Continuous use of separation media

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 continuously using a separation medium according to the present invention comprises a separation medium packing step (S1) for packing a separation medium in a capillary, a first pre-running step (S2) for performing a pre-running, a first sample injection step (S3) for injecting a first-round sample into the capillary, a first electrophoresis step (S4) for applying a voltage to perform a first-round electrophoresis, a second pre-running step (S5) for performing a pre-running after the first electrophoresis step (S4), a second sample injection step (S6) for injecting a next-round sample into the capillary that has been subjected to the second pre-running step (S5), and a second electrophoresis step (S7) for performing a next-round electrophoresis, in which the second pre-running step (S5), the second sample injection step (S6) and the second electrophoresis step (S7) are performed repeatedly preset times.

Description

[Technical Field]

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

[0002] The proportion of the population aged 60 and over to the total population will continue to 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 has been 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, DNA sequencers 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 various sequencing techniques have been developed. Among these, a technology called next-generation sequencing is characterized by the ability to perform sequencing 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 why capillary sequencers have established a firm position in research fields is the application of liquid polymers, which enable separation media within the capillary. Before capillary sequencers became widespread, DNA separation was typically performed using a gel solidified between two gel plates. However, preparing this gel required a lot of effort, which was 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 microfluidic 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 the user. Given this situation, the ability to easily replace liquid polymers 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 reason why repeated analyses cause deterioration of liquid polymers 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 are typically made of unmodified glass, with their surfaces covered with hydroxyl groups. The buffer used in capillary sequencing is an aqueous solution with a pH of approximately 8.0, which negatively charges the glass surface of the capillary. Positive ions in the aqueous solution adsorb to the glass surface. When a high voltage is applied to the capillary array in this state, electroosmotic flow occurs, pulling the positive ions from the glass surface. This electroosmotic flow degrades DNA separation performance, so the effectiveness of electroosmotic flow suppression significantly affects analytical results. Liquid polymers function as a DNA separation medium while also adsorbing to the glass surface and preventing electroosmotic flow. However, according to Non-Patent Document 3, urea decomposition products inhibit 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 process 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] Of 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, the running costs of 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 that keeps running costs high 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, they are limited to performing one analysis using that liquid polymer. In other words, customers cannot perform multiple analyses using a single liquid polymer fill. Meanwhile, because liquid polymer is the most expensive reagent required for capillary sequencing analysis, it has been difficult for customers to reduce their analysis running costs.

[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 electrode 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 prevents gel deterioration and allows repeated electrophoretic analysis using the same gel.

[0015] Furthermore, 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 migration of a sample migrating through a capillary (a migration completion determination means). This migration completion determination means calculates the intensity of the signal peak corresponding to each component in the sample detected by the detector, and determines that migration of the sample through the capillary has completed 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 electrophoretic gel, which is the electrophoretic separation medium, is used repeatedly for multiple analyses without replacement. 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 migration time, enabling multiple accurate analyses to be performed 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 so that the polynucleotide with the slowest electrophoretic mobility in the previous sample passes through the detection window of the electrophoresis system before the polynucleotide with the fastest electrophoretic mobility in the next sample. Patent Document 3 describes 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. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] Japanese Patent Application Laid-Open No. 2004-191247 [Patent Document 2] Japanese Patent Application Laid-Open No. 2000-314720 [Patent Document 3] Special Publication No. 2006-504099 [Non-patent literature]

[0018] [Non-Patent Document 1] J. Braz. Chem. Soc., Vol. 15, No. 3, 413-420, 2004 [Non-patent document 2] J Forensic Sci. 2005 Jul;50(4):842-8. [Non-patent document 3] J Chromatogr A. 1998 May 8;806(1):157-64. [Non-patent document 4] J Applied Polymer. 2010 Jan, 48994 Summary of the Invention [Problem to be solved by the invention]

[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, in

[0017] of Patent Document 3, it is stated that "the same capillary polyacrylamide gel is used for electrophoresis of all samples" and that "sequential electrophoresis is performed in the same capillary electrophoresis gel without washing or replacing the capillary gel," but there is no mention of performing the pre-run described above.

