JPH09127058A - Cataphoresis device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To bring the excitation time of a fluorescent labeled molecule which is subjected to molar amount separation close to an optimum excitation time where sensitivity can be maximized in a laser induction fluorescent measurement cataphoresis device. SOLUTION: A fluorescent labeled sample is eluted from the lower edge of gel into a buffer liquid by cataphoresis and is subjected to fluorescent measurement in the buffer liquid. The strength of electric field applied to both edges of gel is periodically changed with a cycle of T1+T2+T3, where T1, T2, and T3 can be arbitrarily set. The strength of electric field is set to a constant value at the time zone of T1 and no excitation laser is applied. The strength of electric field is set to zero at the time zone of T2 and excitation laser is applied and fluorescent exposure is made. The strength of electric field continuously set to zero at the time zone of T3, no excitation laser is applied, and a fluorescent sample remaining at a measurement position is eliminated. The above is repeated for continuos measurement.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrophoresis device for DNA, RNA or protein.

[0002]

2. Description of the Related Art Analysis techniques for DNA, RNA, etc. are becoming more and more important in the fields of medicine and biology including gene analysis and gene diagnosis. In particular, recently, development of a high-speed, high-throughput DNA analysis device has been advanced in relation to the genome analysis plan.

DNA analysis is carried out by separating the molecular weight of a fluorescently labeled sample by gel electrophoresis and detecting it by laser-induced fluorescence. For gel electrophoresis, a flat gel in which acrylamide is polymerized between two glass plates with a gap of about 0.3 mm is often used. The sample applied to the upper end of the slab gel is electrophoresed toward both ends of the slab gel while the molecular weight is separated by applying an electric field to both ends of the slab gel. All the migration paths are irradiated at once from the side of the flat gel with a laser at the position where a certain distance has been migrated, and the separated fragments of the fluorescence-labeled sample passing through the beam are excited. Fluorescence exposure is performed at fixed time intervals, and continuous cycle measurement is performed with the sum of the exposure time and the non-exposure time as the cycle.
The DNA base sequence is determined by analyzing this result.

Recently, instead of a flat gel, a capillary gel in which a gel is polymerized in a fused silica capillary tube has been used. Capillary gel electrophoresis is faster than slab gel electrophoresis because it can apply a larger electric field.
Analytical Chemistry 62,900 (1), which has been attracting attention as a method capable of high resolution.
990)). Usually, one capillary tube is used in the capillary gel electrophoresis apparatus, and on-column measurement is performed by detecting fluorescence in the capillary near the lower end of the capillary tube. Since the entire outer surface of the capillary is coated with polyimide, the coating at the detection position is removed to leave a glass-exposed window. The DNA base sequence is determined by irradiating a laser at this position and exciting the emitted fluorescence of the separated fragment of the fluorescence-labeled sample that migrates inside the capillary by electrophoresis and measuring and analyzing the continuous fluorescence. ing.

However, in the on-column measuring device, the scattering of the laser beam on the capillary surface is large, and therefore, one laser beam is measured at a time.
There is a problem that only the capillaries of books can be handled and the throughput cannot be increased. Therefore, recently, a capillary-ray gel electrophoresis device has been developed that analyzes multiple samples simultaneously by arraying multiple capillaries (Nature (N
ature), 361, 565-566 (1993), JP-A-6
-138037) In this device, the lower end of the capillary array is immersed in a buffer solution, the fragments whose molecular weights have been separated by gel electrophoresis are eluted as they are in the buffer solution, and fluorescence is detected in the areas where there are no capillaries. There is. Further, by slowly flowing the buffer solution in the migration direction, the separated fragments eluted from different capillary gels are mixed with each other in the buffer solution, or the two fragments separated in one capillary gel are separated by the buffer solution. I try not to mix them inside. Avoiding the problem of laser beam scattering on the capillary surface by irradiating the buffer solution in the vicinity of the exit of the capillary array where there is no capillary, to excite the fragments eluted from multiple capillaries collectively and perform simultaneous fluorescence analysis. Is possible.

