JP2000258392A - Cataphoresis device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cataphoresis device that is suited for obtaining an accurate cataphoresis spectrum and is also suited for comparing the measurement results of a sample that migrates in a plurality of migration paths even if their length differs. SOLUTION: After a fluorescence-labeled sample is injected, voltage is applied to a capillary end 90a. The sample migrates and a DNA fragment is separated according to molecular weight. When a laser beam is applied from a laser 108 to a capillary array, fluorescence is generated from each fragment, and a DNA fragment image for each capillary is detected by a two-dimensional detector 118. The detection signal is analyzed and displayed by fluorescence analysis parameters that differ for each capillary by a data-processing unit 119. Also, the detection result is displayed after being subjected to processing for correcting a migration time difference that is generated since a capillary length differs, or electric field strength being generated in the capillary is set essentially to the same so that the time difference can be canceled.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an electrophoresis apparatus, and more particularly to an electrophoresis apparatus suitable for determining the base sequence of DNA.

[0002]

2. Description of the Related Art DNA and protein analysis techniques are important in the fields of medicine and biology, including gene analysis and genetic diagnosis. Particularly, recently, high-throughput DNA analyzers have been developed. These analyzes are mainly performed by gel electrophoresis.

[0003] Gel electrophoresis uses a gel-like substance as a separation medium for electrophoresis, and is usually detected by laser-induced fluorescence detection. For example, when the measurement sample is DNA, a fluorescently labeled DNA is prepared, and a fluorescently labeled DN is placed on the upper end of an acrylamide gel (flat gel) formed between two glass plates.
Inject A sample. Thereafter, an electric field is applied to both ends of the gel to cause each component to migrate toward the lower end while separating the molecular weight, irradiate a position where the gel has migrated by a certain distance with a laser, and detect a fluorescent component passing through the position. By analyzing this result, D
The NA base sequence can be determined and the polymorphism of the restriction enzyme fragment can be identified.

[0004] Recently, capillary gels obtained by polymerizing and filling gels in quartz capillaries (capillaries) have been used instead of flat gels. Capillary gel electrophoresis has attracted attention as a method capable of high speed and high separation because a larger electric field can be applied compared to slab gel electrophoresis (Analytical Chemistry 62,900 (199
0)). Usually, a capillary gel electrophoresis apparatus uses one capillary tube, irradiates the inside of the capillary near the lower end with a laser, and performs on-column measurement for fluorescence detection.

[0005] Further, a capillary array gel electrophoresis apparatus has been reported in which a plurality of capillaries are arrayed to simultaneously analyze a large number of samples for the purpose of improving the throughput. One of them is a capillary array scanning method (Nature, 359, 167 (1992)) in which a plurality of capillary arrays are sequentially moved, and the capillaries are sequentially irradiated with lasers one by one to measure on-column fluorescence. Others are capillary array sheath flow method (Nature, 361, 565-5
66 (1993) and JP-A-06-138037). In this device, the lower end of the capillary array is immersed in a sheath liquid, and the sample components whose molecular weight has been separated by gel electrophoresis are eluted as they are in the sheath liquid. Are simultaneously detected, and sample components separated by a plurality of capillaries are simultaneously measured.

[0006]

In the capillary array gel electrophoresis apparatus, the arrangement at the sample injection section is mainly a microtiter plate-like (9 mm intervals).
The detection units are densely arranged (0.2 mm intervals, 0.4 mm intervals, etc.), and the length of each capillary gel differs. Therefore, when the same voltage is applied as usual,
Since the electric field strength and the like in each capillary gel are different and the migration speed changes depending on the length of the capillary gel (length of the migration path), it is difficult to compare the measurement results for each sample,
It is difficult to make evaluations such as determining abnormal measurements, etc.
The measurement results were difficult to understand.

Further, when detecting a plurality of wavelength components of fluorescence emitted from the phosphor by light irradiation, and calculating each component of the plurality of phosphors from the plurality of wavelength components of the fluorescence,
Normally, one type of operation coefficient is used, but the optimum value differs for each migration path, and as a result, the operation accuracy is likely to be reduced.

One object of the present invention is to provide an electrophoresis apparatus suitable for obtaining an accurate migration spectrum.

Another object of the present invention is to provide an electrophoresis apparatus suitable for facilitating the comparison of the measurement results of a sample migrated in a plurality of migration paths even if the lengths of the plurality of migration paths are different. Is to do.

