system comprising a hydrophobic separation matrix
component and a hydrophilic wall coating component wherein said
system sequences of from between 500 to 800 bases in 12 minutes or
less.
11. The system of claim 10 wherein said hydrophobic separation
matrix component comprises a poly(N,N-dimethylacrylamide) and said
hydrophilic wall coating component comprises a
poly(N-hydroxyethylacrylamide).
12. The system of claim 11 further comprising a microchannel
substrate selected from a micro-dimensioned capillary, said
capillary defining an internal dimension ranging from about 10
microns to about 150 microns, and a microfluidic electrophoresis
chip comprising a microchannel ranging from about 10 microns to
about 150 microns.
13. The system of claim 12, wherein said matrix component comprises
about 3% (w/v) poly(N,N-dimethylacrylamide), with a weight average
molar mass ranging from about 3 to about 5 MDa, and about 1% (w/v)
to about 2% (w/v) poly(N,N-dimethylacrylamide), with a weight
average molar mass ranging from about 200 to about 300 kDa.
14. The system of claim 13, wherein said
poly(N-hydroxyethylacrylamide) is contacted with said
substrate.
15. The system of claim 10 wherein said system sequences of from
between 500 to 600 bases in less than 7 minutes.
16. The system of claim 10 wherein said system sequences up to 630
bases in about 9 minutes.
17. The system of claim 10 wherein said system sequences about 800
bases in about 12 minutes.
Description
[0001] This application is a continuation of and claims priority
benefit from application Ser. No. 11/591,915 filed Nov. 1, 2006,
which claims priority benefit from application Ser. No. 60/732,398
filed Nov. 1, 2005, each of which is incorporated herein by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] Microfluidic chip-based electrophoresis for DNA sequencing
represents the future for high-throughput sequencing projects due
to reductions in cost, time and reagent consumption and the
possibility of integrating sequencing with other steps of genetic
analysis into a total micro-analytical system. To this end, the
development of optimal polymeric separation matrices and wall
coatings for DNA sequencing on microfluidic chips is crucial.
[0004] Hydrophilic separation matrices, e.g., linear
polyacrylamides ("LPA"), have been used along with covalent
hydrophilic coatings to achieve read lengths greater than 500 bases
by microchannel electrophoresis; however, the separations have all
taken greater than 15-18 minutes. Poly(N,N-dimethylacrylamide)
("pDMA") is a hydrophobic separation matrix that due to the hybrid
separation mechanism achieves similar read lengths as polymers of
the prior art but in much faster times. While covalent coatings
have been used almost exclusively for all published microchannel
DNA sequencing results, dynamic coatings, being much less expensive
and also much easier to implement, are greatly preferred for the
microchannel format. However, all dynamic coatings demonstrated for
DNA sequencing have been somewhat hydrophobic leading to loss of
separation efficiency due to interactions of the DNA fragments and
the wall coatings. Previous work has shown that
poly(N-hydroxyethylacrylamide) ("pHEA") is a suitable hydrophilic
dynamic coating for capillaries for both protein separation and DNA
sequencing, but only when pHEA is also the separation matrix. While
most published data on microchip-based DNA sequencing have reported
read lengths of greater than 400 bases, sequencing times on chips
generally have ranged from 18-30 minutes. And, capillary
electrophoresis requires about 60-90 minutes to give comparable
results. Time and read length considerations present ongoing
concerns in the art relating to electrophoretic separations.
SUMMARY OF THE INVENTION
[0005] In light of the foregoing, it is an object of the present
invention to provide one or more polymeric compositions, systems
and/or methods for use in microchannel separation, thereby
overcoming various deficiencies and shortcomings of the prior art,
including those outlined above. It will be understood by those
skilled in the art that one or more aspects of this invention can
meet certain objectives, while one or more aspects can meet certain
other objectives. Each objective may not apply equally, in all its
respects, to every aspect of this invention. As such, the following
objects can be viewed in the alternative with respect to any one
aspect of this invention.
[0006] It can be an object of the present invention to provide a
dynamic wall coating polymer to better employ the advantages
associated with hydrophobic separation matrices.
[0007] It can be another object of the present invention to
provide, alone or in conjunction with the preceding objective, a
hydrophilic wall coating polymer to reduce the incidence or effect
of electroosmotic flow and/or analyte-wall interactions.
[0008] It can be another object of the present invention to provide
a matrix/wall coating system, together with related methods of use,
to increase sequence read lengths over shorter times as compared to
the prior art.
[0009] It can be another object of the present invention to provide
one or more matrix/wall coating systems affording longer, quicker
sequence reads, flow microchannel electrophoresis.
[0010] Other objects, features, benefits and advantages of the
present invention will apparent from this summary and the following
descriptions of certain embodiments, and will be readily apparent
to those skilled in the art of various electrophoretic methods and
techniques. Such objects, features, benefits and advantages will be
apparent from the above as taken in conjunction with the
accompanying examples, data, figures and all reasonable inferences
to drawn therefrom.
[0011] The present invention relates to a novel system that can
enable ultra-fast DNA sequencing or genotyping by microchip
electrophoresis under an applied electric field, with a relatively
short separation channel. Such a system can comprise a polymeric
separation component and a polymeric coating component. In certain
embodiments, a DNA separation matrix comprising a high-molecular
weight poly(N,N-dimethylacrylamide) (pDMA) or a copolymer thereof,
can be used in conjunction with a hydrophilic water-soluble polymer
component, poly(N-hydroxyethylacrylamide) (pHEA), to provide
extraordinarily fast separation of DNA sequencing fragments by
microchannel electrophoresis. Without limitation, pHEA can be
considered a dynamic (i.e., physically adsorbed) polymer wall
coating component. It can be pre-coated on a microchannel wall
and/or provided as part of a composition comprising a polymeric
separation matrix component. In certain other embodiments, such a
system or composition of this invention can comprise such
separation and coating components at molecular weights and/or in
amount(s) sufficient to provide a novel mode of DNA migration i.e.,
a novel DNA separation mechanism that combines DNA reptation with
transient entanglement coupling (TEC).
[0012] More generally, in part, the present invention can be
directed to a DNA sequencing or genotyping composition for
microchannel electrophoresis. Such a composition can comprise a
hydrophobic separation matrix component comprising a polyacrylamide
of a formula CH.sub.2C(R)C(O)NR'R'' wherein R can be selected from
H and methyl, and R' and R'' can be independently selected from
C.sub.1 to about C.sub.8 linear alkyl moieties, C.sub.1 to about
C.sub.8 alkoxy-substituted linear alkyl moieties, C.sub.1 to about
C.sub.8 branched alkyl moieties, and C.sub.1 to about C.sub.8
alkoxy-substituted branched alkyl moieties, copolymers thereof, and
combinations of such polymers and/or copolymers; and a hydrophobic
wall coating component comprising a poly(N-hydroxyethylacrylamide),
more hydrophilic than such a matrix component.