[0006]

The fluorescent molecule which is being continuously irradiated with laser repeats excitation and emission, but with a certain probability, the fluorescent molecule is decomposed and no more emission occurs. Normal,
In fluorescence measurement, the sensitivity increases as the number of times that one fluorescent molecule emits light increases. Generally, the number of times of light emission increases as the laser output increases and the excitation time increases. However, since the fluorescent molecule decomposes with a certain probability, there is an optimum excitation time Top that increases the sensitivity at a constant laser output.

In flat-plate gel electrophoresis and on-column capillary gel electrophoresis, the excitation time of one fluorescent molecule in a fragment whose molecular weight has been separated is obtained by multiplying the time required for one molecule to pass through a laser beam by the exposure time ratio, It is proportional to the laser beam diameter and exposure time ratio, and inversely proportional to the migration speed of the molecule. Here, the exposure time ratio is defined as the ratio of the exposure time to the fluorescence measurement cycle. The time period in which no exposure is performed in one cycle measurement is T1, the exposure time is T2, and the measurement cycle is T0 = T1.
When + T2 is set, the exposure time ratio becomes T2 / T0. Therefore, the excitation time Tex is given by Equation 1 where L is the migration distance (distance from the sample injection end to the laser beam), T is the migration time (time from the start of migration to detection), and ΔL is the laser beam diameter. .

[0008]

(Equation 1)

That is, since T has a small T, Tex <Top is inadequate for a molecule having a small molecular weight, and Tex> Top has an overexcitation for a molecule having a large molecular weight because T is large. However, the maximum sensitivity cannot be increased beyond the optimum excitation time.

On the other hand, the situation is different in off-column capillary gel electrophoresis in which the separated fragment is eluted from the lower end of the capillary gel into a buffer solution and fluorescence is measured. The buffer solution is constantly flowed in the migration direction so that sample mixing due to diffusion or the like does not occur in the buffer solution. When the buffer solution flow rate is Vf, the excitation time Tex becomes the equation 2, and it can be seen that it does not depend on the migration time, that is, the molecular weight.

[0011]

(Equation 2)

However, in order to suppress the mixing of the sample in the buffer solution, the buffer solution flow rate Vf needs to be higher than the migration speed of the separated fragments in the capillary gel. Therefore, the excitation time Tex is shorter than in the case of flat plate gel electrophoresis and on-column capillary gel electrophoresis, and the difference becomes larger as the migration time, that is, the larger the molecular weight of the molecule.
That is, in off-column capillary gel electrophoresis, Tex <Top is set regardless of the molecular weight, and the excitation is insufficient.

An object of the present invention is to provide a method for improving sensitivity in a laser-induced fluorescence measurement electrophoretic device by correcting a state where the excitation time is insufficient or excessive with respect to an optimum value.

[0014]

In the present invention, the electric field strength applied to both ends of a flat gel or a capillary gel is periodically changed, and laser excitation and fluorescence are performed in a state where the electric field strength during the cycle is zero or relatively low. Expose. In the flat plate gel electrophoresis and the on-column capillary gel electrophoresis, for example, the case of applying the electric field strength and the case of setting the electric field strength to zero are alternately repeated, and laser excitation and fluorescence exposure are performed in a time zone when the electric field strength is zero. In off-column capillary gel electrophoresis, for example, the case where the electric field strength is applied and the case where the electric field strength is set to zero are alternately repeated without flowing the buffer solution that elutes the sample, and it remains without being decomposed with fluorescence exposure in the time zone when the electric field strength is zero. A time period for removing the fluorescent light is provided. The fluorescence is removed, for example, by flowing a buffer solution only in that time zone or by irradiating a strong laser beam to decompose the fluorescent molecules in a short time.

It is considered that the case of applying a constant electric field strength and the case of setting a constant electric field strength are alternately repeated. Slab gel electrophoresis,
In on-column capillary gel electrophoresis, the electric field application time width is T1, the electric field zero time width is T2, laser excitation fluorescence exposure is performed in the time zone of T2, and the cycle measurement of cycle T1 + T2 is performed. The excitation time of one fluorescent molecule in the fragment whose molecular weight has been separated by electrophoresis is different from the conventional technique, regardless of the laser beam diameter, the exposure time ratio, and the migration speed of the molecule,
It becomes a fixed value T2. However, in order to prevent the same fluorescent molecule from being excited in different fluorescence exposure time zones, it is necessary to satisfy the condition of Expression 3.

