JPH07209251A - Electrophoretic device - Google Patents

Abstract

PURPOSE:To provide a DNA sequencer having an ultrahigh throughput. CONSTITUTION:Gel electrophoretic paths are provided separately and the DNA pieces slipping out of the gel are irradiated collectively with laser light 4. Fluorescent light from the DNA piece in each electrophoretic path is coupled with a line sensor or an area sensor 13 through an optical fiber 11. This constitution realizes a system wherein the number of electrophoretic paths is not limited.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for separating and analyzing biological materials such as DNA, particularly a capillary electrophoresis apparatus.

[0002]

2. Description of the Related Art Conventionally, DN for determining DNA base sequences has been used.
Gel electrophoresis was used for the analysis of A. Recently, with the increasing need for DNA sequencing such as genomic analysis, a DNA sequencer that labels a DNA fragment with a fluorescent substance and measures the DNA fragment in real time while performing gel electrophoresis separation is commercially available and put into practical use. This DNA sequencer uses a slab gel (polyacrylamide gel), secures a migration path of 30 to 40 on the gel plate, irradiates the lower part of the gel plate with laser light, and the fluorescence emitted by the fluorescently labeled DNA fragment passing therethrough. Is to detect.

However, the throughput of a commercially available DNA sequencer is as small as 10 K bases to 20 K bases / day, which is one of the difficulties in genome analysis. For high throughput, it is effective to increase the migration speed and to increase the number of samples that can be analyzed at a time by providing a large number of migration paths.
Recently, a capillary gel electrophoresis apparatus capable of performing electrophoretic separation in a short time has been developed. Furthermore, a capillary array electrophoresis apparatus has been developed which arranges a large number of capillaries and measures them at the same time, and high throughput has become possible.

Two types of capillary array electrophoresis devices have been reported. The first type of apparatus is one in which a flat plate array in which capillaries are bundled is fixed on an XY table, laser light is irradiated from above, and fluorescence is received in the laser light irradiation direction. Laser light can irradiate only one capillary gel at a time, but signals from all capillaries are measured by repeatedly moving the capillary gel at a constant speed (Nature 359, 167, (1992)).

In the second type of apparatus, gel capillaries are bundled in a sheet form, the ends of which are placed in a sheath flow formed in a spectroscopic cell, and laser light is incident from the side to simultaneously irradiate all migration paths. Then, the obtained fluorescence image is CCD
It is measured with a camera, etc. (Nature 361,565, (199
3)).

[0006]

The above-mentioned capillary array electrophoresis device greatly improves the throughput, but the number of migration paths that can be secured is about 100. This is because, in the case of the first type device, if the number of capillaries is increased, the measurement time per capillary becomes shorter and the sensitivity becomes insufficient, and it is necessary to lower the migration speed because fast scanning cannot be performed. Because it will be. Further, the second type of device is for reducing and forming the fluorescence line image of the capillary array by the lens, and therefore, there is a limitation in the field of view of the image forming optical system, so that the number of capillaries is increased. This is also because if the reduction ratio is increased, the amount of received light decreases, the sensitivity becomes insufficient, and signal separation from each migration path becomes difficult.

An object of the present invention is to provide an electrophoretic device which overcomes these problems of the prior art and can perform measurement without impairing the sensitivity and the migration speed even if the number of capillaries increases. .

[0008]

In order to achieve the above object, in the present invention, a plurality of gel capillaries are bundled into one or a plurality of sheets, and the ends thereof are aligned along a straight line to form a buffer solution. Put it in a filled optical cell,
The area where the fluorescent labeled DNA is eluted from the end of each gel capillary is simultaneously irradiated with light. An optical fiber is arranged in close proximity to each migration path, and the fluorescence emitted from the fluorescence labeled DNA is guided through the optical fiber to the individual light receiving elements of the line sensor or area sensor for detection.

The optical fiber may have a condenser lens or an optical filter. The output end of each optical fiber is one of line sensor or area sensor.
Optically coupled to one light receiving element or several light receiving elements.

[0010]

[Function] DNA is eluted regardless of the number of capillaries by aligning the ends of the capillaries in a straight line and arranging a plurality of capillaries in a sheet shape and irradiating laser light from the side of the sheet along the width direction of the sheet. Since all the migration paths in the vicinity of the end of the capillary can be simultaneously irradiated with one laser beam, efficient light irradiation is possible.

Unlike the case where the light receiving unit forms an image of a plurality of migration paths with a single lens, an optical fiber with a condenser lens is installed for each migration path, so that efficient light detection is performed regardless of the number of capillaries. You can Further, since the output end of each optical fiber is detected by being matched with the light receiving element of the line sensor or the area sensor, it is possible to perform measurement using the capillaries corresponding to the number of light receiving elements. Since the line sensor has about 1000 light-receiving elements and the area sensor has more than 100,000 light-receiving elements, the present invention makes it possible to measure an array of virtually unlimited capillaries. become.

