Electrophoresis gel migration apparatus

A gel capillary electrophoresis apparatus has gel capillaries (2) filled with gel (2a) that are fixed at both ends thereof on an upper plate 5 and a lower plate (6). The gel capillaries (2) are arranged coarse on the upper plate (5) for sample injection and dense on the lower plate (6) for fluorescence detection. The apparatus is made easy in the sample injection and high in the fluorescence detection efficiency so that throughput of analysis of DNA and the like can be increased, and is available for three-dimensional electrophoresis.

BACKGROUND OF THE INVENTION 
The present invention relates to an electrophoresis gel migration apparatus 
for electrophoresis separation of a DNA or protein. 
Conventionally, base sequence of a DNA has been determined in the way that 
the DNA was labeled by a radio isotope element and subjected to the 
electrophoresis gel separation before the separation pattern was 
transferred onto a film. However, the prior art technique mentioned above 
has the disadvantage that it is not only troublesome to use the 
radioactive label, but also it needs too much labor and time. To overcome 
such problems, a new real-time fluorescent label method has been recently 
used, as disclosed in the Japanese Patent Application Laid-Open 61-62843. 
The fluorescent label method mentioned above uses slab gel, while a further 
new gel capillary electrophoresis method is now attracting attention. The 
gel capillary electrophoresis method provides a high-speed, high-sensitive 
analysis with use of a capillary filled with gel (hereinafter referred to 
as the gel capillary), as disclosed in the Analytical Chemistry, vol. 62, 
pp. 900-903, 1990. 
The gel capillary electrophoresis is ordinarily made in the way that one 
capillary tube and a detection lens are built in a package to integrate. 
The capillary tube can be used repeatedly. However, it is usually 
discarded whenever it is used a few times as the gel is distorted during 
the operation. To make possible analysis of many samples at a time, there 
has been a disclosure that a multiple of gel capillaries are arranged for 
measurement. In the measurement, the multiple of gel capillaries are 
retained by the respective holders as disclosed in the BioTechniques, vol. 
9, p. 74, 1990. 
Any of those measurements having the capillary gel is made in the way that 
light is irradiated around the end of the capillaries to excite the 
fluorescent-labeled DNA passing there to emit fluorescence for detection 
of sample fragments. To make measurement at a high sensitivity and 
accuracy, the capillaries have to be set precisely. 
In order to measure many DNA samples at a time to increase throughput, 
numbers of gel capillaries have to be arranged. The gel capillaries have 
to be replaced after a few times of measurement. This means that it must 
be easy to attach or detach the numbers of gel capillaries and to align 
their positions. The injection of sample into the gel capillaries has been 
made with the use of electric field into the ends of the gel capillaries 
put in sample wells. However, no reports have been made for good 
workmanship of injecting the sample if the numbers of gel capillaries 
should be arranged. This is one of the problems to be solved. 
Distances of the gel capillaries should be longer with respect to easiness 
of the sample injection. But, they should be shorter for efficient 
measurement of the sample fragments. It therefore has been needed to 
develop an apparatus meeting both of these requirements. 
SUMMARY OF THE INVENTION 
In view of the foregoing, it is an object of the present invention to 
provide a gel capillary electrophoresis separation portion integrated in 
such a way that numbers of gel capillaries are coupled at both ends 
thereof with sample well side and a detection end for detection of sample 
fragments, in order to make easy attachment or detachment and alignment of 
the gel capillaries. It is the other object of the present invention to 
provide a highly sensitive gel capillary electrophoresis apparatus 
providing good workmanship of sample injection. 
Briefly, the foregoing objects are accomplished in accordance with aspects 
of the present invention by a gel capillary electrophoresis apparatus. The 
apparatus is of gel capillary cartridge type that, as shown in FIGS. 1a 
and 1b, can separate the gel capillary electrophoresis separation portion 
from a sample injection plate 3 having sample wells 4 to be injected with 
samples and a detector portion 7 for detecting fluorescent light from 
sample fragments. The apparatus includes a gel capillary cartridge 1 
having, in combination, an upper plate 5 of wide area, the gel capillaries 
2, and a lower plate 6 of narrow area. The upper plate 5 couples the gel 
capillaries 2 with the sample injection plate 3 at capillary terminuses 
thereof from which the samples are injected. The lower plate 6 couples the 
gel capillaries 2 with the detector portion 7 at detection ends at which 
the samples migrated are detected. The gel capillary cartridge 1 can be 
replaced with every measurement. This does not only increase throughput, 
but also make the sample injection easy for simple setting of the gel 
capillaries 2. The gel capillaries 2 are made dense on the narrow area of 
the detector portion 7 so that any of the lights emitted from the sample 
fragments can be focused on an high-sensitivity image sensor on the 
detector portion 7 without shrinking any of remaining image. The detector 
portion 7 thus provides a highly efficient photodetection. 