[0016]

(Equation 3)

T1 may be changed according to the migration time so as to satisfy this condition. Of course, T2 can be set arbitrarily, so that it can be matched with or close to the optimum excitation time Top, and high sensitivity can be achieved.

On the other hand, in off-column capillary gel electrophoresis, the electric field application time width is T1, and the electric field zero time width is T2 +.
T3, laser-excited fluorescence exposure is performed in the time period of T2,
Fluorescence is removed during the period T3, and the cycle is T1 + T2 + T3.
The period is measured. Also in this case, the excitation time of one fluorescent molecule in the fragment whose molecular weight has been separated by electrophoresis is fixed to T2 regardless of the laser beam diameter, the exposure time ratio, and the migration speed of the molecule. Become. Further, in this method, since the fluorescence is removed in the time zone T3, the same fluorescent molecule is not excited in different fluorescence exposure time zones. Therefore, there is no condition for T1. Of course T2
Can be set arbitrarily, so that it can be matched with or close to the optimum excitation time Top, and high sensitivity can be achieved. Further, T1 + T2 may be the fluorescence exposure time zone.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION

(Example 1) In this example, DNA by slab gel electrophoresis was used.
The basic configuration is shown in Fig. 1 for the purpose of nucleotide sequencing. The slab gel 1 has a length of 35 cm, a width of 20 cm, and an interval of 0.
6% T (Total monomer concentratio) containing 7 M urea as a modifier between two 3 mm quartz glass plates.
n), 5% C (Crosslinking material concentration)
It was prepared by polymerizing acrylamide gel of the following concentration.

The fluorescence excitation laser 2 is a He-Ne laser 63.
3 nm (25 mW) was focused on a beam diameter of 0.3 mm, and a position 30 cm from the top of the slab gel was made incident vertically from the side of the slab gel in the migration direction, and all migration paths were excited at one time for fluorescence measurement. went. Bandpass filter 3 (center wavelength 6
Excitation light was completely cut by an interference filter having a half-value width of 40 nm at 65 nm), and fluorescence from all migration paths was exposed and measured at once by a cooling type one-dimensional CCD camera 5 through a camera lens 4.

The DNA sequencing reaction followed the conventional Sanger method. Using M13 universal primer labeled with fluorescent substance Cy5 (emission maximum wavelength 665 nm) sold by Biological Detection System (USA), a sequence reaction is performed for each terminal base species A, C, G, T. It was The four reaction products were set in different migration paths at the top of the slab gel.

The electric field strength applied between the upper and lower ends of the slab gel was periodically changed with time as shown in FIG. That is, the constant electric field strength of the time width T1 is 30 V / cm (1 k
V) section and zero electric field strength section of time width T2 were alternately repeated. The time width T1 was changed stepwise according to the migration time as shown in FIG. 2 so that the same fluorescent molecule was not laser-excited in different exposure time zones. This is because the fragments whose molecular weights have been separated are electrophoresed at a migration distance of 30 cm, and are sufficiently excited in sequence in a unit of a width of 0.3 mm corresponding to the laser diameter to measure fluorescence. Unlike the measurement time, the migration time is defined as the sum of the times during which the electric field is applied (the sum of the time width T1 up to that time).

For example, T1 = 1 second when the migration time is 0 to 1000 seconds, and T1 is 1000 seconds to 2000 seconds.
= 2 seconds, T1 = 3 seconds from 2000 seconds to 3000 seconds, such as 1000 × n seconds to 1000 × (n + 1)
Until the second, T1 = n + 1 second (where n is an integer from 0 to 35) and the electrophoresis was continued for about 10 hours. The time width T2 was fixed at 1 second regardless of the migration time as shown in FIG.
Irradiation of the excitation laser onto the slab gel and fluorescence exposure measurement by a cooling type one-dimensional CCD camera were performed only in this zero second electric field intensity section.

Since the output of the pump laser is sufficiently large (2
5 mW), the fluorescent molecules of the fragment existing on the laser beam having a laser diameter of 0.3 mm were excited by laser irradiation for 1 second until they were almost decomposed. Fluorescence from four migration paths corresponding to different base species was independently measured, and the change over time was analyzed to determine the DNA base sequence of the sample.

