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Claims  |
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What is claimed:
1. A scanner for exciting and detecting radiation from a plurality of
adjacent capillary passages comprising
a plurality of side-by-side capillary passages disposed in a plane,
a source of radiant energy of a first wavelength,
an objective lens for receiving and focusing said radiant energy at an
excitation volume in the plane of said plurality of side-by-side capillary
passages,
means for moving said plurality of side-by-side capillary passages so that
said excitation volume sequentially and repetitively is within one of said
plurality of side-by-side capillary passages to excite material in said
one passage and cause the material to radiate energy at a different
wavelength,
said objective lens serving to collect the radiated energy of said
different wavelength and direct it to an optical system which includes
confocal spatial and spectral filter means to transmit said radiated
energy at said different wavelength and reject radiation at other
wavelengths,
a detection system for receiving said radiated energy and generating a
signal, and
computer means for receiving and processing said signal to provide an
output representative of the material at the excitation volume in each of
said plurality of side-by-side capillary passages.
2. A scanner as in claim 1 in which said means for moving said plurality of
side-by-side capillary passages is controlled by said computer, whereby
the output is correlated with the individual passages in said plurality of
side-by-side capillary passages.
3. A scanner as in claim 2 in which each of the capillary passages of the
plurality of side-by-side capillary passages is part of an elongated
cylindrical capillary.
4. A scanner as in claim 3 including means for holding a region of the
cylindrical capillaries in side-by-side co-planar relationship for
presentation to the focused radiant energy.
5. A scanner as in claim 4 in which the ends of said capillaries are
separable for individual manipulation and loading.
6. A scanner as in claim 1 in which said means for moving the plurality of
side-by-side capillary passages moves them continuously whereby to scan a
band in each capillary of said plurality of side-by-side capillary
passages.
7. A scanner as in claim 1 in which said means for moving said plurality of
side-by-side capillary passages steps the individual capillary passages
into said excitation volume.
8. An apparatus for exciting and detecting radiation from sample material
in capillary passages comprising
means for presenting a plurality of capillary passages in side-by-side
coplanar relationship,
radiation means for exciting sample material in said plurality of
side-by-side coplanar capillary passages with radiation of a first
wavelength;
means for moving said plurality of side-by-side coplanar capillary passages
to sequentially and repetitively excite sample material in each passage of
said plurality of side-by-side coplanar capillary passages with said
radiation,
means for collecting radiation emitted from said sample material,
means for detecting radiation emitted by the sample material in said
plurality of side-by-side coplanar capillary passages responsive to said
radiation at a first wavelength, and
computer means for processing the detected emitted radiation and for
controlling said moving means to providing a two-dimensional output
representative of the sample material in said plurality of side-by-side
coplanar capillary passages as a function of time and position.
9. A scanner as in claim 8 in which each of said capillary passages of said
plurality of said capillary passages is part of an elongated cylindrical
capillary.
10. A scanner as in claim 9 including means for holding a region of said
elongated cylindrical capillaries in side-by-side coplanar relationship
for presentation to the radiant energy.
11. A scanner as in claim 8 in which the ends of said cylindrical
capillaries is independently manipulatable.
12. A scanner for detection of fluorescently labeled analytes which can be
separated in small diameter capillaries comprising
a plurality of capillaries each for separating a fluorescently labeled
analyte
a source of radiant energy having an excitation wavelength which excites
fluorescence from said labeled analyte,
lens means for focusing said radiant energy to a small volume and for
collecting fluorescence from said volume
means for sequentially and repetitively presenting a region of said
plurality of capillaries to said small volume of radiant energy whereby to
cause fluorescence of the fluorescently labeled analyte at said volume,
and;
means for receiving the collected fluorescence and providing an output
representative of the analyte at said small volume of the presented region
of said plurality of capillaries.
13. A scanner as in claim 12 in which said lens means comprises an
objective lens forming a part of a confocal optical detection assembly
including a spatial filter.
14. A scanner as in claim 13 in which said optical detection system
includes spectral filters for rejecting energy at said excitation
wavelength.
