Method for continuous use of separation media

Abstract

Provided is a method for continuously using a separation medium, which is capable of reducing the operating cost of analysis when a liquid polymer is used as the separation medium. A method for continuously using a separation medium according to the present invention comprises: a separation medium filling step (S1) in which a capillary tube is filled with a separation medium; a first pre-operation step (S2) in which pre-operation is performed; a first sample injection step (S3) in which a first sample is injected into the capillary tube; a first electrophoresis step (S4) in which a voltage is applied to perform first electrophoresis; a second pre-operation step (S5) in which pre-operation is performed after the first electrophoresis step (S4); a second sample injection step (S6) for injecting a next amount of sample into the capillary on which the second pre-operation step (S5) has been performed; and a second electrophoresis step (S7) in which the next amount of electrophoresis is performed. The second pre-operation step (S5), the second sample injection step (S6), and the second electrophoresis step (S7) are repeated for a preset number of times.

Description

Technical Field

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

[0002] The proportion of people over 60 years old in the total population will increase in the future, and all developed countries will usher in a super-aged society. Against this background, people's concern for health has been very high in recent years, and health care technologies that maintain health and longevity have attracted attention. Among them, genomic medicine is one of the most popular technologies. Genomic medicine is to use personal genetic information like text for diagnosis and apply medical treatment suitable for each patient. The device used to read this gene sequence information is a DNA sequencer. In other words, the DNA sequencer can be said to be one of the representative analysis devices.

[0003] The Human Genome Project, which planned to analyze the entire base sequence of the human genome, was completed in 2003. Technologies related to DNA sequences have made great progress since then, and various technologies related to various sequences have been developed. Among them, the technology called next-generation sequencing is characterized by ultra-parallel sequence interpretation. Through this technological innovation, it is now possible to interpret the human genome for less than $1,000.

[0004] However, the DNA reading method called Sanger method used in capillary sequencing, which is still adopted in the Human Genome Project, is considered the gold standard in DNA sequencing. The reason is that the reading base accuracy is high and the analysis cost per sample is advantageous compared to other methods such as next-generation sequencing. Therefore, the Sanger method used in this capillary sequencing has now become an indispensable technology in research sites around the world.

[0005] One of the reasons why capillary sequencers have established a firm position in the research field is the use of liquid polymers that can be used as separation media in capillaries. Before the popularization of capillary sequencers, DNA separation was usually carried out using a gel solidified between two gel plates. However, the production of this gel requires labor and therefore becomes a burden for the user. However, by using a liquid polymer, electrophoresis can be carried out by filling the flow path with the liquid polymer without making a gel. The liquid polymer can continue to measure by replacing the liquid polymer in the flow path in each measurement. That is, the labor of making the gel for the user can be saved.

[0006] If it is a liquid polymer, the capillary remains as it is, and if only the separation medium, i.e., the liquid polymer, is replaced, the measurement can be continuously implemented. In addition, in the microfluidic chip using a solid separation medium (gel), since the gel solidifies, it is necessary to discard the chip itself at each measurement and prepare a new chip. This has led to an increase in running costs and has become a burden on users. In this case, the liquid polymer that can be easily replaced becomes one of the advantageous features of the capillary sequencer.

[0007] In addition, as one of the biggest requirements of users of capillary sequencers, there is a reduction in running costs. The reagents used in the capillary sequencer include anode side buffer, cathode side buffer, liquid polymer, etc., among which the most expensive reagent is the medium for separating DNA, i.e., liquid polymer. In order to reduce the running cost of the most expensive liquid polymer, in non-patent literature 1, a capillary sequencer of Beckman Co. was used to verify whether multiple analyses can be performed on a single liquid polymer filling of the capillary. In this verification, the results of the deterioration of analytical performance with the overlap of the number of analyses in a single liquid polymer filling were shown. The conclusion mentions "Replacement of the separation matrix is ​​simple and is not worth compromising the results with its reuse" (replacing (capillary) liquid polymer is simple, and the experimental results should not be damaged by the reuse of liquid polymer). In other words, the reuse of liquid polymer damages analytical performance, so it is strongly recommended to replace the liquid polymer in each analysis.

[0008] The reason why the liquid polymer deteriorates after multiple analyses is the decomposition of urea contained in the liquid polymer. Generally, electrophoresis analysis in a capillary sequencer is performed by applying a high voltage of about 15 kV to the end of the capillary at a temperature of 60°C. Urea has the effect of dissociating two strands of DNA into one strand, but according to Non-Patent Document 2, urea has the problem of decomposing at high temperatures.

[0009] In addition, the capillary array used in DNA sequencing usually uses non-staining glass that is not chemically modified, and its surface is covered with hydroxyl groups. On the other hand, the buffer used in capillary sequencing is an aqueous solution near pH 8.0, and the glass surface of the capillary is negatively charged. Positive ions in the aqueous solution are adsorbed on the glass surface, but when a high voltage is applied to the capillary array in this state, the positive ions on the glass surface are dragged and electroosmotic flow is generated. Since this electroosmotic flow has the effect of deteriorating the separation performance of DNA, the quality of electroosmotic flow suppression has a great influence on the analysis results. Here, the liquid polymer is the separation medium of DNA, and it also has the effect of being adsorbed on the glass surface and preventing electroosmotic flow. However, according to non-patent literature 3, the decomposition products of urea have the effect of preventing liquid polymers from being adsorbed on the glass surface. As a result, as urea decomposes, electroosmotic flow increases, and the separation performance of DNA decreases.