[0013] Whether a homopolymer, random or block copolymer, or in any
such combination, in certain embodiments, R can be H and R' and R''
can comprise a C.sub.1 to about C.sub.4 alkyl moiety, whether
substituted (e.g., methoxy or ethoxy, propoxy, etc.) or
unsubstituted. In certain other embodiments, R' and R'' can be
methyl, and such a component can comprise pDMA or a pDMA copolymer.
With respect to such a formula, .sub.n is an integer greater than 1
corresponding to the average molar mass of such a polymer. In
certain embodiments, such a polymer can be moderately hydrophobic,
as would be understood in the art, and at least partially soluble
in water or an aqueous medium. Accordingly, such components can
comprise pDMA, poly(N-methoxyethylacrylamide) or
poly(N-ethoxyethylacrylamide), combinations thereof, as well as
copolymers thereof with respect to monomers corresponding to any
one or more of the polymeric components described or inferred
herein (e.g., without limitation, a copolymer of
N,N-dimethylacrylamide and N,N-dihexylacrylamide). Regardless, any
combination of R' and R'' is limited only by hydrophobic character
imparted to such a polymeric component and ability to, either
intra- or intermolecularly, physically interact and/or associate
with other such moieties or polymeric components to an extent at
least partially sufficient to provide a functional effect of the
sort described more fully below.
[0014] Such a hydrophilic wall coating component can be considered
in the context of hydrophilic character sufficient to at least
partially reduce electroosmotic flow and/or reduce deleterious
analyte-wall interactions. pHEA can have a molecular weight ranging
from about 600,000 to about 4 million g/mol (MDa) or to about 5
million g/mol (MDa) or more. Regardless, depending upon molecular
weight and/or end-use application, pHEA can be present in a fluid
medium at less than 0.5% (w/v) of the medium. In certain other
embodiments, pHEA can be present at about 0.1 to about 0.4% (w/v)
in such a medium (e.g. aqueous) as would be understood by those
skilled in the art. Regardless, certain embodiments, a pHEA coating
component can also be used compositionally in conjunction with or
added to one or more of the aforementioned hydrophobic
polyacrylamide separation matrix components.
[0015] Regardless, the resulting matrix component can be present in
a composition comprising a fluid medium, of the sort described
herein, e.g., in water or an aqueous medium (e.g., without
limitation, a buffer solution), in a concentration at a percent
(w/v) ranging up to about 5% or greater, such a concentration as
can depend upon average molar mass. In certain embodiments, up to
about 3% (w/v) of such a matrix component can comprise pDMA, with a
weight average molar mass ranging from about 3 to about 5 MDa. In
other such embodiments, the matrix component can comprise an
additional about 1% to about 2% (w/v) of a pDMA, with a lower
weight average molar mass, e.g., without limitation, ranging from
about 200 to about 300 kDa. Various other matrix components are
available, over a range of concentrations, determined and limited
only by choice of monomeric component(s) and corresponding
moieties.
[0016] Unless otherwise indicated, all numbers expressing
properties such as molar mass, percent and the like, used herein,
are to be understood as being modified in all instances by the term
"about". Accordingly, unless indicated to the contrary, the
numerical parameters herein are approximations that can vary
depending upon desired polymer or system properties or results to
be achieved using any methods relating thereto, such percentages
and molar masses as can be varied by those skilled made aware of
this invention.
[0017] With respect to any of the compositions, systems, methods
and/or apparatus of the present invention, the polymers described
or inferred herein can suitably comprise, consist of or consist
essentially of any of the aforementioned monomers, regardless of
the percent of any such monomer in any corresponding polymer. Each
such polymeric or copolymeric compound or monomeric component
thereof is compositionally distinguishable, characteristically
contrasted and can be practiced in conjunction with the present
invention, separate and apart from another. Accordingly, it should
also be understood that the inventive compositions, systems,
methods and/or apparatus, as illustratively disclosed herein, can
be practiced or utilized in the absence of any one polymer,
monomeric component and/or step which may or may not be disclosed,
referenced or inferred herein, the absence of which may not be
specifically disclosed, referenced or inferred herein.
[0018] In part, the present invention can also be directed to a
microchannel electrophoresis system for RNA and DNA separations.
Such a system can comprise a hydrophobic separation matrix
component comprising a pDMA and a hydrophilic wall coating
component comprising a pHEA; and a microchannel substrate selected
from a micro dimensioned capillary (e.g., without limitation,
defining an internal diameter ranging from about 10 microns to
about 150 microns), or a microfluidic electrophoresis chip with a
similar such microchannel dimension. Such a system can comprise a
pDMA matrix component of the sort described above. In certain
non-limiting embodiments, the matrix component can comprise about
3% (w/v) pDMA with a weight average molar mass ranging from about 3
to about 5 MDa; and about 1% to about 2% (w/v) pDMA, with a lower
weight average molar mass (e.g., one ranging from about 200 to
about 300 kDa).
[0019] In part, the present invention can also be directed to a
method of using a polymeric wall coating and separation matrix
system for either electrophoretic DNA or RNA separation. Such a
method can comprise providing a system comprising a pDMA component
comprising about 3% (w/v) to about 5% (w/v) of such a system, and a
pHEA component; introducing the system to a substrate of the sort
selected from a microchannel electrophoresis capillary and a
microfluidic sequencing chip; and contacting a mixture of either a
DNA sequencing reaction product or a RNA component with the system,
at an applied voltage and for a time at least partially sufficient
for eletrophoretic separation. Such a method can comprise a system
comprising a pDMA component of the sort described above. In certain
embodiments, the pHEA component can be contacted with the substrate
prior to introduction of the separation matrix system. In such
embodiments, the pHEA component can be used as an aqueous solution,
contacting the substrate for a time sufficient to provide a wall
coating component of the sort described herein. Regardless, such a
method can be used to separate DNA sequences (e.g., single-strand
DNA) of length up to about 800 bases, such separation depending
upon time and microchannel length. Non-limiting examples of such a
separation/sequencing methodology are provided below.
[0020] As can relate to the preceding, this invention can also be
directed to a microchannel electrophoresis apparatus. Such an
apparatus can comprise a substrate and a polymeric system thereon,
with such a substrate selected from a micron-dimensioned capillary
and a microfluidic electrophoresis chip. Without limitation to
microchannel substrate, or apparatus configuration, such a system
can comprise a polymeric system comprising a pDMA component
comprising about 3% to about 5% (w/v) of such a system and pHEA
component of the sorts described above. Such polymeric systems have
demonstrated separation and/or wall-coating performance, and can
also be used in conjunction or combination with other types of
capillary or microchannel matrix or wall coating materials known in
the art.
