Method for continuously using separation medium

Abstract

Provided is a method for continuously using a separation medium, which makes it possible to reduce running cost for an analysis when a liquid polymer is used as the separation medium. The method for c

Description

Continuous use of separation media

[0001] The present invention relates to a method for continuous use of a separation medium.

[0002] The proportion of the population aged 60 and over to the total population will increase in the future, and all developed countries will become super-aged societies. Against this backdrop, interest in people's health has increased significantly in recent years, and there is growing interest in healthcare technologies to maintain health and longevity. Among these technologies, genomic medicine is one of the most notable. Genomic medicine literally applies an individual's genetic information to diagnosis, and aims to apply medical treatment appropriate to each individual patient. The device used to read this genetic sequence information is a DNA sequencer. In other words, a DNA sequencer can be said to be one of the most representative analytical devices.

[0003] The Human Genome Project, which aimed to analyze the entire base sequence of the human genome, was completed in 2003. DNA sequencing technology has made great advances since then, and a variety of sequencing techniques have been developed. Among these, a technology called next-generation sequencing is characterized by the ability to perform sequence decoding in a massively parallel manner. Thanks to this technological innovation, it is now possible to sequence the human genome for less than $1,000.

[0004] However, the Sanger method, a DNA reading method used in capillary sequencing and adopted in the Human Genome Project, remains the gold standard in DNA sequencing. This is due to its high base reading accuracy and its superior analytical cost per sample compared to other methods such as next-generation sequencing. Therefore, the Sanger method used in capillary sequencing remains an indispensable technology in research settings around the world.

[0005] One of the reasons capillary sequencers have established a strong presence in research fields is the use of liquid polymers as a separation medium within the capillary. Before capillary sequencers became widespread, DNA separation was typically performed using a solidified gel between two gel plates. However, preparing this gel required a lot of effort, which placed a burden on users. However, by using liquid polymers, it was discovered that electrophoresis could be performed by simply filling the flow path with the liquid polymer without creating a gel. Liquid polymers allow measurements to be continued by replacing the liquid polymer in the flow path after each measurement. This means that users can avoid the effort of preparing gels.

[0006] With liquid polymers, continuous measurements can be performed by simply replacing the liquid polymer, which is the separation medium, without changing the capillary. However, with microchannel chips that use a solid separation medium (gel), the gel solidifies, so the chip itself must be discarded after each measurement and a new one must be prepared. This increases running costs and places a burden on users. Given this situation, the easily replaceable liquid polymer is one of the advantageous features of capillary sequencers.

[0007] Furthermore, one of the most important demands of capillary sequencer users is the reduction of running costs. Reagents used in capillary sequencers include anode buffers, cathode buffers, and liquid polymers. The most expensive of these is the liquid polymer, which serves as the DNA separation medium. To reduce the running costs of this most expensive liquid polymer, Non-Patent Document 1 uses a Beckman capillary sequencer to verify whether multiple analyses are possible with a single liquid polymer fill. This verification demonstrates that analytical performance deteriorates with each successive analysis. The conclusion is that "Replacement of the separation matrix is ​​simple and is not worth compromising the results with its reuse." In other words, because reusing the liquid polymer impairs analytical performance, it is strongly recommended to replace the liquid polymer after each analysis.

[0008] The cause of deterioration of liquid polymers after multiple analyses is the decomposition of urea contained in the liquid polymer. Generally, electrophoretic analysis in a capillary sequencer is performed by applying a high voltage of about 15 kV to the end of the capillary in a temperature environment of 60°C. Urea has the function of dissociating double-stranded DNA into single strands, but according to Non-Patent Document 2, urea has the problem of decomposing at high temperatures.

[0009] Furthermore, capillary arrays used in DNA sequencing typically use pure glass without chemical modification, and their surfaces are covered with hydroxyl groups. On the other hand, the buffer used in capillary sequencing is an aqueous solution with a pH of approximately 8.0, which causes the glass surface of the capillary to be negatively charged. Positive ions in the aqueous solution adsorb to the glass surface. When a high voltage is applied to the capillary array in this state, the positive ions on the glass surface are drawn, generating electroosmotic flow. This electroosmotic flow degrades DNA separation performance, so the effectiveness of electroosmotic flow suppression significantly affects analytical results. Here, liquid polymers not only function as a DNA separation medium, but also adsorb to the glass surface and prevent electroosmotic flow. However, according to Non-Patent Document 3, urea decomposition products prevent the adsorption of liquid polymers to the glass surface. As a result, electroosmotic flow increases with urea decomposition, resulting in a decrease in DNA separation performance.

[0010] The analytical steps in a capillary sequencer can be divided into four steps: (1) polymer filling into the capillary array, (2) pre-run, (3) sample injection, and (4) electrophoresis.

[0011] Among these, (2) pre-run is a process in which a voltage is applied to the liquid polymer only for about three minutes after filling the capillary array with the liquid polymer. This process is essential for removing impurities from the liquid polymer and is considered necessary for creating an optimal separation medium environment for analysis. Non-Patent Document 4 reports that the application of pre-run significantly improves protein separation in slab gels.

[0012] Currently, Thermo Fisher Science holds 90% of the capillary sequencer market. Thermo Fisher Science sells not only instruments but also reagents and analysis software, providing customers with comprehensive solutions as a "sample to answer." However, due to its powerful oligopoly, running costs for capillary sequencing remain high, and the black box nature of the instruments restricts customer flexibility. On the other hand, Thermo Fisher Science offers a wide variety of electrophoresis instruments, thereby easing customer flexibility.

[0013] A specific factor contributing to the high running costs is that the protocol for replacing the liquid polymer, which is the DNA separation medium, for each analysis is provided to customers in a form that does not allow for changes. In other words, if a customer fills a capillary array with liquid polymer once, the customer is limited to performing one analysis using that liquid polymer. In other words, the customer cannot perform multiple analyses using a single liquid polymer fill. Meanwhile, because liquid polymer is the most expensive reagent required for capillary sequence analysis, it has been difficult for customers to reduce the running costs of their analyses.

[0014] Under these circumstances, several inventions have been proposed that can reduce the running costs of analysis. For example, Patent Document 1 discloses an electrophoretic analysis method in which heat generated by each capillary is dissipated from the electrodes via a thermally conductive medium, and the temperature of each capillary during electrophoresis is maintained at room temperature or higher and 80°C or lower. Patent Document 1 states that this configuration can prevent gel deterioration and enable repeated electrophoretic analysis using the same gel.

[0015] For example, Patent Document 2 discloses a capillary electrophoresis apparatus equipped with a detector for detecting separated components and a means for determining the completion of electrophoresis of a sample migrating through a capillary (electrophoresis completion determination means). The electrophoresis completion determination means calculates the intensity of signal peaks corresponding to each component in the sample detected by the detector, and determines that electrophoresis of the sample in the capillary has ended when the calculated intensity is lower than a predetermined value. Patent Document 2 describes that this configuration allows accurate calculation of the start time of the second and subsequent measurements when the electrophoresis gel, which is the electrophoresis medium, is used repeatedly for multiple analyses without replacing it. Patent Document 2 also describes that this configuration prevents the risk of multiple samples being introduced into the capillary simultaneously and prevents unnecessary prolongation of electrophoresis time, enabling multiple accurate analyses in a minimum amount of time.