15. A method of detecting fluorescence from DNA sequencing fragments
electrophoretically separated in a plurality of capillaries which
comprises
positioning a region of said plurality of capillaries in side-by-side
coplanar relationship
exciting a predetermined volume sequentially and repetitively in individual
capillaries with light energy of predetermined wavelength focused therein
by an objective lens to cause fragments to fluoresce and emit light at a
different wavelength,
collecting the fluorescently emitted light from said predetermined volumes
in each of said capillaries with said objective lens,
spectrally and spatially filtering said fluorescently emitted light of
different wavelength to reject light at said predetermined wavelength and
pass said fluorescently emitted light; and,
applying the filtered fluorescently emitted light to a detector to generate
an output signal representative of the fluorescence from said fragments in
said predetermined volume in each of said capillaries.
16. The method of claim 15 in which the ends of the capillaries are
separated for rapid parallel loading of sequencing fragments into the
capillaries.
17. A scanner for exciting and detecting radiation from a plurality of
adjacent capillary passages comprising
a plurality of side-by-side capillary passages disposed in a plane,
a source of radiant energy of a first wavelength,
an objective lens for receiving and focusing said radiant energy at an
excitation volume in the plane of said passages,
a beamsplitter for directing said radiant energy to the objective lens to
excite said excitation volume with energy at said first wavelength,
means for moving said passages so that said excitation volume sequentially
and repetitively is within one of said plurality of side-by-side capillary
passages to excite material in said passage and cause the material to
radiate energy at a different wavelength,
said objective lens serving to collect said the radiated energy of said
different wavelength and direct it through said beamsplitter which passes
radiated energy at said different wavelength to an optical system which
includes a confocal spatial filter and spectral filter to transmit said
radiated energy at said different wavelength and reject radiation at other
wavelengths,
a detection system for receiving said radiated energy and generating a
signal, and
computer means for receiving and processing said signal to provide an
output representative of the material at the excitation volume in each of
said plurality of side-by-side capillary passages.
18. A scanner as in claim 17 in which said means for moving said passages
is controlled by said computer, whereby the output is correlated with each
of said capillary passages of said plurality of side-by-side capillary
passages.
19. A scanner as in claim 18 in which said means for moving the plurality
of side-by-side capillary passages moves said plurality of side-by-side
capillary passages continuously whereby to scan a band in each of said
capillary passages.
20. A scanner as in claim 18 in which each of the capillary passages of
said plurality of side-by-side capillary passages is part of an elongated
cylindrical capillary.
21. A scanner as in claim 20 including means for holding a region of the
cylindrical capillaries in side-by-side co-planar relationship for
presentation to the focused radiant energy.
22. A scanner as in claim 21 in which the ends of said cylindrical
capillaries is separable for individual manipulation and loading.
23. A scanner as in claim 17 in which said means for moving said plurality
of side-by-side capillary passages steps the plurality of side-by-side
capillary passages into said excitation volume.
24. A method of detecting fluorescence from DNA sequencing fragments
electrophoretically separated in a plurality of capillaries which
comprises
positioning a region of said plurality of capillaries in side-by-side
coplanar relationship
exciting a predetermined volume in each of said capillaries sequentially
and repetitively with light energy of predetermined wavelength focused
therein by an objective lens to cause fragments to fluoresce and emit
light at a different wavelength,
collecting the fluorescently emitted light from said predetermined volumes
in each of said capillaries with said objective lens,
spectrally and spatially filtering said fluorescently emitted light energy
of different wavelength to reject light at said predetermined wavelength
and scattered light, and pass said emitted light of different wavelength;
and,
applying the filtered emitted light of different wavelength to a detector
to generate an output signal representative of the fluorescence from said
fragments in said volume of each of said capillaries.
25. The method of claim 24 in which the ends of the capillaries are
separated for rapid parallel loading of sequencing fragments into the
capillaries.