[0010]

According to an aspect of the present invention, there is provided an electrophoresis apparatus comprising: a plurality of migration paths through which a sample labeled with a fluorescent material migrates; Means for irradiating the migration path with light, means for detecting each of the plurality of wavelength components of the generated fluorescence, and means for analyzing and displaying the detection result, wherein the analysis display means displays the detection result as the It is characterized by analyzing and displaying with different parameters for each migration path.

According to another aspect of the electrophoresis apparatus of the present invention, a plurality of migration paths on which a sample labeled with a fluorescent substance migrates, and the electrophoresis path are arranged so as to generate fluorescence from the fluorescent substance. -Means for irradiating the light, means for detecting a plurality of wavelength components of the generated fluorescence, and means for analyzing and displaying the intensity of the plurality of wavelength components of the detected fluorescence, and the analysis display The means corrects the intensities of the plurality of wavelength components of the fluorescence with different fluorescence analysis parameters for each of the migration paths.

According to still another aspect, the electrophoresis apparatus of the present invention includes a plurality of migration paths on which a sample labeled with a fluorescent substance migrates, and a plurality of migration paths for generating fluorescence from the fluorescent substance. Means for irradiating laser light, means for detecting a plurality of wavelength components of the generated fluorescence, and means for analyzing and displaying the intensities of the plurality of wavelength components of the detected fluorescence. The display means corrects and displays a migration time difference of the sample based on a difference in length of the plurality of migration paths.

According to another aspect of the electrophoresis apparatus of the present invention, there are provided a plurality of migration paths on which a sample labeled with a fluorescent substance migrates, and means for forming an electric field for causing the sample to migrate on the plurality of migration paths. Means for irradiating the migration path with laser light so as to generate fluorescence from the phosphor, and means for respectively detecting a plurality of wavelength components of the generated fluorescence,
The electric field forming means includes means for generating a voltage for forming the electric field and generating an independent voltage for each of the plurality of migration paths.

According to still another aspect, the electrophoresis apparatus of the present invention forms a plurality of migration paths on which a sample labeled with a fluorescent substance migrates, and an electric field for causing the sample to migrate on the plurality of migration paths. Means, means for irradiating the migration path with laser light so as to generate fluorescence from the phosphor, and means for respectively detecting a plurality of wavelength components of the generated fluorescence, wherein the electric field forming means includes The apparatus further includes means for generating a voltage for forming the electric field and generating an independent voltage for each migration path group in which the plurality of migration paths are divided according to their length.

According to still another aspect, the electrophoresis apparatus of the present invention forms a plurality of migration paths on which a sample labeled with a fluorescent substance migrates, and an electric field for causing the sample to migrate on the plurality of migration paths. Means for applying a voltage to each of the plurality of migration paths via an external resistor, and means for irradiating the migration paths with laser light so as to generate fluorescence from the phosphor,
Means for detecting a plurality of wavelength components of the generated fluorescence, respectively, wherein the resistance value of each external resistance is the sum of the external resistance value and the internal resistance value of the migration path corresponding to the external resistance value. Are set to be substantially the same.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION As an embodiment of the present invention, an electrophoresis apparatus capable of determining a base sequence of DNA is used. This embodiment uses a plurality of capillaries filled with gel, that is, a capillary array, and performs measurement in a sheath flow cell.

Prior to description of an electrophoresis apparatus showing an embodiment based on the present invention, a method for preparing a sample for DNA sequencing (fluorescently labeled DNA fragment) will be described first. In order to identify four base species in one migration path (gel capillary), a primer labeled with a different fluorescent substance for each of A (adenine), C (cytosine), G (guanine) and T (thymine) fragments was used. use. The fragment A has a primer (abbreviated as primer A) labeled with a fluorescent substance having a maximum fluorescence wavelength of about 550 nm, and the C, G, and T fragments each have a fluorescence maximum wavelength of about 520, 575, and 605, respectively.
Primers labeled with a phosphor of nm (abbreviated as primer C, primer G and primer T, respectively) are used. Using these, a DNA polymerase reaction is carried out by the well-known dideoxy method of Sanger et al. To prepare a fluorescence-labeled DNA fragment. These fragment groups are finally mixed in a single sample tube, and after ethanol precipitation, dissolved in deionized formamide (including a trace amount of Tris buffer or EDTA solution) to obtain a sample solution. Before injecting the sample solution into the gel capillary, the sample solution is heat-denatured by heating at 95 ° C. for 2 minutes, and then immediately cooled on ice and used for the injection operation. In addition,
When a reagent labeled with a terminator instead of a fluorescent label is used as a primer, a sample can be prepared by a well-known method and used similarly for measurement.

