JP3450947B2 - Fluorescence detection type capillary array electrophoresis device - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a capillary array electrophoresis apparatus for analyzing biological materials such as DNA using a laser induced fluorescence method.

[0002]

2. Description of the Related Art Large amounts of DNA have been developed with the progress of the genome project.
Nucleotide sequencing has become an issue. Autoradiography using a radioisotope label has been used to determine the nucleotide sequence of DNA. Recently, instead of this, an automatic DNA sequencer using a fluorescent label, namely D
An NA sequencer has been developed and used.

The DNA sequencer consists of 4
It is a device for determining the sequences of the three bases A, C, G and T.
The principle is that DNA is labeled with a fluorescent substance corresponding to each of the four bases at the end of the DNA sample, the labeled DNA sample is electrophoresed and separated in a gel, and the separated DNA sample is irradiated with laser light. Then, the fluorescent label in the sample is excited by laser light, the luminescence due to this excitation is measured, the terminal base species are sequentially discriminated from the short DNA from the emission wavelength, and the sequence is determined from that sequence. . The number of samples that can be sequenced at one time with this DNA sequencer is 2
8 to 4 to 36 samples to determine 400 to 500 bases
I need 10 hours. Therefore, it is desired to develop a DNA sequencer having a larger processing capacity.

In order to process a large amount of DNA sample, it is effective to increase the speed of electrophoresis, increase the number of lanes for electrophoresis, and electrophorese a plurality of DNA samples in the same lane. The applied voltage may be increased to increase the speed of electrophoresis. However, as the voltage increases, the generation of Joule heat also increases, so the heat dissipation efficiency must be increased. The plate gel improves the heat dissipation efficiency by making the gel thin, but there is a limit. On the other hand, the capillary gel used by filling the capillary with gel has a diameter of 0.05 to 0.1 mm.
Its thin shape makes it possible to apply a high electric field without causing a temperature rise due to Joule heat due to its high heat dissipation efficiency, which is suitable for increasing the speed of electrophoresis (An
al. Chem. 62, 900-903, 1990). Therefore, in order to achieve high throughput, a device with a large number of capillaries arranged has been devised (Nature 359,167, 1992; Nature 361,
565, 1993).

In order to perform measurement using a capillary array in which a large number of capillaries are arranged, it is necessary to irradiate a large number of capillaries at the same time and receive and detect fluorescence emitted, but a method using sheath flow is an effective method. There is. That is, a large number of gel-filled capillaries are used as an analysis part, and the ends thereof are put in a buffer solution and the eluted DNA fragments are irradiated with light to detect fluorescence. A sheath flow of a buffer solution is formed in the vicinity of the end of the gel so that the DNA fragments eluted from the gel do not spread and spread to reduce the separation ability. Using this method, 100 samples can be analyzed at one time, and the time required for measurement is 2
It takes about time, and a large throughput can be obtained.

Ar ⁺ laser light (488 nm) is usually used as an excitation light source for irradiating DNA fragments with light, but four kinds of fluorescent substances having different fluorescence maximum wavelengths (and hence different optimum excitation wavelengths) are used. When excited by laser light,
A phosphor whose optimum excitation wavelength is far from the laser wavelength has low excitation efficiency and cannot obtain high sensitivity. Therefore, it is advantageous to use two types of laser light to excite two sets of phosphors having a relatively high excitation efficiency corresponding to the respective laser lights. For example, with Ar ⁺ laser light, FITC (fluoresceine
-5-isothiocyonate emission maximum wavelength 515nm) and FI
TC-Cl2 (dichloro-FITC emission maximum wavelength 52
He--Ne laser light (594n)
m) in Texas Red® (Sulforhodami
ne 101 emission maximum wavelength 607 nm) or Cy-5
(Registered trademark) (emission wavelength 667 nm) or the like is excited. Of course, there is a case where three kinds of phosphors can be efficiently excited by one laser beam, and the present invention is not limited to this. As an example of exciting three types of phosphors with one laser light, FITC is excited with Ar ⁺ laser light, and YAG laser light (532 nm)
3 types of phosphor JOE (registered trademark) (emission maximum wavelength 5
55 nm), TAMRA (registered trademark) (Tetra Methil R
hodamine emission wavelength 585 nm), ROX (registered trademark)
(Rhodamine X emission maximum wavelength 615 nm) may be excited.

