« PreviousContinue »
BACKGROUND OF THE INVENTION
The present invention relates to a capillary electrophoresis 5 apparatus for separating and analyzing DNA, protein, and the like.
Techniques of analyzing DNA, protein, and the like are important in the medical and biological fields including gene 1Q analysis and gene diagnosis. A DNA analyzing apparatus having high throughput is being developed recently. Those analyses are mainly performed by a gel electrophoresis. In the gel electrophoresis, a substance in a gel state is used as a separating medium of the electrophoresis, DNA labelled 15 with fluorophore is usually prepared and the fluorescence labelled DNA sample is injected into the upper end of polyacrylamide gel formed between two glass plates (slab gel). After that, an electrical field is applied to both ends of the gel, components (fragments) of the sample DNA are 2Q allowed to migrate to the lower end, a predetermined position from the upper end is irradiated with laser light, and fluorophore is detected. The result is analyzed, and DNA base sequence is determined or polymorphism of a restriction fragment is identified. Recently, in place of the slab gel, 2J a capillary gel obtained by polymerizing gel in a capillary is often used. Attention is paid to a capillary electrophoresis as a method which enables high speed and high separation since an electric field larger than that used in the slab gel electrophoresis can be applied. In a capillary electrophoresis 3Q apparatus, on-column measurement in which one capillary column is used, capillaries near the lower end are irradiated with laser light, and the fluorophore is detected, is usually used ("Bunseki", 1995, No. 5, pp. 342-349).
A capillary array gel electrophoresis apparatus for simul- 35 taneously analyzing a number of samples by using a plurality of capillaries in order to improve the throughput has been reported. According to a first example, on-column fluorescent measurement is performed in such a manner that a plurality of capillaries are arranged on a moving stage and 40 the moving stage is moved, thereby sequentially irradiating the capillaries one by one with laser light ("Analytical Biochemistry", 215, pp. 163 to 170, 1993). A second example relates to a method of using a sheath flow (Japanese Patent Application Laid-Open No. 06-138037 and U.S. Pat. 45 No. 5,529,679). In an apparatus according to the second example, the lower end of a capillary array is soaked in a buffer solution, components of the sample separated by the gel electrophoresis are eluted in the buffer solution, and fluorescent light from the sample component is detected in 50 a part where no capillary exists.
SUMMARY OF THE INVENTION
In a capillary gel electrophoresis, both ends of a capillary are directly soaked into an electrolyte solution in an elec- 55 trode vessel (positive electrode and negative electrode) and a voltage is applied. Similarly, when a sample to be measured is injected into a capillary gel, a vessel such as a sample tube is filled with a measurement sample solution and one end of the capillary is directly soaked into the 60 measurement sample solution, and a voltage is applied across the one end and the other end. That is, the sample injecting end of the capillary is arranged almost vertically above the electrolyte solution in the electrode vessel or the measurement sample solution in the sample vessel and is 65 almost vertically inserted. Similarly, the capillary is bent in the middle, and the other end of the capillary is arranged so
as to be almost vertically inserted into the electrolyte solution in the electrode vessel. With respect to the moving direction of the sample to be measured in the capillary, it is moved vertically upward at the injecting end of the capillary and it is contrarily moved vertically downward at the other end of the capillary, so that the moving directions are opposite. With such arrangement, the capillary itself serves as a pipet, so that the sample solution can be easily injected into the capillary. Since the capillary is bent, however, a certain length of the capillary which can be used is necessary. For example, it is difficult to make the total length of the capillary equal to or shorter than 20 cm, so that there is a problem that it is not suitable for high-speed electrophoresis.
When a plurality of capillaries are arranged and used, the group of capillaries cannot help but be arranged in a threedimensional space and air has to be circulated in order to adjust the temperature of the group of capillaries, so that there is a problem that the structure of the apparatus is enlarged. Further, by bending the capillary, there is the possibility that the separating performance deteriorates. The capillary is generally covered by brown-colored polyimide which emits fluorescent light at the time of fluorescent detection or the like in which Argon laser light or the like is used as excitation light. The emitted fluorescent light becomes background light, so that there is a problem that measurement sensitivity tends to deteriorate. Further, in a capillary array apparatus using sheath flow, there is the possibility that dusts or bubbles in the buffer solution become noises, so that it is necessary to pay attention at the time of installation and measurement.
It is an object of the invention to provide a high-speed and high-precision capillary electrophoresis apparatus which solves the above problems and in which operations of the temperature adjustment and sample injection are easy.