[0016] In Patent Document 3, the next sample is sequentially taken at time n (T Ｓ -T Ｆ ) into the injection end of a capillary electrophoresis gel, and performing electrophoresis on the next sample such that the polynucleotide having the slowest electrophoretic mobility in the previous sample passes through the detection window of the electrophoresis system before the polynucleotide having the fastest electrophoretic mobility in the next sample. Patent Document 3 discloses that by using this configuration, in a typical embodiment, a total of up to about 25 electrophoresis runs can be performed on the same capillary electrophoresis gel, such that the same capillary polyacrylamide gel is used for electrophoresis of all samples without washing or replacing the capillary gel.

[0017] Japanese Patent Laid-Open No. 2004-191247 Japanese Patent Laid-Open No. 2000-314720 Japanese Patent Laid-Open No. 2006-504099

[0018] J. Braz. Chem. Soc., Vol. 15, No. 3, 413-420, 2004J Forensic Sci. 2005 Jul;50(4):842-8.J Chromatogr A. 1998 May 8;806(1):157-64.J Applied Polymer. 2010 Jan, 48994

[0019]

[0008] of Patent Document 1 states that "electrophoretic analysis can be performed repeatedly using the same gel," but does not state that the above-mentioned pre-run is performed. Similarly,

[0017] of Patent Document 3 states that "the same capillary polyacrylamide gel is used for electrophoresis of all samples," and that "electrophoresis is performed sequentially using the same capillary electrophoresis gel without rinsing or replacing the capillary gel," but does not state that the above-mentioned pre-run is performed.

[0020] On the other hand, Patent Document 2 describes preliminary electrophoresis (pre-run) in

[0020] , sample introduction in

[0022] , and electrophoresis in

[0024] . Furthermore, Patent Document 2 describes in

[0026] that sample introduction and electrophoresis are performed in the above-mentioned procedure after the completion of analysis. Patent Document 2 also describes in

[0020] that the standard sample and the analytical sample are not introduced into the capillary in the preliminary electrophoresis. However, Patent Document 2 does not clarify whether the above-mentioned procedure includes gel filling and preliminary electrophoresis. Furthermore, Patent Document 2 does not clarify the timing of gel filling in the description of the overall operation from

[0020] onwards.

[0021] Furthermore, the inventions described in Patent Documents 1 to 3 all use gel as a separation medium, and do not describe or suggest anything about a configuration in which a liquid polymer is used as a separation medium. Therefore, although the inventions described in Patent Documents 1 to 3 have been proposed, the problem of reducing the running costs of analysis when a liquid polymer is used as a separation medium remains unsolved.

[0022] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for continuous use of a separation medium that can reduce the running costs of analysis when a liquid polymer is used as the separation medium.

[0023] The method for continuous use of a separation medium according to the present invention, which solves the above-mentioned problems, comprises a separation medium filling step of filling a capillary with a separation medium, a first pre-run step of applying a voltage to the capillary filled with the separation medium to perform a pre-run, a first sample injection step of injecting a first sample into the capillary that has undergone the pre-run, a first electrophoresis step of applying a voltage to the capillary into which the first sample has been injected to perform a first electrophoresis, a second pre-run step of applying a voltage to the capillary after the first electrophoresis step to perform a pre-run, a second sample injection step of injecting a next batch of sample into the capillary that has undergone the second pre-run step, and a second electrophoresis step of applying a voltage to the capillary into which the next batch of sample has been injected to perform a next electrophoresis, and the second pre-run step, the second sample injection step, and the second electrophoresis step are repeated a predetermined number of times.

[0024] According to the present invention, when a liquid polymer is used as a separation medium, a method for continuous use of the separation medium can be provided, which can reduce the running costs of analysis. Problems, configurations, and effects other than those described above will become clear from the description of the following embodiments.

[0025] 3A is a schematic diagram illustrating the configuration of an electrophoresis device to which a method for continuous use of a separation medium according to one embodiment of the present invention can be applied. FIG. 3B is a schematic top view diagram illustrating the configuration of an electrophoresis device to which a method for continuous use of a separation medium according to one embodiment of the present invention can be applied. FIG. 3C is a cross-sectional view taken along IIIa-IIIa in FIG. 2. FIG. 3D is an enlarged view of part IIIb in FIG. 3A. FIG. 3E is a schematic diagram illustrating the control configuration of an electrophoresis device to which a method for continuous use of a separation medium according to one embodiment of the present invention can be applied. FIG. 3F is a flowchart illustrating the content of a method for continuous use of a separation medium according to a first embodiment of the present invention. FIG. 3G is a schematic diagram illustrating an example of a graphical user interface (GUI) for setting the refilling frequency of a separation medium in a separation medium refilling step. FIG. 3H is a flowchart illustrating processing and electrophoresis in a microcomputer after a user inputs the refilling frequency of a separation medium via an input / output device, with respect to the method for continuous use of a separation medium according to the first embodiment of the present invention. FIG. 3I is a flowchart illustrating the content of a conventional method in which a separation medium is refilled for each analysis. FIG. 3J is a flowchart illustrating the content of a method for continuous use of a separation medium according to a second embodiment of the present invention. FIG. 3I is a flowchart illustrating processing and electrophoresis in a microcomputer after a user inputs the refilling frequency of a separation medium via an input / output device, with respect to the method for continuous use of a separation medium according to the second embodiment of the present invention. 1 is an explanatory diagram showing DNA base sequences and waveform data around 400 bp in the first run (1st), third run (3rd), and eighth run (8th) of Example 1. 2 is an explanatory diagram showing DNA base sequences and waveform data around 600 bp in the first run (1st), third run (3rd), and eighth run (8th) of Example 1. 3 is a graph showing statistical values ​​of adjacent read length (CRL) for each sample injection number (Injection ID), i.e., run numbers 1 to 6, in Examples 1 and 2. 4 is a graph comparing statistical values ​​of total CRL for sample injection numbers 1 to 6 (up to Injection 6) in Examples 1 and 2. 5 is a graph continuously showing the progression of CRL in DNA sequence analysis for sample injection numbers (Injection ID) 1 to 16, i.e., run numbers 1 to 16, in Example 4.1 is a graph continuously showing the transition of EQ in fragment analysis when the number of sample injections (Injection ID) is 1 to 12, i.e., the number of runs is 1 to 12, in Example 4.

[0034] FIG. 1 is an explanatory diagram comparing the difference in analysis time for the second run between Examples 1 and 3, in which a pre-run is performed for each run, and Examples 2 and 4, in which a pre-run is performed only when the first run is performed after filling with liquid polymer, and no pre-run is performed for the second or subsequent runs.

[0035] FIG. 1 is a table showing the runtimes of the first to tenth runs of four capillaries (Cap1 to Cap4) in Example 4.

[0026] Hereinafter, a method for continuous use of a separation medium according to one embodiment of the present invention (hereinafter, sometimes simply referred to as "this method") will be described in detail with reference to the accompanying drawings. In the description of the embodiment, substantially identical or similar components will be designated by the same reference numerals, and redundant descriptions may be omitted.

[0027] (Electrophoresis Apparatus) Before describing this method of use, an electrophoresis apparatus to which this method can be applied will be described. FIG. 1 is a schematic diagram illustrating the configuration of an electrophoresis apparatus 1 to which this method can be applied. FIG. 2 is a schematic top view illustrating the configuration of an electrophoresis apparatus 1 to which this method can be applied. FIG. 3A is a cross-sectional view taken along IIIa-IIIa in FIG. 2. FIG. 3B is an enlarged view of part IIIb in FIG. 3A. FIG. 4 is a schematic diagram illustrating the control configuration of an electrophoresis apparatus 1 to which this method can be applied.