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

[0011] The pre-run of (2) is a process in which a voltage is applied to the liquid polymer for only about 3 minutes after the capillary array is filled with the liquid polymer. This process is considered necessary to remove impurities in the liquid polymer and is necessary to adjust the separation medium environment suitable for analysis. In non-patent document 4, it is reported that the protein separation in the plate gel is greatly improved by applying the pre-run.

[0012] Currently, Thermo Fisher Scientific holds 90% of the capillary sequencer market. Thermo Fisher Scientific sells not only devices, but also reagents and analysis software, providing customers with a comprehensive solution from sample to answer. However, due to its strong monopoly, the operating costs of capillary sequencing remain high, and the black box nature of the device also hinders the freedom of customers. On the other hand, Thermo Fisher Scientific seeks to ease the freedom of customers by providing a variety of electrophoresis devices.

[0013] The specific reason for the high running costs is that the protocol for changing the liquid polymer, the separation medium for DNA, for each analysis is provided to customers in a form that does not allow for changes. In other words, when a customer fills the capillary array with liquid polymer once, the customer's analysis using the liquid polymer is limited to one time. In other words, customers cannot perform multiple analyses for one liquid polymer filling. On the other hand, liquid polymers are the most expensive reagents required for capillary sequencing analysis, so it is difficult for customers to reduce the running costs of the analysis.

[0014] Under such circumstances, several inventions have been proposed that can reduce the running cost of analysis. For example, Patent Document 1 discloses an electrophoresis analysis method in which the heat generated by each capillary is dissipated from the electrode via a heat conductive medium, and the temperature of each capillary during electrophoresis is maintained at a temperature above room temperature and below 80° C. Patent Document 1 states that by adopting such a structure, gel degradation can be prevented, and electrophoresis analysis can be repeatedly performed using the same gel.

[0015] In addition, for example, Patent Document 2 discloses a capillary electrophoresis device, which includes a detector for detecting the separated components and a unit for determining the end of the electrophoresis of the sample electrophoretically moving in the capillary (electrophoresis end determination unit). The electrophoresis end determination unit calculates the intensity of the signal peak corresponding to each component in the sample detected by the above-mentioned detector, and when the calculated intensity is lower than a specified value, it is determined that the electrophoresis of the above-mentioned sample in the above-mentioned capillary has ended. Patent Document 2 records that by adopting such a structure, when the electro-separation medium, i.e., gel, is repeatedly used for multiple analyses without replacing it, the start time of the measurement after the second time can be calculated without being too much or too little. In addition, Patent Document 2 records that by adopting such a structure, the danger of introducing multiple samples into the capillary at the same time and the electrophoresis time being unnecessarily prolonged can be avoided, and multiple accurate analyses can be performed in a minimum time.

[0016] Patent document 3 discloses a method for performing electrophoresis, which comprises the following steps: injecting the next sample into the injection end of the capillary electrophoresis gel at time n (TS-TF) in sequence, so that the polynucleotide with the slowest electrophoresis speed in the previous sample passes through the detection window of the electrophoresis system before the polynucleotide with the fastest electrophoresis speed in the next sample. Patent document 3 records that by adopting such a structure, in a typical embodiment, electrophoresis is performed up to a total of about 25 times in the same capillary electrophoresis gel, so that the same capillary polyacrylamide gel is used for electrophoresis of all samples without washing or replacing the capillary gel. Prior art literature Patent Literature

[0017] Patent Document 1: Japanese Patent Application Publication No. 2004-191247 Patent Document 2: Japanese Patent Application Publication No. 2000-314720 Patent Document 3: Japanese Patent 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 Technical problem to be solved by the invention

[0019]

[0008] of Patent Document 1 states that "electrophoresis analysis can be repeatedly performed using the same gel", but there is no description of performing the above-mentioned pre-run. Similarly, in

[0017] of Patent Document 3, there is a description that "the same capillary polyacrylamide gel is used for electrophoresis of all samples" and "electrophoresis is performed sequentially using the same capillary electrophoresis gel without washing or replacing the capillary gel", but there is no description of performing the above-mentioned pre-run.

[0020] On the other hand, in

[0020] of Patent Document 2, pre-elution (pre-run) is described, in

[0022] , sample introduction is described, and in

[0024] , electrophoresis is described. Then, in

[0026] of Patent Document 2, it is recorded that after the analysis is completed, sample introduction and electrophoresis are performed according to the above steps. In

[0020] of Patent Document 2, it is recorded that in pre-elution, standard samples and analytical samples are not introduced into the capillary. However, whether the above steps include gel filling and pre-boiling is not clear in Patent Document 2. In addition, in Patent Document 2, when to perform gel filling is not clear in the description of the overall operation after

[0020] .

[0021] Furthermore, the inventions described in Patent Documents 1 to 3 are all inventions using gel as a separation medium, and there is no description at all about the structure when a liquid polymer is used as a separation medium, nor is there any description as a suggestion, etc. Therefore, although the inventions described in Patent Documents 1 to 3 are proposed, the problem of achieving a reduction in the running cost of the analysis when a liquid polymer is used as a separation medium has not been solved.