The object can be achieved by keeping the direction of the sample injecting end of each capillary almost vertically and arranging a plurality of capillaries without bending or bending back. That is, it is arranged in such a manner that the position of a sample detection point is set higher than a sample injecting end, the sample injecting end is directed vertically downward, and the sample detection point is directed vertically upward or almost horizontally. With such a construction, a single capillary or a plurality of capillaries can be arranged without being bent largely in the analysis region.
In an electrophoresis apparatus in which sample elution ends of a plurality of capillaries are arranged in a straight line in a sheath flow cell and are terminated and components eluted from the capillaries in the sheath flow cell are detected, the capillaries are arranged in a manner similar to the above. The termination faces of the sample elution ends of the capillaries are arranged near the lower part of the sheath flow cell so as to face the upper part of the cell, and a buffer solution flows from the lower part of the cell toward the upper part of the cell.
In a case of on-column detection, the detecting portion is placed upward of the sample injecting end and the capillaries from the injecting ends to the detecting points are arranged in a straight line.
Most parts in the longitudinal direction of the plurality of capillaries at least in an analyzing region are arranged almost in a vertical plane, the sample injection end of each capillary is arranged in the lower part and the sample is allowed to migrate upward to the detection portion, so that the migration direction of the sample is not reversed.
Further, by using black for the color of the coat at least near one end of each capillary, the background light is prevented from being generated from the capillary, so that high sensitive detection can be performed.
The features of the present invention will be described in further detail. According to the invention, a capillary electrophoresis apparatus comprises: a plurality of capillaries filled with migration media, having first ends into which samples are injected and second ends in which components included in the samples are eluted; a sheath flow cell in which the second ends are arranged in a straight line at first predetermined intervals and terminated; means for flowing a buffer solution from the lower part to the upper part of the sheath flow cell; and means for detecting components separated from the capillaries by electrophoresis near the second ends in the sheath flow cell, wherein the buffer solution flows from the lower part to the upper part in the sheath flow cell.
According to further characteristics of the invention, in the capillary electrophoresis apparatus, most parts of the capillaries are arranged almost in the same plane in the analysis region, a plurality of capillaries or rod members each having an outer shape of almost the same size as that of the capillary are arranged at the first predetermined intervals on both sides of an array of the second ends of the plurality of capillaries, the migration medium includes either gel or polymer, the first ends are arranged lower than the detecting positions or the second ends, a sample vessel having a plurality of wells which are arranged in a straight line at second predetermined intervals, at which the first ends are arranged, in parallel to the array of the first ends and house the plurality of samples, respectively, and means for moving the sample vessel.
Further, in a capillary electrophoresis apparatus using a plurality of capillaries each having a first end into which a sample is injected, a second end in which a component included in the sample is eluted, and a light irradiation part which is located between the first end and the second end and is irradiated with laser light, parts from the first ends to the light irradiation parts of the plurality of capillaries are arranged in the same plane, the first ends are arranged lower than the light irradiation parts, and the light irradiation part is irradiated with laser light in a direction parallel to the plane. According to the characteristics, since the plurality of capillaries are housed in a small space, the temperature can be controlled with a simple method and the plurality of capillaries can be held at almost the same temperature.
The present invention will be summarized as follows with reference to FIG. 1.
Both ends of each of a plurality of capillaries 90 are fixed to capillary holders 91 and 92, both of sample injecting ends 90a and sample elution ends 90fc are cut so as to have the same length, and the sample elution ends 90fc are attached together with the capillary holder 92 to the lower part of a sheath flow cell 83. In the capillary holder 92, dummy glass rods 93 are attached on both sides of each capillary 90 at the same intervals as those of the capillaries 90. A buffer solution flows in a path from a buffer solution vessel 80 via a tube 81, a holder 82 for holding the sheath flow cell, the sheath flow cell 83, a holder 84 for holding the sheath flow cell, and a tube 85 into a drain vessel 86. Sample components eluted from the capillary 90 are excited by a laser beam emitted to a position around the sample elution ends 90fc and are detected by fluorescent substances. With such a structure, it is almost unnecessary to bend the capillaries and the most part of the capillary is arranged in the same plane.
In a case of on-column detection, the sample injecting points are placed below the detecting points and the capillaries in the analyzing region are arranged in a straight and in the same plane. According to the invention, a capillary electro5 phoresis in which operations such as temperature adjustment, sample injection, and the like are easy and high sensitive and prompt measurement can be performed can be realized.