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

[0020] , sample introduction in paragraph

[0022] , and electrophoresis in paragraph

[0024] . Furthermore, Patent Document 2 describes in paragraph

[0026] that after the analysis is completed, sample introduction and electrophoresis are performed in the above-mentioned procedure. Furthermore, Patent Document 2 also describes in paragraph

[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 filling of the gel and preliminary electrophoresis. Furthermore, Patent Document 2 does not clarify when filling of the gel is performed in the description of the overall operation from paragraph

[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 the configuration when 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. [Means for solving the problem]

[0023] The method for continuous use of a separation medium according to the present invention, which has solved the above-mentioned problems, comprises a separation medium filling step of filling a capillary with a separation medium, a first pre-run step of performing a pre-run by applying a voltage in the same direction as the direction of electrophoresis to the capillary filled with the separation medium, a first sample injection step of injecting a first sample into the capillary that has been subjected to the pre-run, a first electrophoresis step of performing a first electrophoresis by applying a voltage to the capillary into which the first sample has been injected, a second pre-run step of performing a pre-run by applying a voltage in the same direction as the direction of electrophoresis to the capillary after the first electrophoresis step, a second sample injection step of injecting a next sample into the capillary that has been subjected to the second pre-run step, and a second electrophoresis step of applying a voltage to the capillary into which the next sample has been injected to perform the 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 to complete the analysis using the separation medium filled in one separation medium filling step, Thereafter, the process returns to the separation medium filling step, the separation medium in the capillary is replaced, and the first pre-run step and subsequent steps are carried out with new separation medium a desired number of times. [Effects of the Invention]

[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 apparent from the following description of the embodiments. [Brief explanation of the drawings]

[0025] [Figure 1] 1 is a schematic diagram illustrating the configuration of an electrophoresis apparatus to which a method for continuous use of a separation medium according to one embodiment of the present invention can be applied. [Figure 2] 1 is a schematic top view illustrating the configuration of an electrophoresis apparatus to which a method for continuous use of a separation medium according to one embodiment of the present invention can be applied. [Figure 3A] FIG. 3 is a cross-sectional view taken along line IIIa-IIIa in FIG. 2. [Figure 3B] FIG. 3B is an enlarged view of part IIIb in FIG. 3A. [Figure 4] FIG. 1 is a schematic diagram illustrating the control configuration of an electrophoresis apparatus to which a method for continuous use of a separation medium according to one embodiment of the present invention can be applied. [Figure 5] 1 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. [Figure 6] FIG. 1 is a schematic diagram showing an example of a graphical user interface (GUI) for setting the frequency of packing the separation medium in the separation medium packing step. [Figure 7] 1 is a flowchart illustrating the processing and electrophoresis in a microcomputer after a user inputs the frequency of separation medium filling using an input / output device, in relation to a method for continuous use of a separation medium according to a first embodiment of the present invention. [Figure 8] 1 is a flowchart illustrating the conventional method of filling a separation medium for each analysis. [Figure 9] 10 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. [Figure 10] 10 is a flowchart illustrating the processing and electrophoresis in a microcomputer after a user inputs the frequency of refilling the separation medium via an input / output device, in relation to a method for continuous use of a separation medium according to a second embodiment of the present invention. [Figure 11] FIG. 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. [Figure 12] FIG. 1 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. [Figure 13] 1 is a graph showing the number of sample injections (InjectionID) in Examples 1 and 2, that is, the statistical values ​​of the adjacent read length (CRL) for each of the first to sixth runs. [Figure 14] 1 is a graph comparing the total CRL statistics for sample injections 1 to 6 (up to Injection 6) in Example 1 and Example 2. [Figure 15] 1 is a graph continuously showing the transition of CRL in DNA sequence analysis in Example 4, where the number of sample injections (Injection ID) is 1 to 16, that is, the number of runs is 1 to 16. [Figure 16] 1 is a graph continuously showing the transition of EQ in fragment analysis from 1 to 12 sample injection numbers (Injection ID) in Example 4, that is, from 1 to 12 run numbers. [Figure 17] FIG. 10 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. [Figure 18]10 is a table showing the runtimes of the first to tenth runs of four capillaries (Cap1 to 4) in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[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 device) Before describing this method of use, an electrophoresis apparatus to which this method of use can be applied will be described. FIG. 1 is a schematic diagram illustrating the configuration of an electrophoresis device 1 to which this method can be applied. FIG. 2 is a schematic top view illustrating the configuration of an electrophoresis device 1 to which this method can be applied. FIG. 3A is a cross-sectional view taken along line 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 device 1 to which this method can be applied.

