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Description  |
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FIELD OF THE INVENTION
The present invention relates to DNA sequencing methods based on separation
of DNA fragments in a DNA fragment mixture by capillary electrophoresis.
REFERENCES
Altria, K. D., et al., Electrophoresis 11:732 (1990).
Applied Biosystems, Model 370A DNA Sequencing System/Tag Polymerase
Technical Manual, Applied Biosystems, Foster City, Calif. (1989).
Ausubel, F. M., et al., Current Protocols in Molecular Biology, John Wiley
& Sons, Inc., Media, Pa.
Bergot, J. B., et al. PCT Publication No. WO 91/05060, published Apr. 18,
1991.
Cobb, K. A., et al., Anal. Chem. 62:2478 (1990).
Cohen, A. S., et al., J. Chrom. 516:49 (1990).
Grossman, P. D., and Colburn, J. C., Eds., Capillary Electrophoresis,
Academic Press, Inc., San Diego, Calif. (1992).
Guttman et al., Anal. Chem. 62:137 (1990).
Heiger, D. N., et al., J. Chrom. 516:33 (1990).
Hiemenz, P. H., Principles of Colloid and Surface Chemistry, 2nd Ed.,
Marcel Dekker, Inc., NY (1986).
Huang, X. C., et al., Anal. Chem. 64:967 (1992a).
Huang, X. C., et al., Anal. Chem. 64:2149 (1992b).
Huang, X. C., et al., J. Chrom. 600:289 (1992c).
Karger, B. L., and Cohen, A. S. U.S. Pat. No. 4,865,706 (1989).
Mathies, R. A., and Huang, X. C. Nature 359:167 (1992).
Maxam, A. M., and Gilbert, W., Proc. Natl. Acad. Sci. USA 74:560 (1977).
Pentoney, S. L., et al., J. Chrom. 480:259 (1989a).
Pentoney, S. L., et al., Anal. Chem. 51:1642 (1989b).
Pentoney, S. L., et al., Electrophoresis 13:467 (1992).
Sambrook, J., et al., Molecular Cloning, 2nd Ed., Cold Spring Harbor
Laboratory Press, NY (1989).
Sanger, F., et al., Proc. Natl. Acad. Sci. USA 74:5463 (1977).
Smith et al., Nucl. Acids Res. 13:2399 (1985).
Sudor, J., et al., Electrophoresis 12:1056 (1991).
Swerdlow, H, et al., Nucleic Acids Res. 18:1415 (1990a).
Swerdlow, H., et al., J. Chrom. 516:61 (1990b).
BACKGROUND OF THE INVENTION
Electrophoresis is widely used for fractionation of a variety of
biomolecules, including DNA species, proteins, peptides, and derivatized
amino acids. One electrophoretic technique which allows rapid,
high-resolution separation is capillary electrophoresis (CE) (Grossman and
Colburn, 1992). Typically, CE employs fused silica capillary tubes whose
inner diameters are between about 10-200 microns, and which can range in
length between about 5-100 cm or more.
One use for CE that has received much attention has been in the separation
and identification of DNA sequencing fragments. Such separations have been
carried out previously using slab gel configurations requiring painstaking
preparation of crosslinked polyacrylamide matrices between glass plates.
More recently, methods have been described for carrying out such
separations by CE, providing the advantages of shorter separation times,
reduced sample sizes, potential automation of sample loading, and
potentially higher resolution of sample peaks.
Guttman et al. (1990) have reported single-base separation of
polynucleotides containing on the order of 160 bases by CE using
crosslinked polyacrylamide gels containing 3-6% T and 5% C.
Cohen et al. (1990) have demonstrated use of CE with a crosslinked
polyacrylamide gel (3% T, 5% C) to separate DNA sequencing fragments
differing in length by a single base from 18 to about 330 bases in total
length.
Swerdlow et al. (1990a) have compared the separation of DNA sequencing
fragments achieved by CE with that achieved by slab gel electrophoresis
using identical crosslinked polyacrylamide matrices (6% T, 5% C). The
separations afforded by CE were said to be 3-fold faster and to provide
2.4-fold better resolution and 5-fold better separation efficiency than
provided by a conventional slab gel configuration.
Although crosslinked polyacrylamide matrices such as above have been shown
to be useful in DNA sequencing analysis, certain limitations have
remained. One limitation has been that bubbles can form in the
polyacrylamide matrix during polymerization in the capillary tube,
compromising peak resolution and necessitating rejection of some
acrylamide-filled tubes following polymerization (Swerdlow, 1990a,b).