[0022] The present invention has been made in view of the above-mentioned circumstances. An object of the present invention is to provide a method for continuously using a separation medium that can reduce the running cost of analysis when a liquid polymer is used as a separation medium. Technical solutions to technical problems

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

[0024] According to the present invention, a method for continuously using a separation medium capable of reducing the running cost of analysis when a liquid polymer is used as the separation medium is provided. Technical problems, structures, and effects other than those described above will become more apparent through the following description of the embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic structural diagram illustrating the structure of an electrophoresis apparatus to which the continuous use method of a separation medium according to one embodiment of the present invention can be applied. Figure 2 It is a schematic plan view illustrating the structure of an electrophoresis apparatus to which the continuous use method of a separation medium according to one embodiment of the present invention can be applied. Figure 3A yes Figure 2 IIIa-IIIa cross-sectional view. Figure 3B yes Figure 3A Enlarged view of part IIIb. Figure 4 This is a schematic diagram for explaining a control structure of an electrophoresis apparatus to which the continuous use method of a separation medium according to one embodiment of the present invention can be applied. Figure 5 This is a flowchart for explaining the contents of the method for continuously using a separation medium according to the first embodiment of the present invention. Figure 6 This is a schematic diagram showing an example of a graphical user interface (GUI) for setting the filling frequency of the separation medium in the separation medium filling step. Figure 7This is a flowchart for explaining the process and electrophoresis in the microcomputer after the user inputs the filling frequency of the separation medium through the input / output device, in the continuous use method of the separation medium according to the first embodiment of the present invention. Figure 8 This is a flow chart illustrating the contents of a conventional method for filling a separation medium for each analysis. Fig. 9 This is a flowchart for explaining the contents of the method for continuously using a separation medium according to the second embodiment of the present invention. Fig.10 This is a flowchart for explaining the process and electrophoresis in the microcomputer after the user inputs the filling frequency of the separation medium through the input / output device, in the continuous use method of the separation medium according to the second embodiment of the present invention. Fig.11 It is an explanatory diagram showing the DNA base sequences and waveform data around 400 bp in the 1st run (1st), the 3rd run (3rd), and the 8th run (8th) of Example 1. Fig.12 It is an explanatory diagram showing the DNA base sequences and waveform data around 600 bp in the 1st run (1st), the 3rd run (3rd), and the 8th run (8th) of Example 1. Fig.13 It is a graph showing the statistical values ​​of the number of sample injections (InjectionID) of Example 1 and Example 2, that is, the adjacent lead length (CRL) of each of the first to sixth operation numbers. Fig.14 This is a graph comparing the statistical values ​​of the total CRL of Example 1 and Example 2 when the number of sample injections is 1 to 6 times (up to Injection 6). Fig.15 This is a graph continuously showing the transition of CRL in the DNA sequence analysis of Example 4 when the number of sample injections (InjectionID) is 1 to 16, that is, the number of runs is 1 to 16. Fig.16 This is a graph continuously showing the transition of EQ in the fragment analysis in which the number of sample injections (InjectionID) of Example 4 is 1 to 12, that is, the number of runs is the 1st to 12th times. Fig.17 This is an explanatory diagram for comparing the difference in analysis time of the second run in Examples 1 and 3 in which a pre-run was performed for each run, and Examples 2 and 4 in which a pre-run was performed only in the first run after filling with a liquid polymer and no pre-run was performed in the second and subsequent runs. Fig.18This is a table showing the operation time and the like of the 1st to 10th operations of the four capillaries (Cap1 to Cap4) in Example 4. DETAILED DESCRIPTION

[0026] Hereinafter, a method for continuous use of a separation medium according to an embodiment of the present invention (hereinafter sometimes referred to as "the present method of use") is described in detail with reference to the accompanying drawings as appropriate. In the description of the embodiments, substantially the same or similar structures are denoted by the same reference numerals, and their description is sometimes omitted when the description is repeated.

[0027] (Electrophoresis device) Before describing the present method of use, an electrophoresis device to which the present method of use can be applied will be described. Figure 1 It is a schematic structural diagram for explaining the structure of an electrophoresis device 1 to which the present method of use can be applied. Figure 2 It is a schematic plan view for explaining the structure of an electrophoresis device 1 to which the present method of use can be applied. Figure 3A yes Figure 2 IIIa-IIIa cross-sectional view. Figure 3B yes Figure 3A Enlarged view of part IIIb. Figure 4 It is a schematic diagram for explaining the control structure of the electrophoresis apparatus 1 to which the present method of use can be applied.

[0028] like Figure 1 As shown, the electrophoresis apparatus 1 to which the present method of use can be applied (hereinafter sometimes referred to as "the present apparatus 1") can be roughly divided into two units: an automatic sampler unit 150 located at the lower part of the apparatus and an irradiation detection / constant temperature chamber unit 160 located at the upper part of the apparatus.

[0029] In the automatic sampler unit 150, the Y-axis driver 85 is mounted on the sampler base 80 and can be driven along the Y-axis. The Z-axis driver 90 is mounted on the Y-axis driver 85 and can be driven along the Z-axis. The sample tray 100 is mounted on the Z-axis driver 90. Then, as shown in FIG. Figure 1 and Figure 2 As shown, the user sets the separation medium container 20, the anode side buffer container 30, the cathode side buffer container 40, and the sample container 50 on the sample tray 100. The sample container 50 is set on the X-axis driving body 95 installed on the sample tray 100, and only the sample container 50 can be driven along the X-axis on the sample tray 100. The liquid feeding mechanism 60 is also installed on the Z-axis driving body 90. The liquid feeding mechanism 60 is arranged below the separation medium container 20.