The gel capillary cartridge 1 can practically hold 20 to 500 gel 
capillaries 2 so that the throughput of the sample measurement can be 
increased. The sample injection can be made easy in the way that the gel 
capillaries 2 are made coarse at the ends arranged and fixed on the upper 
plate 5 of the gel capillary cartridge 1 and the ends are aligned with a 
sample holder or injection jig. The gel capillary electrophoresis can be 
made high in sensitivity as the detector portion 7 can be increased in the 
detection efficiency in the way that the gel capillaries 2 are made dense 
at the other ends fixed on the lower plate 6. 
The capillaries available for the gel capillaries 2 are not limited in 
their inside diameter, wall thickness, and length. The inside diameter 
should be smaller than 0.3 mm, ordinarily 0.1 to 0.2 mm, for convenience 
of bending. The wall thickness should be usually made 0.1 to 0.2 mm. The 
length should be practically 10 to 100 cm, ordinarily 30 to 50 cm. Outside 
diameter of the capillaries should be ordinarily made 0.3 to 0.4 mm. 
The detector portion 7 has a gap between bulkheads 7a and 7b provided in 
parallel, the gap width being virtually equal to the inside diameter of 
the capillaries. The gap is filled with buffer solution or gel. The buffer 
solution or gel is irradiated by an excitation light. The excitation light 
has to be made to irradiate all the migration paths at the same time. If 
the excitation light is not scanned, it cannot directly irradiate all the 
capillary tubes at a time. For the reason, the excitation light should 
irradiate positions at which the samples elute from the capillaries. If 
the irradiation positions are too close to the lower ends of the 
capillaries, scattering at the ends of the capillaries affect the 
measurement. If they are too far from the lower ends, on the other hand, 
the samples eluted may mix with one another. Both cases are undesirable. 
In the present invention, it is effective that the excitation light should 
irradiate positions 0.5 to 1 mm away from the lower ends of the 
capillaries. If the excitation light, such as laser beam, is scanned for 
measurement, it may irradiate the capillary tubes themselves because 
diverged light beams having passed through the capillary tubes are not 
re-used. It is good that distance between the bulkheads should be 
virtually equal to the inside diameter of the capillaries of 0.1 to 0.2 
mm. 
The upper ends of the gel capillaries 2 can be coarse that is spaced not to 
disturb injection of the samples, say, intervals of 2 to 10 mm. The lower 
ends should be as dense as possible to increase the fluorescence detection 
efficiency, say 0.3 to 1 mm. The densities however are limited to those.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Embodiment 1 
The following describes an embodiment 1 of the electrophoresis gel 
migration apparatus according to the present invention by referring to 
FIGS. 1a, 1b, 2a, 2b, 2c, 3a, and 3b. FIG. 1a is a perspective view 
illustrating major parts of a gel capillary electrophoresis migration 
apparatus in the embodiment 1. 
There is provided a gel capillary cartridge 1 in the apparatus. An upper 
plate 5 of the gel capillary cartridge 1 is coupled with a sample 
injection plate 3. Sample wells 4 on the sample injection plate 3 are 
immersed in an upper buffer solution 18 in an upper buffer vessel 17 shown 
in FIG. 3a. A lower plate 6 of the gel capillary cartridge 1 is coupled 
with a detector portion 7 that can detect light emitted by sample 
fragments being eluted from a gel 2a in the gel capillaries 2 of the gel 
capillary cartridge 1. Distance between the upper plate 5 and the lower 
plate 6 can be changed. In order to protect the gel capillaries 2 from 
crash and to mechanically reinforce the gel capillary cartridge 1, 
however, the upper plate 5 and lower plate 6 are tied together at their 
sides with a plastic ribbon (not shown) of a predetermined length. The 
plastic ribbon is a sheet-like ribbon of around 0.5 mm thick, around 2 cm 
wide, and 20 to 30 cm length. The gel capillaries 2 are made of silica 
covered with polyimid resin on its surface. The gel capillaries 2 can be 
bent as they are thin in the diameter and has the polyimid covered on the 
surface. The gel capillaries 2 are bonded to the upper plate 5 and lower 
plate 6 as shown in FIG. 3a or are fixed with rubber rings 14 for holding 
capillary tubes as shown in FIG. 2a, with the upper plate 5 and sample 
injection plate 3 mechanically aligned with faucet joint and screwed 
together. Similarly, the lower plate 6 and a detector portion 7 are 
connected together. 