By performing the fluorescence measurement by the above method,
The fragments separated by electrophoresis can be excited efficiently without waste,
High-sensitivity measurement has become possible.

In the present embodiment, the time width T1 was changed according to the migration time to control the exposure time of the fragment whose molecular weight was separated. However, other than this method, for example, the time width T1 is kept constant and the applied electric field strength is migrated. You may change according to time.

(Example 2) This example is intended for DNA base sequence determination by 10 capillary gel electrophoresis, and the basic constitution is shown in FIG. Capillary gel is
4% T (Total of 5 cm in length, outer diameter 0.35 mm, inner diameter 0.1 mm) containing 7 M urea as a modifier in a fused silica tube
monomer concentration), 5% C (Crosslinking mate
It was prepared by polymerizing acrylamide gel at a concentration of rial concentration). The polyimide coating was removed at a position 30 cm from the injection end of the capillary to prepare a fluorescence measurement window in advance, and the migration distance was fixed at 30 cm. As shown in 6 of FIG. 3, ten of these capillary gels were arranged in a two-dimensional array with a pitch of 0.35 mm and fixed to the horizontally movable stage 8. However, in FIG. 3, only the vicinity of the measurement window of the capillary gel is drawn. The measurement window was aligned in the direction of the capillary gel array, and this part of the stage was a quartz glass plate.

The fluorescence excitation laser 7 is an Ar ion laser 48.
Beam diameter 0.1 mm at 8 nm and 515 nm (40 mW)
Then, the sample was focused on the sample and was irradiated onto the measurement window of one capillary gel from the direction obliquely above the stage at 45 degrees. Movable stage is 0.
The capillary gel was moved in units of 35 mm in the direction of the capillary gel arrangement (the direction of the arrow in FIG. 3), and all the capillary gel was sequentially irradiated with laser light, and fluorescence was measured from below the stage. As shown in FIG. 3, the emitted fluorescence is converted into a parallel light flux by the objective lens 9 and then transmitted through the excitation light cut filter 10 and is separated into four wavelength components by the three dichroic mirrors 11,
Measurement was performed at once with four sets of photomultipliers 12.

The sequence reaction of 10 kinds of samples was carried out using a reaction kit manufactured by Perkin Elmer Co. according to the Sanger method. The prepared DNA fragment contains terminal base species A, C,
Corresponding to G and T, four types of phosphor JOE (maximum emission wavelength 5
50 nm), FAM (maximum emission wavelength 520 nm), TA
MRA (maximum emission wavelength 580 nm) and ROX (maximum emission wavelength 605 nm) were labeled. Three sets of dichroic mirrors were designed so that these four types of fluorescence were wavelength-selected and incident on the four sets of photomultipliers shown in FIG. Four kinds of reaction products corresponding to the terminal base species were mixed for each sample species and then concentrated 10 times by ethanol precipitation to replace the sample solvent with formamide. The injection ends of 10 capillary gels were immersed in 10 kinds of sample solutions, and a constant electric field strength of 100 V / cm (5 kV) was applied to both ends of the capillary gel for 5 seconds to perform sample injection.