[0028] As shown in FIG. 1, an electrophoresis apparatus 1 to which this method can be applied (hereinafter, sometimes simply referred to as "the apparatus 1") can be broadly divided into two units: an autosampler unit 150 located at the bottom of the apparatus, and an irradiation / detection / constant temperature bath unit 160 located at the top of the apparatus.

[0029] The autosampler unit 150 has a Y-axis driver 85 mounted on the sampler base 80, allowing for drive in the Y axis. A Z-axis driver 90 is mounted on the Y-axis driver 85, allowing for drive in the Z axis. A sample tray 100 is mounted on the Z-axis driver 90. As shown in FIGS. 1 and 2 , the user places the separation medium container 20, the anode-side buffer solution container 30, the cathode-side buffer solution container 40, and the sample container 50 on the sample tray 100. The sample container 50 is placed on an X-axis driver 95 mounted on the sample tray 100, allowing only the sample container 50 to be driven in the X axis on the sample tray 100. A liquid delivery mechanism 60 is also mounted on the Z-axis driver 90. This liquid delivery mechanism 60 is located below the separation medium container 20.

[0030] As shown in FIG. 1 , the irradiation / detection / thermostat unit 160 includes a thermostat unit 110 and a thermostat door 120, which can maintain a constant temperature inside. An irradiation / detection unit 130 is mounted behind the thermostat unit 110, allowing detection during electrophoresis. The user places a capillary array 10, which includes multiple capillaries 125 (see FIG. 3B ), in the thermostat unit 110. Electrophoresis is performed while the thermostat unit 110 maintains the capillary array 10 at a constant temperature, and detection is performed by the irradiation / detection unit 130. The thermostat unit 110 also includes an electrode 115 for grounding when a high voltage is applied for electrophoresis.

[0031] As shown in FIG. 3A, the capillary array 10 includes a load header 126, a capillary head 127, a detection unit 128, and the like. The load header 126 is attached to one end of the capillary array 10. The load header 126 serves as the cathode end of the capillary array 10. The capillary head 127 is attached to the other end of the capillary array 10. The capillary head 127 is a pressure-tight, detachable member in which capillaries 125 are bundled together in a pressure-tight manner. The capillary head 127 serves as the anode end of the capillary array 10. Inside the detection unit 128, the capillaries 125 are arranged at regular intervals in a plane. The irradiation and detection unit 130 irradiates light onto the capillaries 125 arranged in the detection unit 128. The detection unit 128 detects fluorescence and the like emitted from the sample undergoing electrophoresis in each capillary 125 by irradiating the sample with light.

[0032] As described above, the capillary array 10 is set in the thermostatic bath unit 110. The separation medium container 20, the anode side buffer container 30, the cathode side buffer container 40, and the sample container 50 can be driven in the Y and Z axes by the autosampler unit 150, and only the sample container 50 can be driven in the X axis. The separation medium container 20, the anode side buffer container 30, the cathode side buffer container 40, and the sample container 50 can be automatically connected to any position on the set capillary array 10 by the movement of the autosampler unit 150.

[0033] 2, the anode buffer container 30 set on the sample tray 100 includes an anode washing tank 31, an anode electrophoresis buffer tank 32, and a sample introduction buffer tank 33. The cathode buffer container 40 includes a waste liquid tank 41, a cathode washing tank 42, and a cathode electrophoresis buffer tank 43.

[0034] The separation medium container 20, the anode side buffer container 30, the cathode side buffer container 40, and the sample container 50 are positioned in the positional relationship shown in FIG. 2 . That is, the anode side buffer container 30 is positioned on the left side of the front of the device (the side where the thermostatic chamber door 120 opens and closes) on the sample tray 100. The separation medium container 20 is positioned behind the anode side buffer container 30. The sample container 50 is positioned on the right side of the front of the device on the sample tray 100. The cathode side buffer container 40 is positioned behind the sample container 50. As a result, the positional relationships between the anode side and the cathode side when connecting to the capillary array 10 are "separation medium container 20-waste tank 41," "anode side washing tank 31-cathode side washing tank 42," "anode side electrophoresis buffer tank 32-cathode side electrophoresis buffer tank 43," and "sample introduction buffer tank 33-sample container 50."

[0035] As shown in FIG. 3A , the separation medium container 20 is inserted into a guide 101 embedded in the sample tray 100 and set therein. The separation medium container 20 contains a separation medium. A liquid polymer, for example, is preferably used as the separation medium. In this manner, the liquid delivery mechanism 60 in the device 1 can efficiently and automatically fill and discharge the separation medium into and from the capillary 125 (see FIG. 3B ). The liquid delivery mechanism 60 is positioned so that a plunger 61 built into the liquid delivery mechanism 60 is below the separation medium container 20. When the plunger 61 pushes up a cylinder (not shown) provided in the separation medium container 20, the separation medium (not shown) in the separation medium container 20 is introduced into the capillary 125 via the capillary head 127.

[0036] 3A of the capillary array 10 is the anode side, and the right side is the cathode side. The autosampler unit 150 moves to the position of "anode side electrophoresis buffer tank 32-cathode side electrophoresis buffer tank 43," and a high voltage is applied to the cathode side capillary array 10, and the voltage is passed through the cathode side buffer tank 40 and the anode side buffer tank 30 to GND at the electrode 115, thereby performing electrophoresis.

[0037] As shown in FIG. 3B , each of the capillaries 125 constituting the capillary array 10 is fixed via a metal hollow electrode 124. The hollow electrode 124 is provided on a portion of the capillary 125 (from the cathode end to the load header 126). As shown in FIG. 3B , the tip 125 a of each capillary 125 protrudes from the hollow electrode 124 by approximately 0.5 mm. The length of the capillary 125 protruding from the hollow electrode 124 is not limited to 0.5 mm. Furthermore, all of the hollow electrodes 124 provided on each capillary 125 are attached together to the load header 126 (see FIG. 3A ). All of the hollow electrodes 124 are connected to the high-voltage power supply 122 via the load header 126. The high-voltage power supply 122 applies a negative voltage to the hollow electrode 124, so that when a voltage is applied to the hollow electrode 124 during electrophoresis, sample injection, or the like, the hollow electrode 124 operates as a cathode electrode.

[0038] As shown in FIG. 4 , the voltage control mechanism includes a microcontroller (MCU) 141, a controller 142, a high-voltage power supply 122, a first ammeter 121, and a second ammeter 123. The MCU 141 uses the separation medium filled in the capillary array 10 to check the current flow state of the anode-side electrophoresis buffer tank 32 and the cathode-side electrophoresis buffer tank 43. The MCU 141 then outputs the results of the checked current flow state to an input / output device (not shown). The input / output device may be, for example, a touch panel. The controller 142 controls the high-voltage power supply 122 to apply voltage to the current flow path, move the sample tray 100, and move the sample container 50 using the X-axis driver 95.