26. A scanner for exciting and detecting radiation from a plurality of
adjacent capillary passages comprising
a plurality of side-by-side capillary passages disposed in a plane,
a source of radiant energy of a first wavelength,
an objective lens for receiving and focusing said radiant energy at an
excitation volume in the plane of said plurality of side-by-side capillary
passages,
means for moving said plurality of side-by-side capillary passages so that
said excitation volume sequentially and repetitively is within one of said
plurality of side-by-side capillary passages to excite material in said
one passage and cause the material to radiate energy at a plurality of
different wavelengths,
said objective lens serving to collect the radiated energy of said
different wavelengths and direct it to an optical system including a
plurality of dichroic beam splitters for selectively directing the
radiated energy of each of said different wavelengths to a different
location, a plurality of sets of spatial and spectral filter means one for
each of said different wavelengths, and a plurality of detection means,
one for each set of spatial and spectral filter means, for receiving the
radiated energy of each different wavelength and providing corresponding
output signals, and
computer means for receiving and processing said signals to provide an
output representative of the material at the excitation volume in each of
said plurality of side-by-side capillary passages.
27. A scanner for exciting and detecting radiation from a plurality of
adjacent capillary passages comprising
a plurality of side-by-side capillary passages disposed in a plane,
a source of radiant energy of a first wavelength,
an objective lens for receiving and focusing said radiant energy at an
excitation volume in the plane of said passages,
a beamsplitter for directing said radiant energy to the objective lens to
excite said excitation volume with energy at said first wavelength,
means for moving said passages so that said excitation volume sequentially
and repetitively is within one of said plurality of side-by-side capillary
passages to excite material in said passage and cause the material to
radiate energy at different wavelengths,
said objective lens serving to collect the radiated energy of said
different wavelengths and direct it through said beamsplitter which passes
radiated energy at said different wavelengths to an optical system which
includes a plurality of additional beamsplitters, spatial and spectral
filter means for selectively directing the radiated emitted energy of
different wavelengths to different locations and a plurality of detection
means each receiving emitted energy at one of said different wavelengths
and providing corresponding output signals, and
computer means for receiving and processing said signals to provide an
output representative of the material at the excitation volume in each of
said plurality of side-by-side capillary passages.
28. A method of detecting fluorescence from DNA sequencing fragments
electrophoretically separated in a plurality of capillaries which
comprises
positioning a region of said plurality of capillaries in side-by-side
coplanar relationship,
exciting a predetermined volume in each of said capillaries sequentially
and repetitively with light energy of predetermined wavelength focused
therein by an objective lens to cause fragments to fluoresce and emit
light at different wavelengths,
collecting the fluorescently emitted light at different wavelengths from
said predetermined volumes in each of said capillaries with said objective
lens,
spectrally and spatially filtering said fluorescently emitted light energy
of different wavelengths to reject light at said predetermined wavelength
and scattered light, and pass said emitted light of different wavelengths;
and,
applying the filtered emitted light of different wavelengths to detectors
to generate an output signal representative of the fluorescence from said
fragments in said volume of each of said capillaries at each of said
different wavelengths. |
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Claims |
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Description |
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to provide a continuous sampling of the capillary volume.
These and other objects of the invention are achieved by a laser-excited
capillary array scanner including a plurality of capillaries having a
parallel, side-by-side, coplanar relationship and a laser-excited confocal
fluorescence detector for detecting fluorescence from a selected interior
volumes of each of said capillaries sequentially and repetitively during
electrophoresis or other separation method. The invention also relates to
a method of analyzing a plurality of capillaries, with a single scanner,
by scanning a plurality of capillary passages in side-by-side
relationship, and periodically and repetitively detecting fluorescence
from each capillary passage during electrophoresis or any other separation
procedure.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects of the invention will be more clearly
understood from the following description when read in connection with the
accompanying drawings, wherein:
FIG. 1 is a schematic diagram of a confocal-fluorescence capillary array
scanner in accordance with one embodiment of the invention;
FIG. 2 is a view of a holder for supporting a region of the capillaries in
side-by-side relationship;
FIG. 3 is an enlarged view of the focal zone;
FIGS. 4A and 4B illustrate how the excitation beam is focused to a volume
in the interior of a cylindrical capillary;
FIG. 5 is an image obtained by scanning a four-capillary array during a DNA
separation;
FIGS. 6 (A-D) are an electropherogram of the DNA separation of FIG. 5;
FIGS. 7 (A-D) are an expanded view of the indicated regions of the
electropherograms of FIGS. 6 (A-D);
FIG. 8 is an image obtained by scanning a twenty-four capillary array; and
FIG. 9 is a schematic diagram of a four-color confocal-fluorescence
capillary scanner.