FIG. 1 shows a capillary array and a sheath flow cell section serving as a fluorescence measuring section of an electrophoresis apparatus according to an embodiment of the present invention.

Using forty-eight capillaries filled with gel inside, a capillary array is prepared in which one ends thereof are arranged in a straight line. The capillary used here can have various inner and outer diameters. For example, a capillary having an inner diameter of 0.075 mm, an outer diameter of 0.2 mm and a length of about 20 cm is used, and a well-known method is used so as to function as a migration path. Then, use acrylamide gel after filling it. The gel concentration is 4% T (ratio to the total solution amount of (acrylamide + bisacrylamide)) and 5% C (ratio of bisacrylamide to (acrylamide + bisacrylamide)). In addition, the surface of the capillary is coated with polyimide for the purpose of preventing breakage at the time of bending or the like. In this embodiment, a black coating obtained by mixing carbon into polyimide is used. Of course, other coatings besides polyimide can be used. Further, the gel to be filled in the capillary can have various concentrations other than those described above, and may be filled with a substance such as a polymer instead of the gel.

FIG. 1 shows a capillary array array composed of eight capillaries for easy understanding. Parts such as an O-ring for sealing the liquid are omitted. Capillary holders 91 and 92 are formed with mounting grooves at 3 mm intervals and 0.4 mm intervals, respectively.
Capillary arrays can be easily attached. First, forty-eight capillaries 90 are arranged on the same plane and fixed to the capillary holders 91 and 92 so that one end is spaced by 3 mm and the other end is spaced by 0.4 mm.
Cut and align their edges. Capillary ends 90a fixed at 3 mm intervals are ends on the sample injection side, and 90b on the opposite side is a capillary end from which the electrophoretically separated sample fragments are discharged. Capillary end 90b is capillary
Attached to the sheath flow cell section together with the holder 91.

The capillary of the capillary holder 92 is
On the holder 91 side, in addition to the 48 capillaries 90, six dummy glass rods 93 are attached to the outside at the same intervals as the capillaries 90. The dummy glass rod 93 is provided to make the individual environments of the ends 90b of the capillary 90 all the same, and may have an outer diameter substantially equal to that of the capillary 90. In this embodiment, the capillary is the same as the capillary 90, and the inside is filled with a gel. The number of dummy glass rods 93 depends on the width of the cross section of the flow path of the sheath flow cell, and is adjusted so that the capillaries are uniformly present over the entire width. With such a dummy glass rod 93, the environment of each sheath flow at the capillary end 90b becomes the same, and the capillary 9
Thus, a stable and uniform flow can be formed from the first 0 to the 48th. The dummy glass rod is not indispensable in the configuration of the apparatus, and the measurement itself can be performed without the dummy glass rod.

In the sheath flow cell section, the cross section of the flow channel is 0.1 mm.
A sheath flow cell 83 made of quartz glass having a size of 23 mm × 24.2 mm is included, and upper and lower portions thereof are fixed by holding members 82 and 84 for fixing the sheath flow cell. Holding tool 82,
Reference numeral 84 has a space in which a liquid for sheath (sheath liquid) flows. The sheath liquid is a buffer equivalent to the one prepared from acrylamide gel. The sheath liquid forms a flow path from the sheath liquid container 80 via the tube 81, through the holder 82, the flow cell 83, and the holder 84, and flows into the waste liquid container 86 via the tube 85.

By arranging the sheath liquid container 80 higher than the waste liquid container 86, the sheath liquid flows slowly and stably through the flow channel according to the siphon principle, and a sheath flow is formed near the end 90b of the capillary 90. Become. Attachment of the capillary 90 to the sheath flow cell portion is performed after the inside of the sheath flow cell is filled with the sheath liquid in order to reduce as much as possible damage due to drying of the capillary end face 90b. Similarly, the capillary end face 90a is held in a buffer solution after cutting in order to reduce damage due to drying as much as possible.

FIG. 2 shows an overall configuration of an electrophoresis apparatus according to one embodiment of the present invention. The capillary holder 92, the capillary 90, the capillary holder 91, and the sheath flow cell 83 are arranged in the same plane, and they are fixed vertically upright so that the sheath flow cell 83 side faces upward. It flows upward from below in the flow cell 83. At the lower part of the capillary 90, a sample container 100 having 48 wells formed at the same interval as the ends 90a arranged in a line (each well holds a sample solution to be measured), and an electrode tank for storing a buffer solution 101, a cleaning liquid tank 102 for storing a cleaning liquid is arranged side by side on a moving table 103, and these are driven up and down, left and right (sample container 100, electrode tank 101, (The direction in which the washing liquid tanks 102 are arranged), and the capillary end 90a can be inserted into a sample solution, a buffer solution, or a washing solution depending on the purpose.