[0012]

EXAMPLES Examples of the present invention will be described in detail below. [Example 1] An example in which the present invention is applied to DNA nucleotide sequence determination will be described. In DNA sequencing (DNA sequencing), complementary strand synthesis is performed using the DNA strand to be analyzed as a template to form a DNA fragment group. That is,
Oligomers or complementary chains with specific sequences called primers are synthesized to prepare fragments of various lengths ending with adenine (A), thymine (T), cytosine (C), or guanine (G). Fragments with A at the end are called A family, those of T are called T family, those of C are called C family, and those of G are called G family.

Each family is labeled with a different fluorophore, DNA fragments are separated by length by gel electrophoresis, and the shorter DNA fragments are passed through the light irradiation part in order. Fluorescent label D
The DNA fragment terminal base species can be known from the fluorescence wavelength emitted by the NA fragment, and the length of the DNA fragment can be known from the migration time to the irradiation part. The DNA base sequence is determined from this information. In this case, it is necessary to wavelength-select and detect a plurality of fluorescences (4 is convenient) in order to identify the 4 types of terminal bases. Further, the separation and detection of DNA by gel can be used not only for DNA sequencing but also for gene diagnosis, and in that case, only one kind of fluorescent substance may be used.

FIG. 1 is a schematic view of a capillary array device according to this embodiment. A group of DNA fragments for sequence analysis made from various sample DNAs is injected into the upper part of the capillary 1 for each sample. The injection is performed electrically by immersing the upper end of the capillary 1 in each sample bottle containing the fragment group, and applying a voltage of about 10 kV between the electrode immersed in the sample solution and the lower end of the capillary. After injecting the sample, the upper ends of the capillaries are put together into the buffer solution in the upper buffer tank 19, and the electrode 17 immersed in the upper buffer solution and the electrode 17 immersed in the buffer solution in the lower buffer tank 18 with the lower part of the capillary immersed A voltage of 10 to 15 kV is applied during the migration. The length of gel capillary 1 is usually 2
The length is 0 cm to 40 cm, but may be 10 cm to 15 cm if analysis of short DNA or high resolution is unnecessary. Also,
50 cm when long DNA analysis and high resolution are required
A gel capillary with a length of -100 cm may be used.

The lower part of the gel capillary is irradiated with laser light 4 from a laser light source 5 to detect fluorescence, but when the capillary tube is directly irradiated with light, the laser light is scattered or refracted on the surface of the capillary tube, and It is not possible to illuminate the capillaries at the same time. Therefore, the lower ends of the gel capillaries 1 are aligned and immersed in the buffer solution of the optical cell 2, and all the DNA fragments eluted from the lower ends of the gel capillaries are simultaneously irradiated with the laser light 4. In order to prevent the DNA band 3 from spreading due to diffusion etc. after elution, a hollow capillary 6 is installed below the gel capillary 1 to let the buffer solution flow out from the sheath solution buffer tank 7 into the optical cell 2, As shown in an enlarged view in the lower left circle of 1, a sheath flow (buffer liquid flow) 40 is formed around each migration path. In addition, even if the hollow capillaries 6 are not installed corresponding to the gel capillaries 1 one by one, there is no problem in practice if a flow is created in the light irradiation part.

A quartz tube having an inner diameter of 0.1 mm was used for the gel capillary 1 of the present embodiment.
Tubes with various inner diameters such as 0.3 mm can be used depending on the purpose. The gel is polyacrylamide (Total)
The concentration is 5% and the crosslink ratio is 3%), and the manufacturing method thereof is described in, for example, Analyt. Chem. 64 , 1221-1225 (1992).

The buffer liquid flow for the sheath is 50 nl / sec.
If the number is equal to or more than this, there is no problem, but it is desirable to examine the shape of the DNA band and the flow rate dependence of the fluorescence intensity and optimize it according to the optical cell. The optical cell 2 was connected to the external buffer tank 7, and the height of the external buffer tank 7 was changed to control the flow rate. The optical cell 2 is provided with a hollow capillary tube 6 through which the laser light 4 is incident from the side surface and the sheath buffer solution flows out.

The gel capillary 1 is set on the upper part of the optical cell 2 together with the holder 9, and the lower end of the capillary is adjusted to be located about 0.5 mm above the laser beam irradiation part. The distance from the lower end of the gel to the optical path of the laser beam 4, that is, the light irradiation portion is preferably 2 mm or less. If this distance is too long, problems such as mixing between migration paths may occur.

A condenser lens or an optical fiber 11 with a condenser lens is attached to each migration path by an optical fiber array holder 10 on a line including an irradiation position in a direction perpendicular to a plane formed by a capillary and a laser optical path. The distance from the migration path to the incident end of the optical fiber 11 is 2-3 mm, and the distance between adjacent capillaries is 0.3.
The length is set to 5 mm to 2 mm so that each optical fiber 11 does not receive fluorescence from the adjacent migration path.