[0039] The high-voltage power supply 122 applies a voltage to the current path under the control of the controller 142. The current path includes the hollow electrode 124, the electrophoresis buffer solution filled in the cathode-side electrophoresis buffer tank 43 (or the sample in the sample container 50), the electrophoresis buffer solution filled in the anode-side electrophoresis buffer tank 32 (or the buffer solution filled in the sample introduction buffer tank 33), and the electrode 115. More specifically, the high-voltage power supply 122 generates a voltage (negative voltage) lower than the GNDs 129a and 129b. Therefore, when a negative voltage is applied by the high-voltage power supply 122, the current flows as shown by the dashed arrows in FIG. 4. That is, the current flows in the following order: GND 129a → second ammeter 123 → electrode 115 → capillary array 10 → hollow electrode 124 → first ammeter 121 → high-voltage power supply 122. Therefore, the current path is GND 129 a → second ammeter 123 → electrode 115 → capillary array 10 → hollow electrode 124 → first ammeter 121 → high-voltage power supply 122 .

[0040] A high-voltage power supply 122 is electrically connected to a hollow electrode 124 via a first ammeter 121 and to an electrode 115 via a second ammeter 123. When a voltage of several tens of kilovolts is applied across these terminals, an electric field is generated in the direction from the hollow electrode 124 to the electrode 115. This electric field causes a negatively charged sample such as nucleic acid to move from a tip 125a (see FIG. 3B) on the cathode side of the capillary 125 toward a detection unit 128 (see FIG. 3A). This causes a negatively charged sample (e.g., DNA) to migrate in the direction of arrow A1 in FIG. 4.

[0041] At this time, the first ammeter 121 detects the current flowing from the high-voltage power supply 122 to the hollow electrode 124 and transmits the current value to the microcomputer 141. The second ammeter 123 detects the current flowing from the electrode 115 to the GND 129a and transmits the current value to the microcomputer 141. The second ammeter 123 is typically used to check the current value and its fluctuations. This is because the second ammeter 123 more directly reflects the current value flowing through the electrophoresis path. If there is a leakage current or the like between the first ammeter 121 and the second ammeter 123, the value indicated by the first ammeter 121 includes the leakage current value, whereas the value indicated by the second ammeter 123 does not include the leakage current. In other words, the net amount of current flowing through the electrophoresis path is detected. The area between the first ammeter 121 and the second ammeter 123 is a portion where a medium with a relatively high resistance compared to metal, such as a buffer solution or a separation medium (e.g., a liquid polymer), exists, and there are also many connecting parts such as blocks and capillaries 125. Therefore, the circuit passing through the first ammeter 121 can be said to be a portion where leakage current is likely to occur.

[0042] (Method for Continuous Use of Separation Medium) Next, this method will be described. Fig. 5 is a flowchart illustrating the content of the method for continuous use of separation medium according to the first embodiment of the present invention. This method is applied to the electrophoresis apparatus 1.

[0043] 5, the method according to the first embodiment includes a separation medium filling step S1, a first pre-run step S2, a first sample injection step S3, a first electrophoresis step S4, a second pre-run step S5, a second sample injection step S6, and a second electrophoresis step S7. In this method, the second pre-run step S5, the second sample injection step S6, and the second electrophoresis step S7 are repeated a predetermined number of times (n times).

[0044] The separation medium filling step S1 is a step of filling the capillaries 125 with the separation medium. The capillaries 125 can be filled with the separation medium using the liquid delivery mechanism 60 described above. The first pre-run step S2 is a step of performing a pre-run by applying a voltage to the capillaries 125 filled with the separation medium. The pre-run refers to applying a voltage to the separation medium prior to sample electrophoresis. By performing the pre-run, impurities in the separation medium can be removed, creating an optimal separation medium environment for analysis. The pre-run is performed without injecting a sample. The first pre-run step S2 can be performed by appropriately driving the sample tray 100 along the X-axis, Y-axis, and Z-axis to set the positional relationship between the anode side and the cathode side in the connection with the capillary array 10 to "anode side electrophoresis buffer tank 32 - cathode side electrophoresis buffer tank 43," and then applying a voltage. The first pre-run step S2 can be performed under typical conditions, for example, 60° C.×3 minutes×15 kV.

[0045] The first sample injection step S3 is a step of injecting a first sample into the capillary 125 that has undergone the pre-run. The first sample injection step S3 can be performed by appropriately driving the sample tray 100 along the X-axis, Y-axis, and Z-axis so that the positional relationship between the anode side and the cathode side in connection with the capillary array 10 is "sample introduction buffer tank 33-sample container 50," and then applying a voltage. The first sample injection step S3 can be performed, for example, at 60°C for 4 seconds at 1.2 kV.

[0046] The first electrophoresis step S4 is a step of applying a voltage to the capillary 125 into which the first sample has been injected to perform the first electrophoresis. The first electrophoresis step S4 can be performed by appropriately driving the sample tray 100 along the X-axis, Y-axis, and Z-axis so that the anode-cathode positional relationship in connection with the capillary array 10 is "anode-side electrophoresis buffer tank 32 - cathode-side electrophoresis buffer tank 43," and then applying a voltage. The first electrophoresis step S4 can be performed, for example, at 60°C for 30 minutes at 7.5 kV.

[0047] The second pre-run step S5 is a step of applying a voltage to the capillary 125 after the first electrophoresis step S4 to perform a pre-run. In other words, in this method, the separation medium in the capillary 125 is not discarded and re-injected (i.e., the separation medium is replaced), as is typically done after the previous electrophoresis (run), but the pre-run (second pre-run step S5) for the next sample analysis is performed using the separation medium used in the previous run. Note that a run refers to the generation of a potential difference across the capillary 125 by the high-voltage power supply 122, causing the sample added to the separation medium to migrate through the capillary 125. During the run, the sample is detected by the irradiation and detection unit 130.

[0048] The second sample injection step S6 is a step of injecting the next batch of sample into the capillary 125 that has been subjected to the second pre-run step S5. The second sample injection step S6 can be performed in the same manner as the first sample injection step S3. The second electrophoresis step S7 is a step of applying a voltage to the capillary 125 into which the next batch of sample has been injected to perform the next batch of electrophoresis. The second electrophoresis step S7 can be performed in the same manner as the first electrophoresis step S4.

[0049] As described above, this method of use involves repeating the second pre-run step S5, the second sample injection step S6, and the second electrophoresis step S7 a predetermined number of times (n times). After repeating the series of steps of the second pre-run step S5, the second sample injection step S6, and the second electrophoresis step S7 a predetermined number of times, this method ends the analysis using the separation medium filled in one separation medium filling step S1. The process then returns to the separation medium filling step S1, replaces the separation medium in the capillary 125, and performs each step from the first pre-run step S2 onward with the new separation medium, as described above. This is repeated a desired number of times in this method of use.

[0050] Here, the separation medium filling frequency (f times) in this specification refers to the filling of a new separation medium (refilling frequency of the separation medium) each time the total number of runs performed on a single filled separation medium reaches a predetermined number (f times). In other words, the separation medium filling frequency (f times) refers to the total number of runs, including one first electrophoresis step S4 and n repeated runs of the second electrophoresis step S7, for each separation medium filling. In other words, f times = 1 + n times. In other words, the number of repetitions (n ​​times) of the second pre-run step S5, the second sample injection step S6, and the second electrophoresis step S7 can be calculated and set as "the separation medium filling frequency by the separation medium filling step S1 - 1 time" (f - 1 times). For example, if the separation medium filling frequency (f times) is set to 4 times, the setting includes one run of the first electrophoresis step S4 and three runs of the second electrophoresis step S7 as one set. In this manner, in this embodiment, setting the separation medium filling frequency (f times) sets the number of repetitions (n ​​times) of the second pre-run step S5, the second sample injection step S6, and the second electrophoresis step S7. Meanwhile, the number of repetitions (n ​​times) of the second pre-run step S5, the second sample injection step S6, and the second electrophoresis step S7 refers to the number of subsequent runs performed using the separation medium used in the first run.