A sample liquid for measurement is injected into the 48-well of the sample container 100 with a pipette. A buffer solution is injected into the electrode tank 101, and distilled water is injected into the cleaning liquid tank 102 as a cleaning liquid. First, the capillary end 90a is immersed in the cleaning liquid tank 102 by the moving table 103, and the dust or the buffer solution that may be attached to the tip is washed. Then
The sample liquid in the sample container 100 is brought into contact with the capillary end 90a by the moving table 103, and about 25 V / cm
A voltage of -500 V (negative electrode side) and a sheath liquid side of the sheath flow cell 83 of 0 V (positive electrode side) are applied to apply a voltage to the sample, and the sample is injected into the capillary. In the figure, the positive electrode, the negative electrode, and the high voltage power supply are omitted. The negative electrode can be easily realized by providing a platinum electrode for each of the 48 wells of the sample container 100. As the positive electrode, the sheath flow cell holder 84 is made of stainless steel and the holder 84 itself is used as an electrode, a platinum electrode is provided on the upper part in the sheath flow cell 83, or the waste liquid container 8 is provided.
For example, it is possible to provide a platinum electrode in 6.

After the sample is injected, the moving table 103 is operated, and the capillary end 90a is moved to the electrode bath 101 and inserted into the buffer. A platinum electrode is held in the buffer solution in the electrode tank, and a voltage is applied to the platinum electrode. The voltage to be applied is a voltage -500 at which an electric field intensity of about 25 V / cm is obtained for the first 5 minutes.
V, and then a voltage -1 at which an electric field strength of about 50 V / cm is reached.
000 V, and then a voltage of -1500 V at which an electric field intensity of about 75 V / cm is gradually increased. Capillary 90
When the sample is electrophoresed in the migration path, DNA fragments are separated for each molecular weight and eluted from the capillary end 90b into the sheath flow cell. The eluted sample fragments are subjected to fluorescence measurement in a sheath flow state. By using the sheath flow, the sample fragments eluted from each capillary end 90b flow so as not to be mixed. With this configuration, simultaneous injection of 48 samples, simultaneous electrophoresis and analysis can be realized. In addition, if the sample liquid is previously placed in the sample container 100, the sample injection does not need to be performed manually by pipetting one sample at a time as in the case of the conventional flat gel, thereby improving operability. be able to.

The fluorescence measurement method will be described. As a light source for exciting the sample, an argon laser device 108 having a wavelength of 514.5 nm is used. Argon laser light 112
Is reflected by a mirror 110, and is radiated to the sheath flow cell 83 with a lens (not shown) having a focal length of about 70 mm. The generated fluorescence is imaged on a two-dimensional detector 118 by a condenser lens 114, four types of spectral filters 115a to 115d, an image splitting prism 116, and an imaging lens 117, and is separated from a DNA fragment of each sample separated by electrophoresis. The time waveform of the fluorescence intensity is analyzed and displayed by the data processing unit 119.

In the above configuration, the lengths of the 48 capillaries are different from each other (accurately, symmetrically). This is because, when a large number of capillaries are arranged side by side on the same plane, the capillaries must be densely packed at the detection end in order to obtain a high light collection rate of the fluorescence from the emission point of each capillary. On the other hand, the capillaries cannot be so densely packed at the sample injection end from the viewpoint of ease of sample injection (collection). Specifically, when the length of the center capillary is approximately 200 mm, the length of the outermost capillary is approximately 220 mm, and a difference of about 10% occurs between them. Therefore, when all the capillaries are electrophoresed at the same voltage, the electric field strength in each capillary is different, and the electrophoresis speed of the sample is different for each capillary, so that the sample fragment of the same base length is detected. Will be different. In general, the migration speed is proportional to the electric field strength (when the capillary length is constant), and the time during which the sample fragment is detected (when the electric field strength is constant) is proportional to the capillary length up to the detection position. Therefore, the decrease in the electric field intensity caused by the increase in the capillary length and the delay in the detection time are corrected, and the display is performed with the corrected migration time (described later). Thereby, the difference in the detection time between the capillaries (between the electrophoresis paths) is calibrated, and the comparison for each sample becomes easy. In addition, it is possible to directly compare the migration speed, the peak interval, and the like, and it is easy to determine the abnormality of the sample and the abnormality of the migration including the gel.