[0030] like Figure 1As shown, in the irradiation detection / constant temperature bath unit 160, there are a constant temperature bath unit 110 and a constant temperature bath door 120, which can maintain a certain temperature. The irradiation detection unit 130 is installed behind the constant temperature bath unit 110 to perform detection during electrophoresis. In the constant temperature bath unit 110, the user sets a plurality of capillaries 125 (refer to Figure 3B ), electrophoresis is performed while the capillary array 10 is kept at a constant temperature in a constant temperature bath unit 110, and detection is performed by an irradiation detection unit 130. In addition, an electrode 115 is installed in the constant temperature bath unit 110, and the electrode 115 is used to drop to GND when a high voltage for electrophoresis is applied.

[0031] like Figure 3A As shown, the capillary array 10 includes a loading head 126, a capillary head 127, a detection unit 128, and the like. The loading head 126 is mounted on one end of the capillary array 10. The loading head 126 becomes the end of the capillary array 10 on the cathode side. The capillary head 127 is attached to the other end of the capillary array 10. The capillary head 127 is a detachable member obtained by bundling the capillary tubes 125 in a pressure-tight manner. The capillary head 127 is the end of the capillary array 10 on the anode side. The capillaries 125 are arranged in a plane at regular intervals inside the detection section 128. The irradiation detection unit 130 irradiates light to the capillaries 125 arranged in the detection section 128. Then, the detection section 128 detects fluorescence or the like generated from the sample electrophoresed in each capillary 125 by irradiating light.

[0032] As described above, the capillary array 10 is disposed in the thermostatic bath unit 110. The separation medium container 20, the anode buffer container 30, the cathode buffer container 40, and the sample container 50 can be driven along the YZ axis by the automatic sampler unit 150, and only the sample container 50 can be further driven along the X axis. The separation medium container 20, the anode buffer container 30, the cathode buffer container 40, and the sample container 50 can be automatically connected to any position of the capillary array 10 by the operation of the automatic sampler unit 150.

[0033] like Figure 2 As shown, the anode side buffer container 30 provided on the sample tray 100 includes an anode side cleaning tank 31, an anode side electrophoresis buffer tank 32, and a sample introduction buffer tank 33. In addition, the cathode side buffer container 40 includes a waste liquid tank 41, a cathode side cleaning tank 42, and a cathode side 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 arranged Figure 2 The positional relationship shown in FIG. That is, the anode side buffer container 30 is arranged on the front side of the device on the sample tray 100 (the side where the constant temperature chamber door 120 is opened and closed) and is arranged on the left side. The separation medium container 20 is arranged deep in the anode side buffer container 30. The sample container 50 is arranged on the front side of the device on the sample tray 100 and is arranged on the right side. The cathode side buffer container 40 is arranged deep in the sample container 50. Therefore, the positional relationship between the anode side and the cathode side when connected to the capillary array 10 is "separation medium container 20-waste liquid tank 41", "anode side cleaning tank 31-cathode side cleaning tank 42", "anode side electrophoresis buffer tank 32-cathode side electrophoresis buffer tank 43", "sample introduction buffer tank 33-sample container 50".

[0035] like Figure 3A As shown, the separation medium container 20 is inserted and arranged in the guide 101 embedded in the sample tray 100. The separation medium is contained in the separation medium container 20. As the separation medium, for example, a liquid polymer is preferably used. Thus, the liquid delivery mechanism 60 in the present device 1 can preferably and automatically deliver the liquid to the capillary 125 (see Figure 3B ) is filled with separation medium, or the separation medium is discharged from the capillary 125. The liquid delivery mechanism 60 is configured so that the plunger 61 built in the liquid delivery mechanism 60 is located below the separation medium container 20. The plunger 61 pushes the cylinder (not shown) provided in the separation medium container 20 upward, so that the separation medium (not shown) in the separation medium container 20 is introduced into the capillary 125 via the capillary head 127.

[0036] During electrophoresis, the capillary array 10 is Figure 3A The left side 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", applies a high voltage to the capillary array 10 on the cathode side, and flows GND through the cathode side buffer tank 40 and the anode side buffer tank 30 in the electrode 115, thereby performing electrophoresis.

[0037] like Figure 3B As shown, each capillary 125 constituting the capillary array 10 is passed through and fixed to a metal hollow electrode 124. The hollow electrode 124 is provided at a portion of the capillary 125 (from the cathode side end to the loading head 126). like Figure 3BAs shown, the front end 125a of the capillary 125 is in a state of protruding about 0.5 mm from the hollow electrode 124. The length of the capillary 125 protruding from the hollow electrode 124 is not limited to 0.5 mm. Moreover, all the hollow electrodes 124 provided in each capillary 125 are mounted on the loading head 126 in an integrated state (see Figure 3A ). In addition, all hollow electrodes 124 are connected to the high voltage power supply 122 via the loading head 126. Since the high voltage power supply 122 applies a negative voltage to the hollow electrodes 124, when a voltage is applied to the hollow electrodes 124 during electrophoresis or sample injection, the hollow electrodes 124 act as cathode electrodes.

[0038] like Figure 4 As shown, 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 uses the separation medium filled in the capillary array 10 to confirm the power-on state of the anode-side electrophoresis buffer tank 32 and the cathode-side electrophoresis buffer tank 43. Then, the microcomputer 141 outputs the result of the confirmed power-on state to an input-output device (not shown). The input-output device is composed of, for example, a touch panel. The controller 142 controls the high voltage power supply 122 to control application of voltage to the current path, movement of the sample tray 100 , movement of the sample container 50 by the X-axis driving body 95 , and the like.