[0021] In part, this invention can also be directed to a method of
using a hydrophobic polymer matrix to enhance DNA separations
speed. Such a method can comprise providing a microchannel
substrate selected from a micro-dimensioned capillary and a
microfluidic electrophoresis chip; coupling or applying a
hydrophilic pHEA wall coating component to such a substrate;
introducing a hydrophobic separation matrix to the substrate, such
a matrix component selected from the polyacrylamides described
herein, copolymers thereof and combinations of such polyacrylamides
and/or such copolymers; and contacting a mixture of DNA sequence
components and such a matrix component, at an applied voltage and
for a time at least partially sufficient for electrophoretic
separation of such a mixture, the matrix component(s) of a
molecular weight and at a concentration at least partially
sufficient for at least one of transient entanglement coupling and
reptation of the DNA components within the mixture. In certain
embodiments, separation can be a combination of transient
entanglement coupling and reptation. In certain such embodiments,
the DNA components can migrate by transient entanglement coupling
about 50% of the migration time and by reptation about 50% of the
migration time. Regardless, such migration dynamics can be
monitored by epifluorescent videomicroscopy of fluorescently
stained DNA molecules. In certain embodiments, such a method can
provide separation up to about 3 times faster, compared to
separations using LPA matrices of the prior art.
[0022] Without limitation, in certain embodiments, the matrix
component of such a methodology can be selected from pDMA and/or
copolymers thereof. With regard to the former, such a matrix
component can comprise about 3% (w/v) pDMA, with a weight average
molar mass ranging from about 3 to about 5 MDa, and about 1% (w/v)
to about 2% (w/v) pDMA, with a lower weight average molar mass,
e.g., one ranging from about 200 to about 300 kDa. Regardless, in
such embodiments, migration can be characterized by a linear region
of a log-log plot of DNA electrophoretic mobility versus DNA
molecular size through the matrix, where molecular size can be
gauged in terms of bases and/or base pairs. For instance, without
limitation, a DNA molecular size range can be from about 200 bases
to about 800 bases, with the linear region of such a log-log plot
having a slope between about -4.40 and -0.60.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1. Characterization of pDMA by GPC-MALLS. Molar mass
distribution of pDMA used in several non-limiting examples. The
distribution is an average of three runs.
[0024] FIG. 2. The viscosity of pDMA matrices increase dramatically
at concentrations suitable for sequencing. However, the viscosity
is still much lower than LPA matrices, of the prior art, for easier
microchannel loading.
[0025] FIG. 3. ssDNA separations for pDMA matrices compared to a
prior art LPA matrix. All 37 peaks were resolved for 3% and 4%
pDMA. Loss of resolution was observed for 5% pDMA and the LPA
matrices. The DNA fragment migration times are dependent on
concentration.
[0026] FIGS. 4 A-B. A) The selectivities
(.mu..sub.i-.mu..sub.i-1/.mu..sub.avg) of the peaks from the
separation in FIG. 2. Both matrices show similar peak separations.
B) The peaks in the LongRead.TM. matrix get much wider than in the
pDMA matrix past 250 bases. (This band broadening is a major cause
of lower sequencing performance of this matrix.)
[0027] FIG. 5. Mobility data for ssDNA ladder in pDMA matrices.
Conditions are the same as in FIG. 1. The dashed lines mark the DNA
sizes where the plot is linear. Slopes of the linear region are
-0.54, -0.59, and -0.50 for the 3%, 4%, and 5% matrices,
respectively.
[0028] FIG. 6. Digital images captured from DNA imaging videos. A
series of frames at the shown time intervals show two DNA molecules
moving through a representative network of this invention. The top
molecule reptates through the entire viewing frame in the given
time. The lower molecule is reptating at first and then hooks and
drags the polymer network through the viewing frame in a mechanism
similar to transient entanglement coupling.
DETAILED DESCRIPTIONS OF CERTAIN EMBODIMENTS
[0029] Entangled polymer solution properties that can influence DNA
separations in electrophoresis.
[0030] To further understand certain embodiments of this invention,
consider the following: Developing optimal polymer matrices for
sequencing is one of the greatest limitations for commercializing
microchannels systems. DNA sequencing is only possible in
semi-dilute polymer solutions where the polymer chains overlap and
form and entangled network. The overlap concentration, c*, is
defined as the bulk solution concentration that exactly matches the
concentration of the polymer inside a coil. Equation 1 provides an
estimate of c* based on this definition
c * .apprxeq. 3 M w 4 .pi. N A R g 3 ( 1 ) ##EQU00001##
where M.sub.w is the weight average-molar mass, N.sub.A is
Avagadro's number, and R.sub.g is the polymer radius of gyration.
From Eq (1), the overlap concentration can be calculated by
measuring both the M.sub.w and R.sub.g by techniques such as light
scattering. Alternatively, the overlap concentration can be
determined by measuring the zero-shear viscosity of a series of
polymer concentrations and plotting viscosity versus polymer
concentration on a log-log scale. The concentration where the plot
becomes non-linear is the overlap concentration the extent at which
the network is entangled can be expressed as the c/c* ratio.
[0031] As DNA moves through entangled polymer networks, the
critical parameter for defining the network is the polymer
screening length, .xi.. For polymer solutions, the screening length
is dependent on polymer R.sub.g and concentration
.xi. = 0.5 R g ( c c * ) - 3 4 ( 2 ) ##EQU00002##
[0032] In gel electrophoresis, the average pore size was critical
for determining separation mechanism and in an attempt to connect
the theories regarding separation mechanism from gels to entangled
networks. It has been proposed to use the screening length as a
"pore size" in these solutions, with this view altered slightly by
defining the "blob" size, .xi..sub.b, as the average chain length
between entanglement points. This adjustment only introduced a new
prefactor in the definition and is defined as
.xi. b = 2.86 .xi. = 1.43 R g ( c c * ) - 3 4 ( 3 )
##EQU00003##
Thus, the polymer network in general can be defined in terms of
this blob size which depends on both polymer concentration and coil
size.
[0033] DNA separation mechanisms in entangled polymer
solutions.
[0034] The polymer network parameters described above can be used
to describe the different mechanisms of DNA migration through the
entangled network. The mechanism for separation depends on the
R.sub.g of the DNA molecules relative to the size of the blobs in
the network. DNA sizes smaller than the pore size of the network
migrate through the solution by a mechanism similar to one
described by Ogston for spheres moving though a dense array of
fibers. This is generally referred to as Ogston sieving and the
mobility of DNA relative to the free solution mobility can be
described mathematically by
.mu. .mu. 0 = exp ( - K r c ) ( 4 ) ##EQU00004##
where K.sub.r is the retardation coefficient which is proportional
to the square of the DNA coil radius and c is the gel or polymer
concentration. This mechanism is analyzed by Ferguson plots which
plot the natural log of the analyte mobility versus the gel of
polymer concentration. The slope of the line, then, gives the value
for the retardation coefficient.
[0035] The Ogston model for DNA mobility would be expected to fail
as the coil size of DNA molecules approach and surpass the pore
size of the network. The observation that DNA larger than the pore
size led to the biased reptation model for DNA mobility. This model
was re-evaluated by taking into account that the length of the DNA
molecule is subject to fluctuations and named biased reptation with
fluctuations. These models predict that the mobility of DNA snakes
through the network similar to the reptation theory for entangled
polymer melts of solutions. A critical parameter in this mechanism
is the reduced electric field and this is given by
= .eta. s .xi. b .mu. 0 E k b T ( 5 ) ##EQU00005##
where .eta..sub.s is the solvent viscosity, E is the electric field
strength, k.sub.b is Boltzmann's constant, and T is the absolute
temperature. These models were generally derived for instances of
low fields, or .epsilon.<<1. The resulting form of the
mobility for the BRF model is given by
( .mu. .mu. 0 ) BRF = [ ( 3 N ) - 2 + ( 2 5 + 2 .alpha. ) 2 ] 1 2 (
6 ) ##EQU00006##
where N is the DNA size in bases and .alpha. is an adjustable
factor. For this model a critical DNA size exists for which DNA
sizes below the first term dominates and DNA mobility is size
dependent.