The detection of a fluorescent image will be described in more detail with reference to FIGS. The capillary end 90b is arranged in the sheath flow cell 83, and the fluorescent light emitting points 220 and 230 are formed above the capillary end 90b along the laser light path. The fluorescent light emitted from the fluorescent light emitting point 220 passes through the condenser lens 114, is divided into four by the image dividing prism 116, and is imaged by the image forming lens 117 (221a to 221a).
221d). At this time, the four types of spectral filters 115a to 115a
By arranging 15d on the front surface (or the rear surface) of the image splitting prism, it is possible to make each split light to have a different wavelength component. The same applies to the fluorescent light emitting point 230. As can be seen from the figure, the respective light emitting points 220, 2
The fluorescent light from 30 is imaged in the direction parallel to the laser light path (221, 231), and the fluorescent light of different wavelength components from the same light emitting point is emitted by the image splitting prisms 115a to 115d for each light emitting point. An image is formed in a direction perpendicular to the optical path (221a to 221d, 231a to 231d).
1d). The situation is the same for all the light emitting points other than the fluorescent light emitting points 220 and 230 shown.

As is apparent from the above, the fluorescence splitting of each wavelength is performed simultaneously for all the light emitting points. That is,
For example, since there is no time division as in the case of laser beam scanning or mechanical scanning of the detector, the measurement interval of the fluorescence intensity does not become long, and high-speed measurement can be performed. It is also possible to measure continuously in real time.

In this embodiment, all the samples to be migrated on the capillary array can be simultaneously divided into four parts and measured with high accuracy. Therefore, four types of spectral filters 11
By adjusting 5a-d so that the fluorescence of the primer A, the primer C, the primer G, and the primer T is mainly transmitted, it is possible to identify and measure the primer A, the primer C, the primer G, and the primer T. become.

However, as shown in FIG.
The fluorescence of each fluorescent-labeled primer is broad, and the fluorescence of the four fluorescent-labeled primers overlap. Therefore, by correcting the overlap by a matrix operation as described below, primer A, primer C, primer G,
A time waveform of the fluorescence intensity of the primer T alone is obtained. These are the electrophoretic spectra of the A, C, G, and T fragments, respectively, from which the nucleotide sequence of DNA can be determined by an ordinary method.

(Y (t)) = (M) (x (t)) (y (t)): a 4 × 1 matrix showing the fluorescence intensity as a function of time for each base (each element is y1 (t) , y2 (t),
(x (t)): a 4 × 1 matrix showing the measured fluorescence intensities as a function of time, where each element is x1 (t), x2 (t),
(M): 4 × 4 matrix for correction (operation coefficient matrix) (Each element is Mab (a = 1, 2, 3, 4; b = 1, 2, 3, 4)) This matrix (M) has different values (arithmetic coefficients) of its elements depending on the migration path. As can be seen from FIG. 4, the fluorescence emitted from the fluorescence emission point 220 substantially at the center with respect to the entire plurality of migration paths is collimated by the condenser lens 114, and then the light from the spectral filters 115 a to 115 d and the Fluorescence emitted from the fluorescent light emitting point 230 at the end is obliquely incident on the surfaces of the spectral filters 115a to 115d and the image splitting prism 116, while being incident on the surface almost perpendicularly. That is, the angle of incidence of the fluorescent light on the imaging system is different due to the difference in the position of the fluorescent light emitting point. For this reason, the fluorescent light generated from each fluorescent light emitting point differs in a light collection range, a transmission wavelength range, a transmittance, and the like.
Therefore, each element of the operation coefficient matrix (M) (operation coefficient)
Is the fluorescence analysis parameter for each migration path (capillary
Each) will have a different value. That is, it is necessary to perform an operation using an operation coefficient matrix optimized for each migration path. The optimum value for each migration path can be calculated by separately and sequentially electrophoresing the four types of fluorescent labels for each migration path and measuring the fluorescence with the above-mentioned apparatus. FIG. 6 shows an example of the optimum value of the fluorescence analysis parameter, which is each element (calculation coefficient) of the actually calculated calculation coefficient matrix with respect to the migration path position. FIG. 6 shows an example in which another type of terminator labeling reagent is used. The optimum value can be set more finely for each migration path as compared with the case of calculating with a single matrix.

As described above, the fluorescence intensity (y (t)) is obtained by calculation based on the calculation coefficient which is a fluorescence analysis parameter optimized for each migration path.
The migration spectra of the G and T fragments can be calculated more accurately, and a highly accurate base sequence can be determined.