The electric field strength applied to both ends of the capillary gel was periodically changed with time as shown in FIG. That is, the constant electric field strength section of the time width T1 is 200 V / cm (10
kV) and the electric field intensity zero section of the time width T2 were alternately repeated. The time width T1 was changed stepwise according to the migration time as shown in FIG. 4 so that the same fluorescent molecule was not laser-excited in different exposure time zones. This is because the fragments whose molecular weights have been separated have a migration distance of 30 cm, and are sufficiently excited in order to measure fluorescence in a unit of a width of 0.1 mm corresponding to the laser diameter without leakage. Unlike the measurement time, the migration time is defined as the sum of the times during which the electric field is applied (the sum of the time width T1 up to that time). For example, T1 = 0.5 seconds, 1500 when the migration time is from 0 seconds to 1500 seconds
From 1 second to 3000 seconds T1 = 1 second, 3000 seconds to 450 seconds
T1 = 1.5 seconds until 0 seconds, 1500 × n
From 1 second to 1500 × (n + 1) seconds, T1 = 0.5 × (n
+1) seconds (where n is an integer from 0 to 5) about 2
The electrophoresis was continued for half an hour. The time width T2 was fixed at 15 seconds regardless of the migration time as shown in FIG. Only during this zero second electric field intensity section, movement of the movable stage, irradiation of the excitation laser to the capillary gel, and fluorescence exposure measurement by four sets of photomultipliers were performed. The movable stage was moved in units of 0.35 mm at intervals of 1.5 seconds, and 10 capillary gels were sequentially excited. The fluorescence exposure time per capillary gel was fixed at 1 second, and during the remaining 0.5 seconds, the stage was moved and data processing by the computer was performed. The output of the pump laser is large enough (40 m
W), the fluorescence from the fragment existing on the laser beam with the laser diameter of 0.1 mm was excited by laser irradiation for 1 second until it was almost decomposed. The time changes of the four types of signals obtained by the four sets of photomultipliers were analyzed by a computer for every 10 capillary gels, and the DNA base sequences of the 10 types of samples were determined. By performing the fluorescence measurement by the above method, the fragments electrophoretically separated can be efficiently excited without waste, and high-sensitivity measurement becomes possible.

In this embodiment, the laser diameter is 0.1 mm.
The electric field intensity was turned on and off in order to excite fluorescence sufficiently without leaking in order with the width of the unit as a unit, but the following method is also possible other than this method. By keeping the electric field strength constant and irradiating a pulse of a high-power laser every time the fluorescent-labeled fragment migrates by a width of 0.1 mm corresponding to the laser diameter, various fluorescent lights are sequentially migrated to the position of the laser beam. The labeled fragments can be sequentially excited without any omission and fluorescence can be measured.

(Embodiment 3) This embodiment is aimed at determining the DNA base sequence by electrophoresis of 20 capillary gels, and the basic constitution is shown in FIGS. Capillary gel is a 4% T, 5 containing a 7M urea as a modifier in a fused silica tube having a length of 30 cm, an outer diameter of 0.2 mm and an inner diameter of 0.1 mm.
It was prepared by polymerizing an acrylamide gel having a concentration of% C. The inner surface of the capillary was chemically silanized by subjecting the inner surface of the capillary to a silanization treatment in advance. As shown in 13 of FIGS. 5 and 6, the 20 capillary gels are arranged in a two-dimensional array with 0.4 mm pitch, and the elution ends of the separated fragments are aligned and immersed in the buffer solution 20 in the measuring cell 15. did. However, in the capillary gels of FIGS. 5 and 6, only the elution end portion was drawn.

The fluorescence excitation laser 14 is a YAG laser 532.
nm (20 mW) and He-Ne laser 594 nm (1
(0 mW) is made coaxial, then the beam diameter is focused to 0.2 mm, and 0.2 mm below the elution end of the capillary gel in the buffer solution is irradiated in the direction of the capillary gel array, and 20 capillary gels are electrophoresed. The fragments eluted in buffer were excited at once. That is, a space of 0.1 mm was provided between the laser beam diameter and the capillary gel elution end to prevent the laser beam from being scattered by the capillary gel. The fluorescence measurement was performed in the direction perpendicular to the plane of the capillary gel array. Corresponds to an image splitting prism that splits an image into four in the vertical direction and a light flux that connects four images by collimating 20 fluorescent light emission point groups arranged in a row with a width of 8 mm in the horizontal direction by the first camera lens 16 The four types of combination filters 17 thus made were transmitted and an image was formed by the second camera lens 18. The fluorescence selection method is described in detail in JP-A-2-269936. The fluorescent emission point group developed in a two-dimensional manner into 20 × 4 = 80 pieces was subjected to exposure measurement at one time by a cooling type two-dimensional CCD camera 19.