The electrophoresis gel migration apparatus can be made small as numbers of 
the gel capillaries 2 can be bundled together and bent so that they can be 
contained in a narrow space for their long gel capillary migration paths. 
For the accommodation, it is effective that the numbers of the gel 
capillaries 2 should be gathered in a way that the gel capillaries 2 are 
sandwiched between two sheets of polymer film. In the way, if the gel 
capillary migration path used is 50 cm long, for example, an 
electrophoresis plate needed is around 60 cm long for slab gel. The gel 
capillaries 2 then can be bent to a length shorter than 20 cm for 
accommodation. 
The upper plate 5, as described above, is tightly coupled with the sample 
injection plate 3 having sample wells 4 of 0.3 mm diameter, four in a 
column thereof and 25 in a row thereof at a pitch of 5 mm. It is designed 
that each of the gel capillaries 2 and their respective sample wells 4 
should be aligned. The upper plate 5, as described above, has upper ends 
of the gel capillaries 2 arranged roughly at thereon in the two dimensions 
of columns and rows at the 5 mm pitch, while the lower plate 6 has the 
lower ends aligned closer than the above on a straight line at a pitch of 
1 mm. The lower plate 6 is attached to and coupled with detector portion 
7. The gel capillary cartridge 1 can be attached with or detached from the 
sample injection plate 3 and detector portion 7. Couplings of the gel 
capillaries 2 with the lower plate 6 are protected by rubber rings 14 for 
holding capillary tubes as shown in FIG. 2a. Similarly, couplings of the 
gel capillaries 2 with the sample injection plate 3 are protected by the 
rubber rings 14 for holding capillary tubes. The rubber rings 14 for 
holding capillary tubes are ignored and not shown in FIGS. 1b, 2b, 2c, 3a, 
3b, and 5. The rubber rings 14 for holding capillary tubes can be made of 
TEFLON or rubber as well. A DNA sample that has been separated by the gel 
capillaries 2 that is an electrophoresis separator of the sample, is 
eluted from gel 2a in the gel capillaries 2 and enters the detector 
portion 7. FIG. 1b is a cross section A of FIG. 1a in the vicinity of the 
sample injection plate 3. As any of the gel capillaries 2 is coupled with 
the upper plate 5, the sample injected into the corresponding one of the 
sample wells 4 contacts the gel 2a. The gel capillaries 2 thus can be 
immersed in the upper buffer solution 18 as shown in FIG. 3a. 
The embodiment 1 uses a sample adjusting titer plate having holes of 3 mm 
diameter aligned at the same intervals of as the sample injection plate 3 
in addition to the sample injection plate 3 to inject the gel 2a into the 
sample. The titer plate has thin silicon rubber film lined on a bottom 
thereof. The titer plate is laid on the sample injection plate 3. The 
silicon rubber film can be broken with a needle or the like to make holes 
of around 0.5 mm diameter. In this way, the sample in the holes of the 
titer plate can be easily injected into the gel 2a. 
The upper plate 5 of the gel capillary cartridge 1 in the embodiment 
described so far has the upper ends of the gel capillaries 2 arranged in 
the two dimensions, but may have them in on dimensions, or in a straight 
line. 
The detector portion 7 is coupled with the lower plate 6 of the gel 
capillary cartridge 1 as shown in FIGS. 2a, 2b, or 2c which is a cross 
section B of FIG. 1a. The migrated DNA sample elutes from the gel 2a 
before migrating in a lower buffer solution 7d or a hollow portion filled 
with the gel. An excitation light 8a is irradiated to excite a 
fluorescence label of the DNA sample in a direction parallel with the line 
of the lower ends of the gel capillaries 2. The hollow portion that is an 
irradiation light path for the excitation light 8a is formed of bulkheads 
7a and 7b of two silica plates. In the detector portion 7, as shown in 
FIGS. 2a and 2b, the excitation light source 8 for exciting the 
fluorescence label irradiates at a position around 0.5 mm in front of the 
lower ends of the gel capillaries 2 in a gap of 0.1 mm formed by the 
bulkheads 7a and 7b of the two silica plates placed in parallel. FIG. 2c 
is a variation of the example in FIG. 2b that the lower buffer solution 7d 
or the gel can be easily immersed and contact the lower ends of the gel 
capillaries 2 from which the migrated DNA sample. The gap mentioned above 
is filled with the lower buffer solution 7d or the gel and serves as the 
path for the excitation light 8a. The excitation light 8a of the 
excitation light source 8, for example, a laser beam, reflected by a 
reflection mirror 9 can irradiate at the position around 0.5 mm above the 
lower ends of the gel capillaries 2 so that it can irradiate the DNA 
sample eluting from all the gel capillaries 2 at substantially the same 
time. So that in FIGS. 2b and 2c, the excitation light 8a is irradiated in 
a direction perpendicular to the drawing. 