The above-described filter is used to image fluorescent light from different light-emitting points along the laser light path and form light of different wavelengths from each light-emitting point in a direction perpendicular to the laser light path. Instead of the spectral method using & prism, for example, a spectral method using a diffraction grating may be used.

In the embodiment, the method of performing measurement in the sheath flow cell has been described. However, a portion of the capillary is removed, the portion is irradiated with laser light, and the fluorescence in the portion is measured. The same can be applied to the case.

As described above, since the capillary spacing differs between the sample injection side end and the detection side end of the capillary array (migration path array), the migration path lengths, ie, the capillary lengths, are different from each other. The migration speed of the sample is different for each migration path (capillary), and there is a difference in the time for detecting a sample fragment having the same base length for each capillary. According to the present invention, this correction is possible, and one embodiment thereof will be described below.

The migration speed v of a certain DNA fragment in the capillary is approximately proportional to the electric field strength E in the capillary (migration path). The electric field strength E is proportional to a voltage V applied to both ends of the capillary to generate the electric field,
Since it is inversely proportional to the capillary length L (corresponding to the distance from the sample injection end to the detection position in the case of the embodiment), after all, DN
The detection time t of the fragment A is the square of the capillary length L and the voltage V
Are proportional to the reciprocal of. Therefore,
The length of the center capillary is Lc, the length of the other capillaries is Lx, the detection time of the same DNA fragment in the center capillary is tc, and the detection time in the other capillaries is tc. Assuming that the detection time is tx, tx
Is equal to the product of tc and (Lx / Lc) ² . That is, t
x is (Lx / Lc) ² times tc. Therefore, when tx obtained by the measurement is multiplied by (Lc / Lx) ² , the result becomes equal to tc. This is (Lc / Lx) ² tx
= Tc and therefore capillaries of different lengths
The DNA fragments of the same length migrated inside (in the migration path) can be displayed at the same time.

FIG. 7 shows a display example of the sample signal pattern without time correction and the signal pattern with time correction displayed on the data processing unit 119 for the same measurement sample. 7 (A) and 7 (B) show that the sample was pGEM.
(The display is the display of a G fragment.)
An example when the length is about 20 cm is shown. The vertical axis indicates time, and the horizontal axis indicates the position of the capillary. 7A shows data without time correction, and FIG. 7B shows data with time correction based on the embodiment of the present invention. By performing the time correction in this way, it is possible to easily and accurately analyze a base sequence for each sample.

In the above-described embodiments, the voltage V applied to the capillaries is fixed, but the applied voltage is adjusted so that the electric field strength E of each capillary and therefore the migration speed v are substantially constant. By making V different for each capillary, the time at which the same sample fragment is detected,
The difference between the capillaries (the migration path) can be corrected.

FIG. 8 shows a capillary voltage application section of another embodiment of the electrophoresis apparatus according to the present invention. Other parts and functions thereof are substantially the same as those in FIGS.
In this embodiment, the applied voltage V for generating the electric field intensity E is made different for each capillary (each electrophoresis path) so that the electric field intensity E of each capillary and therefore the migration speed v is substantially constant. Thereby, the difference in the time at which the same sample fragment is detected for each capillary is corrected.

In the figure, an electrode tank 101 into which a buffer solution enters
Is divided into small electrode tanks 1011 for each capillary, a buffer solution is filled in each small electrode tank, and an electrode (platinum electrode) 1012 is provided. Similarly, an independent electrode (platinum electrode) is provided in each well of the sample container 100 (not shown). The electrode of each small electrode tank 1011 and the electrode of the corresponding well are respectively connected to the corresponding electrode terminal 1013.
, And their electrode terminals are connected to corresponding independent high-voltage power supplies 1014 via switches 1015, respectively. It is desirable that the electrode 1012 be configured so that it can be used for the sample container 100.

With such a configuration, a voltage corresponding to the length of each capillary (electrophoresis path) can be independently applied. The voltage applied to the central capillary is V
c, assuming that the voltage applied to the other capillaries is Vx, in order to satisfy the condition of E = constant (hence, v = constant), Vx is set to Vx according to the length of each capillary.
= (Lx / Lc) Vc may be set substantially. Specifically, for example, the electric field strength E is set to 100 V /
In order to apply a constant voltage of 2000 V to the central capillary having a length of about 20 cm, it is necessary to set about 22 cm.
The high-voltage power supply may be set so that a voltage of 2200 V is applied to the capillary at the end having an m length.