( .mu. .mu. 0 ) = 1 3 N for N < N crit ( 7 ) ##EQU00007##
This regime of DNA reptation is known as unoriented reptation and
most DNA separations including sequencing is carried out in this
regime. For DNA above the critical size, the second term dominates
and size based separation vanishes. This regime of reptation is
known as oriented reptation. The critical size of DNA depends on
both pore size and electric field strength.
N.sub.crit.about..epsilon..sup.-2 (8)
The critical size is predicted to be independent of mesh size, but
prior work showed that the transition from un-oriented to oriented
reptation depends on mesh size. Such results show that smaller pore
sizes tend to shift oriented reptation to smaller DNA sizes. So the
electric field strength as well as the blob size of the entangled
network are critical parameters in determining the upper limit for
DNA size based separation by electrophoresis.
[0036] While the mechanisms of Ogston sieving and biased reptation
are applicable to entangled solutions, Barron et al. discovered a
different separation mechanism prevalent in polymer solutions below
the overlap concentration. In ultra-dilute solutions of
hydroxyethlycellulose (HEC) separation of large dsDNA molecules
(>1 kbp) was possible and that the theories for entangled
polymer solutions were inadequate to explain the results.
Therefore, a new mechanism termed transient entanglement coupling
was proposed to account for the separation. In this mechanism, the
DNA encounters the polymer chains during migration and the coupling
of the two increases the drag on the DNA molecule. The probability
of encountering polymer chains is DNA size dependent and thus
separation is possible.
[0037] Polymer physical properties have an impact on sequencing
performance. Generally, hydrophilic, high molar mass polymers are
needed for longer DNA sequencing read lengths. At the same time,
lower viscosities and the ability to coat a glass surface provide
technical and cost advantages to operating the system. The
synthesis conditions targeted pDMA and pHEA molar masses in excess
of 1.times.10.sup.6 g/mol. Typically this is the threshold to
provide longer DNA sequencing reads while also meeting the
requirement for a stable dynamic coating. FIG. 1 displays the molar
mass distribution for the pDMA used in this study as measured by
GPC-MALLS. Polymer properties measured at room temperature for pDMA
and pHEA are summarized in Table 1. The zero-shear viscosity of
pDMA solutions are shown in FIG. 2. Many LPA matrices have
viscosities on the order of 100,000 cP.sup.28 pDMA matrices in the
concentration range useful for sequencing will be much easier to
load into microchannels.
TABLE-US-00001 TABLE 1 Polymer properties of pDMA and pHEA
synthesized for matrix and coating Polymer M.sub.w (MDa) R.sub.g
(nm) PDI pDMA 3.4 124 1.6 pHEA 4.0 135 3.2
ssDNA Separations
[0038] Although pDMA has been explored as an effective sequencing
matrix in capillary electrophoresis instruments, this polymer has
not been tested for sequencing in microchannel systems. Separation
of ssDNA fragments was carried out in a microchannel
electrophoresis system using the pDMA in concentrations from 3-5%
along with a commercial LPA matrix as a control. The sample is a 25
base ladder containing 37 DNA fragment sizes. The electropherograms
from these separations are presented in FIG. 3. While the 3% and 4%
pDMA matrices are able to separate all 37 peaks, the 5% pDMA and
the LPA matrices do not fully resolve the largest DNA fragments. So
in addition to having lower viscosities, pDMA solutions separate a
25 base ladder up to 900 bases better than a 4% LPA matrix. The
effective separation distance in this system is 7.5 cm, much less
than in commercial CAE instruments. Since microchannel systems
employ a cross injector design that enables a much more efficient
separation, the required channel length is greatly reduced and thus
the separation is faster.
[0039] For the pDMA matrices, a dependence of DNA migration times
on the polymer concentration is evident, and migration times are
generally shorter for these pDMA matrices than the LPA matrix.
Since most microchannel sequencing studies have also used longer
channels as well as different electric field strengths and
temperatures in an effort to optimize their individual systems, it
is difficult to directly compare migration times. However, the
matrix of choice for many microchannel studies, 4% LPA, results in
the longer migration times than the pDMA matrices used here, under
the same electrophoresis conditions.
DNA Sequencing Results
[0040] In addition to increased separation speed compared to LPA,
pDMA can act as a self-coating matrix eliminating the need to
covalently coat the channel walls, which can be expensive and
time-consuming. Dynamically adsorbed polymeric wall coatings are
more attractive than a covalent coating for high throughput
environments due to their reduced cost and simpler implementation.
The pDMA coating, however, is somewhat hydrophobic and is likely to
interact with the analyte. The hydrophilic character of pHEA makes
it an excellent wall coating since it can reduce electroosmotic
flow and analyte-wall interaction.
[0041] Sequencing results for the various concentrations of pDMA
and the commercially available Pop-5.TM. and Beckman LongRead.TM.
matrices are presented in Table 2 along with the results using
different dynamic coatings. The chemistry of the polymer coating
greatly influences the read length. For the 4% pDMA matrix, using
pDMA as a dynamic coating can achieve read lengths of 420 bases.
When pHEA is applied as the dynamic coating the read length is
extended by 130 bases. This increase in read length is attributed
to the reduction of wall-analyte interactions that can increase
band-broadening during the separation.
TABLE-US-00002 TABLE 2 DNA sequencing results on glass microchips
Average Read Sequencing Matrix Coating Polymer Length.sup.a (n = 3)
Long Read Length.sup.a Time.sup.b (s) 3% pDMA pHEA 349 377 225 4%
pDMA pHEA 512 550 340 5% pDMA pHEA 489 530 388 Pop-5 .TM. pHEA 384
430 355 LongRead .TM. LPA pHEA <300 318 N/A.sup.c 4% pDMA pDMA
372 420 290 Pop-5 .TM. Pop-5 .TM. <50 N/A.sup.c N/A.sup.c *All
runs conducted at 50.degree. C. and 235 V/cm (~3 .mu.A current)
.sup.aAt 98.5% accuracy .sup.bTime to reach average read length
.sup.cCould not be determined
[0042] Another interesting result is that the Pop-5.TM. polymer
cannot be used as a self-coating matrix in contrast to its
commercial use in ABI CAE instruments. This might be explained by a
fundamental difference between the glass chemistry used to
fabricate the microfluidic chips in this study and the fused silica
glass used in capillaries. The presence of salts and other
impurities that increase the bonding properties of glass chips
glass could alter the coating ability of the Pop-5.TM. polymer.