A number of the gel capillaries 2 used in the embodiment 1 is 100. 
Fluorescent signals can be obtained from an range of around 10 cm on the 
basis of the elution of the DNA sample as the excitation light 8a is 
irradiated. The fluorescent signals are detected by a photodetector 11, 
such as a line sensor, through a lens system 10 and a filter (not shown) 
at substantially the same time. The detected fluorescent signals are 
processed by a data processor 12 before fed out to an output device 13, 
such as a display. 
If directly irradiated to the gel capillaries 2, the excitation light 8a 
(the laser beam here) is diverged, so that it cannot irradiate the number 
of the gel capillaries 2 at the same time. To solve such a difficulty, 
there can be a method that portions of the gel capillaries 2 to be 
irradiated are immersed in the lower buffer solution 7d to make 
diffraction differences little so that scatter of the light at the tube 
interface of the gel capillaries 2 as the excitation light 8a is 
irradiated for the detection of the fluorescent signals. It, however, is 
not always sufficient. There could be a better method that the detector 
portion 7 has no capillary tubes provided therefor. In the embodiment 1, 
the DNA sample is eluted from the gel capillaries 2, and the excitation 
light 8a is irradiated in a state of no capillary tubes or a state similar 
to it before the fluorescent signals are detected. 
As shown in FIG. 3a, a detector is kept in a lower buffer vessel 7c, and a 
voltage is applied between an upper electrode 19 and a lower electrode 20 
in the upper buffer vessel 17 filled with the upper buffer solution 18 
before migration starts. The lower part of the electrophoresis gel 
migration apparatus shown in FIG. 3a can be modified as shown in FIG. 3b. 
In FIGS. 3a and 3b, the excitation light 8a for exciting the fluorescence 
label is irradiated in a direction perpendicular to the drawing so that 
the DNA sample eluting in the path for the excitation light 8a can 
generate fluorescent light. The fluorescent light is detected in a 
direction C or C' of a plane formed of transparent silica or a direction 
D. If the fluorescent light is detected in the direction C or C', for 
example, a fluorescent image of around 10 cm long is made small to one by 
four before detected by an image line sensor, for example, of 25 mm long 
of the S3902, the Hamamatsu Photonix Inc., a diode array equipped with an 
image amplifier, or a CCD detector. If the fluorescent image is detected 
in the direction C shown in FIG. 3b, it has the advantage that it has less 
effect due to reflection of the light by the surface than the one C in 
FIG. 3a. 
Embodiment 2 
In turn, the following describes an embodiment 2 of the present invention 
by referring to FIG. 4. In the embodiment 2, lower ends of a gel 
capillaries 2 are arranged and fixed in two dimensions of columns and rows 
on a lower plate 6 of a gel capillary cartridge 1 so that numbers of the 
lower ends of the gel capillaries 2 can be collected in a narrow area. DNA 
sample eluting from the lower ends of the gel capillaries 2 are detected 
by a two-dimensional detector 7. In the figure, the lower ends of the gel 
capillaries 2 are arranged and fixed at a pitch of 1 mm in the columns and 
rows on the lower plate 6. The detector portion 7 has a group of detectors 
any of which is constructed as shown in FIG. 2a or 2b. That is, as shown 
in FIG. 5, an excitation light 8a irradiates at a position around 0.5 mm 
below the ends of the gel capillaries 2 in a space filled with a lower 
buffer solution 7d or gel in a direction perpendicular to the drawing. In 
the embodiment 2, bulkheads 30a through 30z for forming the space of 
around 0.1 mm gap as paths for the excitation light 8a may be 
non-transparent as lights generated from the DNA sample can be detected 
below the detector portion 7. The detector portion 7 is provided in the 
lower buffer solution 7d or the gel in a lower buffer solution 7d. The 
excitation light 8a is reflected by a reflection mirror 9 to irradiate 
below all the lower ends of the gel capillaries 2 in the lower buffer 
vessel 7c (not shown in FIG. 4) at substantially the same time by means 
of prisms 16a and 16b provided one on each sides of the detector portion 
7. Fluorescent signals from DNA samples eluted from the gel capillaries 2 
are all detected at substantially the same time by the two-dimensional 
detector portion 7 through an image reflection mirror 15, a lens system 
10, and a filter (not shown). A bottom plate of the lower buffer vessel 7c 
(not shown in FIG. 4) is made of silica plate. Of course, for example, the 
reflection mirror 9 can be moved to sequentially scan the line excitation 
light 8a over the space around the area from one side of the detector 
portion 7 to a point at which the lower buffer solution 7d or the gel is 
filled with and the DNA sample elutes. Alternatively, the reflection 
mirror 9 can be made to irradiate at the same time the whole line of the 
space around the area from the one side of the detector portion 7 to the 
point at which the lower buffer solution 7d or the gel is filled with and 
the DNA sample elutes. The prisms 16a and 16b can be provided either on an 
inside or the outside of the lower buffer vessel 7c (not shown in FIGS. 4 
and 5). 