By doing so, the capillary
Correction of the difference in migration time between (electrophoresis paths) becomes accurate,
Comparison of measurement results for each sample becomes easy. In addition, it is possible to directly compare the migration speed and peak interval, etc.
This makes it easy to determine abnormalities in electrophoresis including gels.

FIG. 9 conceptually shows a capillary voltage applying section of another embodiment of the electrophoresis apparatus according to the present invention. This embodiment uses 48 capillaries.
Are divided into a plurality of groups having similar lengths, and different voltages are applied to the capillaries in each group so that the intensity of the electric field becomes substantially constant. Portions not shown can be considered to be the same as those in the above-described embodiment. In the figure, 1013a indicates nine electrode terminal groups, and these electrode terminal groups correspond to nine corresponding electrode terminal groups.
The switches are connected to corresponding high-voltage power supplies 1014a via switches 1015a so as to apply different voltages to the individual capillary groups (electrophoresis path groups). Capillary groups are represented by capillary numbers (migration path numbers) 1-3, 4-6, 7-10, 11-14, 15-3 in order from the left side of the figure.
4, 35-38, 39-42, 43-45, 46-48
, And each of these groups of capillaries has a capillary length that is close to each other.
(Migration path). The average capillary length of the central group of capillaries is the shortest, and the average capillary length gradually increases from the central group of capillaries to the end groups of capillaries, and therefore the average of the end groups of capillaries. The capillary length is the longest.

A high voltage power supply 1014 is connected to the 48 capillaries.
If different voltages shown in the following table are applied in units of capillary groups (units of migration path groups) from a, the electric field strength of the 48 capillaries can be made approximately 100 V / cm.

Capillary group Applied voltage (average capillary length) (shown by capillary number) 1-3, 46-48 2190V 21.9 cm 4-6, 43-45 2140V 21.4 cm 7-10, 39-42 2100 V 21.0 cm 11 to 14, 35 to 38 2060 V 20.6 cm 15 to 34 2010 V 20.1 cm The embodiment of FIG. 9 has an advantage that the number of high voltage power supplies can be reduced as compared with the embodiment of FIG.

FIG. 10 conceptually shows a capillary voltage application section of an electrophoresis apparatus according to still another embodiment of the present invention. Portions not shown can be considered to be the same as those in the above-described embodiment. The electrical resistance of a capillary (electrophoresis path) filled with a gel or the like is 500 MΩ to 1000 MΩ in a steady state (depending on components). Therefore, in the embodiment of FIG. 10, the electrode terminals 1013 are connected to the high voltage power supply 1014b via the external resistors 1016, and the resistance of each external resistor is determined by the resistance value and the corresponding capillary ( It is set so that the sum with the resistance value of the migration path) is substantially the same. As a result, the electric field intensity of each capillary becomes substantially uniform.
With this configuration, the difference in migration time for each (migration path) is further reduced, and correction is facilitated.

The high-voltage power supply 101 will be described with reference to FIGS.
4, 1014a, 1014b and the resistor 1016 are preferably variable.

[0050]

According to the present invention, there is provided an electrophoresis apparatus suitable for obtaining an accurate migration spectrum. According to the present invention, there is also provided an electrophoresis apparatus suitable for facilitating comparison of measurement results of a sample migrated in the migration paths even if the lengths of the migration paths are different.

[Brief description of the drawings]

FIG. 1 is a configuration layout diagram of a capillary array of an electrophoresis apparatus and a sheath flow cell unit serving as a fluorescence measurement unit according to an embodiment of the present invention.

FIG. 2 is a three-dimensional view showing the overall configuration of an electrophoresis apparatus showing one embodiment according to the present invention.

FIG. 3 is a view in the direction A of FIG. 2;

FIG. 4 is a view as viewed in a direction B in FIG. 2;

FIG. 5 is a diagram showing the relationship between the transmission characteristics of the spectral filter of FIG. 2 and the fluorescence characteristics of a fluorescent labeling primer.

FIG. 6 is a diagram showing an example of an optimum value of each element (operation coefficient = fluorescence analysis parameter) of an operation coefficient matrix used when calculating the fluorescence intensity.

FIG. 7 is a view showing a display example of a sample signal pattern without time correction and a signal pattern with time correction for the same measurement sample.

FIG. 8 is a conceptual diagram of a capillary voltage application section of an electrophoresis apparatus showing another embodiment based on the present invention.

FIG. 9 is a conceptual diagram of a capillary voltage application section of an electrophoresis apparatus showing another embodiment according to the present invention.

