U.S. patent application number 12/349448 was filed with the patent office on 2010-03-04 for denaturant-free electrophoresis of biological molecules under high temperature conditions.
This patent application is currently assigned to Applied Biosystems, LLC. Invention is credited to Kevin J. Levan, Qingbo Li, Heide Monroe.
Application Number | 20100051459 12/349448 |
Document ID | / |
Family ID | 22737302 |
Filed Date | 2010-03-04 |
United States Patent
Application |
20100051459 |
Kind Code |
A1 |
Li; Qingbo ; et al. |
March 4, 2010 |
Denaturant-Free Electrophoresis of Biological Molecules Under High
Temperature Conditions
Abstract
The present invention relates to a method of separating a sample
comprising biological compounds, such as nucleic acids. The nucleic
acids are subjected to electrophoresis using a matrix that is
essentially free of denaturants and having at least one random,
linear copolymer comprising a first comonomer of acrylamide and at
least one secondary comonomer. A temperature of at least a portion
of the matrix is at least about 80.degree. C.
Inventors: |
Li; Qingbo; (State College,
CA) ; Levan; Kevin J.; (State College, PA) ;
Monroe; Heide; (Pittsburgh, PA) |
Correspondence
Address: |
LIFE TECHNOLOGIES CORPORATION;C/O INTELLEVATE
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
Applied Biosystems, LLC
|
Family ID: |
22737302 |
Appl. No.: |
12/349448 |
Filed: |
January 6, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10661558 |
Sep 15, 2003 |
7473341 |
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12349448 |
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10258547 |
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PCT/US01/13336 |
Apr 25, 2001 |
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10661558 |
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60199389 |
Apr 25, 2000 |
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Current U.S.
Class: |
204/450 |
Current CPC
Class: |
G01N 27/44704 20130101;
G01N 27/44747 20130101 |
Class at
Publication: |
204/450 |
International
Class: |
B01D 57/02 20060101
B01D057/02; G01N 27/26 20060101 G01N027/26 |
Claims
1. A method of separating a first sample comprising nucleic acids,
the method comprising: providing a matrix that is essentially free
of denaturing agents; raising a temperature of a first portion of
the matrix to at least about 80.degree. C.; subjecting the nucleic
acids to electrophoresis through at least the first portion of the
matrix while the temperature of the first portion is at least about
80.degree. C.; and deliberately cooling a second portion of the
matrix to less than about 30.degree. C., the nucleic acids
migrating through the second portion after they have first migrated
through the first portion.
2. The method of claim 1, wherein the first portion of the matrix
is raised to a temperature between 80.degree. C.-90.degree. C.
3. The method of claim 1, wherein the matrix comprises at least one
random, linear copolymer comprising a first comonomer of acrylamide
and at least one secondary comonomer.
4. The method of claim 1, wherein the second portion of the matrix
is cooled to less than about 25.degree. C.
5. The method of claim 1, wherein the matrix is completely free of
denaturing agents.
6. The method of claim 1, further comprising subjecting a second
sample of nucleic acids to electrophoresis within the same matrix,
after the first sample has been electrophoresced.
7. The method of claim 6, comprising subjecting a total of at least
25 additional samples of nucleic acids, one at a time, without
replacing the matrix.
8. The method of claim 7, wherein the temperature of at least a
portion of the polymer matrix in which the second sample is
electrophoresced is at least about 80.degree. C.
9. A method of separating a first sample comprising nucleic acids,
the method comprising: subjecting the nucleic acids to
electrophoresis using a matrix that is essentially free of
denaturants, the matrix having at least one random, linear
copolymer comprising a first comonomer of acrylamide and at least
one secondary comonomer, wherein a temperature of at least a
portion of the matrix is at least about 80.degree. C.
10. The method of claim 9, wherein the comonomers are randomly
distributed along the copolymer, and wherein the at least one
secondary comonomer is selected from the group consisting of vinyl
monomers, monomers of acrylamide derivatives, monomers of acryloyl
derivatives, monomers of acrylic acid derivatives, monomers of
polyoxides, monomers of polysilanes, monomers of polyethers,
monomers of derivatized polyethylene glycols, monomers of cellulose
compounds, or mixtures thereof, each having between 2-24 carbon
atoms.
11. The method of claim 9, wherein the at least one secondary
comonomer is N,N-dimethylacrylamide monomer.
12. The method of claim 11, wherein the polymer is a copolymer
polymerized using about a 1:1 ratio of acrylamide and
N,N-dimethylacrylamide monomer.
13. A method of sequencing a sample comprising nucleic acids,
comprising: providing a matrix that is essentially free of
denaturing agents, the matrix having at least one random, linear
copolymer comprising about a 1:1 ratio of acrylamide and
N,N-dimethylacrylamide monomer, and a buffer having a pH of at
least about 8, a temperature of at least a portion of the matrix
being at least about 80.degree. C.; subjecting the nucleic acids to
electrophoresis through said matrix; and prior to detecting the
nucleic acids, deliberately cooling a second portion of the matrix
to less than about 25.degree. C., the second portion of the matrix
receiving nucleic acids from the heated portion of the matrix.