[0051] As described above, in this method, a single-filled separation medium is used in multiple runs, which reduces the frequency of filling the separation medium, e.g., liquid polymer, which is the most expensive reagent required for capillary sequence analysis, and thereby reduces the running costs of the analysis.

[0052] In this embodiment, the user can arbitrarily set the number of repetitions (n) of the second pre-run step S5, the second sample injection step S6, and the second electrophoresis step S7. The number of repetitions of these steps (i.e., the number of repetitions of the run by the second electrophoresis step S7) can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, but may also be 26 or more. Since there is one run of the first electrophoresis step S4, if the set number of repetitions is, for example, 5, a total of 6 runs will be performed using a separation medium filled once. In this manner, in this embodiment, by setting in advance the set number of times (n times) to repeat the second pre-run step S5, the second sample injection step S6, and the second electrophoresis step S7, it is possible to perform one run of the first electrophoresis step S4 and an arbitrarily set number of n runs of the second electrophoresis step S7 as one set for each filling of the separation medium.

[0053] The number of times (n) the above steps are repeated can be set arbitrarily depending on the condition of the sample, such as the deterioration of the separation medium and the length of the DNA strand. For example, if the DNA strand is about 300 bp long, the set number can be 4 to 5 times per filling of the separation medium (5 to 6 total runs, i.e., 5 to 6 separation medium filling frequencies). For example, if the DNA strand is about 600 bp long, the set number can be 3 times per filling of the separation medium (4 total runs, i.e., 4 separation medium filling frequencies).

[0054] The input of the separation medium filling frequency (f times) in the separation medium filling step S1 can be performed, for example, by an input / output device (not shown) connected to the microcomputer 141. FIG. 6 is a schematic diagram showing an example of a graphical user interface (GUI) for setting the separation medium filling frequency in the separation medium filling step S1. As shown in FIG. 6, the input / output device can display a GUI for inputting the separation medium filling frequency (f times). The user can set the separation medium filling frequency to 4 times by entering, for example, "every 4 injection" or "4" in the "Polymer exchange frequency" field in the lower column of the GUI shown in FIG. 6, or by selecting "4" using the radio button.

[0055] Although not shown in FIG. 6, the set number of repetitions (n ​​times) of the process can be input in the same manner as the separation medium filling frequency (f times). For example, a GUI for inputting the set number of repetitions (n ​​times) of the process may be displayed on the input / output device. In order to input the set number of repetitions (n ​​times) of the process, a column called "Number of repetitions of 2nd electrophoresis" is provided in the GUI. For example, by inputting "3 times" or "3" or selecting "3" using the radio button, the set number of repetitions of the process can be set to 3 times.

[0056] In this method of use, when the separation medium filling frequency reaches a predetermined number (f times), in other words, when the total number of runs reaches f times (f times) (f-1 times, calculated and set as the set number of repetitions of the above-mentioned steps (n times)) (Yes in step S8 of FIG. 5), all analyses (runs) performed with one separation medium filling are terminated. Then, return to the separation medium filling step S1, replace the separation medium in the capillary 125, and perform each step from the first pre-run step S2 onwards with the new separation medium as described above. On the other hand, if the frequency of the separation medium filling step S1 does not reach the predetermined number (f times), in other words, when the total number of runs does not reach f times (f-1 times, calculated and set as the set number of repetitions of the above-mentioned steps (n times)) (No in step S8 of FIG. 5), return to the second pre-run step S5, and perform the second pre-run step S5, the second sample injection step S6, and the second electrophoresis step S7.

[0057] FIG. 7 is a flowchart illustrating the processing and electrophoresis performed by the microcomputer 141 after the user inputs the separation medium refill frequency via the input / output device, in relation to the method for continuous use of a separation medium according to the first embodiment of the present invention. As shown in FIG. 7 , the user inputs the separation medium refill frequency (step S11). In the example of FIG. 7 , for example, the separation medium refill frequency is input as "4" (f=4). This causes the microcomputer 141 to set the following: when a total of four runs has been performed, the old separation medium is discarded from the capillary 125 and a new separation medium is refilled into the capillary 125. The microcomputer 141 also calculates "separation medium refill frequency - 1" and sets the number of repetitions of the above process (n=3).

[0058] Then, when the user presses the start key, analysis begins (step S12). The microcomputer 141 determines whether or not this is the first electrophoresis (step S13). If this is the first electrophoresis (Yes in step S13), the microcomputer 141 issues an instruction to fill the separation medium (step S14), causing the device 1 to perform the separation medium filling step S1. Next, the microcomputer 141 issues an instruction to perform a pre-run (step S15), causing the device 1 to perform the first pre-run step S2. Next, the microcomputer 141 issues an instruction to perform sample injection (step S16), causing the device 1 to perform the first sample injection step S3. Next, the microcomputer 141 issues an instruction to perform electrophoresis (step S17), causing the device 1 to perform the first electrophoresis step S4.

[0059] After the electrophoresis is completed, the microcomputer 141 determines whether the total number of runs has reached the entered separation medium filling frequency (f = 4) (step S18). If the total number of runs has not reached the separation medium filling frequency (f = 4) (No in step S18), the microcomputer 141 returns to the time immediately after the key input to start the run and determines whether the next electrophoresis run is the first run (step S13). Since the next electrophoresis run is not the first run (No in step S13), the microcomputer 141 issues an instruction to perform a pre-run (step S15), and the second pre-run step S5 is performed by the device 1. Next, the microcomputer 141 issues an instruction to perform sample injection (step S16), and the second sample injection step S6 is performed by the device 1. Next, the microcomputer 141 issues an instruction to perform electrophoresis (step S17), and the second electrophoresis step S7 is performed by the device 1.

[0060] After the electrophoresis is completed, the microcomputer 141 determines whether the total number of runs has reached the entered separation medium filling frequency (f=4) (step S18). If the total number of runs has not reached the separation medium filling frequency (f=4) (No in step S18), the microcomputer 141 returns to the time immediately after the start of the run keystroke and determines whether the next electrophoresis is the first run (Yes or No in step S13). Based on this determination, the microcomputer 141 issues the above-described instruction to the apparatus 1 (proceeding to step S14 or step S15). If the total number of runs has reached the separation medium filling frequency (f=4) (Yes in step S18), the microcomputer 141 issues an instruction to end the run to the apparatus 1 (step S19), and the apparatus 1 ends the run.

[0061] The microcomputer 141 performs the above-described series of steps shown in FIG. 7 for the number of times set by the user to analyze the sample.

[0062] In this way, the method of use does not require filling of the separation medium at a set filling frequency unless the electrophoresis is the first run, and therefore can reduce the filling frequency of the separation medium, such as the liquid polymer, which is the most expensive reagent required for capillary sequence analysis, thereby reducing the running costs of the analysis.