When pHEA, however, is applied prior to Pop-5.TM. matrix loading
into the chip, the read length is increased from very low reads
(<50 bases) to approximately 380 bases. Comparing the results
for the pDMA matrices with the Pop.TM. matrix demonstrates that
polymer matrices developed for capillary systems are not
necessarily the best matrices for microchannel systems. This result
is further confirmed by the better sequencing read lengths obtained
in the pDMA matrices versus the Beckman LongRead.TM. matrix, an
optimized LPA matrix for capillary systems.
[0043] The combination of optimal formulations of pDMA sequencing
matrix with the pHEA dynamic coating deliver longer read lengths
than the commercial matrices in a shorter time. The 4% pDMA matrix
delivered an average of 512 bases at 98.5% accuracy including a
long read of 550 bases. Comparing different pDMA concentrations
reveals that the 3% pDMA matrix results in a 25% improvement in
migration time for the 500 base fragment in ssDNA separations;
however, only 349 bases could be accurately called on average for
sequencing. Thus, for longer read lengths, concentrations greater
than 3% for this particular pDMA molar mass are required. However,
the 4% formulation represents an optimal concentration, as the read
lengths decrease at a concentration of 5%.
[0044] From the separations in FIG. 3, the 3% pDMA matrix separated
large DNA fragments quickly and with high resolution, but did not
obtain sequencing read lengths comparable to the higher pDMA
concentrations. As higher polymer concentration is an important
parameter for separating short DNA fragments, and lower
concentrations favor separating larger fragments, polymer matrices
were formulated by blending polymers with high average molar mass
and low average molar mass. Table 3 shows the results of two
"blends" of pDMA polymers (at 98.5% accuracy). The pDMA with high
average molar mass (3.4 MDa) was used at a concentration of 3%
(w/v) while the lower average molar mass pDMA (240 kDa) was used at
both 1% and 2% (w/v) so that the total pDMA concentrations for the
two matrices were 4% and 5% (w/v). Although the matrices with a
single average molar mass perform very well, the mixed molar mass
matrices perform even better. The 4% blended matrix had the highest
average read length at 560 bases (with a long read of 587 bases in
6 minutes), while the 5% blended matrix achieved the longest
individual read at 601 bases, taking only 6.5 minutes to achieve.
(The four-color sequencing electropherogram for the longest
sequencing run is not shown.)
[0045] The separations in pDMA matrices and the LongRead.TM. LPA
matrix can be further analyzed to determine the source of the
poorer performance of this commercial matrix. From the ssDNA
separations, both the separation selectivity and the peak widths
are plotted for the various DNA fragment sizes in FIGS. 4A-B. The
selectivity is a metric of the separation power of the matrix,
defined as the difference in mobilities between adjacent peaks
normalized by their average mobility. The peak width measures the
combined effect of all band broadening sources in the system. From
FIG. 4A, it is apparent that the selectivities of the 4% pDMA
polymers and the LongRead.TM. matrix are similar and cannot explain
the difference in sequencing performance. However, FIG. 4B shows
that at DNA sizes greater than approximately 250 bases, there is a
large increase in the peak widths in the LPA based matrix. It is
believed that this band broadening effect is the main cause of poor
performance in this system and that the much higher peak
efficiencies (smaller widths) in the pDMA matrices result in the
increased sequencing read lengths of these polymers. Furthermore,
the wider peaks of the LongRead.TM. LPA system will probably
require much longer separation channels to achieve similar read
lengths (which also will require longer times).
TABLE-US-00003 TABLE 3 Sequencing comparison of blended molar mass
pDMA matrices using pHEA coatings.sup.a Average Read Longest Read
Sequencing Matrix.sup.b Length.sup.c (n = 3 Length.sup.c Time (min)
3% high MWpDMA + 560 587 6 1% low MW pDMA 3% high MW pDMA + 542 601
6.5 2% low MW pDMA .sup.aRun Conditions identical to Table 2
.sup.bHigh molar mass pDMA is 3.4 MDa; low molar mass pDMA is 240
kDa .sup.cRead length at 98.5% accuracy
Investigating DNA Separation Mechanisms in the pDMA Matrices
[0046] DNA separation through an entangled polymer solution is
believed to proceed via two the mechanisms described above. While
small DNA fragments tend to sieve through the polymer network by
Ogston sieving, DNA reptation is the separation mechanism for
larger fragments. Reptation provides higher resolution separations
and should be the desired mechanism for high performance
sequencing. This can explain why higher concentrations are needed
to sequence smaller DNA sizes, since the smaller mesh sizes at
higher concentrations shifts the reptation mechanism start point to
smaller DNA sizes. Similar reasoning can explain why lower
concentrations are needed for longer reads. As the mechanism shifts
to oriented reptation, all resolving power in the matrix is lost.
Since this transition depends on the mesh size (see Eq 8) DNA tends
to be oriented at smaller sizes for higher polymer
concentrations.
[0047] When plotting the mobility of DNA versus the fragment size
on a logarithmic scale, separation by un-oriented reptation can be
observed in the linear portion of the data, as shown in FIG. 5 for
the separation of the ssDNA ladder from FIG. 1. The data presented
in FIG. 5 is similar to data presented earlier for pDMA matrices in
capillary systems. In particular, smooth transitions exist between
Ogston-type sieving to unoriented reptation and for the transition
from unoriented reptation to oriented reptation.
[0048] For long DNA sequencing reads, the extension of the linear
region of the plots in FIG. 5 to higher DNA sizes can be important.
The mobilities in 4% pDMA matrix remain in the linear region for
larger DNA sizes relative to the 5% pDMA, so that the transition to
oriented reptation is shifted to smaller DNA sizes. Thus, the 4%
pDMA should give longer read lengths than the 5% matrix, and the
results in this study show that even for DNA sizes below this
limit, the 4% matrix provides better sequencing results. It should
be noted, however, that the mobility plots do not account for band
broadening effects that may reduce single-base resolution needed
for sequencing, so sequencing read lengths are less than might be
expected just from the mobility plots of the ssDNA ladders.
Entangled Network Rupture
[0049] The 3% pDMA matrix shows a linear region for the mobility
plot that extends further than the two higher concentrations, yet
the read lengths for this matrix are generally much lower. One
reason for this is that higher polymer concentrations are needed
for better separation of small DNA sizes as discussed above.
Another factor influencing the separation performance for larger
DNA sizes is the strength of the entanglements of the polymer
network. Generally, at a given molecular weight, the strength of
the entangled network increases as the polymer concentration is
increased and the chains are better entangled, thus higher molar
mass polymers are more effective when mesh size needs to be large
to sequence larger DNA sizes. Entanglement strength is also
affected by the coil size of the individual chains. pDMA chains
generally have smaller coil sizes than more hydrophilic polymers
such as LPA.