14. A method of separating a plurality of samples of biological
compounds, comprising: providing a matrix that is essentially free
of denaturing agents; subjecting a first sample to electrophoresis
through said matrix, the first sample comprising nucleic acids, and
wherein a temperature of a first portion of the matrix is
sufficient to substantially denature the nucleic acids; and
subjecting a second sample to electrophoresis in a separate step
but through the same matrix, the second sample comprising a complex
of at least two biological compounds.
15. The method of claim 14, wherein the temperature is from about
80.degree. C. to about 99.degree. C.
16. The method of claim 15, wherein the temperature is from about
80.degree. C. to about 90.degree. C.
17. The method of claim 15, further comprising deliberately cooling
a second portion of the matrix to less than about 30.degree. C.,
the first and second samples migrating through the second portion
after each has first migrated through the first portion.
18. The method of claim 17, wherein the second portion of the
matrix is cooled to less than about 25.degree. C.
19. The method of claim 15, wherein the complex comprises at least
one of a nucleic acid-protein complex and a protein-protein
complex.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
10/661,558, filed Sep. 15, 2003, which is a continuation of
application Ser. No. 10/258,547, filed Oct. 25, 2002, now
abandoned, which is a national phase application of application no.
PCT/US01/13336, filed Apr. 25, 2001, which claims priority to U.S.
Provisional Application No. 60/199,389, filed Apr. 25, 2000, which
are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to methods and devices for
sequencing nucleic acids in separation matrices that are
essentially free of chemical denaturants.
BACKGROUND
[0003] A conventional DNA sequencing gel matrix typically contains
3-9 M urea, or a combination of urea and formamide as a denaturant.
The function of a denaturant in a gel is to help keep DNA molecules
denatured during electrophoresis in order to achieve accurate base
calling. The existence of urea or formamide in a gel matrix
represents a distinctive difference between denaturing gel
electrophoresis for DNA sequencing (separating single-stranded DNA)
and non-denaturing gel electrophoresis for separating double
stranded DNA. The denaturing power of a gel matrix is generally
proportional to the concentration of a denaturant in the gel
matrix. Higher denaturing powers minimize compression, a
self-folding behavior, of single stranded DNA fragments in a DNA
sequencing sample.
[0004] When urea is used, however, the denaturing power is limited
by the saturation concentration of urea, which is about 9M. When
formamide is used, there is also a limit, which is the manageable
viscosity of a matrix and separation speed. For example, because
the viscosity of the matrix increases significantly with the
percentage of formamide in a gel, separation speed decreases with
higher percentages of formamide. Sometimes, the denaturing power of
a gel with a maximum concentration of urea or formamide still does
not provide sufficient denaturing power to resolve some compression
bands in GC-rich DNA samples.
[0005] A popular method to overcome the above-mentioned problem of
insufficient denaturing power is to heat up a gel during
electrophoresis, typically 35-70.degree. C., and add a denaturant.
The combination of high temperature electrophoresis and high
concentration denaturant typically provides sufficient denaturing
power to resolve difficult compression bands.
[0006] There are several issues, however, associated with
electrophoresis using a matrix containing urea or formamide. First,
urea and formamide degrade in the basic solution that is typically
used for DNA sequencing (pH 8.0-8.5). Higher temperatures
accelerate such degradation. The degradation of urea or formamide
has adverse effects on the gel and separation columns. The
degradation products include ammonia, uric acid, and formic acid.
These products increase the ion concentration and pH of the matrix.
They may also cause bubble formation in a matrix at higher
temperatures. When an uncoated capillary is used, these degradation
products reduce the adhering affinity between the polymer dynamic
coating and the capillary wall. This allows electroosmotic flow to
occur, which consequently reduces separation efficiency. Capillary
lifetime is also shortened because the decreased coverage of
polymer coating on the capillary wall allows biomolecules to attack
and adsorb onto the capillary wall, which in turn degrades
separation efficiency.
[0007] To minimize these problems, one can take several approaches:
a) optimize the electric field strength and column temperature so
that the degradation products can be consistently driven out of the
separation column at a rate that is equivalent to or higher than
the rate of generation; b) develop better dynamic coating polymers
that adsorb onto capillary wall more efficiently under high
temperature; c) reduce the pH value of the gel matrix, e.g., from
pH 8.3 down to pH 7.6, in order to reduce the degradation rate of
urea or formamide at high temperatures, and to enhance the
adsorbing efficiency of polymer on the capillary wall. These
methods, however, have limitations.
SUMMARY OF THE INVENTION
[0008] The present invention relates to a method of separating a
mixture of nucleic acids, comprising subjecting the biological
molecules to electrophoresis using a matrix that is essentially
free of denaturants, the matrix having at least one random, linear
copolymer comprising a first comonomer of acrylamide and at least
one secondary comonomer, wherein a temperature of at least a
portion of the polymer matrix is at least about 75.degree. C. and
more preferably at least about 80.degree. C. The maximum
temperature of the matrix should be less than the boiling point of
a fluid within the matrix, but preferably is less than about
95.degree. C. In one embodiment, the matrix is completely free of
denaturants.