In this embodiment, a set of four fibers is provided for each migration path. The four optical fibers are optical filters 12 having different transmission wavelength bands.
Through the line sensor 13 or the area sensor, each light receiving element is optically coupled. For this, a CCD with an optical fiber in which a light receiving element and an optical fiber are combined at a ratio of 1: 1 is used, or the optical fiber is fixed with an adhesive while aligning under a microscope. Alternatively, the tip of the optical fiber array may be aligned in advance using a jig and then fixed to the line sensor or area sensor with an adhesive.

As the phosphor, for example, ABI (Applie
d BioSystems Inc.) FAM (emission wavelength 519 nm), JOE (emission wavelength 548 nm), T
AMRA (emission wavelength 578 nm), ROX (emission wavelength 6
05 nm) or the like, and the excitation laser light source 5 is A
An r ⁺ laser (wavelength 488 nm) or a YAG laser (532 nm) is used. The optical filter has a transmission wavelength bandwidth of about 20 centered on the emission wavelength of each phosphor.
4 band pass filters of nm are used.

If the number of capillaries is about 100, one line sensor can be divided into four and four color separation data can be obtained. It is convenient to use an area sensor or four line sensors for each color when there are many capillaries or when you want to detect the light from one optical fiber using a thick optical fiber with several light receiving elements. Is. FIG. 1 shows a case where an optical filter 12 for each color and four line sensors 13 are used in combination.

A photodetector such as a line sensor or an area sensor is controlled by a driver 14, and the detection signal is processed by a data processor 15 to identify the type of the fluorescent substance and to detect the temporal change of the fluorescent intensity, thereby detecting the DNA. The sequence is determined. The output of the data output device 15 is output to the output device 16 such as a CRT, a recorder or a plotter. There are several methods for coupling the light receiving section and the photodetector with an optical fiber. FIG. 2 shows an example thereof.

In FIG. 2A, the fluorescence from the light emitting point 31 is condensed by the lens 8 into parallel light, and four optical fibers 11 each having an optical filter 32 having a different light transmission band on the end face are coupled. It is an example. Optical filter 32
Is formed by depositing a dielectric film or the like on the incident end of the optical fiber 11. The optical fiber 11 reaching the photodetector is used by narrowing the tip as necessary or using coupling fibers having different areas on both end surfaces in order to match the size of the light receiving element constituting the line sensor or the like.

In FIG. 2B, the fluorescence from the light emitting point 31 is condensed by the lens 8 and passed through one optical fiber 11. Optical fibers that transmit fluorescence from each capillary are bundled in a line, and the light rays emitted from the narrowed optical fiber ends are imaged on the area sensor 13 by the lens 20,
At this time, an image division prism 21 with a four-color filter and an imaging lens 2 in which an image division prism and a color filter are combined
0 is used for wavelength selective detection.

FIG. 2 (c) shows a system in which the four optical fibers 11 are guided as they are to a light receiving element such as a line sensor, but a lens 20 and a filter 22 are interposed in the middle,
It is again connected to the light receiving element such as a line sensor through the optical fiber 23. The number of optical fibers is not limited to four, but may be two, six, eight or the like depending on the purpose.

[Embodiment 2] Although one optical cell is used in the above embodiment, the number of optical cells is not limited to one. FIG. 3 shows an embodiment in which signals from many capillaries are simultaneously detected by dividing laser light or connecting a plurality of optical cells in series. Although not shown in FIG. 3, FIG.
Similarly, the upper end of the gel capillary 1 is immersed in the upper buffer tank, the lower ends of the hollow capillaries 6 corresponding to the individual gel capillaries 1 are immersed in the lower buffer tank, and an electrophoretic voltage is applied to both ends of the gel capillary. There is. A buffer tank for sheath liquid is connected to the optical cell 2, and a sheath flow is formed around each migration path.

The number of capillaries 1 handled collectively is DNA.
Same as the number of holes in the titer plate used for sample adjustment 96
Although it is set to (12 × 8), a multiple of 24, which is twice as much as one line on the titer plate, is good for matching with a sample adjusting robot and the like, which is convenient for operation. Therefore, an optical cell is made by using an array in which 96 capillaries are bundled as one unit, and the number of optical cells 2 is increased as necessary.

The plurality of optical cells 2 are arranged in tandem, and even if the laser light having passed through the laser light irradiation window 24 of the adjacent optical cells is overlapped and irradiated with laser light, the laser light is divided and irradiated in parallel. May be. In the example of FIG.
The laser light from the laser light source 27 is applied to the half mirror 25.
The laser light transmitted through the half mirror is changed in direction by the mirror 26, and a plurality of optical cells 2 are arranged in tandem in the optical path of each laser light, thereby significantly improving the throughput. The optical fibers from each cell are collectively coupled to a photodetector 13 such as a line sensor or area sensor.