In order to excite these many phosphors with two kinds of laser light, the migration path irradiation is performed along the capillary array surface in the vicinity of the ends of the capillaries where the two laser lights are superposed and arranged in a plane. In this way, the fluorescence emitted from a large number of DNA fragments simultaneously irradiated is spectrally received, and D
The terminal base species of the NA fragment are discriminated, and the base sequence is determined from the order. This type of device can be widely used for analysis of fluorescently labeled bio-related substances, in addition to its use as a DNA sequencer.

[0008]

There are two methods of irradiating the capillary array with light: one in which laser light is scanned to irradiate one capillary one by one, and one in which aligned capillaries are simultaneously irradiated from the side. The latter has the advantage of being able to use the laser light more effectively, but it is necessary to keep the ends of the capillaries forming the capillary array in the vicinity of the irradiated area on the same plane. However, it was difficult to keep the tip free and keep it flat because the capillary was soft. As a result, a DNA fragment often passed through a portion deviated from the laser light irradiation portion, and a measurement error often occurred.

Further, when a plurality of laser beams are used, the laser beams are superposed and used, which is used when a rigorous analysis is carried out by comparing the ferrograms of DNAs labeled with fluorescent substances excited by different laser beams. Needed for. In this case, the excitation laser light is superposed using a half mirror, but the amount of light is halved by this, and the background light caused by both excitation lights is also superposed and becomes large, making it difficult to obtain high detection sensitivity. was there.

The present invention has been made in view of these problems of the prior art, and an object of the present invention is to provide a capillary array electrophoresis apparatus in which a capillary array is accurately positioned with respect to an optical system. The present invention also provides
An object is to provide an electrophoretic device having a low background light signal intensity and high sensitivity. It is another object of the present invention to provide a capillary array electrophoresis device capable of flowing a uniform sheath flow.

[0011]

According to the present invention, the elution end of the gel-filled capillary array is held in the same plane with sufficient accuracy by sandwiching it between two flat plates. The two flat plates can be used as the inner wall of the optical cell, and the operation of sandwiching the elution end of the gel-filled capillary array with the inner wall of the optical cell is performed in the inner wall gap of the optical cell having a dimension approximately equal to the outer diameter of the gel-filled capillary. This can be done by inserting the elution end of a gel-filled capillary array. Alternatively, a movable plate may be provided in the optical cell, and the inner wall of the optical cell and the movable plate may sandwich the elution end of the gel-filled capillary array. Also, by fixing and holding the vicinity of the elution end of the gel-filled capillary array on a flat plate, the gel-filled capillary array can be held in the same plane with sufficient accuracy.

By holding the elution end of the gel-filled capillary array in the same plane, it is possible to improve the measurement accuracy in the measurement method in which the laser light is irradiated from the same plane direction as the direction in which the gel-filled capillary arrays are arranged. The light irradiation position for exciting fluorescence is set on the downstream side of the elution end of the gel-filled capillary, and the DNA fragment eluted from the gel-filled capillary flows in a sheath liquid composed of a buffer solution,
That is, it can be transported to the light irradiation position by the sheath flow. At this time, by arranging the gel-filled capillaries and the passages through which the sheath liquid flows alternately, a uniform and stable sheath flow can be formed. The sheath liquid passage may be realized by hollow capillaries alternately arranged in contact with the gel-filled capillaries.

When a plurality of excitation lights having different wavelength characteristics are used, the irradiation position of each excitation light is set to a different distance from the elution end of the gel-filled capillary. With this, when the laser light is superimposed, the laser light is attenuated, the background light is increased,
It is possible to avoid an increase in noise. Therefore, a wide dynamic range can be used for measurement,
There is no decrease in S / N. DNA in sheath flow
The moving speed of the sample is determined not by the electric field but by the speed of the sheath flow, and the DNA sample always moves between a plurality of light irradiation positions in a fixed time regardless of the base length. Therefore, by correcting the deviation of the fluorescence generation timing due to the difference in the irradiation position of the excitation light,
It is easy to compare sequence patterns.

Further, by filling the inside of the optical cell with a capillary material or a substance having a refractive index similar to that of the substance in the capillary, light scattering or light scattering occurs even when the capillary is irradiated with light without using a sheath flow. Refraction can be suppressed and highly accurate measurement can be performed.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described in detail below with reference to the drawings. [First Embodiment] FIG. 1 is a diagram showing an example of the overall configuration of a multi-capillary DNA sequencer according to the present invention. The multi-capillary DNA sequencer comprises a gel-filled capillary 1, a buffer solution tank 2, an upper electrode tank 3, a lower electrode tank 4, a power source 5, an optical cell 6, a hollow capillary 7, mirrors 8 and 9, laser light sources 10 and 11, and a lens 12. , 13,
It is composed of a fluorescence filter 14, an image division prism 15, a fluorescence detector 16, and a computer 17, and forms a sheath flow in the measurement section, and irradiates different positions of the sheath flow section with two laser beams 40 and 41.