[0050] Strongly entangled networks are needed to separate DNA by
reptation, and thus disruptions in the polymer network by migrating
DNA should tend to reduce separating power and read lengths. In
weakly entangled networks, disruptions are more frequent,
especially by larger DNA. However, the phenomenon of DNA molecules
entangling with the polymer chains as the network is broken apart
would still tend to be size-dependent. This mechanism is related to
transient entanglement coupling (TEC), a phenomenon well understood
and established in the art, discovered by Barron et al. for the
separation of dsDNA in dilute polymer solutions. (See, e.g.,
Barron, A. E., Soane, D. S. & Blanch, H. W. (1993) J. Chrom. A
652: 3-16; Barron, A. E., Blanch, H. W. & Soane, D. S. (1994)
Electrophoresis 15: 597-615; and Barron, A. E., Sunada, W. M. &
Blanch, H. W. (1996) Biotech. Bioeng. 52: 259-270, each of which is
incorporated herein by reference in its entirety.) It is possible,
then, that although un-oriented reptation is the dominant mechanism
of DNA separation in these matrices, network disruption and
DNA-polymer entanglement is also contributing to the separation.
Since this network disruption with hooking is expected to be faster
than reptation, this mechanism may account for the increased speed
of separation in a more weakly entangled solution of pDMA compared
to LPA sequencing matrices.
[0051] This hybrid mechanism of DNA separation is further suggested
by videomicroscopy studies of dsDNA in the pDMA matrices. FIG. 6
shows the time evolution of two DNA molecules in a 3.0% pDMA matrix
at 25.degree. C. where one molecule is migrating via reptation
while the other molecule has broken the network and is dragging
polymer chains through the solution. The lower DNA molecule is
reptating through the solution and then hooks onto the network and
drags it along while the upper DNA molecule continues to reptate
for the duration of the frame series. This demonstrates that these
two mechanisms can occur simultaneously in the same matrix.
[0052] As shown below, a novel polymer matrix/polymer wall coating
system for capillaries and microchips, comprising the combination
of a hydrophobic polymer, e.g., poly(N,N-dimethylacrylamide) as a
DNA separation matrix with a hydrophilic polymer, e.g.,
poly(N-hydroxyethylacrylamide) as a hydrophilic dynamic wall
coating, results in an ultra-fast separation of DNA sequences by
capillary and especially, microchip electrophoresis. The
combination of a hydrophobic sequencing polymer along with a
dynamic, hydrophilic polymer wall coating has not previously been
demonstrated on a microfluidic electrophoresis device. Sequencing
of 500 to 600 bases, with high accuracy of base-calling, can be
achieved in less than 7 minutes specifically by the combination of
two such polymers on a microchannel electrophoresis format with an
effective separation distance of just 7.5 cm and other conditions
such as (optimally) applied electric field strength (e.g., 235
V/cm) and temperature (e.g., 50.degree. C.). PDMA, optimally at a
M.sub.w of about 3-about 4 MDa, dissolved to a concentration of
3-5% w/v in a 1.times.TTE+7 M urea buffer provides a sequencing
matrix while a pHEA dynamic coating is pre-applied for application
in microfluidic chips Additionally, adding between about 1 and
about 2% low-molar mass pDMA (.about.200-300 kDa) to 3% (w/v)
high-molar mass pDMA (3-4 MDa) solutions (total polymer
concentration 4% (w/v)) results in even longer sequencing reads of
up to 600 bases in 6.5 minutes of electrophoresis. Specifically,
without limitation to any one theory or mode of operation, such
conditions provide ultra-fast sequencing of DNA via a hybrid DNA
separation mechanism somewhere between transient entanglement
coupling and reptation, which has never been demonstrated as a
mechanism for the sequencing of DNA. The mechanism can be deduced
by looking at a log-log plot of DNA mobility versus fragment size,
and is corroborated by single-molecule epifluorescent microscopic
imaging experiments. On the log-log plot of DNA mobility vs. DNA
size, the slope of the linear region under ultra-fast sequencing
conditions is between -0.40 and -0.60.
[0053] Further optimization of such separation media with respect
to polymer properties such as molecular weight, composition, and
solution concentration, as well as optimization of the wall-coating
polymers, can allow for even longer reads at this reduced time. The
results obtained represent the fastest sequencing time for such a
long read reported to date, and hence provide a step forward in the
development of microchannel-based sequencing technologies.
EXAMPLES OF THE INVENTION
[0054] The following non-limiting examples and data illustrates
various aspects and features relating to the compositions, systems,
methods and/or apparatus of the present invention, including the
use of hydrophobic separation matrix polymer components and
hydrophilic wall coating polymer components, for microchannel
electrophoresis. In comparison with the prior art, the present
compositions, methods, systems and/or apparatus provide results and
data which are surprising, unexpected and contrary thereto. While
the utility of the invention is illustrated through the use of
several polymeric components, compositions and apparatus which can
be used therewith, it will be understood by those skilled in the
art that comparable results are obtainable with various other
polymeric components, compositions and apparatus, as are
commensurate with the scope of this invention.
Example 1
[0055] PDMA Synthesis
[0056] The high-molecular weight pDMA separation matrix polymers
were synthesized from the monomer N,N-dimethylacrylamide, purchased
at 99+% purity from Monomer-Polymer & Dajac Labs (Feasterville,
Pa. USA). A 4 wt % solution of the monomer in DI water was degassed
with flowing N.sub.2 for 30 minutes in a water bath set to
47.degree. C., followed by initiation with V-50
(2,2'-azobis(2-amidinopropane) dihydrochloride, Wako Chemicals,
Richmond, Va. USA). After 16 hours, the viscous polymer solution
was transferred to 100,000 MW cutoff dialysis membranes where they
were dialyzed with frequent water changes for 10 days. Following
dialysis, the samples were lyophilized to recover the solid pDMA
polymer, with a molar mass in excess of 1 MDa. Various other matrix
polymers and copolymers of this invention are either commercially
available or can be prepared in analogous fashion from the
corresponding monomers, as described above, or by using known
synthetic techniques or modifications thereof as would also be
known to those skilled in the art made aware of this invention.
[0057] Additionally, adding 5 mL isopropyl alcohol to the reaction
mixture described above, reduces the molar mass of pDMA suitable
for blending into mixed molar mass matrices. By including 5 mL
isopropyl alcohol into the monomer solution, polymerization and
purification result in a polymer product with a molar mass of about
200 to about 300 kDa.
Example 2
pHEA Synthesis
[0058] To synthesize the wall-coating polymer, pHEA, the
N-hydroxyethylacrylamide monomer was purchased as a 45% solution
from Cambrex (sold under trade name Duramide.TM.). Some of the
monomer solution was diluted to a volume of 100 mL so that the
final concentration of the monomer was 0.5 wt %. The monomer
solution was degassed under N.sub.2 for 30 minutes in a water bath
set to 25.degree. C. Following degassing, the reaction was
initiated by adding 100 uL of a 10 wt % ammonium persulfate
solution (Amresco Inc, Solon, Ohio USA) and 10 uL of TEMED
(Amresco). The reaction proceeded for 16 hours after which the
polymer solution was transferred to 100,000 MW cutoff dialysis
membranes where it was dialyzed with frequent water changes for 10
days. Following dialysis, the solutions were lyophilized to recover
the solid polymer, with a molar mass in excess of 1 MDa.