Another limitation has been the formation of bubbles near the injection end
of the capillary tube during electrophoresis of the sample (Swerdlow,
1990b).
A third limitation has been that at high voltages, electroosmosis can
occur, leading to extrusion of the gel matrix from the tube. To counter
such extrusion, crosslinked matrices have been covalently attached to the
inside wall of the tube (Karger et al., 1989). However, such covalent
linkage can lead to the formation of voids in the matrix due to
contraction during polymerization or electrophoresis (Grossman et al.,
1992, at pp. 140-142).
A fourth limitation, in DNA sequence analysis, has been the fouling of the
capillary inlet by the sequencing template. Accumulation of a large,
essentially immobile template at the inlet can limit the degree of
resolution achievable with subsequently loaded samples, thereby limiting
use of the capillary to a few uses at most.
Another limitation has been that, when polymerization of the matrix is
carried out in the capillary tube, the polymerization procedure must be
performed individually for each tube and typically requires a significant
delay (e.g., overnight polymerization) before the capillary can be used
for electrophoresis.
Linear (non-crosslinked) polyacrylamide matrices have also been found
useful in the separation of DNA fragments. Heiger et al. (1990) have shown
that linear polyacrylamide matrices containing 6, 9, 12% T were useful in
the separation by CE of restriction fragments ranging in size from about
75-12,000 basepairs in length (non-denaturing conditions), and further,
that a higher % T (e.g., 9% T) was useful in resolving, under denaturing
conditions, a mixture of polydeoxyadenylate fragments ranging from 40-60
bases in length. The authors suggested that polymerization of the polymer
matrix be performed inside the capillary tube to obtain high viscosity and
to minimize the difficulties of handling viscous solutions.
Sudor et al. (1991) reported separation of DNA fragments using linear
polyacrylamide solutions containing 3-10% T (weight percent of total
acrylamide) and 7M urea. Polyacrylamide-filled capillary tubes were
prepared by forming the polyacrylamide solution outside of the tube and
then forcing the polymerized solution into the tube by syringe, while
taking care not to break the syringe due to excessive pressure. Comparison
of CE separations performed with solutions containing 3, 5, and 10% T
showed that 10% T gave the best resolution of oligonucleotide test
fragments (poly-dC) 10 to 36 bases in length.
More recently, Mathies, Huang, and coworkers (Huang et al., 1992a,b,c;
Mathies et al., 1992) have described a linear polyacrylamide matrix (9% T
containing 7M urea) for DNA sequence analysis by CE. The matrix is said to
typically allow sequencing of up to 300-350 bases per capillary (Huang et
al., 1992b), and as high as 500 bases beyond the primer (Huang et al.,
1992a). The highly viscous matrix is polymerized in the capillary tube and
is said to be physically stable, allowing multiple (e.g., three or four)
sample injections.
Ideally, a matrix for use in separating DNA sequencing fragments should (i)
provide single-base resolution for DNA sequencing fragments of 300 bases
in length, preferably 500 bases in length, or greater, and (ii) have a
sufficiently low viscosity to allow rapid filling and re-filling of the
capillary tube.
SUMMARY OF THE INVENTION
The present invention provides an improved method and apparatus for
achieving single-base resolution of DNA sequencing fragments, by providing
a low-viscosity medium that can readily be loaded into and removed from a
capillary electrophoresis tube.
In one aspect, the invention includes an improved automated method of
determining the nucleotide sequence of a target polynucleotide, in which
DNA fragments in a DNA sequencing mixture are separated by size in a
capillary electrophoresis tube. The improvement includes separating the
DNA fragments in an aqueous denaturing solution comprising between about 4
and about 7 weight percent polyacrylamide molecules having an average
molecular weight of between about 20 and about 100 kDa. The solution
viscosity allows filling of a capillary electrophoresis tube by applying a
low pressure differential across the tube, e.g., no more than about 100
psi. After fragment separation, the polyacrylamide solution is removed
from the tube and fresh aqueous denaturing solution is introduced into the
tube by applying a pressure differential across the ends of the tube.
In another aspect, the invention includes an improved method of determining
the nucleotide sequence of a target polynucleotide, wherein DNA fragments
in a DNA sequencing mixture are separated by size. The improvement
includes separating the DNA fragments by capillary electrophoresis in an
aqueous denaturing solution comprising between about 4 and about 7 weight
percent linear polyacrylamide molecules.
The linear polyacrylamide molecules preferably have an average molecular
weight between about 20 kDa and about 100 kDa, more preferably about 55
kDa.