Each gel-filled capillary 1 has a sample injection end immersed in a sample in a sample container (not shown) to apply a voltage to inject an electric field into the DNA sample, and then insert the sample injection end into the upper electrode tank 3. To do. Then, the power supply 5 is operated to apply a voltage between the upper electrode tank 3 and the lower electrode tank 4, so that the DNA sample injected with the electric field is electrophoresed in the gel-filled capillary 1 to be separated. The buffer solution is sent from the buffer solution tank 2 to fill the inside of the optical cell 6, flows into the lower hollow capillary 7, and is discharged to the lower electrode tank 4. When the separated DNA sample passes through this optical cell 6,
Laser light 4 guided by mirror 8 and mirror 9
The irradiation of 0, 41 excites the fluorescent substance labeling the sample to emit fluorescence. This fluorescent light is emitted from the condenser lens 1
2. Multiple fluorescent filters 1 arranged in the vertical direction
4. After passing through the image dividing prism 15 and the image forming lens 13 that divide the image for each fluorescence, the fluorescence reaches the fluorescence detector 16 and is detected.

The fluorescence detector 16 is a cooled CCD camera or the like.
The three-dimensional imaging device. In the illustrated example, the fluorescence filter 14 is composed of four filters that separate and transmit the fluorescence wavelengths of four types of phosphors.
The images of the four types of phosphors separated by are vertically separated by the image separation prism 15 and the image forming lens 13 and formed on the image pickup surface of the two-dimensional image pickup device. Fluorescence detector 1
The signal detected by 6 is sent to the computer 17 for analysis, and the base sequence of the sample is determined.

FIG. 2 is a detailed explanatory view of the portion of the optical cell 6, and FIG. 3 is a side sectional view thereof. The distance between the non-fluorescent transparent glass plates 6a and 6b before and after the optical cell 6 filled with the buffer solution is 0.
It is 22 mm, and the gel-filled capillary 1 having an outer diameter of 0.2 mm is inserted into this gap from above. The gel-filled capillaries 1 are adhered and held on a flat plate 18 in an array shape, for example, at a pitch of 0.4 mm, and hollow capillaries 7 are arranged in the lower part so as to face the gel-filled capillaries 1. Gel-filled capillaries 1 arranged in a plane
The distance between the hollow capillary 7 and the hollow capillary 7 is about 5 mm, and the two parallel laser beams 40 and 41 pass through this gap. In this way, the tip of the gel-filled capillary 1 is held in the glass gap about the outer diameter of the capillary, so that it is held in the same plane with sufficient accuracy. The optical cell 6 need only be transparent in at least the laser light incident region and the fluorescence extraction region, and it is not always necessary to form all the wall surfaces with transparent members.

As shown in FIG. 3, a buffer solution container 50 is provided above the optical cell 6, and the buffer solution sent from the buffer solution tank 2 enters the buffer solution container 50 and then moves upward in the optical cell. Flows down from. In addition, guide members 51a and 51b having guide grooves are attached to the side wall of the buffer solution container 50, and the end portions of the flat plate 18 holding a large number of gel-filled capillaries 1 are connected to the guide grooves of the guide members 51a and 51b. By inserting, the tips of the gel-filled capillaries 1 aligned in a plane are arranged at predetermined positions in the optical cell 6.

DN separated by gel-filled capillary 1
The A fragment 38 tends to spread due to diffusion as it elutes from the gel-filled capillary 1 into the buffer solution, but moves downward while maintaining a laminar flow state due to gravity-induced natural fall (buffer solution flow). Before being diffused by the laser beam 39, it traverses the optical paths of the laser beams 40 and 41 and is irradiated with light to emit fluorescence. D separated by each gel-filled capillary 1
Since the NA fragments 38 are carried by the sheath flow 39 without being mixed with each other while maintaining their planarity, they can be simultaneously irradiated with the laser beams 40 and 41. DNA fragment 3
8 and the buffer solution pass through the hollow capillary 7 and the lower electrode tank 4
Is discharged to.