Example 3
Polymer Molecular Weight Characterization
[0059] Molecular weight distributions were determined by tandem
gel-permeation chromatography-multi angle laser light scattering
(GPC-MALLS). The dilute polymer solutions were first fractionated
on the GPC (Waters Corp, Milford, Mass. USA) using Shodex Ohio-Pak
columns SB-806 HQ, SB-804 HQ, and SB-802.5 HQ connected in series.
After fractionation, the effluent from the GPC flows directly into
the DAWN DSP laser photometer and then into the Optilab DSP
interferometric refractometer (both instruments are from Wyatt
Technologies, Santa Barbara, Calif. USA). During characterization,
100 uL of dilute (1 mg/mL) solution (with a mobile phase consisting
of 0.1 M NaCl, 50 mM Na.sub.2HPO.sub.4, and 200 ppm NaN.sub.3) is
injected into the instruments and flows through at 0.300 mL/min.
GPC-MALLS data were processed using ASTRA software from Wyatt
Technologies. The polymers synthesized were all characterized by
molar mass, radius of gyration and polydispersity index.
Example 4
Applying the pHEA Coating
[0060] Following the protocol described by Albarghouthi et al the
microchannel is first filled with 1 M HCl solution and left for 15
minutes. Following the removal of the 1 M HCl solution, the channel
is rinsed with de-ionized water and then filled with the pHEA
coating solution and left for 15 minutes. The pHEA coating solution
is a 0.1% w/v aqueous solution of the polymer in de-ionized water.
The coating results in channels that give an electroosmotic flow of
less than 1.times.10.sup.-5 cm.sup.2/Vs.
Example 5
Microchannel/Microchip Electrophoresis
[0061] Analysis of ssM13 mp18 sequencing fragments and ssDNA ET-900
ladder (both Amersham Biosciences, Piscataway, N.J. USA) was
carried out on a microchannel electrophoresis system that was
custom-built for our laboratory by ACLARA Biosciences (Mountain
View, Calif. USA). This system allows sensitive multi-color
detection through laser-induced fluorescence (LIF). The system is
comprised of two subsystems; an electrical system supplies voltage
to the microfluidic device, and an optical subsystem that allows
detection of fluorescent molecules as they pass the point in the
channel the laser is focused. These two subsystems can both be
controlled using a single program written in LabView software.
Example 6
[0062] The electrical system is powered by a high voltage power
supply that allows independent control of the four electrodes. Each
electrode can be set from between 0 and 4.5 kV or can be
disconnected from the circuit ("floated"). The software allows the
user to set the voltage of all four electrodes to the desired
voltage set point for a set duration, and multiple voltage and time
steps can be used sequentially for complex chip functions or
different separation strategies. The optical subsystem is comprised
of a confocal, epifluorescence system where fluorophores are
excited by a JDS Uniphase Series 2214-30s1 single-line, 488-nm
argon ion laser (San Jose, Calif. USA). Mirrors are used to direct
the laser beam into a TE200 inverted, epifluorescence microscope.
The beam is then passed through a band-pass filter, and is
reflected off of a dichroic mirror and focused through a Nikon
10.times./0.45 microscope objective into the center of the
microfluidic channel producing a laser spot approximately 10 .mu.m
in diameter. The emitted fluorescence is collected through the same
objective and is passed through the dichroic mirror, followed by a
second, wide-band-pass filter (Chroma Technology, Brattleboro, Vt.
USA). The spectrum of the filtered light is measured by directing
it through a transmission grating and focusing it onto a high
quantum-efficiency, 532.times.64 pixel charge-coupled device (CCD)
cooled to -15.degree. C. (Hamamatsu Corp., Brigewater, N.J. USA).
Pixel binning is applied to the data from the CCD camera to
quantify the intensity of the emission over a particular range of
wavelengths, calibrated by Raman scattering lines of solvents. Data
collection can be accomplished at rates from 10 to 50 Hz. The CCD
output is collected, binned, low-pass filtered, and stored using a
program written in LabView.
Example 7
[0063] Experiments were conducted using single channel glass
microchips with 7.5-cm effective separation distance purchased from
Micronit Microfluidics BV (Enschede, The Netherlands). Channels
were coated with either pDMA or pHEA by first rinsing with 1 M HCl
for 15 minutes followed by filling the channel with a 0.1% (w/v)
polymer solution for 15 minutes. Separations of ssDNA and
sequencing were carried out in pDMA (M.sub.w=3.4 MDa) and LPA
(M.sub.w=2.5 MDa) matrices with concentrations ranging from 3-5%
(w/v) in TTE buffer (50 mM Tris, 50 mM TAPS, and 2 mM EDTA) with 7M
urea, Pop-5.TM. matrix (Applied Biosystems, Inc, Foster City,
Calif. USA) and Beckman LongRead.TM. LPA matrix (Amersham). For
each run, a 235 V/cm electric field is applied for 60 seconds prior
to sample injection. Also, mixed molar mass pDMA matrices were
formulated with 1% or 2% of a low molar mass pDMA (about 200-about
300 kDA) and 3% to 4% of high molar mass pDMA. An offset T injector
with 100 .mu.m offset was used and the sample was injected for 40
seconds at 100 V/cm. Separation was carried out at 235 V/cm with 38
V/cm back biasing applied to the sample and sample waste wells. The
chip was maintained at 50.degree. C. for the duration of the run.
Basecalling was completed using NNIM Basecaller (NNIM, LLC, Salt
Lake County, Utah USA) and Sequencher v 4.0.5 (Gene Codes Corp.,
Ann Arbor, Mich. USA). Such pDMA matrices resulted in sequencing
read lengths of about 500 to about 600 bases in 6-7 minutes,
whereas commercial matrices do not sequence more than about 400
bases.
Example 8
Rheology
[0064] The zero-shear viscosity was determined using an Anton Paar
Physica MCR 300 (Ashland, Va., USA) with the temperature maintained
by a peltier controller connected to a digitally controlled
recirculating water bath (Julabo USA Inc., Allentown, Pa., USA).
Controlled shear stress and shear rate sweeps were performed with a
cone-and-plate (model CP50-1) fixture and a double gap Couette
(model DG26.7) fixture over a 25-50.degree. C. temperature range.
Zero-shear viscosity versus polymer concentration curves were
created using this instrument. (See, FIG. 2.)
Example 9
Video Microscopy
[0065] DNA was fluorescently labeled with YOYO-1 (Molecular Probes,
Eugene, Oreg. USA). All protein and sugar reagents were purchased
through Fisher Scientific (Pittsburgh, Pa. USA), 48 kbp ds-.lamda.
DNA was purchased through Invitrogen (San Diego, Calif. USA) and
betamercaptoethanol (BME) was purchased through (Sigma-Aldrich, St.