FIG. 10 is a conceptual diagram of a capillary voltage application section of an electrophoresis apparatus showing still another embodiment according to the present invention.

[Explanation of symbols]

83: Flow cell, 90: Capillary, 90a, 90
b: Capillary end, 100: sample container, 101: electrode tank, 102: cleaning liquid tank, 103: moving table, 108: argon laser device, 114: condenser lens, 115a to d ...
Spectral filter 116, image splitting prism 117, imaging lens 118, two-dimensional detector 119, data processing unit 220, 230 ... fluorescent light emission points, 221a-d,
231a-d: imaging, 1011: small electrode tank, 1012 ...
Electrode, 1013 ... electrode terminal, 1014, 1014a, 1
014b: High voltage power supply, 1015, 1015a: Switch, 1016: Resistance.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kazunari Imai 882, Omo, Oaza-shi, Hitachinaka-shi, Ibaraki Prefecture Inside the Measurement Instruments Division, Hitachi, Ltd. (72) Inventor Masao Kamahori 1-280 Higashi Koigakubo, Kokubunji-shi, Tokyo Inside the Central Research Laboratory, Hitachi, Ltd. (72) Inventor Susumu Hiraoka 1-280 Higashi Koigakubo, Kokubunji-shi, Tokyo Hitachi, Ltd. Central Research Laboratory F term (reference) 2G043 AA03 BA16 CA03 DA02 EA01 EA19 FA01 FA06 GA04 GB01 GB03 HA01 HA02 HA15 JA03 KA05 KA09 LA03 NA01

Claims (7)

[Claims] 1. A plurality of migration paths on which a sample labeled with a phosphor migrates, means for irradiating the migration path with light so as to generate fluorescence from the phosphor, and a plurality of wavelengths of the generated fluorescence. Means for detecting each of the components, and means for analyzing and displaying the detection result, wherein the analysis and display means analyzes and displays the detection result with different parameters for each of the migration paths. Electrophoresis device. 2. A plurality of migration paths through which a sample labeled with a fluorescent substance migrates, means for irradiating the migration path with laser light so as to generate fluorescent light from the fluorescent substance, Means for respectively detecting a plurality of wavelength components, and means for analyzing and displaying the intensities of the plurality of wavelength components of the detected fluorescence, wherein the analysis and display means determines the intensities of the plurality of wavelength components of the fluorescence by the electrophoresis. An electrophoresis apparatus, wherein correction is performed using different fluorescence analysis parameters for each path. 3. A plurality of migration paths through which a sample labeled with a fluorescent substance migrates, means for irradiating the migration path with laser light so as to generate fluorescent light from the fluorescent substance, Means for detecting each of the plurality of wavelength components, and means for analyzing and displaying the intensity of the plurality of wavelength components of the detected fluorescence, wherein the analysis and display means is based on a difference in the length of the plurality of migration paths. An electrophoresis apparatus, wherein the electrophoresis apparatus corrects and displays a difference in migration time of the sample. 4. A plurality of migration paths on which a sample labeled with a fluorescent substance migrates, means for forming an electric field for causing the sample to migrate on the plurality of migration paths, and a device for generating fluorescence from the fluorescent substance. Means for irradiating the migration path with laser light, and means for detecting a plurality of wavelength components of the generated fluorescence, respectively, wherein the electric field forming means is a voltage for forming the electric field and An electrophoresis apparatus including means for generating an independent voltage for each migration path. 5. The electrophoresis apparatus according to claim 4, wherein the voltage varies depending on the length of the plurality of migration paths. 6. A plurality of migration paths on which a sample labeled with a fluorescent substance migrates, means for forming an electric field for causing the sample to migrate on the plurality of migration paths, and a plurality of fluorescent light generating means for generating fluorescence from the fluorescent substance. Means for irradiating the migration path with laser light, and means for detecting a plurality of wavelength components of the generated fluorescence, respectively, wherein the electric field forming means is a voltage for forming the electric field and An electrophoresis apparatus including means for generating an independent voltage for each migration path group in which the migration path is divided according to its length. 7. A plurality of migration paths through which a sample labeled with a fluorescent substance migrates, and each of the plurality of migration paths is connected via an external resistor so as to form an electric field for causing the sample to migrate in the plurality of migration paths. Means for applying a voltage, means for irradiating the migration path with laser light so as to generate fluorescence from the phosphor, and means for respectively detecting a plurality of wavelength components of the generated fluorescence, The resistance value of each external resistor is set so that the sum of the external resistance value and the internal resistance value of the migration path corresponding to the external resistance value is substantially the same. Electrophoresis device.