The separation medium (polymer solution) for use in the methods of the
invention is effective to achieve a single-base resolution for fragments
of at least about 300 bases in length, and preferably at least 500 bases
in length, although the method can be used for smaller selected ranges,
e.g., for achieving single-base resolution for fragments in the range of
about 30 to about 100 or 200 bases in length.
These and other objects and features of the invention will become more
fully apparent when the following detailed description of the invention is
read in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a schematic view of a capillary electrophoresis apparatus in
accordance with the invention.
FIGS. 2A-2E show selected time segments of a single electropherogram
obtained by the method of the invention with a mixture of DNA sequencing
fragments. The fragments were generated by the Sanger method and terminate
at their 3'-ends with the dideoxy form of cytidine. The length of each
fragment is indicated by the number at the top of each peak.
DEFINITIONS
"Acrylamide" and "acrylamide monomer" refer to a structure having the form
NH.sub.2 C(.dbd.O)CR.sub.1 .dbd.CR.sub.2 R.sub.3, where R.sub.1 is
hydrogen, and R.sub.2 and R.sub.3 can be hydrogen or a methyl group.
"Polymer" is used in its traditional sense of a large molecule composed of
smaller monomeric subunits covalently linked together to form a chain.
"Linear polyacrylamide" or "linear polyacrylamide polymer" refers to a
polymer formed from acrylamide monomers in the absence of a crosslinking
agent.
"Crosslinked polyacrylamide" refers to a polymer formed from acrylamide
monomers in the presence of a crosslinking agent (e.g., bis-acrylamide) to
produce a 3-dimensional, covalently crosslinked gel.
"Aqueous denaturing solution" refers to an aqueous solution containing a
denaturing agent (e.g., urea) at a concentration effective to maintain
polynucleotides in a single-stranded state largely or entirely devoid of
secondary structure.
The term "polymer solution" refers to any solution containing linear
polyacrylamide molecules of the invention, i.e., having an average
molecular weight of between about 20 kDa and about 100 Kda. The polymer
solution may contain a denaturing agent as defined in the preceding
paragraph.
"Average molecular weight" refers to the number-average molecular weight
(M.sub.n) of a sample population made up of polymer species having a
multiplicity of molecular weights. This quantity is defined by the
equation:
M.sub.n =(.SIGMA.n.sub.i .times.MW.sub.i)/.SIGMA.n.sub.i
where n.sub.i indicates the number of molecules of species i, and MW.sub.i
is the molecular weight of species i.
"Electrophoretic mobility" refers to the steady-state velocity induced per
unit field strength for a selected molecular species. Electrophoretic
mobility can be measured in terms of the time required for a molecular
species to pass a particular point in the tube, or in terms of distance of
a molecular species from a reference point along the length of the tube at
a selected time. "Relative electrophoretic mobility" refers to the
electrophoretic mobility of a molecular species in comparison to that of
another molecular species.
The term "DNA sequencing fragments" refers to DNA polynucleotides generated
for the purpose of obtaining sequence information about a selected DNA
target sequence. Such fragments can be generated enzymatically, e.g., by
the Sanger dideoxy method, or chemically, e.g., by the approach of Maxam
and Gilbert, for example. In addition, the fragments may originate from a
single sequencing reaction (e.g., a dideoxy sequencing reaction performed
in the presence of dideoxycytidine triphosphate), or from more than one
sequencing reaction (e.g., from four different dideoxy sequencing
reactions which include suitably labeled 5'-primers to identify the
3'-terminal base of each fragment).
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a linear polyacrylamide preparation having
an average molecular weight between about 20 kDa and about 100 kDa, which
is useful in a sieving medium (polymer solution) for separating
polynucleotides, and particularly, DNA sequencing fragments. For
electrophoresis, the polymer solution contains the linear polyacrylamide
at a concentration of between about 4 and about 7 weight %, along with a
denaturant and a selected buffer for controlling the pH of the solution.
The polymer solution of the invention can be introduced into the capillary
tube by applying a small pressure differential across the two ends of the
tube. The polynucleotide sample is introduced into one end of the
capillary tube and an electric field is applied across electrode
reservoirs in which the ends of the tube have been immersed. As the
polynucleotide fragments move through the electric field, they are
fractionated on the basis of length by differential migration through the
sieving matrix established by the polymer solution.
While the polymer solution of the invention is particularly useful in
determining a contiguous nucleotide sequence in a target polynucleotide
(DNA or RNA) molecule, other uses of the solution include detection of
differences (e.g., mutations) between two or more sequences, assessment of
the purity of synthetic oligonucleotides, and resolution and sizing of
restriction fragments.