10 BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram showing the construction and arrangement of a capillary array and a sheath flow cell serving as a fluorescent measurement unit according to a first embodiment of the invention; 15 FIG. 2 is a diagram showing the construction of an electrophoresis apparatus using the capillary array shown in FIG. 1;
FIG. 3 is a diagram illustrating the construction of an 20 image splitting fluorescent detecting unit in the first embodiment of the invention;
FIG. 4 is cross sections from two directions which perpendicularly cross with each other, showing the schematic construction of a capillary array and an electrophoresis unit 25 according to a second embodiment of the invention; and
FIG. 5 is front and side views including a partial section showing schematic constructions of a capillary array and an electrophoresis unit according to a third embodiment of the invention.
DESCRIPTION OF THE PREFERRED
35 An apparatus and method for using a plurality of capillaries (that is, a capillary array) filled with gel and the determining DNA base sequence will be described hereinbelow. DNA sequencing samples (DNA fragments labelled with fluorophore (fluorophore labelled DNA fragment)) are
40 prepared. Primers labelled with fluorophores in which the maximum fluorescent wavelengths are about 550 nm, about 520 nm, 575 nm, and 605 nm (called FA, FC, FG, and FT, respectively) are used for fragment groups of A, C, G, and T, respectively. The fluorophore labelled DNA fragments are
45 prepared by using these labelled primers according to a known dideoxy chain termination method by Sanger et al. These four fragment groups are finally mixed in a single sample tube, and after ethanol precipation, eluted in de-ionized formamide (including tris buffer solution of Vio
50 quantity), thereby obtaining a sample solution. The sample solution is heated at 90° C. for two minutes to cause heat denaturation before injection into a gel capillary, and after that, immediately cooled with ice, and injected into the sample injection end of the capillary. The sample can be also
55 prepared by labelling a terminator, not the primer, with the fluorophore.
The apparatus will be described hereinbelow. FIG. 1 is a diagram showing the construction and arrangement of a capillary array and a sheath flow cell serving as a fluores
60 cence measuring part. A capillary array in which 48 capillaries filled with a gel are used (for simplicity, only a part of the capillaries is shown in the diagram and parts such as an O-ring for sealing the liquid and the like are omitted) and one ends of the capillaries are arranged in a straight line is
65 produced. Although various inner and outer diameters can be used for the capillary, for example, capillaries each having the inner diameter of 0.075 mm, the outer diameter
of 0.2 mm, and the length of about 20 cm are used. The capillaries are filled with polyacrylamide gel (gel concentration is 4% T (ratio of (acrylamide+bis-acrylamide) to the whole solution quantity and 5% C (ratio of the bisacrylamide to (acrylamide+bis-acrylamide)) according to a 5 known method and are used. Polymer gels such as hydroxyalkyl-cellulose, poly (vinyl alcohol) et al can be used as well.
Although polyimide is usually coated on the surface of the capillary in order to prevent damage when the capillary is 1° bent or the like, the capillary which is coated in black by mixing carbon in polyimide or the like is used here. The black coating is performed at least near one (90fc) of the ends of the capillary.
On the capillary holders 91 and 92, attaching grooves at 15 intervals of 2.25 mm and at intervals of 0.4 mm are formed, respectively. Forty eight capillaries 90 are arranged on the same plane, both ends are fixed to the capillary holders 91 and 92, one ends of the capillaries are held at the intervals of 2.25 mm and the others are held at the intervals of 0.4 20 mm, and both ends of the capillaries are cut so as to be aligned, respectively. The capillary ends 90a fixed at the 2.25 mm intervals are ends on the sample injecting side and the capillary ends 90b on the other side are ends on the sample eluting side where separated sample fragments are 25 eluted.