The sequence reaction of 20 kinds of samples was performed according to the Sanger method. The prepared DNA fragments contained four types of fluorescent substances Cy3 (maximum emission wavelength 565 nm) and TRITC (maximum emission wavelength 580) corresponding to the terminal base species A, C, G, and T.
nm), Texas Red (maximum emission wavelength 615 nm), C
y5 (maximum emission wavelength 665 nm) was labeled. Where C
y3 and Cy5 are sold by Biological Detection Systems, Inc. (US), TRITC, Texas
Red is sold by Molecular Probes. Four types of combination filters were designed so that these four types of fluorescence were wavelength-selected and imaged on a two-dimensional CCD camera. Four kinds of reaction products corresponding to the terminal base species were mixed for each sample species and then concentrated 10 times by ethanol precipitation to replace the sample solvent with formamide. The sample injection ends of 20 capillary gels were immersed in 20 kinds of sample liquids, respectively, and 100 V / cm (3k) was applied to both ends of the capillary gel.
The sample was injected by applying a constant electric field strength of V) for 2 seconds.

The electric field strength applied to both ends of the capillary gel was periodically changed with time as shown in FIG. That is, a constant electric field strength section of time width T1 is 200 V / cm (6 k
V) and the electric field intensity zero section of the time width T2 + T3 were alternately repeated. The time width T1 was fixed at 1 second regardless of the migration time as shown in FIG. Unlike the measurement time, the migration time is defined as the sum of the times during which the electric field is applied (the sum of the time width T1 up to that time). As shown in FIG. 7, the time widths T2 and T3 of the zero electric field strength section were fixed at 0.5 seconds, regardless of the migration time. Irradiation of excitation laser into buffer solution, two-dimensional CC only for T2 section of 0.5 seconds
Fluorescence exposure with a D camera was performed. Further, only in the subsequent T3 section for 0.5 seconds, the buffer solution in the measurement portion was caused to flow in the direction of the arrow in FIG. 6 to completely remove the fluorescence of the fragment eluted and suspended from the capillary gel from the vicinity of the measurement position. . The measurement of the total 2 second cycle was continued for about 4 hours (migration time was about 2 hours). The time change of four kinds of signals corresponding to the four kinds of fluorescence obtained by the two-dimensional CCD camera is calculated by the computer.
D of 20 kinds of samples analyzed by each capillary gel of the book
The NA base sequence was determined. By performing the fluorescence measurement by the above method, the fragments electrophoretically separated can be efficiently excited without waste, and high-sensitivity measurement becomes possible.

(Embodiment 4) This embodiment is intended for DNA base sequence determination by 96 capillary gel electrophoresis, and the basic constitution is shown in FIG. Capillary gel is
4% T, 5% C containing 7M urea as a modifier in a fused silica tube having a length of 30 cm, an outer diameter of 0.2 mm and an inner diameter of 0.1 mm.
It was prepared by polymerizing acrylamide gel of the following concentration. The inner surface of the capillary was chemically silanized by subjecting the inner surface of the capillary to a silanization treatment in advance.

The 96 capillary gels are shown in FIGS.
A quartz cell 24 having 96 holes arranged in a 12 × 8 grid pattern as shown in FIG. However, the capillary gel in FIG. 8 depicts only the elution end side of the sample fragment. The whole cell and each hole are the buffer solution 20 in the buffer solution tank 23.
Filled with All 96 holes of the cell were made the same shape. As shown in Fig. 9, the size of the whole cell is 12 mm in the lattice plane.
The thickness was × 8 mm and the thickness was 10 mm. As shown in FIG. 10, the unit of the lattice was 1 mm × 1 mm and the thickness was 10 mm, and the hole was penetrated in the center with 0.4 mm × 0.4 mm. A capillary gel having an outer diameter of 0.2 mm was placed at the center of the hole, and the elution end was fixed 2 mm before the bottom surface of the cell.

The fluorescence excitation laser 32 is an Ar ion laser 4
Beam diameter of 0.3 mm at 88 nm and 515 nm (40 mW)
As shown in FIG. 11 in which the cell is viewed from the bottom surface, the laser beam is repeatedly turned back by seven right-angle prisms 31 to collect 96 pieces of light, The fragments eluted in the buffer by electrophoresis from the capillary gel were excited at one time. 0.15 m between the laser beam diameter and the elution end of the capillary gel
An interval of m 2 was provided to prevent the laser beam from being scattered by the capillary gel. Also, to prevent the laser beam passing through the cell from being attenuated by reflection, scattering, absorption, etc.,
The inner surface precision of 96 holes was adjusted.

