JPH10300721A - Electrophoresis device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an inexpensive multicolor fluorescence detecting capillary array electrophoresis device which stably operates at a high speed. SOLUTION: A sample DNA is electrophoretically separated by applying a voltage across electrophoresis buffer tanks 22 at both ends of a capillary array 20. An information processor 30 determines the base sequence of the sample DNA by detecting the fluorescence of the DNA carrying a phosphor which appears in a sheath flow cell 21 and is excited by a laser beam 25 by means of a double line sensor 29 through an objective lens 26, a four-divided lens 27 provided with an optical filter, and an image forming lens 28. Since the fluorescence from one light emitting point is divided into four parts in the vertical and horizontal directions by means of the fourdivided lens 27 and passed through the optical filter which transmits the light having the wavelength of the fluorescence corresponding to each base species, the base species can be discriminated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for fluorescently labeling DNA, RNA, or protein and analyzing the label by electrophoresis.

[0002]

2. Description of the Related Art Capillary gel electrophoresis has become widespread as a high-speed and high-sensitivity analysis method, and has attracted attention particularly as a promising method for DNA analysis including DNA sequencing (DNA sequencing). In addition, a capillary array device in which a number of capillary analysis tubes are arranged has been developed in order to simultaneously analyze many samples and increase throughput. In the capillary array, the migration speed of the DNA fragment is different for each capillary. Therefore, when used for DNA sequencing, the DNA fragment is labeled with four kinds of fluorophores corresponding to the four terminal bases of the DNA fragment, and these are labeled with the same migration path. So-called multicolor detection, in which electrophoresis is performed and separation is detected, is effective.

A method using a laser scan and four photomultipliers as an apparatus capable of multicolor detection,
A method has been reported in which all capillaries are simultaneously irradiated with light, the obtained image is divided, and one dotted line image is detected by a two-dimensional detector through filters as four vertically separated dotted line images.

[0004] The latter is an effective method without moving parts, but uses an expensive detector such as a cooled CCD. However, in particular, the method of simultaneously irradiating all migration paths from the side of the capillary array plane provides high sensitivity and is most suitable for a system having many capillaries.

As a method of using a line sensor, a method of using the same number of line sensors as the number of phosphors, or a method of providing a fluorescent filter in front of a detector and rotating the filter in synchronization with signal reading can be considered. The inventor has previously devised a method of performing multi-color detection using an image division method and a line sensor.

[0006]

In a system in which light irradiation is performed by laser scanning, if the number of capillaries is large, there is a problem in terms of sensitivity, and a side incidence system is excellent. However, cooling C
CD area sensors are expensive and have drawbacks. On the other hand, a system using a line sensor is inexpensive, but since the area on the sensor is divided into four, it is not suitable for a system having many capillaries, and 20 to 40 capillaries are the limit. In addition, since the received fluorescence is divided, the amount of received light for each fluorescence is reduced.

Accordingly, the present invention has been made to provide a multicolor fluorescence detection capillary array electrophoresis apparatus which operates stably at low cost.

[0008]

In order to achieve the above object, the present invention uses a plurality of line sensors and an image division method. The cooling line sensor is cheaper than the area sensor and has a faster signal reading speed. By using two cooling line sensors, the substantial length of the light receiving element is increased, and measurement of a system having twice as many capillaries becomes possible. Also, the image separation is combined with a band-pass filter and a dichroic mirror, or four band-pass filters are used to divide the image into four parts, thereby enabling color separation measurement of four colors.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION

(Embodiment 1) This embodiment is an example using a light receiving element having two line sensors. Double line sensor is Thomson C
It is commercially available from the CD company and has two line sensors for one element.

The capillary has an outer diameter of 0.2 mm and an inner diameter of 0.075.
A mm-coated polyimide-coated quartz tube was used. The light irradiating section of the capillary array has a method of irradiating a portion where DNA fragments are eluted into the buffer solution at a time by using a sheath flow, and a method of irradiating through a capillary tube arranged in a line. In the former case, the arrangement pitch of the capillary tubes was set to about 0.4 mm in order to achieve a stable sheath flow. In the latter case, it is 0.4.
mm. The number of capillaries is 60, the length of the fluorescence image is 24 mm, and the length of the line sensor is 25.4 mm.
In each case, the pitch was 0.4 mm and the inside of the fluorescent spot was about 0.1.
A dotted line fluorescent image of mm is obtained.