[0009] The comonomers are preferably randomly distributed along the
copolymer. At least one secondary comonomer is selected from the
group consisting of vinyl monomers, monomers of acrylamide
derivatives, monomers of acryloyl derivatives, monomers of acrylic
acid derivatives, monomers of polyoxides, monomers of polysilanes,
monomers of polyethers, monomers of derivatized polyethylene
glycols, monomers of cellulose compounds, or mixtures thereof each
having between 2-24 carbon atoms.
[0010] In one embodiment, the copolymer is polymerized using about
a 1:1 ratio of acrylamide and another comonomer. The other
comonomer is preferably N,N-dimethylacrylamide monomer. The ratio
of reactivity of the at least one secondary comonomer to said
primary comonomer is preferably between 0.3 and 2.
[0011] In another embodiment, the matrix has a viscosity between
100 and 50,000 Cp. The at least one linear random copolymer has a
molecular weight between 100,000 and 5,000,000 Daltons. In a
preferred embodiment the compolymer comprises a buffer having a
basic pH. Preferably, the buffer comprises about 89 mM Tris, 89 mM
borate, and 2 mM EDTA. In a preferred embodiment, the buffer has a
pH of at least about 8 and preferably from about pH 8 to pH
8.3.
[0012] In yet another embodiment, a second mixture of nucleic acids
is subjected to electrophoresis within the matrix with at least a
portion of the matrix being heated to at least about 75.degree. C.
preferably at least about 80.degree. C. The second mixture of
nucleic acids may be identical to the earlier electrophoresed
mixture or the second mixture may be different. Preferably, the
electrophoresis step can be repeated up to at least about 25 times
so that about 25 mixtures can be electrophoresed without first
providing a new matrix.
[0013] In another embodiment, a cooled portion of the matrix is
cooled to less than about 25.degree. C. The cooled portion is
preferably disposed between a detection zone of the matrix and the
heated portion of the matrix so that the cooled portion receives
nucleic acids from the heated portion of the matrix. The length and
temperature of the cooled portion are preferably selected to allow
DNA that was denatured in the heated portion to substantially
re-anneal prior to being detected.
[0014] Another embodiment of the invention relates to a method of
electrophoresing a plurality of mixtures of biological compounds,
comprising subjecting a first mixture to electrophoresis using a
matrix that is essentially free of denaturing agents, the first
mixture comprising nucleic acids, and wherein a temperature of at
least a portion of the matrix is sufficient to substantially
denature the nucleic acids. During electrophoresis of the first
mixture, the temperature of the matrix preferably is between
80.degree. C.-99.degree. C. More preferably, the temperature is
between 80.degree. C.-95.degree. C. and most preferably is between
80.degree. C.-90.degree. C. The temperature of the matrix is
insufficient to boil a fluid, such as water, present in the matrix
and so this determines the upper temperature limit, subject to the
atmospheric conditions.
[0015] A second mixture is subjected to electrophoresis using
substantially the same matrix, the second mixture comprising a
complex of at least two biological compounds. By substantially the
same matrix it is meant, for example, that the same support is used
to electrophorese both the first and second mixtures without first
replacing more than about 20% of the matrix present in the support.
Preferably, less than about 5%, and more preferably none of the
matrix is replaced.
[0016] In a preferred embodiment, the complex comprises at least
one of a nucleic acid-protein complex and a protein-protein
complex. It should be understood that the first and second mixtures
can be electrophoresed in either order.
[0017] Another embodiment of the present invention relates to a
system for electrophoretically sequencing at least one nucleic acid
sample. The system comprises at least one support suitable for
retaining a matrix in which electrophoretic separation of nucleic
acid samples may be conducted. A heat source is in thermal contact
with the at least one support, the heat source being configured to
heat at least a portion of the at least one support to at least
about 80.degree. C. The support preferably provides sufficient
thermal contact between the heat source and the matrix retained by
the support so that heating the support to at least about
80.degree. C. also heats a portion of the matrix to at least about
80.degree. C.
[0018] In one embodiment, the system further comprises a cooling
device configured and arranged to cool a cooled portion of the at
least one support, the cooled portion receiving samples from the
heated portion of the support and being disposed between the heated
portion and a detection zone of the capillary. The cooling device
is preferably configured to cool the temperature of the cooled
portion of the capillary to less than about 25.degree. C.
[0019] In another embodiment, the support contains a matrix
suitable for electrophoretic separation of a nucleic acid sample,
the matrix being essentially free of denaturing agents.
[0020] Another embodiment of the present invention relates to a
system for electrophoretically sequencing a plurality of nucleic
acid samples, the system comprising a plurality of capillaries,
each capillary having a first end, the first ends being arranged in
a two-dimensional array corresponding to an array of wells of a
microtitre tray, each of said wells configured to contain at least
one of the nucleic acid samples. The system includes an apparatus
to fluidly associate each of said nucleic acids samples with a
respective first end to introduce the nucleic acid samples to the
capillaries. Fluidly associating a sample with the first end of the
capillary with a sample in a well preferably introduces a
sufficient quantity of the sample into the capillary to allow
electrophoretic separation of the nucleic acids in the sample
followed by detection of the separated nucleic acids.
[0021] The device includes a heat source in thermal contact with
said plurality of capillaries and configured to heat at least a
heated portion of each of said capillaries to a temperature of at
least about 80.degree. C., and computer means configured to operate
the heat source to heat the heated portions to at least about
80.degree. C.