[0028] As shown in FIG. 1, the electrophoresis apparatus 1 to which this method can be applied (hereinafter, 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 / constant temperature bath unit 160 includes a constant temperature bath unit 110 and a constant temperature bath door 120, and is capable of maintaining a constant temperature inside. An irradiation / detection unit 130 is mounted behind the constant temperature bath unit 110, and detection can be performed during electrophoresis. The user sets a capillary array 10, which includes multiple capillaries 125 (see FIG. 3B), in the constant temperature bath unit 110, and electrophoresis is performed while the constant temperature of the capillary array 10 is maintained by the constant temperature bath unit 110, and detection is performed by the irradiation / detection unit 130. The constant temperature bath 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 the capillaries 125 are bundled together in a pressure-tight manner. The capillary head 127 forms the anode side end of the capillary array 10. Capillaries 125 are arranged at regular intervals in a plane inside the detection section 128. The irradiation and detection unit 130 irradiates light onto the capillaries 125 arranged in the detection section 128. The detection section 128 then detects fluorescence and the like emitted from the sample undergoing electrophoresis in each capillary 125 due to the light irradiation.

[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-side buffer solution container 30 set on the sample tray 100 includes an anode-side washing tank 31, an anode-side electrophoresis buffer solution tank 32, and a sample introduction buffer solution tank 33. The cathode-side buffer solution container 40 includes a waste solution tank 41, a cathode-side washing tank 42, and a cathode-side electrophoresis buffer solution 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 as shown in Figure 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 container 32-cathode side electrophoresis buffer container 43," and "sample introduction buffer container 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. Preferably, a liquid polymer is used as the separation medium. In this manner, the liquid delivery mechanism 60 in the device 1 can efficiently and automatically fill the capillary 125 (see FIG. 3B) with the separation medium and discharge the separation medium from the capillary 125. 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. Electrophoresis is performed by applying a current to GND at the electrode 115 via the cathode side buffer tank 40 and the anode side buffer tank 30.

[0037] 3B, each of the capillaries 125 constituting the capillary array 10 is fixed through a metal hollow electrode 124. The hollow electrode 124 is provided in a portion of the capillary 125 (from the cathode end to the load header 126). As shown in Figure 3B, the tip 125a of the capillary 125 protrudes from the hollow electrode 124 by approximately 0.5 mm. Note that 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 in each capillary 125 are attached as a unit to a load header 126 (see Figure 3A). All of the hollow electrodes 124 are connected to a high-voltage power supply 122 via the load header 126. The high-voltage power supply 122 applies a negative voltage to the hollow electrodes 124, so that the hollow electrodes 124 function as cathode electrodes when a voltage is applied to the hollow electrodes 124 during electrophoresis, sample injection, or the like.

[0038] As shown in FIG. 4, the voltage control mechanism includes a microcontroller (microcomputer) 141, a controller 142, a high-voltage power supply 122, a first ammeter 121, and a second ammeter 123. The microcomputer 141 checks the energization state of the anode-side electrophoresis buffer solution tank 32 and the cathode-side electrophoresis buffer solution tank 43 using the separation medium filled in the capillary array 10. Then, the microcomputer 141 outputs the results of the checked energization state to an input / output device (not shown). The input / output device is configured, for example, with a touch panel. The controller 142 controls the application of voltage to the current path by controlling the high voltage power supply 122, the movement of the sample tray 100, the movement of the sample container 50 by the X-axis driver 95, and the like.

[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 solution tank 43 (or the sample in the sample container 50), the electrophoresis buffer solution filled in the anode-side electrophoresis buffer solution tank 32 (or the buffer solution filled in the sample introduction buffer solution tank 33), and the electrode 115. More specifically, the high-voltage power supply 122 generates a voltage (negative voltage) lower than 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 order of 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] High-voltage power supply 122 is electrically connected to hollow electrode 124 via first ammeter 121 and to electrode 115 via 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 hollow electrode 124 to electrode 115. This electric field causes a negatively charged sample such as nucleic acid to migrate from tip 125a (see FIG. 3B) on the cathode side of capillary 125 toward detection unit 128 (see FIG. 3A). This causes the 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 GND 129a and transmits the current value to the microcomputer 141. The second ammeter 123 is usually 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 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. 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] (Continuous use of separation media) Next, the method of use will be described. Figure 5 is a flow chart illustrating the details of the method of continuous use of a separation medium according to the first embodiment of the present invention. This method of use is applied to an electrophoresis apparatus 1.