DESCRIPTION OF PREFERRED EMBODIMENT(s)
In accordance with this invention, the throughput in capillary
electrophoresis is increased by employing a large number of capillaries in
parallel. The most important problem confronting capillary array
electrophoresis is detection. In copending patent application Ser. No.
07/531,900 filed Jun. 1, 1990, and incorporated herein by reference, there
is described a laser-excited confocal fluorescence gel scanner which
provides enhanced detection of fluorescently labelled DNA in slab gels.
This detection system uses an epi-illumination format where the laser is
focused on the sample by a microscope objective and the emitted
fluorescence is gathered by the same objective using a 180.degree.
retro-optical geometry followed by confocal detection.
Sensitive detection of fluorescently-labeled analytes separated in small
diameter capillaries is a difficult task. Because the capillaries have a
100 .mu.m I.D. or less, a small focal volume is needed. The detection
system must reject potentially strong Rayleigh scattering, fluorescence,
and reflections from the capillary walls. Using confocal excitation and
detection, the depth of field of the optical system is sufficiently small
that only the interior of the 100 .mu.m I.D. capillary is probed. The
lateral resolution which is dictated by the scan stage and the laser beam
diameter can be as small as a few microns. Background scattering and
reflections from the capillary wall are rejected by the spatial and
spectroscopic filters in front of the photodetector.
A confocal fluorescence detection system for use with capillary arrays is
shown in FIG. 1. An argon ion laser (Model 2020, Spectra-Physics, Mountain
View, Calif.), not shown, is used as the excitation source. The laser beam
is expanded to 5 mm diameter, collimated, and then directed through a
32.times., N.A. 0.4 infinite conjugate objective 11 (LD Plan-Achromat
440850, Carl Zeiss, West Germany) by a long-pass dichroic beamsplitter 12
(480 DM, Omega Optical, Brattleboro, Vt.). The dichroic beam splitter 12
reflects the excitation laser beam into the objective 11 but transmits
fluorescent light collected by the objective which is Stokes shifted to
longer wavelengths. The objective focuses the exciting laser on the sample
and gathers the fluorescence with very high collection efficiency. The use
of an infinite conjugate objective permits vertical adjustment of the
probe volume by translating the objective with the mount 13 secured to the
base 14 with no significant perturbation of the optical alignment. The
focused 1 mW, 488 nm wavelength beam is focused to a 10 .mu.m beam
diameter and a 25 .mu.m confocal beam parameter. The fluorescence emission
is passed back through the long-pass dichroic beam splitter 12 mounted on
the base 14 to reduce laser interference and to separate the excitation
and detection paths. The fluorescence is then focused by a 75 mm focal
length lens 16 mounted on the base 14 onto a 400 .mu.m pinhole which
serves as the confocal spatial filter. The light passing through the
pinhole is filtered by a 488 nm rejection band filter (488 RB filter,
Omega Optical, Brattleboro, Vt.), a long-pass cutoff filter (Schott
GG-495, Esco, Oakridge, N.J.), a bandpass fluorescence filter (530 DF60,
Omega Optical, Brattleboro, Vt.), all mounted within the housing 17,
followed by detection with a cooled photomultiplier tube 18 (RCA 31034A,
Burle Industries, Lancaster, Pa.). The spatial filter, the optical filters
and photomultiplier tube are mounted on base 14. The output of the
phototube is amplified and filtered with a low-noise amplifier (SR560,
Standford Research Systems, Sunnyvale, Calif.), digitized with a 12 bit
analog-to-digital board (DASH-16 F, metra-Byte, Taunton, Mass.) and stored
in an IBM PS/2 microcomputer. The electronic filter used for the phototube
output was a first-order, active, low-pass filter (DC to 400 Hz) with a 12
dB/octave rolloff.