[Embodiment 3] In the above-mentioned two embodiments, the capillary tubes are bundled and used, but the present invention can also be applied to a capillary gel using grooves formed in a flat plate. FIG. 4 shows an example in which a large number of linear gels 29 are formed between two glass flat plates 28 by using grooves formed in a spacer or a plate and used as migration paths. Each migration path is divided by spacers (made of Teflon or other plastic sheet) and glass (when grooves are dug). Since it is not possible to irradiate laser light along the gap of the glass to irradiate all migration paths at the same time, a fluorescent labeled DNA fragment is put into the buffer solution in the buffer cell / sheath flow optical cell 30 from the glass end. Elute and irradiate simultaneously with the laser light 4 the DNA fragments emerging from all migration paths. As in the case of the above-mentioned capillary array, a buffer solution flow is created near the end of the capillary gel, and DN
Prevents A band diffusion. The migration plate 28 is placed vertically, but it can be placed horizontally. D removed from the gel
The NA fragment runs down. Buffer solution is glass plate 2
8 and the inner wall of the optical cell 30 flow downward. The optical fiber 11 is installed along the light irradiation area,
The fluorescence emitted from each migration path is condensed by the lens 8 and enters the corresponding optical fiber. The following operation is the same as in the above-described first and second embodiments.

In the above-mentioned embodiment, the fluorescent light is received from only one side of the optical cell, but it is also possible to improve the light collecting efficiency by disposing a mirror or the like on the opposite side. In addition to fluorescence, chemiluminescence can be used to detect the DNA band. In that case, the reaction product is put in the sheath liquid, and the chemiluminescence emitted when the labeled compound of the DNA fragment eluted from the gel reacts with the reaction product in the sheath liquid is detected.

[0032]

As described above, according to the present invention, even in the electrophoresis system having the light irradiation portions dispersed in a wide area,
Since light detection can be performed using one or several line sensors or area sensors, it is possible to provide an optimal measurement system for a system with many migration paths and high throughput. Also,
Line sensors and area sensors are compact and inexpensive compared to arranging a large number of photomultiplier tubes.
It is effective in making an inexpensive system. Furthermore, it is possible to freely add a capillary array, etc., and provide a system that is easy to use.

[Brief description of drawings]

FIG. 1 is a partial sectional conceptual view of an embodiment of an electrophoretic device according to the present invention.

FIG. 2 is an explanatory diagram of an optical fiber condensing / wavelength selecting method.

FIG. 3 is a conceptual diagram of a system using a plurality of optical cells.

FIG. 4 is a partial sectional conceptual view of another embodiment of the electrophoretic device according to the present invention.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Gel capillary, 2 ... Optical cell, 3 ... DNA band, 4 ... Irradiation laser light, 5 ... Laser light source, 6 ... Hollow capillary, 7 ... Sheath liquid buffer tank, 8 ... Lens, 9 ... Gel capillary holder, 10 ... optical fiber array holder, 11 ... optical fiber, 12 ... optical filter, 13 ... optical line sensor (or area sensor), 14 ... driver, 15 ... data processing device, 16
Output device, 17 ... Electrophoresis electrode, 18 ... Lower buffer tank, 19 ... Upper buffer tank, 20 ... Lens, 21
Image splitting prism with four-color filter, 22 Optical filter, 23 Optical fiber, 24 Laser irradiation window,
25 ... Half mirror, 26 ... Mirror, 27 ... Laser light source, 28 ... Electrophoresis gel holding plate, 29 ... Linear gel, 30 ... Buffer tank and sheath flow optical cell, 31 ... Luminescence point,
32 ... Color separation filter, 40 ... Sheath flow

Claims (5)

[Claims] 1. A plurality of separated gel separation sections whose ends are aligned along a straight line and a migration path following the separation sections, a light source,
A means for passing the light rays from the light source along the straight line so as to intersect all the migration paths, and the same number of light receiving paths as the migration paths arranged with their optical axes aligned at the intersections of the light rays and the migration paths. Electrophoretic device comprising: a light-guiding optical fiber; and a line sensor or area sensor coupled to the other end of the light-receiving optical fiber. 2. The electrophoretic device according to claim 1, wherein the gel separation part is composed of a gel capillary. 3. The electrophoretic device according to claim 1, wherein the gel separation part is composed of a gel migration path formed separately between two transparent substrates. 4. The electrophoretic device according to claim 1, wherein the light receiving optical fiber receives fluorescence. 5. The electrophoresis according to claim 1, wherein the light receiving optical fiber is optically coupled to one or several photocells of a line sensor or an area sensor. apparatus.