[0043] (First embodiment) As shown in Figure 5, the method of use 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 preset number of times (n times).

[0044] The separation medium filling step S1 is a step of filling the separation medium into the capillary 125. Filling the separation medium into the capillary 125 can be carried out by the liquid delivery mechanism 60 described above. The first pre-run step S2 is a step in which a voltage is applied to the capillaries 125 filled with the separation medium. The pre-run involves applying a voltage to the separation medium prior to sample electrophoresis. This pre-run removes impurities from the separation medium and creates 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, Y, and Z axes to position the anode and cathode sides of the capillary array 10 in the "anode side electrophoresis buffer tank 32 - cathode side electrophoresis buffer tank 43" positional relationship, and then applying a voltage. The first pre-run step S2 can be performed under typical conditions, such as 60°C, 3 minutes, and 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 anode-cathode positional relationship 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-, Y-, and Z-axes 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), which is normally performed 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 between both ends of the capillary 125 by the high-voltage power supply 122, causing the sample added to the separation medium to migrate into 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 sample has been injected to perform the next 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 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). 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 completes 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 to replace the separation medium in the capillary 125, and the first pre-run step S2 and subsequent steps are performed with the new separation medium as described above. This method of use repeats this process a desired number of times.

[0050] Here, the term "separation medium refilling frequency (f times)" used herein refers to the refilling of a new separation medium each time the total number of runs performed on a single separation medium reaches a predetermined number (f times). In other words, the separation medium refilling 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 refill of the separation medium. 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 refilling frequency in the separation medium refilling step S1 - 1 time" (f - 1 times). For example, if the separation medium refilling frequency (f times) is set to four times, one run of the first electrophoresis step S4 and three runs of the second electrophoresis step S7 are included as one set. In this manner, in this embodiment, when the separation medium filling frequency (f times) is set, 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 is set accordingly. On the other hand, 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 runs from the second onwards performed using the separation medium used in the first run.

[0051] As described above, this method allows a single filled separation medium to be used in multiple runs, thereby reducing the filling frequency of the separation medium, e.g., liquid polymer, which is the most expensive reagent required for capillary sequence analysis, and thereby reducing 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 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 single filled separation medium. In this manner, in this embodiment, by presetting the number of times (n) to repeat the second pre-run step S5, the second sample injection step S6, and the second electrophoresis step S7, one run of the first electrophoresis step S4 and an arbitrarily set number of n runs of the second electrophoresis step S7 can be performed 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 degree of degradation 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 frequency (f times) of separation medium filling in the separation medium filling step S1 can be input, for example, by an input / output device (not shown) connected to the microcomputer 141. Figure 6 is a schematic diagram showing an example of a graphical user interface (GUI) for setting the frequency of separation medium filling in the separation medium filling step S1. As shown in Figure 6, the input / output device can display a GUI for inputting the frequency (f times) of separation medium filling. In the GUI shown in Figure 6, the user can set the frequency of filling the separation medium to four times by entering, for example, "every 4 injections" or "4" in the "Polymer exchange frequency" field in the lower column, 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 can be displayed on the input / output device. To input the set number of repetitions (n ​​times) of the process, a field called "Number of repetitions of 2nd electrophoresis" is provided in the GUI. For example, by inputting "3 times" or "3" into this field or selecting "3" using the radio button, the set number of repetitions of the process can be set to 3.

[0056] In this method, when the separation medium filling frequency reaches a preset number (f times), in other words, when the total number of runs reaches f (f times) (further in other words, when the set number of repetitions of the above-mentioned process, calculated and set as f-1 times, is reached (Yes in step S8 of Figure 5), all analyses (runs) performed with one separation medium filling are terminated. After that, return to the separation medium filling step S1 to 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 preset number of times (f times), in other words, if the total number of runs does not reach f times (further in other words, if the set number of repetitions of the above steps (n times) calculated and set as f-1 times) is not reached (No in step S8 of Figure 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 process and electrophoresis in the microcomputer 141 after the user inputs the frequency of refilling the separation medium via the input / output device, in relation to the method for continuous use of the separation medium according to the first embodiment of the present invention. As shown in FIG. 7, the user inputs the separation medium filling frequency (step S11). In the example of FIG. 7, for example, the separation medium filling frequency is input as "4" (f=4). This causes the microcomputer 141 to set that when the total number of runs reaches four, the old separation medium is discarded from the capillary 125 and a new separation medium is filled into the capillary 125. The microcomputer 141 also calculates "separation medium filling frequency - 1" and sets the number of times the above process is to be repeated (n=3).