The capillary array comprises a plurality of capillaries 21 having their
ends 22,23 extending into wells 24, 26 between which a high voltage is
applied for electrophoresis. The ends 22 may be separated for individual
manipulation and loading. A portion 27 of the capillaries is maintained in
side-by-side parallel coplanar relationship by a holder 28, FIG. 2. The
holder 28 includes a window through which the beam can be focused on the
interior volume of the capillaries. FIG. 3 shows the beam 29 focused in an
interior volume of a capillary 21a.
For several reasons, scanning the beam and detection system across the
capillary is better than just probing in the center of the capillary.
First, if the probe laser is fixed at the center of the capillary, the
sample stream will be rapidly photo-bleached by the laser. Scanning the
beam laterally across the capillary interior is much better than sitting
in one spot because all of the band is probed (laterally) and
photo-bleaching is reduced. Also, the off-axis probing is advantageous
because, as shown in FIGS. 4A and 4B, the cylindrical lens effect actually
brings the beam waist back into the capillary gel so the detection system
probes the gel for a longer period of time during the scan than would have
been nominally predicted from the capillary diameter and scan rate.
The holder 28 is mounted on a translation stage 30 (Model 4000, Design
Components, Franklin, Mass.). The stage is programmed to continuously scan
the capillary array back and forth at 20 mm/sec in a direction
perpendicular to the electrophoresis direction. The image acquired in this
way has two dimensions. One is a spatial dimension representing the
physical image of the capillaries. The other is a temporal dimension
proportional to the elapsed time. During a particular sweep, fluorescence
data from the photodetector is sampled at 2000 Hz so the nominal image
resolution is 10 .mu.m/pixel; thus, 10 pixels represent the interior 100
.mu.m width of any given capillary. The electronic low-pass filter cutoff
was set at 300 Hz to provide high frequency noise rejection while still
passing the spatial frequencies required to define the 100 .mu.m I.D. of
the capillaries. An image of the migrating bands is built up as a function
of time by accumulating periodic one-second sweeps of the illuminated
region of the capillaries. The transit time of the migrating DNA past the
probe region, under the conditions employed here, ranges from
approximately 10 seconds for the low molecular weight fragments (40-50
mers) to 14 seconds for the higher molecular weight fragments (380-390
mers). With one-second repeat cycles, this gives 10-14 samples of each
band. The computer processes the data and displays the acquired image in
real time. Image processing can be performed with the NIH program, Image
1.29, and commercial image processing package, Canvas.TM., to provide an
image, FIG. 5. The image data can be reduced to a one-dimensional line
plot or electropherogram by averaging the pixels across the width of each
lane using Image 1.29, FIGS. 6(A-D), and sections can be expanded as shown
in FIGS. 7(A-D).
In one example, zero-crosslinked polyacrylamide gel-filled capillaries were
prepared using a modified method of the procedure described by Cohen, et
al..sup.6,7. A 3 mm wide detection window was produced in each 100 .mu.m
I.D. 200 .mu.m O.D. fused-silica capillaries (Polymicro Technologies,
Phoenix, Ariz.) by burning off the polyimide coating with a hot wire. The
window was burned.about.25 cm from the inlet side of the 40 cm long
capillary. The inner wall of the capillaries was then treated overnight
with a bifunctional reagent, .gamma.-methacryloxypropyltrimethoxy-silane
to prepare the walls for acrylamide adhesion.sup.6. Freshly-made
acrylamide gel solution (9% T, 0% C) in a 1X TBE buffer (tris-boric
acid-EDTA) with 7 M urea was filtered with an 0.2 .mu.m syringe filter and
degassed under vacuum for about one hour. 10% TEMED
(tetraethylmethylenediamine) and 10% APS (ammonium persulfate) solution
were added to the gel solution at a final concentration of approximately
0.03%. The solution was immediately vacuum siphoned into the capillaries
and then allowed to polymerize overnight in a cold room. Prior to use,
both ends of the column were trimmed by about 1 cm and then
pre-electrophoresed for 30 to 60 minutes at 7 kV.