The moving speed of the DNA fragment 38 in the sheath flow 39 is about 0.1 mm / s. At this time, the positions of 0.5 mm and 1 mm from the elution end of the gel-filled capillary 1 are irradiated with the laser beams 40 and 41 from the lasers 10 and 11, and the fluorescence signal is obtained. The time for the DNA fragment 38 to move within the distance of 0.5 mm between these two laser beams is about 5 seconds, and this time is constant regardless of the base length. Figure 4
The ratio of the migration rate of DNA fragments in the sheath flow was set to 20
The length of a DNA fragment with a base length of 1 is 150 bases, and 3
The results of measurement of DNA fragments having a length of 00 bases and a length of 400 bases are shown. From FIG. 4, it is clear that the migration rate of the DNA fragment in the sheath flow is constant regardless of the base length. Therefore, even if the irradiation positions of the laser beams 40 and 41 that excite various phosphors are different, the displacements of the excitation light irradiation positions can be mutually corrected by simply shifting the measurement time by the time determined by the sheath flow velocity. Can
It is possible to determine the base sequence by comparing the relative migration velocities of the respective DNA fragments.

Next, the reason why the optical paths of the two laser beams 40 and 41 are shifted will be described. When the aqueous solution is irradiated with the laser light, a strong signal based on Raman scattering of water is observed on the long wavelength side of about 100 nm from the wavelength of the laser light. When one phosphor is excited by one laser beam, the maximum of the emitted fluorescence usually appears on the long wavelength side of 20 to 30 nm of the optimum excitation wavelength, so a filter that transmits this wavelength portion and blocks the Raman line portion. By receiving light via the, it is possible to detect fluorescence without being affected by Raman scattering.

However, when a plurality of laser beams are used in a superposed manner, the Raman line of the laser beam on the short wavelength side comes to a position close to the maximum fluorescence wavelength of the phosphor excited by the laser beam on the long wavelength side, and the background light intensity is increased. May increase and a high S / N may not be obtained. In fact, when excited at 488 nm, a Raman line appears near 585 nm. This wavelength excites T at 532 nm
Since it is close to the maximum fluorescence wavelength of AMRA, it passes through the transmission filter for TAMRA, and high sensitivity cannot be obtained for TAMRA. Further, the scattering of the excitation light of 532 nm cannot be completely blocked by a FAM (5-carboxyfluorescein) or FITC filter, resulting in an increase in background light, that is, a decrease in sensitivity.

Therefore, two laser beams 4 as in the present invention are used.
These obstacles can be eliminated by spatially shifting the irradiation positions of 0 and 41 and receiving the light with a detector having a position resolving ability. In fact, when the irradiation positions of the two laser lights are shifted, FA
The signal intensity of background light observed with the M filter is 1/2,
The signal intensity of the background light observed by the TAMRA filter can be set to 1/4, and high sensitivity can be achieved.

As an example of a method for aligning the gel-filled capillary array in a plane, the elution end of the gel-filled capillary array is placed in the gap between the glass plates 6a and 6b of the optical cell 6 having a size substantially equal to the outer diameter of the gel-filled capillary. Described how to insert. In this case, if a taper 6c is provided in the gap formed by the glass plate of the optical cell as shown in the cross section of FIG. 5, the gel-filled capillary array can be easily inserted.

Although the elution end of the gel-filled capillary is inserted into the gap of the preassembled optical cell here, an optical cell having a movable plate inside is used, and the gap between the movable plate and the optical cell wall is widened. It is also possible to insert the tip of the gel-filled capillary array into the gap and then move the movable plate toward the cell wall surface so that the elution end of the gel-filled capillary is sandwiched in the optical cell. FIG. 6 is a sectional view of an optical cell portion for explaining this modification.

The optical cell 6 whose schematic sectional view is shown in FIG.
The wall surface 6b on the fluorescence extraction side is made of non-fluorescent transparent glass, and the wall surface 6a on the opposite side is made of stainless steel. A groove-shaped recess extending in the horizontal direction is provided on the wall surface 6a, and a flat plate 61 made of non-fluorescent transparent glass is inserted into the recess. Pressing members 62 are attached to the flat plate 61 at three positions on the back side. The pressing members 62 penetrate the wall surface 6a to extend outside the optical cell, and the compression springs 6 arranged at the ends thereof.
3 is urged in the direction of the arrow. Therefore, the pressing member 62 is moved to the left in the drawing against the compression spring 63,
The tip of the gel-filled capillary array is inserted with the space between the flat plate 61 and the wall surface 6b widened, and then the pressing member 62 is inserted.
The gel-filled capillary 1 is separated from the wall surface 6b.
And sandwiched by the flat plate 61 and aligned on the same plane.