When this image is formed through the four-division lens shown in FIG.
Are observed as two fluorescent point images. As a result, two dotted lines are formed. The number of fluorescent point images included in each line image is double (4 times the total of the upper and lower two lines) by the split lens. The split lens is obtained by cutting a single lens into a cross shape and bonding the lenses again. If there is no cutting allowance, there is one image, but if there is a cutting allowance, the optical axis of each lens fragment is shifted, so that light passing through each lens fragment forms an image at a different position, and four images are formed. Can be.

The greater the cutting margin, the greater the distance between the four images because the optical axis of each lens fragment is greatly shifted. The optical axis of the split lens is adjusted so that the vertically split image comes on the two line sensors. On the other hand, the deviation of the optical axis is reduced so that the left and right images have a small image shift so as not to overlap with the adjacent image. Twice as many point images are observed per line, just as the pitch of the point images is halved. If a filter having a different transmission band is used in accordance with the split lens, it is possible to select the wavelength and receive fluorescence.

FIG. 2 is a schematic diagram of an apparatus using the light receiving detection system. In FIG. 2, 20 is a capillary array,
21 is a sheath flow cell, 22 is an electrophoresis buffer tank,
23 is a laser, 24 is a mirror, 25 is a laser beam, 26 is an objective lens, 27 is a quadrant lens having an optical filter, 28 is an imaging lens, 29 is a double line sensor, 30
Is an arithmetic processing unit.

A DNA sequencing reaction is performed using a sample DNA as a template to prepare a DNA fragment labeled with a fluorescent label. Four groups of fragments are prepared for each base type at the 3 'end of the DNA fragment. The DNA fragments in each group are labeled with fluorescent materials having different emission wavelengths. Each capillary of the capillary array 20 is filled with polyacrylamide gel and used as an analysis unit. Different DN
A sample is injected into each capillary and separated by electrophoresis.

Light is irradiated on the migration path at a certain distance from the injection point, and the fluorescence emitted from the passing DNA fragment is detected.
Shorter DNA fragments migrate faster, so shorter DNA
Fluorescence is emitted in order from the fragments, and the terminal base can be determined from the color of the fluorescence. Based on this information, the base sequence of the DNA is determined, and the length is determined.

The fluorescent images obtained at each measurement point are dot-shaped fluorescent images arranged on a straight line along the laser path. This is received using a lens 26, and four divided images whose wavelengths have been selected using a division lens 27 having a filter and an imaging lens 28 are detected by two line sensors 29. Although the split lens 27 is inserted between the objective lens 26 and the imaging lens 28 in the drawing, the split lens 27 may be used as an objective lens or as an imaging lens.

The four divided image information is arithmetically processed by the arithmetic processing unit 30 to obtain information on the DNA fragment added to each fluorescent substance.

(Embodiment 2) In the first embodiment, a split lens is used. However, as shown in FIG. 3, this embodiment is an example utilizing image division using two separate line sensors and a prism. . In FIG. 3, 40 is a light emitting point, 41 is an objective lens,
42 is a color glass filter, 43 is a dichroic mirror,
44 is a band pass filter for FITC, 45 is JO
E, a band pass filter corresponding to TAMRA, 46 a band pass filter corresponding to ROX, 48 a two-piece prism, 49 an imaging lens,
0 is a line sensor, 51 is an imaging point, and arrow 52 is 560n
The reflected light of m or less, and the arrow 53 indicates the transmitted light of 560 nm or more.