[0058] Then, when the user presses the start key, the analysis begins (step S12). The microcomputer 141 determines whether or not the electrophoresis is the first time (step S13). If the electrophoresis is the first time (Yes in step S13), the microcomputer 141 issues an instruction to fill the separation medium (step S14), and the device 1 performs the separation medium filling step S1. Next, the microcomputer 141 issues an instruction to perform a pre-run (step S15), and the apparatus 1 performs the first pre-run step S2. Next, the microcomputer 141 issues an instruction to inject the sample (step S16), and the device 1 performs the first sample injection step S3. Next, the microcomputer 141 issues an instruction to perform electrophoresis (step S17), and the device 1 performs 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 point immediately after the key input to start the run and determines whether the next electrophoresis is the first time (step S13). Since the next electrophoresis is not the first one (No in step S13), the microcomputer 141 issues an instruction to perform a pre-run (step S15), and the apparatus 1 performs the second pre-run step S5. Next, the microcomputer 141 issues an instruction to inject the sample (step S16), and the device 1 performs the second sample injection step S6. Next, the microcomputer 141 issues an instruction to perform electrophoresis (step S17), and the device 1 performs the second electrophoresis step S7.

[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 point immediately after the start of the run and determines whether the next electrophoresis is the first (Yes or No in step S13), and issues the above-mentioned instructions to the device 1 according to the determination (proceed to step S14 or step S15). If the total number of runs has achieved the separation medium filling frequency (f=4) (Yes in step S18), the microcomputer 141 instructs the device 1 to end the run (step S19), and the device 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 is compared with the conventional method of filling a separation medium for each analysis. FIG. 8 is a flow chart illustrating the conventional method of filling a separation medium 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.

[0064] In the conventional method shown in Figure 8, the separation medium is filled for each analysis, so these steps are performed every time. In other words, the conventional method shown in Figure 8 does not have 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 found that in the method of use according to the first embodiment, 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. The inventors found that the analytical performance obtained in this manner fully meets specifications, although the waveform data is slightly degraded, as described below. Furthermore, the inventors 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 description will be given of the method of use according to the second embodiment. Figure 9 is a flow chart illustrating the content of the method of continuous use of a separation medium according to the second embodiment of the present invention. As shown in Figure 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, the second sample injection step S6 and the second electrophoresis step S7 are repeated a preset 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 present 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 present method of use according to the first embodiment is a step of injecting the next batch of sample into the capillary that has been subjected to the second pre-run step S5. Therefore, the second sample injection step S6 of the present method of use according to the second embodiment and the second sample injection step S6 of the present method of use according to the first embodiment differ in content. Furthermore, in the present method of use according to the second embodiment, the second sample injection step S6 and the second electrophoresis step S7 are repeated a preset number of times (n times). In contrast, in the present method of use according to the first embodiment, the second pre-run step S5, the second sample injection step S6, and the second electrophoresis step S7 are repeated a preset number of times (n times). Therefore, the present method of use according to the second embodiment and the present method of use according to the first embodiment differ in the content of the steps that are repeated. The present method of use according to the second embodiment and the present method of use according to the first embodiment differ in that the present method of use according to the second embodiment does not have the second pre-run step S5, but otherwise have the same configuration. The following describes the method of use according to the second embodiment, focusing on the differences from the method of use according to the first embodiment.

[0068] As described above, the second sample injection step S6 in the method of use according to the second embodiment 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. Specifically, the method according to the second embodiment omits the second pre-run step S5, thereby suppressing the decomposition of urea caused by the pre-run and 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, by eliminating the pre-run, analyses can be completed more quickly. For example, a typical capillary DNA sequencer requires approximately 30 minutes for one analysis, of which approximately 3 minutes is for the pre-run. By omitting the pre-run for the next sample analysis, analysis time can be reduced by approximately 10%. This is an advantageous effect compared to conventional methods, which require 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 in the microcomputer 141 after the user inputs the frequency of refilling the separation medium via the input / output device, in relation to the method for continuous use of the separation medium according to the second embodiment of the present invention. As shown in FIG. 10, the user inputs the separation medium filling frequency (step S11). In the example of FIG. 10, for example, the separation medium filling frequency is input as "4" (f=4). This causes the microcomputer 141 to set that when the total number of runs reaches four, the old separation medium will be discarded from the capillary 125 and a new separation medium will be filled into the capillary 125. The microcomputer 141 also calculates "separation medium filling frequency - 1" and sets the number of times the above process will be repeated (n=3).