[0039] The high-voltage power supply 122 applies voltage to the current path based on the control of the controller 142. The current path is the electrophoresis buffer solution filled in the hollow electrode 124, the electrophoresis buffer solution in the cathode side electrophoresis buffer solution tank 43 (or sometimes the sample in the sample container 50), the electrophoresis buffer solution filled in the anode side electrophoresis buffer solution tank 32 (or sometimes 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 GND 129a, 129b, so the flow of current when the negative voltage is applied by the high-voltage power supply 122 becomes Figure 4 That is, the current flows in the order of GND129a→second ammeter 123→electrode 115→capillary array 10→hollow electrode 124→first ammeter 121→high voltage power supply 122. Therefore, the current flow path becomes GND129a→second ammeter 123→electrode 115→capillary array 10→hollow electrode 124→first ammeter 121→high voltage power supply 122.

[0040] The high voltage power supply 122 is connected to the hollow electrode 124 via the first ammeter 121, and is connected to the electrode 115 via the second ammeter 123. When a voltage of several tens of kilovolts is applied to the two ends, an electric field is generated from the hollow electrode 124 to the electrode 115. By this electric field, negatively charged nucleic acid and other samples are discharged from the front end 125a (see Figure 125) on the cathode side of the capillary 125. Figure 3B ) to the detection unit 128 (refer to Figure 3A ) moves. Therefore, in Figure 4 A negatively charged sample (eg, DNA) migrates in the direction of arrow A1.

[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 sends its current value to the microcomputer 141. In addition, the second ammeter 123 detects the current flowing from the electrode 115 to the GND129a, and sends its current value to the microcomputer 141. The second ammeter 123 is usually used to check the current value and its change. The reason is that the second ammeter 123 more directly reflects the current value flowing through the electrophoresis path. When there is leakage between the first ammeter 121 and the second ammeter 123, the value shown by the first ammeter 121 also includes the current value of the leakage, while the value shown by the second ammeter 123 does not include the amount of leakage. That is, the net current flowing through the electrophoresis path is detected. Between the first ammeter 121 and the second ammeter 123 is a part where there is a medium with a relatively large resistance compared to metal, such as a buffer solution or a separation medium (such as a liquid polymer), and there are more blocks or capillaries 125 and other connecting parts. Therefore, it can be said that the circuit passing through the first ammeter 121 is a portion where leakage easily occurs.

[0042] (Continuous use method of separation medium) Next, the method of use will be described. Figure 5 This is a flowchart for explaining the contents of the method for continuously using a separation medium according to the first embodiment of the present invention. This method of use is applicable to the electrophoresis device 1 .

[0043] (Implementation Method 1) like Figure 5 As shown, the method of use according to Embodiment 1 includes a separation medium filling step S1 , a first pre-operation step S2 , a first sample injection step S3 , a first electrophoresis step S4 , a second pre-operation step S5 , a second sample injection step S6 , and a second electrophoresis step S7 . Then, in the present method of use, the second pre-operation 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 capillary 125 with the separation medium. The filling of the capillary 125 with the separation medium can be performed by the liquid feeding mechanism 60 described above. The first pre-operation step S2 is a step of performing a pre-operation by applying a voltage to the capillary 125 filled with the above-mentioned separation medium. The pre-operation refers to applying a voltage to the separation medium before the electrophoresis of the sample. By performing the pre-operation, impurities in the separation medium can be removed, and the separation medium environment suitable for analysis can be adjusted. The pre-operation is performed without injecting the sample. The first pre-operation step S2 can be performed by driving the sample tray 100 appropriately along the X-axis, Y-axis, and Z-axis so that the positional relationship between the anode side and the cathode side in the connection with the capillary array 10 becomes "anode side electrophoresis buffer tank 32-cathode side electrophoresis buffer tank 43", and applying a voltage. The first pre-operation step S2 can be performed under general conditions, such as 60°C×3 minutes×15kV.

[0045] The first sample injection step S3 is a step of injecting the first sample into the capillary 125 that has been pre-operated. 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 becomes "sample introduction buffer tank 33-sample container 50", and applying a voltage. The first sample injection step S3 can be performed, for example, at 60°C × 4 seconds × 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 is 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, the Y-axis, and the Z-axis so that the positional relationship between the anode side and the cathode side in the connection with the capillary array 10 becomes "anode side electrophoresis buffer tank 32-cathode side electrophoresis buffer tank 43", and applying a voltage. The first electrophoresis step S4 can be performed, for example, at 60°C × 30 minutes × 7.5 kV.

[0047] The second pre-operation step S5 is a step of applying voltage to the capillary 125 after the first electrophoresis step S4 to perform a pre-operation. That is, in the present method of use, the separation medium in the capillary 125 is not discarded and re-injected (i.e., the separation medium is exchanged) as is usually done after the last electrophoresis (operation) analysis, but the pre-operation (second pre-operation step S5) for the next sample analysis is performed while the separation medium used in the last operation is maintained. The operation means that a potential difference is generated at both ends of the capillary 125 by the high voltage power supply 122, so that the sample applied to the separation medium migrates in the capillary 125. During the execution of the operation, the irradiation detection unit 130 detects the sample.

[0048] The second sample injection step S6 is a step of injecting the next amount of sample into the capillary 125 that has undergone the second pre-operation 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 voltage to the capillary 125 into which the next amount of sample is injected, and performing electrophoresis of the next amount. The second electrophoresis step S7 can be performed in the same manner as the first electrophoresis step S4.