[0022] The system preferably includes a light source arranged to
illuminate said samples and a detector arranged to detect
fluoresced light emitted by said samples.
[0023] In one embodiment, the system further comprises a cooling
device configured and arranged to cool a cooled portion of each of
at least some of the capillaries, the cooled portions disposed to
receive nucleic acids from the heated portions of the capillaries.
The cooling device is preferably configured to cool a temperature
of each cooled portion to less than about 25.degree. C.
[0024] In another embodiment, the capillaries contain a matrix
suitable for electrophoretic separation of a nucleic acid sample,
the matrix being essentially free of denaturing agents.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The present invention is described in detail below with
reference to the drawings in which:
[0026] FIG. 1a & 1b show two embodiments for a device utilizing
thermal denaturation in accordance with the present invention;
[0027] FIGS. 2a and 2b show the result of sequencing a PGEMI/U
sample using a 5% copolymer gel in 1xTBE, 7M urea, at room
temperature;
[0028] FIG. 3 shows a graph of Phred score versus called bases for
the separation of FIGS. 2a and 2b;
[0029] FIGS. 4a-4c show the result of sequencing a PGEM/U sample
using 5% copolymer gel in 1xTBE with no urea, at 80.degree. C.
according to the present invention; and
[0030] FIG. 5 shows the Phred score v. called bases for the
separation of FIGS. 4a and 4b.
DETAILED DESCRIPTION OF THE INVENTION
Matrix for Electrophoresis
[0031] The present invention overcomes the problems associated with
the presence of denaturing agents such as, for example, urea or
formamide, in a medium configured to support the electrophoretic
separation of biological molecules. Preferably, the medium is
configured to support the electrophoretic sequencing of nucleic
acids such as DNA under temperature conditions sufficient to at
least partially denature the nucleic acids. The medium is
preferably a matrix, such as a sieving matrix, that is essentially
free of urea, formamide, or other denaturing agents. As used
herein, the term matrix is synonymous with the term gel, which is
often used to describe media used for electrophoretic
separations.
[0032] The matrix of the present invention may be used with any
suitable electrophoresis format, such as, for example, slab gel
electrophoresis, capillary electrophoresis, or microchip
electrophoresis. Preferably, the matrix is retained by a support
that, together with the matrix, defines a path through which a
sample migrates. Suitable supports include, for example, plates for
retaining slab gels, a capillary, or microchip channel. The support
is preferably coated or otherwise modified to minimize
electroosmotic flow, as understood in the art. Modified supports
include supports that are formed materials, such as plastics or
other polymers, that themselves minimize electroosmotic flow.
Alternatively, the matrix itself can provide a "coating" function
reducing the amount of electroosmotic flow. It should be
understood, however, that the present invention is suitable for use
with supports that are either unmodified or uncoated such that
electroosmotic flow occurs.
[0033] By essentially free of denaturing agents it is meant that
denaturing agents, if any, are present in the matrix in an amount
such that the agents themselves or degradation products of the
agents do not adversely affect the matrix or separation support.
For example, separation supports are typically modified or
configured to minimize electroosmotic flow during sequencing. The
denaturing agents, if present, are preferably present in an amount
that is insufficient to increase electroosmotic flow by an amount
sufficient to degrade separation performance. More preferably, the
essentially denaturant free matrix of the present invention is
completely free of urea, formamide, and other denaturing
agents.
[0034] Because the denaturing power of the present matrix
preferably depends upon the temperature of the matrix, rather than
the presence of a denaturing agent, the same matrix can be operated
in denaturing mode for one sample and in non-denaturing mode for a
successive sample. The ability to alternate the same matrix between
denaturing and non-denaturing modes advantageously increases the
rate at which successive samples can be analyzed because time
consuming capillary flushing steps are not required to remove or
add the denaturing agent to the matrix or to change the matrix
itself. For example, the matrix of the present invention may be
used to separate a first sample of double-stranded DNA, without
thermal denaturing, and to separate a second sample of DNA in the
form of single-stranded DNA using thermal denaturing, as in DNA
sequencing.
[0035] The matrix of the present invention also allows binding
assays to be performed in the same matrix that can be used for
sequencing DNA with denaturing. For example, electrophoretic assays
of DNA-protein interactions or assays of protein-protein
interactions require that the temperature of the matrix be raised
sufficiently high to induce electrophoretic mobility differences in
the DNA-protein or protein-protein complex. The present matrix,
being essentially free of denaturing agents, allows the same matrix
to be operated at temperatures sufficiently high to perform a
binding assay on one mixture and sequence DNA with thermal
denaturing in a second mixture. Of course, by reducing the
temperature, the same matrix can be used to separate mixtures
without denaturing. As used herein, the term mixture refers to a
sample comprising compounds to be separated, sequenced, or
otherwise assayed to determine a property of a compound present in
the sample. Assays include both qualitative and quantitative
determinations.