Louis, Mo. USA). The labeling method yielded approximately one
label per 5-10 base pairs of dsDNA. To observe the fluorescently
labeled DNA, the stained solution was combined with catalase stock
solution, glucose oxidase stock solution, BME, 20% glucose buffer,
and polymer solution which had been mixed for 24 hours to allow for
solvation. After gentle mixing overnight, the polymer/DNA solution
was ready to be imaged.
Example 10
[0066] The hybrid chip used to image the DNA was composed of a
Sylgard 184 poly(dimethylsiloxane) (Fisher Scientific, Pittsburgh,
Pa. USA) microchannel and a 1.5-thick glass cover slip (Fisher
Scientific, Pittsburgh, Pa. USA). The PDMS channel was formed by
mixing pre-polymer and curing agent at a 10:1 ratio by weight. The
degassed mixture was poured onto narrow strips of Scotch Magic
Tape.TM. and allowed to cure in a vacuum chamber overnight. The
cured PDMS was cut into 0.5 cm length squares to fit onto a cover
slip and the resulting channels had depths of approximately 50 to
60 microns.
Example 11
[0067] DNA migration was visualized using a homebuilt system
modeling the Morris lab at the University of Michigan.
(Albarghouthi, M. N., Stein, T. M. & Barron, A. E. (2003)
Electrophoresis 24: 1166-1175; de Carmejane, O., Yamaguchi, Y.,
Todorov, T. I. & Morris, M. D. (2001) Electrophoresis 22:
2433-2441.) The imaging system consists of a Nikon TE200 (Nikon
Instruments Inc., Melville, N.Y., USA) inverted epifluorescence
microscope outfitted with a Nikon CFI 100.times./N.A. 1.4 oil
immersion microscope objective. DNA fluorescence was achieved using
a 100 watt mercury lamp light source focused through a heat
absorbing filter in sequence with a blue light excitation filter
cube (460 nm-500 nm) (Chroma Technology, Brattleboro, Vt. USA).
Emitted fluorescence from the DNA was collected with a 0.5 inch
CCD, TM-6710-CL camera (JAI Pulnix, Sunnyvale, Calif. USA) through
a 510 nm long pass filter (Chroma Technology, Brattleboro, Vt. USA)
and a VS4-1845 Generation 3 image intensifier (Videoscope
International, Dulles, Va. USA). The high-speed camera has an
adjustable frame rate capable of 120 frames/sec with a full spatial
resolution of 648.times.484 pixels. All videos were captured at 30
frames/sec directly to computer via a PIXCI control board (EPIX
INC., Buffalo Grove, Ill. USA) utilizing the XCAP-STD (EPIX INC,
Buffalo Grove, Ill. USA) software. Electrophoresis voltages were
achieved using high voltage power supply from Micronit. Single DNA
molecules migrating through the polymer matrices can be imaged
using this technique.
Example 12
[0068] Using a microchannel pre-coated with a dynamic pHEA polymer,
a 4% (w/v) poly(N-methoxyethylacrylamide) (pNMEA) polymer solution
was loaded into the channel as the sequencing matrix. When the
temperature is set at 50.degree. C. and with an electric field of
235 V/cm in the 7.5 cm long separations channel, DNA sequencing
read lengths of 540 bases can be achieved at 98.5% accuracy in
about 6.5 minutes. Basecalling was performed by NNIM basecaller and
Sequencer version 4.0.5. (The sequencing electropherogram is not
shown.)
Example 13
[0069] In addition to sequencing DNA on microchips, the polymer
systems of this invention (e.g., pDMA matrix and pHEA dynamic
coating) has also been used in 22-cm capillaries on an ABI 3100
commercial apparatus. All sequencing runs were carried out at a
temperature of 50.degree. C. and an electric field of 250 V/cm. The
raw sequencing data shows that the 4% mixed molar mass pDMA (1% low
molar mass and 3% high molar mass) allows rapid sequencing versus
commercially available POP.TM.-6 matrix. The separation in such a
pDMA matrix is 3 times faster than in the POP.TM. matrix
demonstrating that more rapid sequencing is also possible in
capillary instruments. The sequencing results are shown in Table 4,
comparing the 4% mixed molar mass pDMA and POP.TM.-6 both in pHEA
coated capillaries. The separation is much faster in the mixed
molar mass pDMA. This matrix sequenced 650 bases in about 22
minutes. Basecalling was completed using NN1M basecaller and
sequencer version 4.0.5 (The sequencing electropherogram for the 4%
mixed molar mass is not shown.)
TABLE-US-00004 TABLE 4 Comparison of sequencing matrices AVG READ
LENGTH Long Read Polymer Coating (n = 4) Length Time (min) 4% pDMA*
pHEA 650 + 10 660 22 POP-6 pHEA 677 + 68 757 60 *3% high molar mass
(2.7 MDa) + 1% low molar mass (280 kDa)
[0070] As demonstrated by the preceding examples, DNA sequencing up
to 600 bases was demonstrated in 6.5 minutes using a pDMA matrix
with a pHEA dynamic coating. The pDMA synthesized for this work
performed much better than the commercially available matrices, and
the choice of the pHEA over more hydrophobic coatings is critical
for extending read lengths. Single stranded DNA fragments possess
higher mobilities in the pDMA matrices used in this study than in
the typical LPA matrices used in previous microchannel studies. The
fact that the pDMA matrices allow sequencing read lengths longer
than 600 bases in only 7.5 cm of separation distance also
contribute to the high speed.
[0071] Rheology data and videomicroscopy studies suggest that
weakly entangled pDMA networks may allow faster sequencing than
more entangled networks since the migrating DNA molecules may
disrupt the network more frequently. Although network disruptions
generally lead to lower separation ability, the DNA can hook and
drag the loose polymer chains, which is similar to the TEC
mechanism where DNA mobility is still size-dependent. While this
hybrid separation mechanism is suggested by videomicroscopy, no
current theory for DNA separation takes these mechanisms into
account concurrently.
[0072] This system can be optimized, and the analysis of mobility
versus DNA size suggests that longer read lengths are possible.
Sequencing has been demonstrated in both 11.5 cm and 15.9 cm
channel lengths. Assuming a diffusion-limited system, the minimum
resolution where the basecaller can accurately call bases scales
with the square root of the channel length, while the migration
time should increase linearly. Therefore, using an 11.5 cm channel,
this system can be used to produce up to a 630 base read in
.about.9 minutes while a 15.9 cm channel will produce an 800 base
read in .about.12 minutes. Such results will be tempered by the
transition to oriented reptation begins near 850 bases for the 4%
pDMA matrix, which will change the mobility dependence on length
and shorter reads may be expected. Nevertheless, the ability of the
pDMA matrix along with the pHEA dynamic coating to provide long
sequencing reads at shorter times represents a significant step
forward for microchip sequencing systems and will advance the
development of the next generation of sequencing technologies.
[0073] The systems/compositions of this invention can extend to
sequencing centers or other sequencing applications where a linear
polymer matrix and dynamic polymer coating could be used. Many
genotyping and forensic applications such that require DNA
separation by electrophoresis may also be interested since a wide
range of DNA sizes can be resolved, for instance, with the matrix
and coating combination as shown in FIG. 1A.
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