[0070] Then, when the user presses the start key, the analysis begins (step S12). The microcomputer 141 determines whether or not the electrophoresis is the first time (step S13). If the electrophoresis is the first time (Yes in step S13), the microcomputer 141 issues an instruction to fill the separation medium (step S14), and the device 1 performs the separation medium filling step S1. Next, the microcomputer 141 issues an instruction to perform a pre-run (step S15), and the apparatus 1 performs the first pre-run step S2. Next, the microcomputer 141 issues an instruction to inject the sample (step S16), and the device 1 performs the first sample injection step S3. Next, the microcomputer 141 issues an instruction to perform electrophoresis (step S17), and the device 1 performs 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 point immediately after the key input to start the run and determines whether the next electrophoresis is the first time (step S13). Since the next electrophoresis is not the first one (No in step S13), the microcomputer 141 issues an instruction to inject a sample (step S16), and the device 1 performs the second sample injection step S6. Next, the microcomputer 141 issues an instruction to perform electrophoresis (step S17), and the device 1 performs the second electrophoresis step S7.

[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 point immediately after the start of the run keystroke and determines whether the next electrophoresis is the first time (Yes or No in step S13), and issues the above-mentioned instructions to the apparatus 1 according to the determination (proceed to step S14 or step S16). If the total number of runs has achieved the separation medium filling frequency (f=4) (Yes in step S18), the microcomputer 141 instructs the device 1 to end the run (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. [Example]

[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 using the flow shown in Figure 5 and 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 a Sequencing Standard, BigDye. TM Terminator v3.1 (Thermo Fisher) was used.

[0075] <Conditions of Example 1> The separation medium filling step S1 was carried out in a manner specified for the capillary DNA sequencer. The first pre-run step S2 was carried out at 60°C for 3 minutes at 15 kV. The first sample injection step S3 was carried out at 60°C for 4 seconds at 1.2 kV. The first electrophoresis step S4 was carried out at 60°C for 30 minutes at 7.5 kV. The second pre-run step S5 was carried out at 60°C for 3 minutes at 15 kV. The second sample injection step S6 was carried out at 60°C for 4 seconds at 1.2 kV. The second electrophoresis step S7 was carried out 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 of Example 2> The separation medium filling step S1 was carried out in a manner specified for the capillary DNA sequencer. The first pre-run step S2 was carried out at 60°C for 3 minutes at 15 kV. The first sample injection step S3 was carried out at 60°C for 4 seconds at 1.2 kV. The first electrophoresis step S4 was carried out at 60°C for 30 minutes at 7.5 kV. The second sample injection step S6 was carried out at 60°C for 4 seconds at 1.2 kV. The second electrophoresis step S7 was carried out 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 in Example 1 are shown in FIGS. 11 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. 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. 12 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. 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 the 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 (InjectionID) in Examples 1 and 2, i.e., the number of runs from 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 the reference value was set to 600 bp, 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 the reference value was set to 400 bp, the CRLs in both Examples 1 and 2 cleared the reference value up to the sixth run, demonstrating that the specifications were fully met. 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, the results shown in FIG. 13 suggest that the specifications can be fully met up to approximately the eighth to tenth runs.

[0083] FIG. 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, which suppressed 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. Except for this, Example 3 was performed in the same manner as Example 1.

[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 runs of analysis. Except for this, Example 4 was performed in the same manner as Example 2.

[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. The CRL 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 CRL standard is set to 600 bp (when the DNA sequence to be analyzed is 600 bp long), a separation medium fill frequency of 4 satisfies the specifications. Furthermore, from the results shown in Figure 15, it was inferred that if the CRL standard is 500 bp or less (when the DNA sequence to be analyzed is 500 bp or less), the separation medium fill frequency can be increased to 5 or more to meet the specifications.