[0036] The matrix of the present invention can also be used in the
quantitation or quality control of products formed during a
polymerase chain reaction (PCR). The PCR products can be very
large, such as greater than about 1000 basepairs. The matrix of the
present invention can be operated with a viscosity sufficiently low
to facilitate separation of the large PCR products. In order to
compensate for the loss of denaturing power due to being
essentially free of denaturants, the temperature of at least a
heated portion of the matrix is preferably sufficient to denature
substantially all of the DNA to be sequenced. The heated portion
preferably comprises substantially all of the migration distance,
which is the distance along the migration path between the region
where the mixture is introduced into the matrix and the region
where components of the mixture are detected. For example, the
heated portion of the matrix comprises at least 50%, preferably at
least 75%, more preferably at least 85%, of the migration distance.
Thus, during electrophoresis, DNA in a mixture preferably remains
denatured for substantially all of the migration time. Preferably,
the DNA remains denatured for at least 50%, of the time required to
migrate from the injection region to the detection region.
[0037] The temperature of the heated portion is at least about
75.degree. C. and preferably is between 80.degree. C.-99.degree. C.
More preferably, the temperature is between 80.degree.
C.-95.degree. C. and most preferably is between 80.degree.
C.-90.degree. C.
[0038] The polymer of the matrix of the present invention may be
any polymer that is suitable for use in electrophoresis and is able
to be operated at temperatures sufficient to denature at least a
portion of the DNA.
[0039] Preferably, the polymer is a copolymer formed of a 1:1 ratio
of acrylamide and N,N-dimethylacrylamide (DMA) monomer. The matrix
of the present invention preferably contains from about 0.2 to
about 10% copolymer by weight. More preferably, the amount of
copolymer is from about 1 to 6%, with about 5% most preferred.
Polymerization techniques suitable to produce polymers of the
present invention and other polymers suitable for use in the
present invention, are described in international application no.
PCT/US00/00793 published Jul. 20, 2000 as publication number WO
0042423, which is incorporated herein by reference in its
entirety.
[0040] In a preferred embodiment, the matrix comprises at least one
random copolymer forming a targeted linear copolymer. The random
copolymer preferably includes more than one monomer, with different
monomer units being distributed along the copolymer chain in no
specific pattern. Preferably, the copolymer is not crosslinked with
other copolymers. The random copolymers are preferably composed of
at least two or more comonomer types. The ratio of comonomers can
be continuously adjusted to optimize the properties for
electrophoretic separation. The ratio of comonomers may be any
ratio that provides the desired properties of the random copolymer.
The comonomers must be sufficiently water soluble to be used in an
electrophoretic separation. Typically, there is a primary comonomer
that gives the random copolymer chain its primary physical,
chemical, and sieving properties. Preferably, the primary comonomer
is an acrylamide or an acrylamide derivative, which contains
between 3-24 carbon atoms, is either saturated or unsaturated, and
is either substituted or unsubstituted.
[0041] Examples of suitable acrylamide derivatives include, but are
not limited to, N,N-dimethacrylamide, N,N-dimethylmethacrylamide,
N-ethylmethacrylamide, N-ethylacrylamide, N-methylacrylamide,
N-methylmethacrylamide, and methacrylamide. The primary comonomers
are available commercially or by simple derivatization of monomer
units.
[0042] The secondary comonomers are selected for their inherent
properties that may be incorporated into the copolymer chains.
These inherent properties include, but are not limited to, one or
more of hydrophilicity, hydrophobicity, self coating properties,
copolymer chain backbone stiffness, stability of copolymer
entanglement structure at different temperature and electric
fields, resistance to hydrolysis, processivity of copolymer chain
extension, gel matrix viscosity, affinity of the copolymer to the
surface of a suitable supporting substrate, such as a coating layer
on the inner surface or exposed bare surface of a capillary tubing,
and chirality. The preferred inherent properties of the secondary
comonomers are hydrophilicity, hydrophobicity, viscosity, and self
coating properties. The selection of the secondary comonomers and
the ratio of secondary comonomers to primary comonomer are based on
predetermined desired properties of the targeted random
comonomer.
[0043] At least one secondary comonomer may be copolymerized with
the primary comonomer to form a random copolymer, wherein each
comonomer unit is distributed along the copolymer chain in no
specific order, and the ratio of the reactivity of the primary
comonomer to the secondary comonomers is between about 0.3 to about
2. The reactivity is the probability that a given monomer is added
to a growing copolymer chain in the presence of other types of
monomers. Formation of the random copolymers is not limited to the
copolymerization of one secondary comonomer with the primary
comonomer. More than one secondary comonomer may be present in the
formation of the random copolymers.
[0044] In another embodiment, the secondary comonomer or comonomers
are vinyl monomers, monomers of acrylamide derivatives, monomers of
acryloyl derivatives, monomers of acrylic acid derivatives and
mixtures thereof. Preferably, the secondary comonomers are vinyl
monomers, monomers of acrylamide derivatives, monomers of acryloyl
derivatives, monomers of acrylic acid derivatives, monomers of
polyoxides, monomers of polysilanes, monomers of polyethers,
monomers of derivatized polyethylene glycols, monomers of cellulose
compounds, and mixtures thereof, each having between 2-24 carbon
atoms, is saturated or unsaturated, and is substituted or
unsubstituted.