I. Capillary Electrophoresis Apparatus
FIG. 1 is a simplified schematic view of a capillary electrophoresis system
20 (Applied Biosystems, Foster City Calif.) suitable for practicing the
method of the invention. The system includes a capillary tube 22 having a
length preferably between about 10-200 cm, typically less than about 100
cm, and an inner diameter (i.d.) of preferably between about 10-200 .mu.m
(microns), typically about 50 .mu.m. In the embodiment shown, the tube is
supported in a horizontal position and has downwardly bent end regions.
The capillary tube is of a type that permits suppression of electroosmosis
during electrophoresis (e.g., such that electroosmosis is below about
2.times.10.sup.-5 cm.sup.2 /sec-V), and which interacts minimally or not
at all with the sample. One preferred capillary tube is a fused silica
tube having an inner diameter of 50 .mu.m (available from Polymicro
Technologies, Phoenix, Ariz.), the inner surface of which is chemically
coated as detailed in section II.B below.
More generally, the capillary tube may be any tube or channel capable of
supporting a column of polymer solution, preferably having an inner
diameter of 200 .mu.m or less. For example, the tube may take the form of
a channel formed in a glass slide or the like.
A cathodic reservoir 26 in system 20 contains an electrolytic polymer
solution 28, described further in the sections which follow. The cathodic
end of the tube, indicated at 22a, is sealed within reservoir 26 and is
immersed in the polymer solution, as shown, during electrophoresis. Second
tube 30 in reservoir 26 is connected to a finely controlled air pressure
system (not shown) which can be used to control the pressure in the head
space above the polymer solution, e.g., for loading polymer solution into
the tube by positive pressure. The pressure system is able to generate a
pressure differential across the ends of the capillary tube of about
100-300 psi or less.
Additionally, the air pressure system can include a vacuum system for
drawing solution through the capillary tube.
A sample reservoir 31 in system 20 contains the sample mixture to be loaded
into the cathodic end of the tube. Preferably, the sample has been
dissolved in a denaturing solution which maintains the sample
polynucleotides in single-stranded form. For example, a dried DNA sample
from a sequencing reaction can be dissolved in a mixture of 1 volume of 5
mM aqueous EDTA, and 12 volumes of formamide, and heated at 90.degree. C.
for 2 minutes prior to sample loading. The sample and cathodic reservoirs
may be carried on a carousel or the like, for placement at a position in
which the cathodic end of the tube can be immersed in the reservoir fluid.
Although not shown here, the carousel may carry additional reservoirs
containing, for example, solutions for cleaning and flushing the tube
between electrophoretic runs, or different polymer solutions.
The opposite, anodic end of the tube, indicated at 22b, is immersed in an
anodic electrolyte solution 32 contained in an anodic reservoir 34. A
second tube 36 in reservoir 34, analogous to tube 30 in reservoir 26, can
be included to control the pressure above solution 32, e.g., for loading
polymer solution into the tube, just as with tube 30 in reservoir 26.
Typically, the compositions of electrolyte solutions 28 and 32 are
identical to the polymer solution in the capillary tube.
For sample loading and subsequent sample separation by electrophoresis, the
filled capillary tube and electrode reservoirs are preferably configured
so that there is little or no net liquid flow through the tube. This can
be effected by keeping the surfaces of the electrode reservoir solutions
at the same height, or by controlling the atmospheric pressures above the
two solutions.
A high voltage supply 40 in the system is connected to the cathodic and
anodic reservoirs as shown, for applying a selected electric potential
between the two reservoirs. The power supply leads are connected to
platinum electrodes 41, 42 in the cathodic and anodic reservoirs,
respectively. The power supply may be designed for applying a constant
voltage (DC) across the electrodes, preferably at a voltage setting of
between 6 kV and 20 kV.
Detector 44 in the system is positioned adjacent the anodic end of the
tube, for monitoring sample peaks migrating through an optical detection
zone 45 in the tube. Typically, the capillary tubing has been treated to
remove a small region of exterior polyimide coating (in the case of a
polyimide-coated capillary tube) to create a small window. The detector
may be designed for UV or visible absorption detection, and/or for
fluorescence emission detection or radioisotope detection, for example.