[0063] Here, this method of use will be compared with a conventional method in which a separation medium is filled for each analysis. FIG. 8 is a flowchart illustrating the details of the conventional method in which a separation medium is filled for each analysis. As shown in FIG. 8, the conventional method includes a separation medium filling step S101, a pre-run step S102, a sample injection step S103, and an electrophoresis step S104. These steps in the conventional method shown in FIG. 8 correspond to the separation medium filling step S1, the first pre-run step S2, the first sample injection step S3, and the first electrophoresis step S4 in the present method of use shown in FIG. 5, respectively.

[0064] In the conventional method shown in Figure 8, the separation medium is filled for each analysis, and these steps are performed every time. In other words, the conventional method shown in Figure 8 does not include the second pre-run step S5, the second sample injection step S6, and the second electrophoresis step S7 of the present method shown in Figure 5, nor does it repeat these steps. Therefore, the conventional method does not reduce the filling frequency of the separation medium, which is the most expensive of the reagents required for capillary sequence analysis, and the running costs of the analysis remain high.

[0065] The inventors conducted research to solve the problems of the present invention and decided to repeat the second pre-run step S5, the second sample injection step S6, and the second electrophoresis step S7 a predetermined number of times in the method of use according to the first embodiment. They found that the analytical performance obtained in this manner fully meets specifications, although the waveform data is slightly degraded, as described below. They also found that the method of use according to the first embodiment achieves an improved turnaround time (TAT) compared to the conventional method shown in FIG.

[0066] (Second Embodiment) Next, a method of use according to a second embodiment will be described. FIG. 9 is a flowchart illustrating the details of a method of continuous use of a separation medium according to a second embodiment of the present invention. As shown in FIG. 9, the method of use according to the second embodiment includes a separation medium filling step S1, a first pre-run step S2, a first sample injection step S3, a first electrophoresis step S4, a second sample injection step S6, and a second electrophoresis step S7. In this method of use, the second sample injection step S6 and the second electrophoresis step S7 are repeated a predetermined number of times (n times).

[0067] The method of use according to the second embodiment differs from the method of use according to the first embodiment, which includes the second pre-run step S5, in that it does not include the second pre-run step S5. Furthermore, the second sample injection step S6 of the method of use according to the second embodiment is a step of injecting the next batch of sample into the capillary after the first electrophoresis step S4. In contrast, the second sample injection step S6 of the method of use according to the first embodiment is a step of injecting the next batch of sample into the capillary that has undergone the second pre-run step S5. Therefore, the second sample injection step S6 of the method of use according to the second embodiment differs from the second sample injection step S6 of the method of use according to the first embodiment in terms of the content of the steps. Furthermore, the method of use according to the second embodiment repeats the second sample injection step S6 and the second electrophoresis step S7 a predetermined number of times (n times). In contrast, the method of use according to the first embodiment repeats the second pre-run step S5, the second sample injection step S6, and the second electrophoresis step S7 a predetermined number of times (n times). Therefore, the present method of use according to the second embodiment differs from the present method of use according to the first embodiment in the content of the repeated steps. The present method of use according to the second embodiment differs from the present method of use according to the first embodiment in that the present method of use according to the second embodiment does not have the second pre-run step S5, but otherwise has the same configuration. Below, the present method of use according to the second embodiment will be described, focusing on the differences from the present method of use according to the first embodiment.

[0068] As described above, the second sample injection step S6 of the second embodiment of the method of use is a step of injecting the next batch of sample (without pre-run) into the capillary 125 after the first electrophoresis step S4. The inventors conducted research to solve the problems of the present invention and surprisingly found that injecting the next batch of sample into the capillary 125 after the first electrophoresis step S4 without pre-run and performing the next electrophoresis, i.e., performing the second sample injection step S6 and the second electrophoresis step S7 after the first electrophoresis step S4, can increase the number of analyses, improve turnaround time (TAT), and improve analytical performance compared to performing a pre-run before the second sample injection step (the second pre-run step S5 in the first embodiment). These effects are achieved by omitting the pre-run and suppressing deterioration of the separation medium. Examples of deterioration of the separation medium include decomposition of urea. That is, the method according to the second embodiment suppresses the decomposition of urea caused by the pre-run by omitting the second pre-run step S5, thereby reducing the effect of urea decomposition products on the adsorption of separation media, such as liquid polymers, to the glass surface. As a result, electroosmotic flow is less likely to increase (is suppressed), thereby improving the number of analyses and analytical performance of samples, such as DNA. Furthermore, since a pre-run is not performed, analyses can be completed more quickly. For example, a typical capillary DNA sequencer requires approximately 30 minutes for one analysis, of which the pre-run requires approximately 3 minutes. By omitting the pre-run for the next sample analysis, the analysis time can be reduced by approximately 10%. This is an advantageous effect compared to conventional methods that perform a pre-run for each analysis, and the method according to the first embodiment.

[0069] FIG. 10 is a flowchart illustrating the processing and electrophoresis performed by the microcomputer 141 after the user inputs the separation medium refill frequency via the input / output device, in relation to the second embodiment of the method for continuous use of a separation medium. As shown in FIG. 10, the user inputs the separation medium refill frequency (step S11). In the example of FIG. 10, for example, the separation medium refill frequency is input as "4" (f=4). This causes the microcomputer 141 to set the following: when a total of four runs has been performed, the old separation medium is discarded from the capillary 125 and a new separation medium is refilled into the capillary 125. The microcomputer 141 also calculates "separation medium refill frequency - 1" and sets the number of repetitions of the above process (n=3).

[0070] Then, when the user presses the start key, analysis begins (step S12). The microcomputer 141 determines whether or not this is the first electrophoresis (step S13). If this is the first electrophoresis (Yes in step S13), the microcomputer 141 issues an instruction to fill the separation medium (step S14), causing the device 1 to perform the separation medium filling step S1. Next, the microcomputer 141 issues an instruction to perform a pre-run (step S15), causing the device 1 to perform the first pre-run step S2. Next, the microcomputer 141 issues an instruction to perform sample injection (step S16), causing the device 1 to perform the first sample injection step S3. Next, the microcomputer 141 issues an instruction to perform electrophoresis (step S17), causing the device 1 to perform the first electrophoresis step S4.

[0071] After the electrophoresis is completed, the microcomputer 141 determines whether the total number of runs has reached the entered separation medium filling frequency (f = 4) (step S18). If the total number of runs has not reached the separation medium filling frequency (f = 4) (No in step S18), the microcomputer 141 returns to the time immediately after the key input to start the run and determines whether the next electrophoresis run is the first run (step S13). Since the next electrophoresis run is not the first run (No in step S13), the microcomputer 141 issues an instruction to inject a sample (step S16), and the second sample injection step S6 is performed in the device 1. Next, the microcomputer 141 issues an instruction to perform electrophoresis (step S17), and the second electrophoresis step S7 is performed in the device 1.

[0072] After the electrophoresis is completed, the microcomputer 141 determines whether the total number of runs has reached the entered separation medium filling frequency (f=4) (step S18). If the total number of runs has not reached the separation medium filling frequency (f=4) (No in step S18), the microcomputer 141 returns to the time immediately after the start of the run keystroke and determines whether the next electrophoresis is the first run (Yes or No in step S13). Based on this determination, the microcomputer 141 issues the above-described instruction to the device 1 (proceeding to step S14 or step S16). If the total number of runs has reached the separation medium filling frequency (f=4) (Yes in step S18), the microcomputer 141 issues an instruction to end the run to the device 1 (step S19), and the device 1 ends the run.