The capillary ends 90b are attached together with the capillary holder 92 to the lower part of the sheath flow cell 83. In the capillary holder 92, six pieces each of the dummy 3Q glass rods 93 are attached on both sides of the 48 capillaries 90 at the same intervals as those of the capillary ends 90fc. The dummy glass rods 93 are provided so that environments of the capillary ends 90fc of the capillaries 90 are uniformed. It is sufficient that the diameter of the dummy glass rod 93 3J is almost equal to that of the capillary 90. The capillary having the same diameter as that of the capillary 90 is used here and is filled with a solid matter. The number of dummy glass rods 93 depends on the width of the cross section of the passage of the sheath flow cell and is adjusted so that the 4Q capillaries are uniformly positioned in the whole width. By using the dummy glass rods 93, the environments of the sheath flow for the capillary ends 90fc are uniformed and a uniform flow of the stable buffer solution can be formed near the capillary ends 90fc of the first to the 48th capillaries 90. 4J
The section of the passage of the sheath flow cell part is 0.22 mmx24.2 mm. The sheath flow cell part is constructed by the sheath flow cell 83 made of quartz glass and the holders 82 and 84 for holding the upper and lower parts of the sheath flow cell 83. The holders 82 and 84 have a space 50 through which the buffer solution for sheath flows. A buffer solution equivalent to that obtained by preparing polyacrylamide gel is used as this buffer solution. The buffer solution flows from the buffer solution vessel 80, via the tube 81, the holder 82, the flow cell 83, the holder 84, and the tube 85 to 55 the drain vessel 86. By arranging the buffer solution vessel 80 so as to be higher than the drain vessel 86, the buffer solution flows slowly and stably by the gravity, a laminate flow is formed, and a sheath flow is formed near the capillary ends 90fc. That is, the buffer solution vessel 80 and the drain 60 vessel 86 are arranged so that the liquid level of the buffer solution vessel 80 is higher than that of the drain vessel 86 and the stable flow of the buffer solution by gravity is formed.
FIG. 2 is a diagram illustrating the construction of an 65 electrophoresis apparatus using the capillary array shown in FIG. 1. The capillary holder 92, the capillaries 90, the
capillary holder 91, the sheath flow cell 83, and the like are arranged in the plane, arranged vertically and fixed so that the sheath flow cell 83 is positioned in the upper part and the buffer solution flows from the lower part to the upper part in the sheath flow cell 83. Below the capillaries 90, a sample vessel 100 having 48 wells formed at the same intervals as those of the capillary ends 90a arranged in a line (each well is filled with the sample solution to be measured), an electrode vessel 101 filled with the buffer solution, and a washing vessel 102 filled with washing solution are arranged, which are moved in the vertical direction 200 and the lateral direction 202 (the direction in which the sample vessel 100, the electrode vessel 101, and the washing vessel 102 are arranged, that is, the direction which perpendicularly crosses a plane formed by the plurality of capillaries 90) by a moving stage 103. Consequently, the capillary ends 90a can be inserted into the sample solution, the buffer solution, and the washing solution in accordance with an object (a movement driving system for the moving stage 103 is omitted in FIG. 2).
The sample solutions for measurement are injected into the 48 wells of the sample vessel 100 by using a pipet. The buffer solution is injected into the electrode vessel 101 and distilled water is injected as the washing solution into the washing vessel 102. First, the moving stage 103 is moved by the movement driving system (the system is omitted in FIG. 2) so that the capillary ends 90a are positioned above the washing vessel 102 and are soaked in the washing solution in the washing vessel 102 by raising the moving stage 103 to wash out dusts, buffer solution, and the like which may be adhered to the capillary ends 90a. Subsequently, the moving stage 103 is lowered and moved by the movement driving system so that the capillary ends 90a are positioned above the sample vessel 100. The moving stage 103 is raised so that the sample solution in the sample vessel 100 comes into contact with the capillary ends 90a, a voltage of -500V is applied to the sample solution side so that an electric field strength is 25V/cm and a voltage of 0V is applied to the buffer solution side in the sheath flow cell 83 and the sample is injected into the capillary ends 90a.
The positive and negative electrodes and a high voltage power source are omitted in FIG. 2. Platinum electrodes arranged for the respective 48 wells of the sample vessel 100 serve as the negative electrodes. The holder 84 is made of stainless steel. Either the holder 84 itself, a platinum electrode arranged in the upper part of the sheath flow cell 83, or a platinum electrode arranged in the drain vessel 86 is used as the positive electrode.
After injecting the samples into the capillary ends 90a, the moving stage 103 is moved by the movement driving system, and the capillary ends 90a are moved into the electrode vessel 101 and are soaked into the buffer solution. A voltage is applied to the platinum electrodes held in the buffer solution in the electrode vessel 101. Various values can be used for the voltage to apply. In this case, a voltage of -500V such that the electric field strength is 25V/cm is applied for first five minutes. Subsequently, a voltage of -2000V such that the electric field strength is lOOV/cm is applied. As a result of the electrophoresis, the DNA fragment is separated every molecular weight and eluted from the capillary ends 90fc into the sheath flow cell. The eluted DNA fragments are fluorescence measured.
Since the sheath flow is formed near the capillary ends 90b, the DNA fragments eluted from the capillary ends 90fc flow without being mixed. Consequently, the fluorescence measurement can be executed by simultaneously injecting 48 samples into the capillaries, respectively, and simulta