The light emitted from the fluorescent light emitting section 40 in the form of a dotted line is collected by the objective lens 41, and after the excitation light component is removed by the color glass filter 42, the light of 560 nm is emitted by the dichroic mirror 43.
The light of m or less is reflected upward (arrow 52) in the figure, and the wavelength components longer than that are transmitted (arrow 53). F as labeling phosphor
ITC (fluorescine isothiocisnate: emission wavelength 520n
m), JOE (trademark of PerkinElmer: 545n)
m), TAMRA (trademark of PerkinElmer: 575n)
m), and ROX (trademark of PerkinElmer: 605)
nm), most of the fluorescence from FITC and JOE passes upward and most of the fluorescence from TAMRA and ROX passes through the dichroic mirror as is. An image splitting prism 48 and band-pass filters 44 to 47 corresponding to the emission wavelengths of the respective phosphors are inserted in the respective optical paths. Image. The distance between the divided images is short so as not to overlap or intersect with the fluorescent point image at a short distance, as in the previous example. The signals from the two line sensors are arithmetically processed to obtain signals from the respective phosphors.

In this embodiment, the number of capillaries is 50, and a sheath flow type irradiation unit is used as in the previous embodiment. Of course, an on-column method of directly irradiating light to the capillary tube may be used. Let the image magnification of the optical system be 1,
A 1-inch (25.4 mm) line sensor was used. The spacing between the capillaries was 0.5 mm, and 100 fluorescent images were arranged on the line sensor at 0.25 mm intervals. The width of the light emitting point was 0.1 mm or less, and each point was detected in a sufficiently separated state.

The line sensor is sufficiently cooled (-10 ° C.), and the dark current can be ignored in this measurement. Reading takes 0.05 seconds. The exposure time is 0.3 to 1 second, and in the high-speed mode, sequencing of up to 400 bases can be performed in 20 minutes. This is a remarkable speed compared to the conventional method that required two to three hours. There is also an advantage that the apparatus can be simplified and reduced in size by using the line sensor.

[0022]

As described above, according to the present invention, a two-line sensor is used in place of the cooled CCD area sensor to reduce the price ratio to 1/5 or less, and to provide a simple multicolor detector by the image division method. It is possible to provide an apparatus having high sensitivity and high throughput.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing a configuration of a four-division lens used in an embodiment of the present invention.

FIG. 2 is a schematic diagram showing an electrophoresis apparatus according to one embodiment of the present invention.

FIG. 3 is a schematic diagram of an optical system of an electrophoresis apparatus according to another embodiment of the present invention.

[Explanation of symbols]

10 lens, 11 ... one of four divided lenses, 12
... Cutting allowance, 14 ... Four-piece lens bonded together, 20 ... Capillary array, 21 ... Sheath flow cell, 22 ... Electrophoresis buffer tank, 23 ... Laser, 24 ... Mirror, 25 ...
Laser light, 26: objective lens, 27: quadrant lens with optical filter, 28: imaging lens, 29: double line sensor, 30: arithmetic processing unit, 40: light emitting point, 41
... Objective lens, 42 ... Color glass filter, 43 ... Diclock mirror, 44 ... Band pass filter (FITC compatible), 45 ... Band pass filter (JOE compatible), 46
.., Band pass filter (for TAMRA), 47, band pass filter (for ROX), 48, two-segment prism, 49, image forming lens, 50, line sensor, 51, image forming point.

Claims (6)

[Claims] 1. An electrophoresis apparatus comprising: a capillary array electrophoresis path or a narrow groove electrophoresis path; a sensor having a plurality of detection lines; and means for dividing an image in horizontal and vertical directions. 2. The electrophoretic device according to claim 1, wherein the line sensor comprises an element having two horizontal line sensors. 3. An electrophoretic device according to claim 1, wherein the light of different wavelength regions is divided and separated by two line sensors and received. 4. The electrophoretic device according to claim 1, wherein an interval between two point images obtained by dividing the image in the horizontal direction is equal to or less than an interval between the migration paths. 5. An electrophoresis apparatus according to claim 2, wherein a lens divided into four in the horizontal and vertical directions is used as the image dividing means. 6. The electrophoresis apparatus according to claim 1, wherein a plurality of detection lines each include a detection element having a different optical filter.