[0073] The microcomputer 141 performs the above-described series of steps shown in FIG. 10 for the number of times set by the user to analyze the sample.

[0074] Next, an example of this method of use will be described. [1] Study 1 <Example 1> In Example 1, capillary sequencing DNA base sequence analysis was performed according to the flow shown in Figure 5 and under the following conditions. The capillary DNA sequencer used was a DS3000 manufactured by Hitachi High-Technologies Corporation. A liquid polymer was used as the separation medium. The liquid polymer used was Spectrum Compact Polymer 7 (CE237A manufactured by Promega). The DNA sample to be analyzed was Sequencing Standard, BigDye. ＴＭTerminator v3.1 (manufactured by Thermo Fisher) was used.

[0075] <Conditions for Example 1> The separation medium filling step S1 was performed using a method specified for a capillary DNA sequencer. The first pre-run step S2 was performed at 60°C for 3 minutes at 15 kV. The first sample injection step S3 was performed at 60°C for 4 seconds at 1.2 kV. The first electrophoresis step S4 was performed at 60°C for 30 minutes at 7.5 kV. The second pre-run step S5 was performed at 60°C for 3 minutes at 15 kV. The second sample injection step S6 was performed at 60°C for 4 seconds at 1.2 kV. The second electrophoresis step S7 was performed at 60°C for 30 minutes at 7.5 kV.

[0076] In Example 1, the separation medium filling frequency was set to 8 (i.e., the number of repetitions of the second pre-run step S5, the second sample injection step S6, and the second electrophoresis step S7 was set to 7), and this was performed twice consecutively in one set, resulting in a total of 16 analysis runs.

[0077] Example 2 In Example 2, capillary sequencing DNA base sequence analysis was performed according to the flow shown in Figure 9 under the following conditions: The capillary DNA sequencer, liquid polymer, and DNA sample were the same as those in Example 1.

[0078] <Conditions for Example 2> The separation medium filling step S1 was performed according to a method specified for the capillary DNA sequencer. The first pre-run step S2 was performed at 60°C for 3 minutes at 15 kV. The first sample injection step S3 was performed at 60°C for 4 seconds at 1.2 kV. The first electrophoresis step S4 was performed at 60°C for 30 minutes at 7.5 kV. The second sample injection step S6 was performed at 60°C for 4 seconds at 1.2 kV. The second electrophoresis step S7 was performed at 60°C for 30 minutes at 7.5 kV.

[0079] In Example 2, the separation medium filling frequency was also set to 8 (i.e., the number of repetitions of the second sample injection step S6 and the second electrophoresis step S7 was set to 7), and this was performed twice consecutively in one set, resulting in a total of 16 runs of analysis.

[0080] Some of the results of the analysis of Example 1 are shown in Figures 11 and 12. Figure 11 is an explanatory diagram showing DNA base sequence and waveform data around 400 bp in the first run (1st), third run (3rd), and eighth run (8th) of Example 1. The first run is the first electrophoresis after one liquid polymer filling. The third run is the third electrophoresis after one liquid polymer filling. The eighth run is the eighth electrophoresis after one liquid polymer filling. Figure 12 is an explanatory diagram showing DNA base sequence and waveform data around 600 bp in the first run (1st), third run (3rd), and eighth run (8th) of Example 1. The first run is the first electrophoresis after one liquid polymer filling. The third run is the third electrophoresis after one liquid polymer filling. The eighth run is the eighth electrophoresis after one liquid polymer filling.

[0081] As shown in Figures 11 and 12, as the number of runs after one liquid polymer filling increased, the waveform data deteriorated slightly, but the DNA base sequence could be analyzed without any problems. Specifically, there was a portion around 645 bp in the eighth run shown in Figure 12 that could not be analyzed with sufficient accuracy, but the rest of the sequence could be analyzed with sufficiently high accuracy. Although illustration of Example 2 is omitted, the results were almost the same as those of Example 1.

[0082] FIG. 13 is a graph showing the statistical values ​​of the adjacent read length (CRL) for each sample injection number (Injection ID) in Examples 1 and 2, i.e., for runs 1 to 6. Note that FIG. 13 shows statistical values ​​obtained by performing the same analysis three independent times. As shown in FIG. 13 , when 600 bp was set as the reference value, the CRLs in both Examples 1 and 2 cleared the reference value up to the fourth run, confirming that the specifications were fully met. If 400 bp was set as the reference value, it was found that the CRLs in both Examples 1 and 2 cleared the reference value up to the sixth run, indicating that the specifications were fully met. Note that the reference value can be arbitrarily changed depending on the length of the DNA sequence to be analyzed. For example, if the DNA sequence to be analyzed is a PCR amplification product and its length can be predicted in advance, such as 300 bp or 400 bp, this can be used as the reference value. For example, when the length of the PCR amplification product is 300 bp, it is estimated from the results shown in FIG. 13 that the specifications can be fully met up to about the 8th to 10th runs.

[0083] 14 is a graph comparing the statistical values ​​of the total CRL for sample injections 1 to 6 (up to Injection 6) in Example 1 and Example 2 (p=0.04721). As shown in FIG. 14, it was confirmed that both Example 1 and Example 2 fully met the specifications. It was also confirmed that Example 2 tended to have a higher CRL than Example 1. It was concluded that this was because the second pre-run step S5 was not performed in Example 2, thereby suppressing the decomposition of urea contained in the liquid polymer.

[0084] [2] Study 2 In Study 2, the packing frequency of the separation medium was changed, and capillary sequencing DNA base sequence analysis was performed under the same conditions as in Examples 1 and 2. Specifically, the analysis was performed as follows.

[0085] Example 3 In Example 3, the separation medium filling frequency was set to 4 (i.e., the number of repetitions of the second pre-run step S5, the second sample injection step S6, and the second electrophoresis step S7 was set to 3), and this was performed four times consecutively in one set, resulting in a total of 16 runs of analysis. In Example 3, the analysis was performed in the same manner as in Example 1 except for this.

[0086] Example 4 In Example 4, the separation medium filling frequency was set to 4 (i.e., the number of repetitions of the second sample injection step S6 and the second electrophoresis step S7 was set to 3), and this was performed four times consecutively in one set, resulting in a total of 16 analysis runs. In Example 4, the analysis was performed in the same manner as in Example 2 except for this.

[0087] Figure 15 is a graph continuously showing the change in CRL in DNA sequence analysis for sample injection numbers (Injection ID) 1 to 16 in Example 4, i.e., for runs 1 to 16. In the figure, 1, 2, and 3 on the right side indicate the capillary numbers. That is, Figure 15 shows the change in CRL in runs 1 to 16 for three capillaries in a line graph. In addition, "▲" in the figure indicates the results of the first run after filling or refilling (replacing) the liquid polymer. "□" in the figure indicates the results of the second, third, and fourth runs using the liquid polymer used in the first run.

[0088] As shown in Figure 15, due to individual differences between capillaries 1 to 3, some performed well and some did not. In some cases, the CRL decreased slightly as the number of runs after one liquid polymer fill increased. However, DNA sequences could be analyzed without any problems. This confirmed that, if the CRL standard is set to 600 bp (when the DNA sequence to be analyzed is 600 bp in length), a separation medium fill frequency of 4 satisfies sufficient specifications. Furthermore, based on the results shown in Figure 15, it was inferred that, for example, if the CRL standard is 500 bp or less (when the DNA sequence to be analyzed is 500 bp or less), sufficient specifications could be met even if the separation medium fill frequency was increased to 5 or more.