Note that parts of the electrophoresis gel migration apparatus above the 
gel capillaries 2 are not shown in FIG. 4. 
In the embodiments described so far, the upper plate 5 of the gel capillary 
cartridge 1 and the sample injection plate 3 can be attached or detached 
together. The lower plate 6 of the gel capillary cartridge 1 and the 
detector portion 7 also can be attached or detached together. 
Alternatively, the upper plate 5 and the sample injection plate 3 can be 
integrated and the lower plate 6 and the detector portion 7 can be 
integrated, and the two integrated couples can be assembled together to 
make another form of gel capillary cartridge. More alternatively, only 
either of the two couples mentioned above can be integrated to make still 
another form of gel capillary cartridge. If the sample injection plate 3 
and the detector portion 7 are integrated with the gel capillary cartridge 
1, the gel capillary cartridge 1 can be attached with or detached from the 
upper buffer vessel 17 and the lower buffer vessel 7c at ends of the 
sample injection plate 3 and the detector portion 7, respectively. 
In usual electrophoresis (one-dimensional electrophoresis), the sample is 
separated in one-dimensional way in a direction x. In the so-called 
two-dimensional electrophoresis, on the other hand, the separated sample 
has an enzyme poured thereon to make some action, is separated again in a 
direction (direction y) perpendicular to the direction x to develop in the 
two dimensions. The two-dimensional electrophoresis provides more detailed 
separation of the sample than the one-dimensional one. It may occur that 
even the two-dimensionally separated pattern on the slab gel is lack of 
amount of information. In this case, it is effective that the slab gel 
having the sample separated thereon should be divided into numbers of 
sections, the sample contained in each of the sections should be made to 
act with enzyme or a DNA probe or the like, and a product made through the 
action should be gel-separated again to obtain more information. For the 
third electrophoresis separation, the present invention can use a 
capillary array distributed in two dimensions. It can be regarded as a 
separation in a direction z in relation to the ones in the directions x 
and y. The final information, or the third dimension information, can be 
obtained in terms of a time-varying signal from measuring point 
distributed in the two dimensions. Alternatively, it can be obtained in a 
way that the capillaries should be rearranged in a line, the signals 
should be obtained, and then data should be stored as if they were 
obtained in the two-dimensional arrangement. 
It is not practical to employ a usual three-dimensional electrophoresis 
having flat or block gel used therein, as the gel cross sections are too 
wide, allowing overcurrent to flow. In the gel capillary electrophoresis, 
current flowing through each of the gel capillaries is so small that no 
problems can be due to heat generation. 
For the purpose of illustration only, the parts of the apparatus shown in 
FIGS. 1 to 5 are drawn different from those of the actual apparatus in 
proportions of shapes. The features of the present invention are as 
follows. The electrophoresis gel migration apparatus according to the 
present invention can increase throughput to a great extent as it can have 
numbers of gel capillary migration paths incorporated in the narrow 
detection end area without affecting sample injection. Also, it can reduce 
sample injection work to a great extent as the arrangement of the sample 
wells on the sample injection plate are matched with that of the sample 
holes on the titer plate. Further, the apparatus can be made small as the 
gel capillaries can be bent so that the long gel capillary migration paths 
can be incorporated in the narrow space. If the gel capillary migration 
paths used are 50 cm long, for example, the slab gel requires a migration 
plate of around 60 cm long, resulting in a large scale apparatus, while 
the gel capillaries can be bent to within 20 cm to contain. In order to 
have the migration paths as much as 100, the slab gel requires the 
migration plate of 40 cm wide or more, while the two-dimensional gel 
capillary arrangement allows the detector area to be made as narrow as 
1.times.1 cm or less.