[0045] More preferably, the secondary comonomer includes at least
one of methacrylamide, N-acryloylmorpholine, N-allylacrylamide,
N-allylmethacrylamide, N, benzylacrylamide, N-benzylmethacrylamide,
N-(iso-butoxymethyl)acrylamide, N-(iso-butoxymethyl)
methacrylamide, N-(tert-butyl)acrylamide,
N-tert-butyl)methacrylamide, N-cyclohexylacrylamide,
N-cyclohexylmethacrylamide, N,N-diethylacrylamide,
N,N-diethylmethacrylamide,
N-[2-(N,N-dimethylamino-)ethyl]acrylamide,
N-[2-(N,N-dimethylamino)ethyl]methacrylamide,
N-[3-(N,N-dimethylamino)propyl]acrylamide,
N-[3-(N,N-dimethylamino)propyl-]methacrylamide,
N,N-dimethylacrylamide, N-methylmethacrylamide, N-methylacrylamide,
N-ethylacrylamide, N-ethylmethyacrylamide, N-phenylacrylamide,
N-phenylmethacrylamide, N,N-diphenylacrylamide,
N,N-diphenylmethacrylamide, N,N-dodecamethylenebisacrylamide,
N-dodecylacrylamide, N-dodecylmethacrylamide,
N-(2-hydroxypropyl)acrylamide, N-(2-hydroxypropyl)methacrylamide,
N,N-methylenebismethacrylamide, N-methylolacrylamide,
N-methylolmethacrylamide, N-propylacrylamide,
N-propylmethacrylamide, N-isopropylacrylamide,
N-isopropylmethacrylamide, N-butylacrylamide,
N-butylmethacrylamide, N-isobutylacrylamide,
N-isobutylmethacrylamide, vinyl acetate, vinylacetic acid,
vinylbenzyl alcohol, vinylcyclohexane, N-vinyl formamide,
1-vinyl-2-pyrrolidinone, vinyl acetonitrile, vinyl acrylate, vinyl
4-tert-butylbenzoate, N-vinylcaprolactam, vinyl crotonate,
vinylcyclopentane, vinyl decanoate, vinyl carbonate, vinyl
2-ethylhexanoate, 1-vinylimidazole, vinyl methacrylate,
2-vinyinaphthalene, 2-vinylpyridine, 4-vinylpyridine, vinyl
sulfone, ethylene glycol vinyl ether, 1,6-hexanediol vinyl ether,
N-vinylphthalimide, vinyl pivalate, 1-vinyl-2-pyrrolidinone, vinyl
trifluoroacetate, 4,4'-vinylidenebis(N,N-dimethylaniline), or
mixtures thereof. In another embodiment, the secondary comonomer
can also include acrylamide alone or in combination with any of the
above comonomers.
[0046] The random copolymers are synthesized by copolymerization of
comonomers using methodology well known to those of ordinary skill
in the art. The preferred method of copolymer synthesis is
free-radical solution polymerization. Any free radical initiator
well known to those of ordinary skill in the art may be used,
including, but not limited to, peroxy compounds, azoalkanes,
photochemical homolysis, biradicals, tin hydrides, alkyl amines,
and heat. Preferably, the free radical initiator is a peroxy
compound, an azoalkane, or alkylamine.
[0047] Typical polymerization initiators known to those of ordinary
skill in the art can be used in the present invention. For
instance, these initiators may be capable of generating free
radicals. Suitable polymerization initiators include both thermal
and photoinitiators. Suitable thermal initiators include, but are
not limited to, ammonium persulfate/tetramethylethylene diamine,
2,2'-azobis-(2-amidino propane) hydrochloride, potassium
persulfate/dimethylaminopropionitrile,
2,2'-azobis(isobutyronitrile), 4,4'-azobis-(4-cyanovaleric acid),
and benzoyl-peroxide. Preferred thermal initiators are ammonium
persulfate/tetramethyethylenediamine and
2,2'-azobisisobutyronitrile ("AIBN"). Suitable photoinitiators
include, but are not limited to, isopropylthioxantone,
2-(2'-hydroxy-5'-methylphenyl)benzotriazole,
2,2'-dihydroxy-4-methoxybenzophenone, and riboflavin. When using
the combination of persulfate and tertiary amine, the persulfate is
preferably added prior to the addition of the non-aqueous medium,
since persulfate is much more soluble in water than in non-aqueous
dispersing media. More preferably, the free radical initiator is
N,N,N',N'-tetramethylethylene-diamine ("TEMED"), or AIBN.
[0048] The matrix of the present invention has a higher long-term
storage chemical stability because the matrix is at least
essentially free of denaturing agents and detrimental degradation
products therefrom. The matrix of the present invention also has a
higher thermal stability and can be operated at higher temperatures
to improve denaturing efficiency than if denaturants were present
in the matrix. Denaturing agents can be thermally unstable and
produce degradation products detrimental to the performance of the
separation support and matrix.
[0049] A matrix of the present invention, which is essentially free
of denaturing agents preferably has a lower viscosity than a matrix
utilizing chemical denaturation, such as formamide, to achieve the
same level of denaturation. This advantage is due at least in part
to the higher level of heating that can be obtained with the
present matrices. The lower viscosity allows faster separation
speeds than if denaturants were used.