Fluorescence emission detection is preferably carried out at one or more
selected excitation wavelengths which are adjustable between about 240-600
nm, depending on the fluorescent species associated with the sample
molecules. Typically, the detector employs an argon laser as an excitation
source. Preferably, a confocal optical arrangement is used (e.g., Huang et
al., 1992, page 968). For recording electrophoretic peaks, the detector is
connected to an integrator/plotter 46, which may take the form of a
computer for data storage on a magnetic medium or the like.
Radioisotope detection may be accomplished by the use of a modified HPLC
isotope detector for .sup.3 H or .sup.14 C (Radiomatic Instruments &
Chemical Co., Inc., Meriden, Conn.). For detection of .sup.32 P-labeled
peaks, a semiconductor or scintillation-based radioisotope detector device
may be used (Pentoney et al., 1989a, pages 2625-2629, 1989b pages
259-270). A detector configured for gamma-ray detection may also be used
(e.g., Altria et al., 1990, pages 732-734).
In operation, the capillary tube is thoroughly washed by flushing suitable
rinsing solutions through the tube by applying positive pressure to the
head space above the appropriate solution reservoir. Alternatively, the
capillary can be washed manually by syringe. In the practice of the
present invention, the polymer-containing electrolyte solution itself can
be used to flush the system between sample runs. If a cleaning solution
different from the polymer electrolyte solution is used, the tube is then
flushed with several volumes of the polymer solution.
The sample is then loaded into the cathodic end of the tube, typically by
electrokinetic injection. The cathodic end of the tube is placed in the
sample solution, and a brief, high voltage pulse is applied across the two
ends of the tube (e.g., 6 kV for 5 seconds). The tube end is then returned
to the solution in cathodic reservoir 26, and a separation voltage (e.g.,
12 kV) is applied until the desired number of fragment peaks have passed
through the detection zone.
For automated electrophoresis of multiple samples, the apparatus may be
adapted to include an array of capillary tubes and suitable detection
means for simultaneous monitoring of sample migration in the tubes. By
such an arrangement, the same sample or a number of different samples can
be analyzed in parallel using such an array.
II. Linear Polyacrylamide Compositions
As indicated above, the present invention is based on the discovery that a
low-viscosity solution containing a selected concentration of linear
polyacrylamide molecules in a selected molecular weight range is useful
for providing single-base resolution of large DNA sequencing fragments in
capillary electrophoresis. The polymer solutions of the invention are
significantly lower in viscosity than the linear polyacrylamide media that
have previously been used for high resolution separation of DNA sequencing
fragments.
II.A Preparation of Linear Polyacrylamide
The linear polyacrylamide molecules of the present invention are
characterized by an average molecular weight between about 20 kDa and
about 100 kDa.
The average molecular weight of the polymer population can be adjusted by a
number of factors. In one approach, the conditions of polymerization are
changed in order to effect changes in the MW of the final polymer
population. The average molecular weight can be decreased by (1)
increasing the reaction temperature, (2) increasing the ratio of radical
initiator to acrylamide monomer, or (3) increasing the amount of chain
transfer reagent. Transfer reagents that can be used include lower alkyl
alcohols, with isopropanol being preferred.
Example 1 describes a procedure for preparing a linear polyacrylamide
preparation in accordance with the invention. In the procedure, a mixture
of isopropanol (6.55 ml) in water (222 ml) is stirred at 35.degree. C. for
10 minutes while being de-gassed with helium. Acrylamide (25 g) is then
added and allowed to fully dissolved. After dissolution of the acrylamide,
polymerization is initiated by the addition of TMED
(N,N,N',N'-tetramethylethylenediamine) and APS (ammonium persulfate), to
final concentrations of about 0.05% (w:v and v:v for TMED and APS,
respectively), and polymerization is allowed to continue for 90 minutes
with vigorous stirring.
Preferably, the polymer solution is dialyzed in 12-14 kDa MW-cutoff
dialysis tubing against multiple changes of buffer over several days to
remove low molecular weight reactants. Following dialysis, the polymer
solution is lyophilized, providing dried polymer in about 40% yield.
A second method for adjusting the average MW of a polymer is by
fractionating the polymer into different MW fractions followed by
isolation and purification. An aqueous solution of polymer is fractionated
by a size-dependent chromatographic separation (such as gel permeation
chromatography), or by fractional precipitations using a water miscible
solvent such as methanol.
Conveniently, the molecular weight distribution of the polymer thus
obtained is represented as a number-average molecular weight as defined
earlier above. The average molecular weight can be determined using gel
permeation chromatography according to procedures which are well known in
the art (e.g., see Hiemenz, 1986, pp. 42-49). In brief, the retention
times of a number of polyacrylamide standards of known molecular weight
composition are determined to establish a standard curve that correlates
molecular weight with elution time. The standard curve is used to
determine the average molecular weight of the sample based on elution
time. An exemplary molecular weight determination is provided in Example
2.