The capillary array was sandwiched in the capillary holder 28 that is
mounted onto the translation stage 30. The capillary holder 28, FIG. 2,
serves the dual purpose of (1) uniformly constraining each capillary in
the array to an identical height above the top of the translation stage,
and (2) exposing a small window through which the confocal zone probed the
capillary interior. Constraining the capillaries to substantially the same
plane is necessary for achieving uniform detection sensitivity from each
capillary because the depth of focus of the microscope objective is
only.about.25-50 .mu.m.
The DNA samples for which the data is shown in FIGS. 5-7D was prepared as
follows: chain-terminated M13mp18 DNA fragments were generated using a
Sequenase 2.0 sequencing kit (U.S. Biochemical Corp., Cleveland, Ohio) and
fluorescein-tagged primer "FAM" (Applied Biosystems, Foster City, Calif.).
The detailed procedure has been published elsewhere.sup.25. Briefly, about
one pmol of the primer and single-stranded M13mp18DNA were heated to
65.degree. C. for three minutes and then allowed to cool (annealing
reaction). Meanwhile, the sequencing extension mixture was added into a
centrifuge tube followed by addition of the dideoxy termination mixture.
When the temperature of the annealing reaction mixture drops below
30.degree. C., a combination of the labeling mixture and diluted enzyme
(Sequenase 2.0.TM.) were added, and the mixture was incubated for five
minutes at room temperature. This mixture was then transferred to the tube
having the termination mixture and allowed to incubate for another five
minutes at 37.degree. C. Instead of adding stop solution, ethanol
precipitation was immediately used to terminate the reaction and recover
the DNA sequencing sample. The high concentration of conductive ions
present in the DNA sequencing sample after the termination step would
reduce the amount of DNA that can be loaded into each capillary by
electrokinetic injection. To counteract this effect, ethanol precipitation
was performed on all DNA samples followed by resuspension in 6 .mu.l of
80% (v/v) formamide to give a concentration about ten-fold higher than
that used in slab gels. The sample was heated at 90.degree. C. for three
minutes to ensure denaturation and then placed on ice until sample
injection.
The flexibility of the capillary columns allows coupling of the individual
capillaries of the array to individual sample wells. In the foregoing
example, since only one DNA sequencing sample was run, the sample was
placed in a single 500 .mu.l centrifuge tube for electrokinetic injection
into the capillaries. The same electric field strength (200 volt/cm) used
during separation was also applied during sample injection. The typical
injection time was ten seconds. After injection, the inlets of the
capillaries were removed from the centrifuge tube and placed into a buffer
reservoir or well 24 filled with fresh running buffer. The 9T gels are
sufficiently stable that four to five sequencing runs could be run on each
capillary.
FIG. 5 presents an image obtained from on-line confocal scanning of a
four-capillary array during electrophoresis of a mixture of DNA sequencing
fragments. The horizontal direction is the physical dimension representing
the geometric arrangement of the array while the vertical direction is
temporal, representing the passage of fluorescent DNA fragments through
the detection window. For lane-to-lane comparison, identical samples of
"G" base DNA fragments were simultaneously, electrokinetically injected
into each capillary. The overall elapsed data acquisition time is
.about.80 minutes after passage of the primer. An expanded region of the
image is included in FIG. 5. The bands in all four lanes are well resolved
and the resolution extends throughout the sequencing run with sufficient
signal-to-noise to detect bands more than 500 bases beyond the primer.
From FIG. 5, one can clearly see that the cylindrical capillaries do not
significantly distort the image.