[0049] Then, as described above, the present method of use repeats the second pre-operation step S5, the second sample injection step S6, and the second electrophoresis step S7 a preset number of times (n times). After the present method of use repeats a series of steps of the second pre-operation step S5, the second sample injection step S6, and the second electrophoresis step S7 a preset number of times, the analysis of the separation medium filled in one separation medium filling step S1 is completed. Then, the process returns to the separation medium filling step S1, the separation medium in the capillary 125 is replaced, and each step after the first pre-operation step S2 is performed as described above using the new separation medium. In the present method of use, the desired number of times is performed.

[0050] Here, the filling frequency (f times) of the separation medium in this specification means that each time the total number of multiple operations performed on the separation medium filled once reaches a predetermined number (f times), a new separation medium is filled (the number of times the separation medium is refilled). That is, the filling frequency (f times) of the separation medium means the total number of operations of the first electrophoresis step S4 once and the second electrophoresis step S7 repeated n times for the filling of the separation medium once. That is, f times = 1 + n times. In other words, the number of repetitions (n ​​times) of the second pre-operation step S5, the second sample injection step S6, and the second electrophoresis step S7 can be calculated and set by "the filling frequency of the separation medium based on the separation medium filling step S1 - 1 time" (f-1 times). For example, if the filling frequency (f times) of the separation medium is set to 4 times, then in its setting content, one set includes one operation of the first electrophoresis step S4 and three operations of the second electrophoresis step S7. Therefore, in the present embodiment, when the filling frequency of the separation medium is set (f times), the set number of repetitions of the second preliminary operation step S5, the second sample injection step S6, and the second electrophoresis step S7 (n times) is set accordingly. On the other hand, the number of repetitions (n ​​times) of the second preliminary operation step S5, the second sample injection step S6, and the second electrophoresis step S7 refers to the number of runs after the second run using the separation medium used in the first run.

[0051] As described above, in the present method of use, the separation medium filled once is used in multiple runs. Therefore, the present method of use can reduce the filling frequency of the most expensive separation medium, such as liquid polymer, among the reagents required for capillary sequencing analysis, thereby reducing the running cost of the analysis.

[0052] Here, in the present embodiment, the user can also arbitrarily set the set number of repetitions (n ​​times) of the second pre-operation step S5, the second sample injection step S6, and the second electrophoresis step S7. The set number of repetitions of these steps (i.e., the number of repetitions based on the operation of the second electrophoresis step S7) can be, for example, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, or 25 times, but can also be 26 times or more. Since the first electrophoresis step S4 is operated once, when the set number of repetitions is set to, for example, 5 times, a total of 6 operations are performed using the separation medium filled once. Thus, in this method, by presetting the set number of repetitions (n ​​times) of the second pre-operation step S5, the second sample injection step S6 and the second electrophoresis step S7, each time the separation medium is filled once, the first electrophoresis step S4 can be run once and the second electrophoresis step S7 can be run arbitrarily set n times in one group.

[0053] The set number of repetitions of the above-mentioned steps (n times) can be arbitrarily set according to the state of the sample such as the degradation condition of the separation medium and the length of the DNA chain. For example, if the length of the DNA chain is about 300 bp, the filling of the separation medium can be set to 4 to 5 times (a total of 5 to 6 operations, i.e., a filling frequency of the separation medium of 5 to 6 times). In addition, for example, if the length of the DNA chain is about 600 bp, the filling of the separation medium can be set to 3 times (a total of 4 operations, i.e., a filling frequency of the separation medium of 4 times).

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

[0055] Although in Figure 6 Not shown in the figure, but the input of the set number of times (n times) of the repetition of the above-mentioned process can also be carried out in the same manner as the filling frequency (f times) of the separation medium. For example, the above-mentioned input-output device can be made to display a GUI for inputting the set number of times (n times) of the repetition of the above-mentioned process. In order to input the set number of times (n times) of the repetition of the above-mentioned process, the "Number of repetitions of 2nd electrophoresis" column is set in the GUI, and in this column, for example, "3 times (3times)", "3" etc. are input, or "3" is selected by a radio button, and the set number of times of the repetition of the above-mentioned process can be set to 3 times.

[0056] In the present method of use, if the filling frequency of the separation medium reaches a preset number of times (f times), in other words, if the total number of operations reaches f times (further in other words, if the set number of repetitions of the above-mentioned step calculated and set as f-1 times (n times)) is reached (in Figure 5 If the result of step S8 is "Yes", all the analysis (operation) performed by filling the separation medium once is completed. Then, the process returns to the separation medium filling step S1, replaces the separation medium in the capillary 125, and performs each step after the first pre-operation step S2 as described above with the new separation medium. On the other hand, when the frequency of the separation medium filling step S1 does not reach the preset number of times (f times), in other words, when the total number of operations does not reach f times (further in other words, when the set number of repetitions of the above step calculated and set as f-1 times (n times)) is not reached ( Figure 5 If the result of step S8 is "No", the process returns to the second preliminary operation step S5, and the second preliminary operation step S5, the second sample injection step S6 and the second electrophoresis step S7 are performed.

[0057] Figure 7 This is a flowchart for explaining the process and electrophoresis in the microcomputer 141 after the user inputs the filling frequency of the separation medium through the input / output device, in the continuous use method of the separation medium according to the first embodiment of the present invention. like Figure 7 As shown in FIG. 1 , the user inputs the filling frequency of the separation medium (step S11). Figure 7In the example of , for example, the filling frequency of the separation medium is input as "4" (f=4). Thus, the microcomputer 141 is set so that when the total number of operations reaches 4, the old separation medium is discarded from the capillary 125 and a new separation medium is filled into the capillary 125. In addition, the microcomputer 141 calculates "filling frequency of the separation medium - 1" and sets the set number of repetitions of the above process (n=3).