II.B Polymer Solution
For separating single-stranded polynucleotides, the polymer solution
includes a denaturant at a concentration effective to maintain
polynucleotides in a single-stranded state that is largely or entirely
devoid of secondary structure. Typically, urea at a concentration of 7-8M
is employed. In general, the polymer solution used for separating DNA
fragments in the present invention has a viscosity between about 10 and
about 300 centipoise, as measured at 30.degree. C.
The solution also includes a buffer for maintaining the pH of the solution
at a selected value between about 4-9. For any given buffer species used,
there exists an optimum concentration for maximum sample peak resolution.
Band-broadening may occur at a too low concentration (low ionic strength)
or at too high a concentration (high ionic strength). In general,
zwitterionic buffers improve resolution and reduce background UV
absorbance variations.
For achieving high resolution of single-stranded polynucleotides, the
polymer of the invention is present in the polymer solution at a
concentration between about 4 and about 7 weight %.
According to an important aspect of the invention, the polymer solution of
the invention is readily flowable, allowing easy, rapid filling and
refilling of the capillary tube. Preferably, the viscosity is sufficiently
low (about 10 to about 300 centipoise, measured at 30.degree. C.) to allow
filling of a 50 cm.times.50 .mu.m i.d. capillary tube within 30 minutes
using a pressure differential across the tube of less than about 100 psi,
preferably less than 50 psi. Alternatively, by use of a syringe and
application of gentle pressure, the polymer solution of Example 4 can be
loaded into such a capillary in less than 60 seconds. The feature of rapid
filling is particularly amenable to an automated capillary electrophoresis
procedure for DNA sequence analysis.
Table 1 below shows calculated fill times for the above capillary tube at
polymer viscosities (.eta.) from 10 to 300 centipoise.
TABLE 1
______________________________________
.eta.*
t.sub.fill (min)
______________________________________
300 23
100 7.7
50 3.9
25 2.0
10 0.8
______________________________________
*length = 50 cm, pressure differential = 100 psi, i.d. = 50 .mu.m.
II.C Preparation and Filling of Capillary Tube
The capillary tube for use in the invention is of a type that permits
suppression of electroosmosis during electrophoresis (e.g., such that
electroosmosis is below about 2.times.10.sup.-5 cm.sup.2 /sec-V), and
which interacts minimally or not at all with the sample. Preferred
capillary tubes are those having internal diameters of less than about 200
microns, preferably less than about 100 microns, and more preferably
within the range of about 25-50 microns, although sizes below 25 microns
are also contemplated.
Typically, the tube is composed of fused silica having an exterior
polyimide coating to confer structural rigidity. Electroosmosis can be
suppressed by masking the negative charge along the inner silica surface
of the tube. Such masking can be accomplished by application of a chemical
coating, which binds to the inner surface covalently or noncovalently,
using approaches known in the art. Details of a procedure for preparing a
suitable covalent coating based on the method of Cobb et al. (1990, page
2479) are provided in Example 3. The selected coating should be stable
under the separation conditions employed, and preferably affords
single-base resolution for fragments up to 300 bases in length, preferably
up to 500 bases in length, or greater, for at least one day of multiple
runs (e.g., through 5 consecutive runs performed over a period of about 8
hours).
In the coating procedure described in Example 3, a fused silica capillary
tube is flushed with 1.0M NaOH and then coated using a three step
procedure. In the first step, the silica surface of the inner wall is
chlorinated using thionyl chloride. Prior to chlorination, the tube is
flushed with about 80 column volumes of dry, distilled tetrahydrofuran
(THF) to dry the inner wall surface. After removal of residual THF (e.g.,
by helium stream), a solution of thionyl chloride containing a trace
amount of dimethylformamide (DMF) is passed through the tube to chlorinate
the silica surface. The reaction is continued overnight by joining the two
ends of the tube to each other and incubating the tube in an oven at
55.degree. C.
In the second step, the tube is flushed with several column volumes of dry,
distilled THF to remove the thionyl chloride solution, and then flushed
with a 1M solution of vinyl magnesium bromide (Grignard reagent) in THF
(.about.100 column volumes over about 5 hours). The tube is heated
intermittently with a heat gun to facilitate the reaction. After the 5
hour flushing step, the ends of the tube are again joined to each other
and heated overnight at 70.degree. C.
In the third step, the surface vinyl groups are reacted with acrylamide.