On the other hand, He--C is used as the fluorescence decomposition laser 33.
d laser 325nm (100mW) beam diameter 0.3mm
Fragments eluted at 0.3 mm below the elution end of the capillary gel in a buffer solution by electrophoresis from 96 capillary gels in the reverse route to the Ar ion laser as shown in FIG. Was irradiated and decomposed at once. As shown in FIG. 8, the fluorescence measurement was performed from the bottom direction of the cell, that is, vertically downward. The 96 fluorescent light emission point groups arranged in a grid pattern of 12 mm x 8 mm are made into a parallel light beam by the first camera lens 25, and an image division prism that divides the image into four in the side direction of 8 mm and a light beam that connects the four images are formed. The corresponding four combination filters 26 were transmitted and an image was formed by the second camera lens 27.

Incidentally, this fluorescence selection method is disclosed in
-269936. 96 x 4 = 384
The fluorescent point groups individually developed in a two-dimensional manner were subjected to exposure measurement at once with a cooling type two-dimensional CCD camera 28.

The sequence reaction of 96 kinds of samples was carried out using a reaction kit manufactured by Perkin Elmer Co. according to the Sanger method. The prepared DNA fragment contains terminal base species A, C,
Corresponding to G and T, four types of phosphor JOE (maximum emission wavelength 5
50 nm), FAM (maximum emission wavelength 520 nm), TA
MRA (maximum emission wavelength 580 nm) and ROX (maximum emission wavelength 605 nm) were labeled. The wavelengths of these four types of fluorescent light are selected and imaged on a two-dimensional CCD camera.
A kind of combinatorial filter was designed. Four kinds of reaction products corresponding to the terminal base species were mixed for each sample species and then concentrated 10 times by ethanol precipitation to replace the sample solvent with formamide. The sample injection ends of the 96 capillary gels were respectively immersed in 96 types of sample liquids, and a constant electric field strength of 100 V / cm (3 kV) was applied to both ends of the capillary gel for 2 seconds for sample injection.

The electric field strength applied to both ends of the capillary gel was periodically changed with time as shown in FIG. That is, a constant electric field strength section of time width T1 is 200 V / cm (6 k
V) and the electric field intensity zero section of the time width T2 + T3 were alternately repeated. The time width T1 was fixed at 1 second regardless of the migration time as shown in FIG. Unlike the measurement time, the migration time is defined as the sum of the times during which the electric field is applied (sum of the time width T1 up to that time). As shown in FIG. 13, the time widths T2 and T3 of the zero electric field strength section were fixed to 1 second, respectively, regardless of the migration time. Irradiation of excitation laser into buffer solution, 2D CCD only for T2 section for 1 second
Fluorescent exposure with a camera was performed. Further, only in the T3 section for 1 second, irradiation of the decomposition laser into the buffer solution, and the sample fluorescence which was eluted from the capillary gel and suspended in the vicinity of the measurement position were completely decomposed and removed. The measurement of the above total 3 second cycle was continued for about 6 hours (migration time was about 2 hours). The time change of four kinds of signals corresponding to four kinds of fluorescence obtained by a two-dimensional CCD camera was analyzed by a computer for each 96 capillary gels, and the DNA base sequences of 96 kinds of samples were determined. By performing the fluorescence measurement by the above method, the fragments electrophoretically separated can be efficiently excited without waste, and high-sensitivity measurement becomes possible.

In the above four embodiments, the applied electric field strength was changed stepwise, but other than this, for example, it may be changed sinusoidally and the valley portion of the sinusoidal wave may be used as the exposure time.

[0044]

EFFECTS OF THE INVENTION According to the present invention, it becomes possible to irradiate a fluorescently labeled sample whose molecular weight has been separated by electrophoresis with a laser beam and to expose it with fluorescence in an optimum excitation time, and to improve the fluorescence measurement sensitivity.

[Brief description of the drawings]

FIG. 1 is a perspective view of an apparatus according to a first embodiment.

FIG. 2 is an explanatory diagram of an electric field strength periodic function according to the first embodiment.

FIG. 3 is an explanatory diagram of an apparatus according to a second embodiment.

FIG. 4 is an explanatory diagram of an electric field strength periodic function according to the second embodiment.

FIG. 5 is a perspective view of the device according to the third embodiment.