[0058] Then, when the user inputs the key to start the operation, the analysis starts (step S12). The microcomputer 141 determines whether 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 preliminary operation (step S15 ), and the first preliminary operation step S2 is performed in the present device 1 . Next, the microcomputer 141 issues an instruction to perform sample injection (step S16 ), and the first sample injection step S3 is performed in the apparatus 1 . Next, the microcomputer 141 issues an instruction to perform electrophoresis (step S17 ), and the first electrophoresis step S4 is performed in the present apparatus 1 .

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

[0060] After the electrophoresis is finished, the microcomputer 141 determines whether the total number of runs has reached the input filling frequency of the separation medium (f=4) (step S18). If the total number of runs has not reached the filling frequency of the separation medium (f=4) ("No" in step S18), the microcomputer 141 returns to the state immediately after the key input for the start of the run, and determines whether the electrophoresis to be performed next is the first time ("Yes" or "No" in step S13), and issues the above-mentioned instruction to the device 1 based on the determination (proceeding to step S14 or step S15). When the total number of operations reaches the filling frequency of the separation medium (f=4) (Yes in step S18), the microcomputer 141 issues an operation termination instruction to the device 1 (step S19), and the device 1 terminates the operation.

[0061] Microcomputer 141 performs the operation at the number of times set by the user. Figure 7 The sample is analyzed by following the above series of processes shown.

[0062] Thus, in the present method of use, within the set filling frequency of the separation medium, the separation medium is not filled when the electrophoresis is not the first time. Therefore, the present method of use can reduce the filling frequency of the most expensive separation medium, such as liquid polymer, among the reagents required for the analysis of capillary sequencing, thereby reducing the running cost of the analysis.

[0063] Here, the present method of use is compared with a conventional method of filling a separation medium for each analysis. Figure 8 This is a flow chart illustrating the contents of a conventional method for filling a separation medium for each analysis. like Figure 8 As shown, the conventional method includes a separation medium filling step S101 , a pre-running step S102 , a sample injecting step S103 , and an electrophoresis step S104 . Figure 8 These steps in the prior art method shown are respectively equivalent to Figure 5 The present method of use shown includes a separation medium filling step S1, a first pre-running step S2, a first sample injecting step S3, and a first electrophoresis step S4.

[0064] Figure 8 In the conventional method shown, since the separation medium is filled for each analysis, these steps are performed each time. Figure 8 The existing methods shown do not have Figure 5 The second pre-running step S5, the second sample injection step S6, and the second electrophoresis step S7 in the present method of use are not repeated. Therefore, the filling frequency of the most expensive separation medium among the reagents required for the analysis of capillary sequencing in the conventional method is not reduced, and the running cost of the analysis remains high.

[0065] The inventors of the present invention have conducted research to solve the technical problem of the present invention. As a result, in the present method of use involved in embodiment 1, the second pre-operation step S5, the second sample injection step S6, and the second electrophoresis step S7 are repeated for a preset number of times. The analysis performance obtained in this way is as described later. Although the waveform data is slightly degraded, it is found that it fully meets the specifications. In addition, it is found that the present method of use involved in embodiment 1 is different from Figure 8 Compared with the existing method shown, an improvement in turnaround time (TAT) can be achieved.

[0066] (Implementation Method 2) Next, the present method of use according to the second embodiment will be described. Fig. 9 This is a flowchart for explaining the contents of the method for continuously using a separation medium according to the second embodiment of the present invention. like Fig. 9 As shown, the present method of use according to Embodiment 2 includes a separation medium filling step S1 , a first pre-running step S2 , a first sample injecting step S3 , a first electrophoresis step S4 , a second sample injecting step S6 , and a second electrophoresis step S7 . Then, in this method of use, the second sample injection step S6 and the second electrophoresis step S7 are repeated a preset number of times (n times).

[0067] The present use method according to the second embodiment is different from the present use method according to the first embodiment having the second pre-operation step S5 in that the second pre-operation step S5 is not included. In addition, the second sample injection step S6 of the present method of use according to Embodiment 2 is a step of injecting the next amount 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 Embodiment 1 is a step of injecting the next amount of sample into the capillary after the second pre-operation step S5. Therefore, the second sample injection step S6 of the present method of use according to Embodiment 2 is different from the second sample injection step S6 of the present method of use according to Embodiment 1 in terms of process content. In addition, the present method of use involved in Embodiment 2 repeats the second sample injection step S6 and the second electrophoresis step S7 for a preset number of times (n times). In contrast, the present method of use involved in Embodiment 1 repeats the second pre-operation step S5, the second sample injection step S6, and the second electrophoresis step S7 for a preset number of times (n times). Therefore, the contents of the repeatedly performed steps are different between the present method of use involved in Embodiment 2 and the present method of use involved in Embodiment 1. 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-operation step S5 , but the other points are the same configuration. Hereinafter, the present method of use according to the second embodiment will be described, centering 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 present method of use involved in Embodiment 2 is a step of injecting the next amount of sample into the capillary 125 after the first electrophoresis step S4 (directly without pre-running). The inventors of the present invention have conducted research to solve the technical problem of the present invention. Surprisingly, if the next amount of sample is directly injected into the capillary 125 after the first electrophoresis step S4 without pre-running, and the next electrophoresis is performed, that is, when the second sample injection step S6 and the second electrophoresis step S7 are performed after the first electrophoresis step S4, compared with the case where a pre-run (the second pre-run step S5 in Embodiment 1) is performed before the second sample injection step, it is found that the number of analyses can be increased, the turnaround time (TAT) can be improved, the analytical performance can be improved, etc. These effects are caused by omitting the pre-run and suppressing the deterioration of the separation medium. As the deterioration of the separation medium, for example, the decomposition of urea can be cited. That is, the present method of use involved in embodiment 2 suppresses the decomposition of urea produced by pre-operation by omitting the second pre-operation process S5, and reduces the effect of preventing separation media such as liquid polymers caused by the decomposition products of urea from being adsorbed on the glass surface. As a result, the electroosmotic flow is not easy to increase (suppressed), thereby, the number of analysis times and analytical performance of samples such as DNA are improved. In addition, since no pre-operation is performed, the analysis can be terminated as soon as possible accordingly. For example, for a general capillary DNA sequencer, one analysis requires about 30 minutes, and the pre-operation requires about 3 minutes. When the sample of the next amount is analyzed, since the pre-operation is omitted, the analysis time can be shortened by about 10%. This can be said to be a favorable effect compared with the existing method of pre-operation for each analysis or the present method of use involved in embodiment 1.