The capillary is first conditioned by sequential flushing with THF (to
remove the Grignard solution), methanol, and water. The tube is then
flushed over 30 min with freshly prepared aqueous polymerization solution
containing 3% (w:v) acrylamide monomer, 0.1% (v:v) TMED, and 0.1% (w:v)
APS. Following this step, the tube is flushed with water and then dried by
helium stream. The dried coated tube may be stored at -20.degree. C. until
use.
Loading of polymer solution into a capillary tube for sample separation is
conveniently accomplished by syringe, or by application of positive
pressure in the head space above a reservoir solution in which one end of
the capillary tube is immersed. For loading by syringe, one end of the
capillary tube is connected to a syringe, and polymer solution is loaded
into the tube using gentle pressure on the syringe plunger. A 50
cm.times.50 .mu.m i.d. capillary tube can be filled within 60 seconds by
syringe, without trapping bubbles in the tube.
Preferably, for use in an automated CE apparatus, the tube is loaded or
reloaded with polymer solution by application of positive pressure in the
head space above a reservoir solution. In this approach, one end of the
capillary tube (e.g., the anodic end) is immersed in polymer solution
contained in a reservoir (e.g. in the anodic reservoir of a CE apparatus
as in section I above). The head space above the reservoir solution is
sealed off from the outside atmosphere by suitable sealing means adapted
to allow one end of the capillary tube to pass into the reservoir
solution. The sealing means further allows access of the reservoir head
space to a controllable gas pressure source (e.g., a compressed-gas
cylinder having suitable regulating means). By applying a positive
pressure to the head space above the anodic solution, a pressure
differential across the tube ends is created, driving anodic reservoir
solution into the tube. As indicated above, the low-viscosity solution
used in the invention can be introduced rapidly into the capillary tube at
low pressure, e.g., 100 psi or less. The pressure differential is
maintained until solution exits the cathodic end of the tube, at which
time the cathodic end is immersed in the cathodic solution.
As mentioned earlier, the filled capillary tube and electrode reservoirs
are preferably configured so that there is little or no net liquid flow
through the tube. This can be effected by keeping the surfaces of the
electrode reservoir solutions at the same height, or by controlling the
atmospheric pressures above the two solutions.
III. Electrophoresis Method
The polynucleotide sample which is to be electrophoretically separated is
prepared by standard methods. Typically, the sample is a mixture of DNA
sequencing fragments derived from one or more sequencing reactions. The
two primary techniques for DNA sequencing are chemical methods (e.g.,
Maxam and Gilbert, 1970), and enzymatic methods (e.g., Sanger et al.,
1977). Routine protocols for both techniques are widely available (e.g.,
Sambrook, Chapter 13, 1989). Although the techniques differ in approach,
both produce populations of polynucleotides (sequencing fragments) which
begin at a defined 5'-terminal base site and end randomly at a selected
base or a combination of selected bases. (The four standard nucleotide
bases for DNA are deoxyadenylate, deoxycytidylate, deoxyguanylate, and
thymidylate, abbreviated as A, C, G, and T, respectively). Typically, the
fragments include a reporter label (referred to interchangeably as
"reporter" or "label") for enhanced detection, and, where appropriate, for
distinguishing fragments which terminate at a selected base from other
fragments which terminate at one or more different selected terminal
bases. Any label suitable for either or both purposes may be used, with a
fluorescent label being preferred. For example, the fragments may be
labeled by use of a fluorescent or radio-labeled 5'-primer, by use of
fluorescent-labeled dideoxy-terminal nucleotides (e.g., Bergot et al.,
1991, pages 1-20), or by use of radioactive nucleotides (e.g., .sup.35
S-labeled dATP).
Usually, the dideoxy (enzymatic) method of Sanger et al. is used. In this
method, a DNA template is annealed to a 5'-primer oligonucleotide in
solution by brief heating (e.g., 60.degree. C. for 10 minutes) of the
solution followed by slow cooling over 20-30 minutes to
4.degree.-20.degree. C. Preferably, the primer includes a fluorescent
label which is covalently bound to the 5'-terminal base in the primer.