[0050] Advantages provided by running the essentially denaturant
free matrix of the present invention also include, for example, an
increased capillary lifetime, a longer sequencing read length, and
a higher confidence level of a called base, as measured by a phred
score, which is described below. For example, when a 5% 1:1
copolymer matrix containing 7M urea is used for room temperature
electrophoresis, a capillary array lasts only up to 14 runs. A
capillary comprising the essentially denaturant free matrix of the
present invention lasts for more than about 25 runs. Thus, for
example, at least about 25 samples can be run in sequence without
providing a new matrix within the separation support. The actual
number of runs that can be obtained with a single capillary before
providing a new matrix depends, for example, upon the type of
coating and the operating temperature.
[0051] The 5% 1:1 copolymer essentially denaturant free matrix
running at 80.degree. C. has a longer sequencing read length than
the same matrix with urea running at about 20.degree. C. This is an
indication of the efficient denaturing power achieved by running
the matrix at 80.degree. C. This is further validated by sequencing
PGEM/U using universal M13 reverse primer, which has been widely
used as a control sample in commercial DNA sequencers. The PGEM/U
sample running at 80.degree. C. using the non-denaturant matrix
shows no sign of compression, whereas the same sample run in a
denaturing matrix does show signs of compression.
[0052] Without urea or formamide present in the matrix, the matrix
is more stable at elevated temperature. Preferably the matrix can
be heated to a temperature sufficiently greater than the
reannealing temperature of the DNA to disrupt the secondary
structure of the DNA, which improves read length Because separation
is faster at higher temperatures, one can lower the running voltage
to further extend the read length. The matrix of the present
invention provides a read length greater than about 600 base pairs
and more preferably greater than about 650 base pairs.
[0053] A Phred score is a standard used widely in the sequencing
community to measure the quality or confidence level of a called
base. It is the negative logarithm of the error probability of a
called base. For example, a Phred score of 20 for a base means the
probability of error for calling this base is 1/100 or 1%. A Phred
score of 20 is a standard cut-off threshold used in popular
sequencing facilities. Only those bases with a Phred score greater
than or equal to 20 are considered reliable and can be accepted
into downstream in a sequence assembling process. As shown below,
the present invention provides a higher phred score than achieved
by using a sieving matrix comprising a denaturant such as urea.
Device for Separations Utilizing Thermal Denaturation
[0054] FIG. 1a shows a preferred arrangement of an embodiment of
the present thermal sequencing device 1. A sample capillary 3 is
provided to electrophoretically separate unknown sample compounds.
As used herein, the term "capillary" collectively refers to any
support or structure configured and arranged to separate a sample
using electrophoresis. Thus, as used herein, the term "capillary"
refers not only to what are commonly called capillaries but to
microfabricated channels, and planar structures, such as used in
slab gel electrophoresis. Capillary 3 is preferably arranged to be
in fluid contact with a sample reservoir 5, which is configured to
contain a volume of sample sufficient to perform an analysis.
Examples of suitable sample reservoirs include the wells of a
microtitre plate, a structure configured to perform PCR
amplification on a volume of sample, a reservoir of a
microfabricated electrophoresis device, and the like.
Alternatively, where planar structures are used, an aliquot of
sample can be added, such as by pipette, to the matrix.
[0055] Device 1 is provided with a power supply (not shown)
suitable for providing a sufficient voltage and current for
electrophoretic separation of a sample. The power supply is
preferably configured to allow at least one of the current or
resistance of the capillary to be monitored during a separation.
Preferably, the current or resistance data is received by the
computing device 17 to allow the electric potential to be varied to
maintain a constant current or resistance.
[0056] A temperature controlled portion 7 of sample capillary 3 is
arranged to be in thermal contact with a heat source such as a hot
plate 9, or the like. Optionally, or in addition, the external heat
source may comprise a wire, filament, or other heating element
arranged external to the capillary. The capillary is preferably
surrounded by a thermally conductive medium 13, to enhance thermal
contact between the heating source and the capillary.
[0057] During electrophoresis, the external heat source, rather
than ohmic heating of the separation medium itself, is preferably
the dominant source of thermal energy to the separation medium
within the capillary. The heat source is configured to heat the
separation matrix to suitable operational temperatures of the
matrix, as discussed above. The temperature is preferably
sufficient to substantially denature DNA in the matrix without
boiling a fluid in the matrix which is either essentially, or
completely, free of denaturing agents. For example, the heat source
is configured to heat the matrix to a temperature of at least about
75.degree. C. and preferably between 80.degree. C.-99.degree. C.
More preferably, the temperature is between 80.degree.
C.-95.degree. C. and most preferably is between 80.degree.
C.-90.degree. C.
[0058] The temperature of the capillary is monitored by a
temperature sensing device in thermal contact with the separation
support, such as a thermocouple 15, which preferably sends data to
a computing device 17. The temperature measured by the temperature
sensing device is considered to be the temperature of the
separation matrix and the sample being electrophoresed. Hot plate 9
is preferably automatically controlled by computing device 17 in
response to temperature data received from sensing device 15. Thus,
device 1 preferably includes computer means comprising at least one
of software or a memory configured to operate the heat source to
heat the capillary to a temperature suitable for separation of
thermally denatured nucleic acids, as described above.