[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 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 as the number of runs after one liquid polymer filling increased. 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 filling frequency of 4 satisfies the 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), the separation medium filling frequency can be increased to 5 or more to meet the 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 and 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 Figure 17, in Examples 1 and 3 (as well as in the conventional method), a pre-run is performed for each run, so in the second run, a voltage is applied for the pre-run shown in part B at the beginning of the analysis, indicated by the two-dot dashed line on the left side in the upper explanatory diagram of Figure 17. In contrast, in Examples 2 and 4, pre-runs are not performed in the second and subsequent runs after filling with the liquid polymer. Therefore, in the second run, the voltage application for pre-runs shown in part B of the upper diagram of FIG. 17 is not necessary, as shown in the lower diagram of FIG. 17. Therefore, it was confirmed that Examples 2 and 4 can shorten the analysis time for the second and subsequent runs after filling with the liquid polymer. Specifically, the horizontal axis in the upper and lower diagrams of FIG. 17 indicates the index of analysis time. In Examples 1 and 3 shown in the upper diagram of 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 of 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 can shorten the analysis time by approximately 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 of the average runtime specification (Average run time spec judge), migration time uniformity (MT Uniformity), migration time uniformity specification (Spec), and judgment of the migration time uniformity specification (MT Uniformity judge).

[0094] As shown in the table in Figure 18, the runtimes for Caps 1 to 4 were longer in the second run, 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 as failing the average runtime specification. The other runs, 1 and 3 to 10, were pass. Also, as shown in the table in Figure 18, the total runtime was between 25 and 30 minutes, with some variation. On the other hand, the uniformity of the migration time met the specification of 5.0% or less for all of the migration times, and all of the first to tenth runs were pass.

[0095] The continuous use method of a 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 the present invention is not necessarily limited to those including all of the described configurations. Furthermore, it is possible to replace part of the configuration of one embodiment with the configuration of another embodiment, or 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. [Explanation of symbols]

[0096] 1. Electrophoresis device (this device) 10 Capillary Array 20 Separation media container 30 Anode side buffer container 31 Anode side cleaning tank 32 Anode side electrophoresis buffer tank 33 Sample introduction buffer tank 40 Cathode buffer container 41 Waste liquid tank 42 Cathode side cleaning tank 43 Cathode side electrophoresis buffer tank 50 Sample container 60 Liquid delivery mechanism 61 Plunger 80 Sampler Bass 85 Y-axis drive body 90 Z-axis drive body 95 X-axis drive body 100 sample trays 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 Roadheader 127 Capillary Head 128 Detector 129a, 129b GND 130 Irradiation detection unit 141 Microcomputer 142 Controller 150 Autosampler Unit 160 Radiation detection / thermostat unit S1 Separation media filling process S2 First pre-run process S3 First sample injection step S4 First electrophoresis step S5 Second pre-run process S6 2nd sample injection process S7 Second electrophoresis step

Claims

1. a separation medium filling step of filling the capillary with a separation medium; a first pre-run step in which a pre-run is performed by applying a voltage in the same direction as the direction of electrophoresis to the capillary filled with the separation medium; 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 in which a pre-run is performed by applying a voltage in the same direction as the direction of electrophoresis to the capillary after the first electrophoresis step; a second sample injection step of injecting the next sample into the capillary that has been subjected to the second pre-run step; a second electrophoresis step of applying a voltage to the capillary into which the next sample has been injected to perform the next electrophoresis; and A method for continuous use of a separation medium, in which the second pre-run step, the second sample injection step, and the second electrophoresis step are repeated a predetermined number of times, and then the analysis using the separation medium filled in one separation medium filling step is completed, and then the process returns to the separation medium filling step to replace the separation medium in the capillary, and each step from the first pre-run step onwards is performed with new separation medium a desired number of times.

2. a separation medium filling step of filling the capillary with a separation medium; a first pre-run step in which a pre-run is performed by applying a voltage in the same direction as the direction of electrophoresis to the capillary filled with the separation medium; 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 sample injection step of injecting a next batch of sample into the capillary after the first electrophoresis step; a second electrophoresis step of applying a voltage to the capillary into which the next sample has been injected to perform the next electrophoresis; and A method for continuous use of a separation medium, in which the second sample injection step and the second electrophoresis step are repeated a predetermined number of times, and then the analysis using the separation medium filled in one separation medium filling step is completed, and then the process returns to the separation medium filling step to replace the separation medium in the capillary, and each step from the first pre-run step onwards is performed with new separation medium a desired number of times.

3. In claim 1 or claim 2, A method for continuous use of a separation medium, wherein the separation medium is a liquid polymer.

4. In claim 1 or claim 2, A method for continuous use of a separation medium, wherein the frequency of packing the separation medium in the separation medium packing step is set by a graphical user interface.

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