Suitable fluorescent dyes and labeling methods which may be used are
described in U.S. Pat. No. 4,855,525, which is incorporated herein by
reference. The template-primer solution is then incubated in the presence
of (i) selected concentrations of the four standard nucleotide bases (ii)
a selected concentration of the dideoxy form of the selected terminal
base, and (iii) a DNA polymerase. The polymerase-catalyzed reaction is
allowed to proceed for a selected time and is then stopped by suitable
means (e.g., by quenching the reaction in cold ethanol). Note that if
desired, one or more of the standard bases can be substituted with a
selected nucleotide analog; e.g., c7dGTP in place of dGTP, for avoiding
peak compression. The action of DNA polymerase is effective to generate
labeled DNA fragments which begin with the (labeled) 5'-primer sequence,
terminate at their 3'-ends with the dideoxy form of the selected terminal
base, and which are complementary in sequence to the template DNA.
Where a sequence of contiguous bases is to be determined, it is preferable
that four separate sequencing reactions be performed, one for each
standard base. Moreover, it is also preferred that for each reaction, a
different label is used, such that fragments formed in each sequencing
reaction can be distinguished from the fragments formed in the other three
reactions. Classes of spectrally resolvable fluorescent dyes suitable for
this purpose have been described (e.g., U.S. Pat. No. 4,855,525; Smith et
al., 1985, pages 2399-2412), thereby allowing fragments from the four
sequencing reactions to be electrophoretically separated in the same lane
(migration path). Four "universal" M13 5'-primers which are labeled
respectively with four spectrally resolvable fluorescent dyes (designated
FAM, JOE, TAMRA, and ROX) are commercially available from Applied
Biosystems.
The DNA sequencing fragments produced in each sequencing reaction are
usually isolated from cold ethanol solution by centrifugation. After the
pellet has been washed with cold 70% ethanol, the resultant pellet is
dried briefly, e.g., by vacuum centrifuge for 3 minutes, and is then ready
for electrophoretic analysis. Alternatively, the sample is stable in this
dried form for several months if stored in the dark at -20.degree. C.
Prior to being loaded in the capillary tube, the dried DNA fragment sample
is resuspended in a denaturing solution and heated briefly to convert all
of the DNA fragments to single-stranded form. For example, the denaturing
solution can consist of a mixture of 1 volume of 5 mM aqueous EDTA, and 12
volumes of formamide. After suspension in the denaturing solution, the
sample is heated at 90.degree. C. for 2 minutes, and then loaded into the
cathodic end of the polymer solution-filled capillary tube, typically by
electrokinetic injection as above. If fragments from more than one
reaction are to be loaded on the capillary tube, the fragments from the
reactions are preferably mixed before loading.
FIGS. 2A-2E show time segments of an electropherogram obtained with a
mixture of sequencing fragments terminating at C (3'-end), generated as
described in Example 4. In these figures, fluorescence intensity (in
arbitrary units) is plotted as a function of elution time past the
detector. The length of each fragment, which was determined from the known
sequence of the m13mp18 (+) strand, is indicated at the top of each peak.
The spacing of elution times of the fragments correlated closely with the
known sequence positions of the cytidylate residues.
FIG. 2A shows the separation of fragments ranging in length from about 20
to 50 bases. As can be seen, peaks differing in length by a single base
were nearly baseline resolved. FIG. 2B shows the separation of fragments
ranging in length from about 220 to 260 bases. Again, single-base
resolution was achieved. FIG. 2C shows the separation of fragments ranging
in length from about 350 to 371 bases. As can be seen, fragments greater
than 300 bases in length are well resolved. FIGS. 2D and 2E show the
separation of fragments in the size ranges of about 405 to 425 bases (FIG.
2D) and about 500 to 525 bases (FIG. 2E), respectively. These figures show
that fragments having lengths up to 400 bases, 500 bases, or greater, can
be resolved in the method.
Electrophoretic separation of the DNA sequencing sample from Example 4 was
also carried out using a conventional slab gel configuration (Applied
Biosystems Model 370A DNA Sequencing System, Applied Biosystems, Calif.).
A crosslinked matrix containing 4% T and 5% C was used. The
electropherogram which was obtained showed peak resolution comparable to
that obtained by the capillary electrophoresis analysis just described.
More importantly, the relative peak intensities were essentially the same
as those found by capillary electrophoresis, demonstrating that the
variable peak heights in FIGS. 2A-2E are attributable to the DNA sample
(i.e., to the actual amount of each fragment in the sample), and not to a
defect in the separation method.
After the mobilities of the desired number of sequencing fragments have
been recorded, electrophoresis is stopped, and the polymer solution in the
capillary tube can be replaced. For example, positive pressure in the head
space above the anodic reservoir solution can be used to drive several
column volumes of new polymer solution through the capillary tube. The
other end of the capillary can be directed to a waste collector for
disposal. The newly filled capillary tube is then ready for separating
another sample.
IV. Utility
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