FIGS. 6(A-D) and 7(A-D) present line plots of the DNA signal integrated
across the width of each capillary. A signal-to-noise ratio of
approximately 20 is observed out to base 385 (.about.65 minutes) and bands
are detected out to base 500 with the present experimental conditions. The
number of theoretical plates is >1.9.times.10.sup.6 (at base 385) over a
24 cm effective column length.
A comparison was made between the signal-to-noise ratio obtained in the
scanning mode and the case where the system is focused in the center of a
single stationary capillary. The latter approach is analogous to
traditional on-column detection from a single capillary. The sensitivity
limits extrapolated for the scanning mode were found to be
.about.2.times.10.sup.-12 M (S/N=3) by flowing 1.times.10.sup.-11 M
fluorescein through an open capillary. The sensitivity limits for the
stationary mode were found to be .about.1.times.10.sup.-12 M. These
detection limits are at least as good as those reported from single
capillaries using the conventional 90.degree. detection geometry.sup.10.
The background from the gel-filled capillaries was .about.2.6 times (n=4)
higher than that from a capillary filled with just TBE buffer. Thus, the
presence of the gel increased the background noise by a factor of
.about.1.6.
This work indicates that the overall throughput performance of CAE can be
very high. In the present study, satisfactory sequencing information is
obtained out to 500 bases for each of four capillaries. The overall
throughput of the system depends upon the total number of capillaries, N,
that can be scanned. The equation, NvT/2D, defines how N depends on the
scan speed (v), the scan repetition period (T), and the capillary outside
diameter (D). For example, one hundred 200 .mu.m wide capillaries can be
easily seen using a scan rate of four cm/sec and a one-sec scan repetition
period. Increasing the array size would require (1) an increase in the
scan speed; (2) the use of smaller O.D. capillaries; and (3) an increase
in the scan repetition period which would reduce the temporal resolution
of the electrophoretic separation. Since reliable systems have velocities
up to ten cm/sec and capillaries with O.D.'s of 150 .mu.m are commercially
available, a limit of approximately 330 capillaries/array can be
projected, assuming a one-second scan repetition period.
To illustrate our ability to extend this system to large numbers of
capillaries we present, in FIG. 8, an array of 24 capillaries that have
been used to separate a different DNA sequencing sample.
Finally, it should be noted that there is a significant difference in the
migration time of a given DNA band from lane-to-lane. This may be caused
by inhomogeneities of the gel matrix or the presence of local non-uniform
variations in the electric field strength. It has previously been
estimated that there is a 5% variation in migration time between identical
samples on different gel columns.sup.4.
The velocity shift of the DNA bands from lane-to-lane may preclude
sequencing DNA with CAE using a single fluorophore and four different
capillaries, one for each base. For DNA sequencing, the present apparatus
must be expanded to a multi-color detection system to sequence all four
bases in a single capillary. Such four-color detection schemes have been
developed for single capillaries.sup.8 and for slab gels.sup.19. The basic
idea is that one is separating four sets of DNA fragments which terminate
with either a G, A, T or C. Each set is labeled with a different
fluorescent tag by any of several procedures and then the fragment sets
are pooled and separated on the same capillary. If the fluorescent tags
emit in a sufficiently distinctive wavelength region, the four sets of
fragments can be uniquely detected by using a four-color detection system.
A schematic diagram of a four-color confocal fluorescence capillary array
scanner is shown in FIG. 9. The scanner includes a laser source such as an
argon laser which projects a beam 30 into the dichroic beamsplitter 31
which directs the beam to the objective 32. The objective collects the
fluorescent energy from the focal volume and directs it through the
beamsplitter. The output of the beam splitter is directed to a first beam
splitter 33 which reflects energy at one wavelength, for example, 540
.mu.m, and passes other, longer wavelengths. The next dichroic
beamsplitter 34, which reflects energy at a second wavelength, for
example, 560 .mu.m, and passes other, longer wavelengths. A third
beamsplitter 36 reflects energy at another wavelength, for example, 580
.mu.m, and passes energy at 610 .mu.m. The energy from each of the
beamsplitters 33, 34, and 36 and the transmitted energy is applied to
confocal, spatial and spec | | |