Further, even when the distance between the glass plates 6a and 6b of the optical cell is larger than the outer diameter of the gel-filled capillary, the flat plate 18 to which the gel-filled capillary array is fixed.
If the length of the free end of the capillary 1 that is projected from is sufficiently short, the elution end of the gel-filled capillary array can be aligned in a straight line with sufficient accuracy only by fixing to the flat plate 18. For example, outer diameter 0.2 mm, inner diameter 0.1 m
In the case of a gel-filled capillary of m, if the length of the capillary protruding from the fixed flat plate 18 is 5 mm or less, the elution end of the gel-filled capillary array is put into practical use without being restricted by the glass plates 6a and 6b of the optical cell. Can be aligned on a straight line with sufficient accuracy.

The fixing method for holding the capillary array in the same plane by the flat plate 18 is not limited to the adhesive method. For example, as shown in the schematic cross-sectional view of FIG. 7, a plurality of gel-filled capillaries 1 are held side by side on a first flat plate 56 made of a stainless steel plate or the like on which a rubber sheet 55 is laid, and the gel-filled capillaries 1 are placed on the first flat plate 56. Alternatively, the second flat plate 57 may be placed, and in that state, both ends of the first flat plate 56 and the second flat plate 57 may be sandwiched and fixed by clips or the like.

In the above example, the two laser beams 40 and 41 are
Irradiation was performed from the side surface of the optical cell 6 in the arrangement direction of the gel-filled capillary array. The excitation laser light can also be irradiated from a direction intersecting the plane formed by the gel-filled capillary array. 8 and 9 are schematic diagrams showing another example of the laser light irradiation method. 8 and 9, upper and lower electrode tanks for applying a migration voltage to the gel-filled capillary,
The parts other than the laser light irradiation optical system such as the buffer tank and the fluorescence detection system are the same as those in FIG.

FIG. 8 shows an example of scanning and irradiating two laser beams from the same side as the fluorescence detection surface of the optical cell. Laser light 40, 41 emitted from two laser light sources 10, 11
Is reflected by the rotating polygon mirror 71, and the optical cell 6 is scanned from one end to the other end. The laser light that has entered the cell through the transparent window of the optical cell 6 sequentially scans the gap between the elution end of each gel-filled capillary 1 and the inlet end of the hollow capillary 7 to elute the DNA eluted from the gel-filled capillary. Irradiate the pieces. The fluorescence emitted from the fluorescent label of the DNA fragment is detected by the fluorescence detector having the above-mentioned positional resolution.

FIG. 9 shows an example of irradiating two sheet-like laser beams 40 and 41 from the same side as the fluorescence detecting surface of the optical cell. Two laser light sources 10, 11 arranged in the vertical direction
The laser beams 40 and 41 emitted from the laser beam are incident on the mirror 72 at a slightly different vertical incident angle, and
After being reflected by 2, the gel-filled capillary 1 in the optical cell 6 is made into a sheet-like beam by the Fθ lens 73.
Is collected between the elution end of the hollow capillary 7 and the entrance end of the hollow capillary 7. The two beams 40 and 41 irradiate the DNA fragments eluted from each gel-filled capillary 1 at positions separated by about 0.5 mm in the sheath flow direction. Even in this case, the fluorescence emitted from the fluorescent labeling of the DNA fragment is
It is detected by the fluorescence detector having the above-mentioned positional resolution.

[Embodiment 2] FIG. 10 is an overall configuration diagram of another example using a capillary array sheet in which gel-filled capillaries and hollow capillaries are alternately arranged. In this example, by arranging the gel-filled capillaries and the hollow capillaries alternately adjacent to each other near the elution end, the pitch accuracy of the gel-filled capillaries is improved, and the buffer solution is supplied to form the sheath flow from the hollow capillaries. By doing so, a stable sheath flow is generated in the glass gap.

The capillary array DNA sequencer of this example comprises a gel-filled capillary 1, a buffer tank 20, an upper electrode tank 21, a lower electrode tank 22, a power source 5, an optical cell 24,
Hollow capillaries 7 for connecting the optical cell and the lower electrode tank, hollow capillaries 26 in which one end side is immersed in a buffer solution tank and the other end side is alternately arranged with gel-filled capillaries, laser light source 10, laser light from laser light source 10. 27 for guiding the light to the optical cell, the laser light source 11, mirrors 28, 29 for guiding the laser light from the laser light source 11 to the optical cell, the fluorescence condensing lens 12, the fluorescence filter 14, the image splitting prism 1.
5, imaging lens 13, fluorescence detector 16, computer 1
It consists of 7.