[0059] Device 1 also includes a light source 23, such as a laser
emitting light having a wavelength suitable to generate
fluorescence from a fluorescent dye. A detector 25 is arranged to
obtain fluorescence intensity data, such as a time-intensity
electropherogram including peaks indicative of the presence of
nucleotides, and send the detected fluorescence intensities to
computing device 17. A detection system such as that disclosed in
U.S. Pat. No. 6,118,127, can be used for this purpose.
[0060] In any embodiment of the present invention, the fluorescence
intensity data of the unknown sample can be obtained simultaneously
with the fluorescence intensity data of a second sample. By
simultaneously, it is meant that the unknown and second samples are
elecrophoresed in a total time at least about 25% less, preferably
about 50% less, than twice the time required to sequentially
electrophorese the samples. Preferably, the unknown sample is
subjected to capillary electrophoresis in the sample capillary 3
and the second sample is simultaneously subjected to capillary
electrophoresis in a second, different capillary 19.
[0061] FIG. 1b shows another embodiment of a thermal denaturation
device 40 in which a temperature control zone 50 of the sample
capillary 3 and optional reference capillary 19 are placed in
thermal contact with a gas, such as air or nitrogen. Device 40 is
provided with a power supply 75, having the same features as the
power supply of device 1 discussed above. Temperature control zone
50 preferably extends for a heated length 64 of the capillaries. At
least one inlet port 52 is provided to introduce the heated gas to
a region 54 between the capillaries and a thermal jacket 56 and at
least one outlet 58 is provided to allow the gas to exit. Thermal
jacket 56 insulates temperature control zone 50 to reduce heat loss
from the temperature control zone 50.
[0062] The gas can be heated using, for example, a resistively
heated filament 60 or a heat exchanger.
[0063] Preferably, a fan 62 or other device to force the gas into
the inlet and out of the exit is provided. Use of a gas, which has
a lower viscosity than other fluids such as liquids, allows the
temperature of the capillary to be changed much more rapidly
because the temperature of the gas can be changed using, for
example, a heated filament much more rapidly than that of a more
viscous liquid. It should be understood, however, that a liquid may
be used to thermostat the temperature of the temperature control
zone.
[0064] A cooled zone 80 having a second, cooled length 66 of
capillaries 3 and 19 can be provided to deliberately reduce the
temperature of the samples being separated after the samples have
passed through the temperature control zone 50. In the context,
`deliberate cooling` means something other than simply allowing the
matrix to cool by simply exposing the capillary, microchip or slab
to room temperature. The temperature in cooled zone 80 can be
controlled using chilled air or other fluid or liquid with an
arrangement similar to that provided in the temperature control
zone. Alternatively, a peltier cooler can be arranged in thermal
contact with this portion of the capillary, to reduce the
temperature. The temperature and length 66 of cooled zone 80 are
preferably low enough and long enough, respectively, to allow a DNA
fragment that was thermally denatured within temperature control
zone 50 to anneal prior to being detected at a reference detection
zone 70 or a sample detection zone 70'. Thus, the cooled zone is
configured and disposed to receive compounds that have migrated
electrophoretically through heated zone 50 of the capillary. The
temperature is reduced to less than about 45.degree. C., more
preferably to less than about 30.degree. C., and most preferably to
less than about 20.degree. C.
Example
[0065] The invention is further illustrated through the following
non-limiting example.
[0066] FIGS. 2a and 2b show the result of sequencing a PGEM/U
sample using a 5% copolymer matrix in 1xTBE, 7M urea, at about
20.degree. C. The copolymer was polymerized using a 1:1 ratio of
acrylamide and N,N-dimethylacrylamide (DMA) monomer.
[0067] As can be seen in FIG. 3, which shows the graph of Phred
score versus called bases for FIGS. 2a and 2b, from base #25 to
base #405, the called bases satisfy the criteria of Phred score 20.
We define the section of the base sequence with Phred score greater
than or equal to 20 as the trim length. For the example in FIG. 3,
the trim length is 405-25=380. The trim length is used as a
measurement of the matrix performance.
[0068] FIGS. 4a and 4b show the result of sequencing a PGEM/U
sample using 5% copolymer gel in 1xTBSE, at 80.degree. C. according
to the present invention. No urea was used. The copolymer was
polymerized from 1:1 acrylamide and DMA monomer. FIG. 5 shows the
Phred score v. called bases. The trim length for this example is
720-34-686, which is an improvement over 380.
[0069] The present invention also provides a higher separation
speed than electrophoresis using a chemical denaturant. For the
example in FIGS. 2a and 2b using urea gel at room temperature, it
takes 120 minutes for 500 bases to pass the detector. FIGS. 4a and
4b, however, show that using non-urea gel at 80.degree. C.
according to the invention, it takes 68 minutes for 500 bases to
pass the detector, and 96 minutes for 800 bases to pass the
detector. Overall, tremendous gain in separation speed and
qualified sequencing length has been demonstrated by using the
non-urea gel at 80.degree. C.
[0070] While the above invention has been described with reference
to certain preferred embodiments, it should be kept in mind that
the scope of the present invention is not limited to these. Thus,
one skilled in the art may find variations of these preferred
embodiments which, nevertheless, fall within the spirit of the
present invention, whose scope is defined by the claims set forth
below.
* * * * *