[0089] FIG. 16 is a graph continuously showing the transition of EQ, which indicates the maximum base length within the range in which single-base separation is possible in fragment analysis for sample injection numbers (Injection ID) 1 to 12 in Example 4, i.e., for runs 1 to 12. In the figure, 1, 2, and 3 on the right side indicate the capillary numbers. That is, FIG. 16 shows the transition of EQ for runs 1 to 12 for three capillaries in a line graph. In addition, "▲" in the figure indicates the results of the first run after filling or refilling (replacing) the liquid polymer. "□" in the figure indicates the results of the second, third, and fourth runs using the liquid polymer used in the first run.

[0090] As shown in Figure 16, due to individual differences between capillaries 1 to 3, some performed well and some did not. The EQ decreased slightly with increasing number of runs after one liquid polymer fill. However, DNA sequences could be analyzed without any problems. This confirmed that if the EQ standard is set to 400 bp (when the DNA sequence to be analyzed is 400 bp long), a separation medium fill frequency of 4 satisfies sufficient specifications. Furthermore, from the results shown in Figure 16, it was inferred that if the EQ standard is 200 bp or less (when the DNA sequence to be analyzed is 200 bp or less), increasing the separation medium fill frequency to 5 or more would also satisfies sufficient specifications.

[0091] 17 is an explanatory diagram illustrating the difference in analysis time for the second run between Examples 1 and 3, in which a pre-run is performed for each run, and Examples 2 and 4, in which a pre-run is performed only when the first run is performed after filling with the liquid polymer, and no pre-run is performed for the second or subsequent runs. Note that Examples 1 and 3 are similar to the conventional method in that a pre-run is performed for each run.

[0092] As shown in FIG. 17 , Examples 1 and 3 (as well as the conventional method) perform a pre-run for each run. Therefore, in the second run, a voltage is applied for the pre-run, indicated by the dashed-two-dot line on the left side of the upper diagram in FIG. 17 , at the beginning of the analysis. In contrast, Examples 2 and 4 do not perform a pre-run for the second or subsequent runs after the liquid polymer is filled. Therefore, as shown in the lower diagram in FIG. 17 , the voltage application for the pre-run, indicated by the dashed-two-dot line in the upper diagram in FIG. 17 , is not necessary for the second or subsequent runs after the liquid polymer is filled. Therefore, it was confirmed that Examples 2 and 4 can shorten the analysis time for the second or subsequent runs after the liquid polymer is filled. Specifically, the horizontal axis in the upper and lower diagrams in FIG. 17 indicates the analysis time index. In Examples 1 and 3 shown in the upper diagram in FIG. 17 , the index from the start to the end of the analysis is approximately 300, whereas in Examples 2 and 4 shown in the lower diagram in FIG. 17 , the index from the start to the end of the analysis is approximately 265. From these results, it was confirmed that Examples 2 and 4 could reduce the analysis time by about 10% compared to Examples 1 and 3 (and the conventional method).

[0093] Fig. 18 is a table showing the runtimes of the first to tenth runs of four capillaries (Cap1 to Cap4) in Example 4. The table shown in Fig. 18 shows these runtimes [min], average runtime (Average run time) [min], average runtime specification (Average run time spec) [min], judgment on the average runtime specification (Average run time spec judge), migration time uniformity (MT Uniformity), migration time uniformity specification (Spec), and judgment on the migration time uniformity specification (MT Uniformity judge).

[0094] As shown in the table of Figure 18, the runtimes for Caps 1 to 4 in the second run were longer, resulting in a longer average runtime. As a result, the second run did not meet the average runtime specification of 30 minutes or less, and was judged to have failed the average runtime specification (Fail). The other runs, 1 and 3 to 10, were judged to have passed (Pass). Also, as shown in the table of Figure 18, the total runtime was 25 to 30 minutes, with some variation. On the other hand, the migration time uniformity met the migration time uniformity specification of 5.0% or less, and all of Runs 1 to 10 passed.

[0095] The continuous use method of the separation medium according to the present invention has been described in detail above through embodiments and examples, but the present invention is not limited to the above-described embodiments and includes various modifications. For example, the above-described embodiments have been described in detail to clearly explain the present invention, and are not necessarily limited to those having all of the described configurations. Furthermore, it is possible to replace part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. Furthermore, it is possible to add, delete, or replace part of the configuration of each embodiment with other configurations.

[0096] 1 Electrophoresis apparatus (this apparatus) 10 Capillary array 20 Separation medium container 30 Anode side buffer container 31 Anode side washing tank 32 Anode side electrophoresis buffer tank 33 Sample introduction buffer tank 40 Cathode side buffer container 41 Waste tank 42 Cathode side washing tank 43 Cathode side electrophoresis buffer tank 50 Sample container 60 Liquid delivery mechanism 61 Plunger 80 Sampler base 85 Y-axis driver 90 Z-axis driver 95 X-axis driver 100 Sample tray 101 Guide 110 Thermostatic chamber unit 115 Electrode 120 Thermostatic chamber door 121 First ammeter 122 High-voltage power supply 123 Second ammeter 124 Hollow electrode 125 Capillary 125a Tip 126 Load header 127 Capillary head 128 Detection section 129a, 129b GND 130 Irradiation detection unit 141 Microcomputer 142 Controller 150 Autosampler unit 160 Irradiation detection / thermostat unit S1 Separation medium filling step S2 First pre-run step S3 First sample injection step S4 First electrophoresis step S5 Second pre-run step S6 Second sample injection step S7 Second electrophoresis step

Claims

1. A method for continuously using a separation medium, the method comprising:a separation medium filling step of filling a capillary with the separation medium;a first pre-run step of applying a voltage to the capillary filled with the separation medium to perform a prerun;a first sample injection step of injecting a first-time sample into the capillary subjected to the pre-run;a first electrophoretic step of applying a voltage to the capillary into which the first-time sample has been injected to perform first electrophoresis;a second pre-run step of applying a voltage to the capillary after the first electrophoretic step to perform a pre-run;a second sample injection step of injecting a next-time sample into the capillary subjected to the second pre-run step; anda second electrophoretic step of applying a voltage to the capillary into which the next-time sample has been injected to perform next electrophoresis,wherein the method repeatedly performs the second pre-run step, the second sample injection step, and the second electrophoretic step a preliminarily set number of times.

2. A method for continuously using a separation medium, themethod comprising:a separation medium filling step of filling a capillary with the separation medium;a first pre-run step of applying a voltage to the capillary filled with the separation medium to perform a prerun;a first sample injection step of injecting a first-time sample into the capillary subjected to the pre-run;a first electrophoretic step of applying a voltage to the capillary into which the first-time sample has been injected to perform first electrophoresis;a second sample injection step of injecting a next-time sample into the capillary after the first electrophoretic step; anda second electrophoretic step of applying a voltage to the capillary into which the next-time sample has been injected to perform next electrophoresis,wherein the method repeatedly performs the second sample injection step and the second electrophoretic step a preliminarily set number of times.

3. The method for continuously using the separation medium according to claim 1 or claim 2,wherein the separation medium is a liquid polymer.

4. The method for continuously using the separation medium according to claim 1 or claim 2,wherein a frequency of filling with the separation mediumin the separation medium filling step is set via a graphical user interface.

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