Each gel-filled capillary 1 has its sample injection end immersed in a sample in a sample container (not shown) to apply a voltage to inject an electric field into the DNA sample, and then the sample injection end is inserted into the upper electrode tank 21. To be done. Next, the power supply 5 is operated to apply a voltage between the upper electrode tank 21 and the lower electrode tank 22, whereby the DNA sample injected with the electric field is electrophoresed and separated in the gel-filled capillary 1. The buffer solution is sent from the buffer solution tank 20 to the hollow capillaries 26 to fill the optical cell 24, flow into the lower hollow capillaries 7, and be discharged to the lower electrode tank 22.

The separated DNA sample is the optical cell 24.
When passing through, the laser light 41 guided by the mirrors 28 and 29 and the laser light 40 guided by the mirror 27 are irradiated, and the fluorescent substance labeling the sample is excited to emit fluorescence. This fluorescence emission passes through a condenser lens 12, a plurality of fluorescent filters 14 arranged in the vertical direction, an image division prism 15 that divides an image for each fluorescence, and an imaging lens 13, and reaches a fluorescence detector 16. To be detected. The fluorescence detection system including the condenser lens 12, the fluorescence filter 14, the image dividing prism 15, the image forming lens 13, and the fluorescence detector is the same as that described in FIG. The detected signal is sent to the computer 17 and analyzed to determine the base sequence of the sample.

FIG. 11 is a detailed explanatory view of a portion of the optical cell, and FIG. 12 is a side sectional view thereof. The optical cell 24 keeps the array of the gel-filled capillaries 1 and the hollow capillaries 26 on the same plane, forms a stable sheath flow 39 over a long distance from the end of the capillary, and forms all DNA.
In order to stably irradiate the sample, the capillary array is sandwiched between two non-fluorescent quartz glass plates 24a and 24b. Internal spacing of the optical cell 24, i.e., the interval of the quartz glass plates 24a and 24 b is approximately 0.21m
In m, it was matched with the outer diameter of the capillary tube. The gel-filled capillary 1 and the hollow capillary 26 are fixed to the flat plate 18. The flat plate 18 is fixed to the device by inserting it into the guide grooves of the guide members 51a and 51b fixed to the frame of the device (not shown). Although the flat plate 18 is drawn so as to be in contact with and fixed to the optical cell 24 in the drawing, the fixed position of the flat plate 18 may be apart from the optical cell 24.

In order to create a stable sheath flow 39, the gel-filled capillaries 1 and the hollow capillaries 26 are alternately arranged and the hollow capillaries 7 are arranged so as to be separated from each other by about 5 mm. Two
They were opposed to each other on the plane sandwiched by 4a and 24b. The upper end of the gel-filled capillary 1 is inserted into the upper electrode tank 21, the upper end of the upper hollow capillary 26 is inserted into the buffer solution tank 20, and the lower end of the lower hollow capillary 7 is inserted into the lower electrode tank 22.

The buffer solution passes from the buffer solution tank 20 through the upper hollow capillaries 26 by a drop, and the DN coming out of each gel-filled capillary 1 inside the optical cell 24.
A sheath flow 39 is formed for the sample A, passes through the lower hollow capillary 7, and is discharged to the lower electrode tank 22. A voltage is applied from the power source 5 to the gel-filled capillary 1, and the separated DNA fragments 38 move in the gel-filled capillary 1 by an electric field and migrate into the sheath flow 39. The DNA fragments 38 that have come out into the sheath flow 39 move to the lower hollow capillary 7 without being mixed with each other by the sheath flow 39. The sheath flow 39 is irradiated with laser beams 40 and 41 without overlapping each other.

The upper hollow capillaries 26 have a function of uniformly flowing a buffer solution between the gel-filled capillaries to form a stable sheath flow 39, and the gel-filled capillaries 1 are arranged alternately so that the gel-filled capillaries 1 are evenly spaced. Has the role of arranging. In FIG. 11, the lower hollow capillary 7
Are arranged side by side without a gap, but the inflow of the sheath flow 39 is made to occur only in the one facing the gel-filled capillary 1. However, unlike this, the sheath flow may flow into all the hollow capillaries 7.

Although the device shown in the figure has a structure in which the sheath flow 39 flows from the upper side to the lower side, the sheath flow flows from the lower side to the upper side, the lateral direction, etc. by inverting the entire apparatus or laying it diagonally or sideways. You may do it. Further, the number of capillaries on the upper side and the number of capillaries on the lower side are not necessarily the same, and further, gel-filled capillaries may be arranged with the same diameter and the same pitch, or with different diameters and the same pitch. The electrodes can be arranged not only in the lower electrode tank 22 but also in the optical cell 24.