FIG. 6 is an explanatory diagram of a measuring cell according to a third embodiment.

FIG. 7 is an explanatory diagram of an electric field strength periodic function according to the third embodiment.

FIG. 8 is a perspective view of the device according to the fourth embodiment.

FIG. 9 is a perspective view of a measuring cell according to a fourth embodiment.

FIG. 10 is an explanatory diagram of a grid unit of the measurement cell according to the fourth embodiment.

FIG. 11 is an explanatory diagram of fluorescence excitation laser irradiation of Example 4.

FIG. 12 is an explanatory diagram of fluorescence decomposition laser irradiation of Example 4.

FIG. 13 is an explanatory diagram of an electric field strength periodic function according to the fourth embodiment.

[Explanation of symbols]

1 ... Flat gel, 2 ... Excitation laser, 3 ... Band pass filter, 4 ... Imaging lens, 5 ... One-dimensional CCD camera.

Claims (14)

[Claims] 1. A laser-induced fluorescence measurement electrophoretic device, comprising: an electric field strength applying means for repeatedly or continuously changing between different kinds of electric field strengths; and a means for receiving fluorescence in synchronization with the repetition. An electrophoretic device comprising means for controlling exposure to measure fluorescence. 2. The electrophoretic device according to claim 1, further comprising means for removing the fluorescence-labeled sample existing at the laser irradiation position. 3. The electrophoretic device according to claim 1, wherein the plurality of types of electric field strengths change according to an electrophoretic time. 4. The electrophoretic device according to claim 1, wherein the applied electric field strength in the fluorescence exposure time zone is zero or substantially zero. 5. The electrophoretic medium according to claim 1, 3 or 4, wherein the electrophoretic medium is a flat gel, and a position in the flat gel where the sample has been electrophoresed for a predetermined distance is irradiated with a laser from the side of the flat gel to cause a plurality of electrophoretic migrations. An electrophoretic device that excites a single path at a time or scans a laser to sequentially excite multiple migration paths to measure fluorescence. 6. The electrophoretic medium according to claim 1, wherein the electrophoretic medium is one or more capillary gels, and the position in the capillary gel where the sample has been electrophoresed for a certain distance is irradiated with laser to obtain one or more capillary gels. An electrophoretic device that excites a capillary gel at a time or scans a laser to sequentially excite one or more capillary gels to measure fluorescence. 7. An electrophoretic device according to claim 2, 3 or 4, wherein the fluorescence removal time period and the fluorescence exposure time period are alternately repeated to perform continuous measurement. 8. The electrophoresis apparatus according to claim 2, 3, 4, or 7, wherein the fluorescence measurement is performed in a buffer solution in which the fluorescence-labeled sample is eluted from the end of the electrophoresis medium. 9. The electrophoretic medium according to claim 8, wherein the electrophoretic medium is a slab gel, and a buffer solution in which the sample is eluted from the end of the slab gel is irradiated with a laser from the side direction of the slab gel to form a plurality of migration paths at once. An electrophoretic device that excites or scans a laser to sequentially excite a plurality of migration paths to measure fluorescence. 10. The electrophoretic medium according to claim 8, wherein the electrophoretic medium is one or more capillary gels, and the buffer solution in which the sample is eluted from the end of the capillary gel is irradiated with a laser to expose the one or more capillary gels once. An electrophoretic device that performs fluorescence measurement by exciting at least one or more capillary gels in sequence by scanning a laser. 11. The electrophoresis apparatus according to claim 9, 10 or 11, wherein the fluorescence removing means causes a buffer solution in the fluorescence measurement region to flow. 12. The fluorescence removing means according to claim 8, wherein the fluorescence removing means irradiates a fluorescence measuring region with a fluorescence removing laser, and the laser decomposes the phosphor at a speed higher than that of the fluorescence measuring excitation laser. Electrophoresis device. 13. The electrophoresis device according to claim 8, 9 or 10, wherein a wall is provided to suppress the diffusion of the sample eluted in the buffer solution. 14. The method of claim 1, 2, 3, 4, 5, 6, 7,
An electrophoretic device in 8, 9, 10, 11, 12, or 13 in which an irradiation time zone to the fluorescence measurement position of the laser synchronized with the fluorescence exposure time zone is provided.