Here, by arranging the gel-filled capillaries 1 and the hollow capillaries 26 for flowing the buffer solution alternately adjacent to each other, the pitch precision of the gel-filled capillaries is improved and a stable sheath flow is generated. The same thing can be realized by using a structural member that also serves as a capillary holding member instead of the hollow capillary. This modification will be described with reference to FIGS. 13 to 15.

In this modification, the vicinity of the elution end of the gel-filled capillary is fixed and held by the capillary holding member. FIG. 13 is a perspective view showing components of the capillary holding member, and FIG. 14 is an overall view of the capillary holding member. As shown in FIG. 13, the capillary holding member 80 is formed by joining two grooved members 81 and 82 in which parallel grooves 83 and 84 are formed, with their grooved surfaces facing each other. The groove 83 has the same dimension as the outer diameter of the gel-filled capillary 1, and the alternate groove 83 of the grooved member 81.
The gel-filled capillary 1 is placed with the elution end slightly protruding from the groove 83. Then, by joining the other grooved member 82, the gel-filled capillary array arranged at a predetermined pitch with the elution ends aligned in a straight line is assembled. The groove 84 is a hole penetrating the holding member 80 in the width direction.

FIG. 15 is a schematic sectional view of a cell portion of a multi-capillary DNA sequencer using a capillary holding member 80. A buffer solution container 50 is fixed to the upper portion of the capillary holding member 80. The lower portion of the capillary holding member 80 has two non-fluorescent quartz glass plates 24a, 2
The optical cell 24 is formed by being sandwiched between 4b. At the lower end of the optical cell 24, a plurality of hollow capillaries 7 are shown in FIG.
1 is connected in the same manner. Capillary holding member 80
The hole formed by the groove 84 connects the buffer solution container 50 and the optical cell 24, and generates a uniform flow of the buffer solution in the optical cell 24. D separated by gel-filled capillary 1
The NA fragment 38 is carried to the irradiation position of the laser beams 40 and 41 by the stable sheath flow thus formed. In any of the examples described above, the excitation laser light can be emitted from a direction intersecting the plane formed by the gel-filled capillary array as shown in FIG. 8 or 9.

[Embodiment 3] The above example relates to a measurement system using a sheath flow in the measuring section, but the present invention is also applicable to a measurement system for simultaneous irradiation of multiple capillaries which does not use a sheath flow in the measuring section. It is valid. For this, there are a case where the coating of the gel-filled capillary is peeled off and the gel-filled capillary portion without the coating is irradiated with light, and a case where the end of the gel-filled capillary is connected to a hollow capillary and the hollow capillary is irradiated with light. Here, an example in which gel-filled capillaries and hollow capillaries are arranged in a column is shown.

FIG. 16 is a schematic diagram of an optical cell portion, and FIG.
Is a sectional view thereof. A large number of gel-filled capillaries 1 held in a plane by the flat plate 18 are arranged in a straight line with the elution end thereof being sandwiched by two non-fluorescent transparent glass plates 91a and 91b. A transparent hollow capillary 92 placed opposite the gel-filled capillary 1 has its coating removed and is lined up in a glass gap filled with glycerin 94. The glycerin 94 is filled in the lower region of the optical cell 90 partitioned by the partition plate 93 and includes the partition plate 9 including the gap between the gel-filled capillary 1 and the hollow capillary 92.
The upper region of 3 is filled with the buffer solution.

After the DNA fragments are separated by the gel-filled capillary 1, they enter the hollow capillary 92 and are irradiated with laser light from the side. The scattering and refraction of the laser beams 40 and 41 on the surface of the capillary 92 are reduced due to glycerin, and all the DNA that moves inside the hollow capillary 92.
The fragment 38 can be stably irradiated with a plurality of laser beams. The material filled around the hollow capillaries 92 has a refractive index of 1., which is close to that of glass, instead of glycerin.
It may be a substance of 2 to 1.6.

In the case of this example, since the laser light passes through the regions having different refractive indexes, it is important that the light beam is sufficiently narrowed and all capillaries are arranged on a plane so that the light can go straight. is there. Arranging all capillaries on a plane is achieved by sandwiching the capillaries with a glass plate or the like. When the periphery of the capillary for irradiating laser light is filled with a substance having a refractive index close to that of glass, it is possible to irradiate a plurality of gel-filled capillaries with laser light at the same time.

[0049]

As described above, according to the present invention, the sensitivity and throughput of the multi-capillary DNA sequencer can be increased by separating a plurality of laser beams and irradiating them on the sheath flow.

[Brief description of drawings]

FIG. 1 is a diagram showing an entire configuration of an example of a multi-capillary DNA sequencer according to the present invention.

FIG. 2 is a detailed explanatory view of a portion of the optical cell of FIG.

FIG. 3 is a cross-sectional view of an optical cell.

FIG. 4 is a diagram showing the relationship between the base length of a DNA fragment and the migration rate.

FIG. 5 is a schematic view of another example of the optical cell.

FIG. 6 is a schematic sectional view of another example of the optical cell.

FIG. 7 is a cross-sectional view illustrating a method of fixing a capillary array.

FIG. 8 is a schematic view showing another example of the excitation light irradiation method.

FIG. 9 is a schematic view showing another example of the excitation light irradiation method.

FIG. 10 is a diagram showing the overall configuration of another example of the multi-capillary DNA sequencer according to the present invention.

11 is a detailed explanatory view of a portion of the optical cell in FIG.

FIG. 12 is a schematic sectional view of an optical cell.

FIG. 13 is a perspective view showing components of a capillary holding member.

FIG. 14 is an overall view of a capillary holding member.

FIG. 15 is a schematic sectional view of a cell portion of a multi-capillary DNA sequencer using a capillary holding member.

FIG. 16 is a detailed view of an optical cell portion of another example of the multi-capillary DNA sequencer according to the present invention.

FIG. 17 is a sectional view of an optical cell.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Gel filling capillary, 2 ... Buffer tank, 3 ... Upper electrode tank, 4 ... Lower electrode tank, 5 ... Power supply, 6 ... Optical cell, 7 ...
Hollow capillaries, 10, 11 ... Laser light source, 12 ... Condensing lens, 13 ... Imaging lens, 14 ... Fluorescent filter,
15 ... Image dividing prism, 16 ... Fluorescence detector, 17 ... Computer, 18 ... Flat plate, 20 ... Buffer tank, 21 ... Upper electrode tank, 22 ... Lower electrode tank, 24 ... Optical cell, 26 ... Hollow capillary, 38 ... DNA fragment, 39 ... Sheath flow, 40, 41 ... Laser light, 50 ... Buffer container, 51
a, 51b ... Guide member, 55 ... Rubber sheet, 56, 5
7 ... Flat plate, 61 ... Flat plate, 62 ... Pressing member, 63 ... Compression spring, 71 ... Polygon mirror, 72 ... Mirror, 73 ... Fθ
Lens, 80 ... Capillary holding member, 81, 82 ... Grooved member, 83, 84 ... Groove, 90 ... Optical cell, 92 ... Hollow capillary, 93 ... Partition plate, 94 ... Glycerin

─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-7-209251 (JP, A) JP-A-3-293557 (JP, A) JP-A-6-94617 (JP, A) (58) Field (Int.Cl. ⁷ , DB name) G01N 27/447 G01N 21/64

Claims (4)

(57) [Claims] An optical cell between gap is formed 1. A by the opposing two flat plates which, the are inserted into the optical cell, sandwiched between the elution end of the sample of the two plates identical Multiple samples held in a plane
Capillary for electrophoretic separation, a fixed plate for fixing the plurality of capillaries, means for irradiating the sample eluted from the elution end into the buffer solution flow in the optical cell with laser light, and fluorescence for labeling the sample have a means for detecting fluorescence emitted from the body, the length of the plurality of capillaries which protrudes from the fixed flat plate
The fluorescence detection type electrophoretic device is characterized by having a length of 5 mm or less . An optical cell between gap is formed wherein by the opposing two flat plates which, the are inserted into the optical cell, sandwiched between the elution end of the sample of the two plates identical Multiple samples held in a plane
, A fixed plate for fixing the plurality of capillaries, and two laser beams parallel to the sample that are eluted from the elution end into the buffer solution flow in the optical cell in a direction parallel to the plane. and means for irradiating the, have a means for detecting fluorescence emitted from the fluorescent material labeling the sample, the length of the plurality of capillaries which protrudes from the fixed flat plate
The fluorescence detection type electrophoretic device is characterized by having a length of 5 mm or less . 3. The plurality of capillaries are the fixed plate.
The fluorescence detection type electrophoretic device according to claim 1, wherein the fluorescence detection type electrophoretic device is held by being adhered thereto . 4. The fixed plate is the plurality of capillaries.
The fluorescence detection type electrophoretic device according to claim 2, wherein the fluorescence detection type electrophoretic device is held by being adhered to the .