[0069] Fig.10 This is a flowchart for explaining the process and electrophoresis in the microcomputer 141 after the user inputs the filling frequency of the separation medium through the input / output device, in the continuous use method of the separation medium according to the second embodiment of the present invention. like Fig.10 As shown in FIG. 1 , the user inputs the filling frequency of the separation medium (step S11). Fig.10In the example of , for example, the filling frequency of the separation medium is input as "4" (f=4). Thus, the microcomputer 141 is set so that when the total number of operations reaches 4, the old separation medium is discarded from the capillary 125 and a new separation medium is filled into the capillary 125. In addition, the microcomputer 141 calculates "filling frequency of the separation medium - 1" and sets the set number of repetitions of the above process (n=3).

[0070] Then, when the user inputs the key to start the operation, the analysis starts (step S12). The microcomputer 141 determines whether 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 preliminary operation (step S15 ), and the first preliminary operation step S2 is performed in the present device 1 . Next, the microcomputer 141 issues an instruction to perform sample injection (step S16 ), and the first sample injection step S3 is performed in the apparatus 1 . Next, the microcomputer 141 issues an instruction to perform electrophoresis (step S17 ), and the first electrophoresis step S4 is performed in the present apparatus 1 .

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

[0072] After the electrophoresis is finished, the microcomputer 141 determines whether the total number of runs has reached the input filling frequency of the separation medium (f=4) (step S18). If the total number of runs has not reached the filling frequency of the separation medium (f=4) ("No" in step S18), the microcomputer 141 returns to the state immediately after the key input for the start of the run, and determines whether the electrophoresis to be performed next is the first time ("Yes" or "No" in step S13), and issues the above-mentioned instruction to the device 1 based on the determination (proceeding to step S14 or step S16). When the total number of operations reaches the filling frequency of the separation medium (f=4) (Yes in step S18), the microcomputer 141 issues an operation termination instruction to the device 1 (step S19), and the device 1 terminates the operation.

[0073] Microcomputer 141 performs the operation at the number of times set by the user. Fig.10 The sample is analyzed by following the above series of processes shown. Example

[0074] Next, an example of the present method of use will be described. [1] Study 1 <Example 1> Example 1 Figure 5 The capillary sequencing DNA base sequence analysis was performed under the following conditions and in the process shown. The capillary DNA sequencer used was DS3000 manufactured by Hitachi High-Tech Corporation. A liquid polymer was used as a separation medium. The liquid polymer used was Spectrum Compact Polymer7 (CE237A manufactured by Promega Corporation). The DNA sample used as the analysis object was Sequencing Standard, BigDye TM Terminator v3.1 (manufactured by Thermo Fisher).

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

[0076] In Example 1, the filling frequency of the separation medium is set to 8 (i.e., the number of repetitions of the second pre-operation step S5, the second sample injection step S6, and the second electrophoresis step S7 is set to 7), which is performed twice in succession as a group, and a total of 16 runs are analyzed.

[0077] <Example 2> Example 2 Fig. 9The capillary sequencing DNA base sequence analysis was performed 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 is performed according to the method specified in the capillary DNA sequencer. The first pre-operation step S2 was performed at 60° C.×3 minutes×15 kV. The first sample injection step S3 was performed at 60° C.×4 seconds×1.2 kV. The first electrophoresis step S4 was performed at 60° C.×30 minutes×7.5 kV. The second sample injection step S6 was performed at 60° C.×4 seconds×1.2 kV. The second electrophoresis step S7 was performed at 60° C.×30 minutes×7.5 kV.

[0079] In Example 2, the filling frequency of the separation medium was set to 8 (ie, 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 in succession as a set, for a total of 16 runs of analysis.

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

[0081] like Fig.11 and Fig.12 As shown in Figure 1, when the number of runs after filling the liquid polymer once increases, the waveform data deteriorates slightly, but the DNA base sequence can be analyzed without any problems. Fig.12In the eighth run shown, there was a portion near 645 bp that could not be analyzed with sufficient accuracy, but other than that, analysis was possible with sufficiently high accuracy. Although illustration of Example 2 is omitted, the results are substantially the same as those of Example 1.

[0082] Fig.13 It is a graph showing the statistical values ​​of the number of sample injections (InjectionID) of Example 1 and Example 2, that is, the adjacent lead length (CRL) of each of the first to sixth operation numbers. Fig.13 The statistical values ​​are shown when the same analysis was performed independently three times. like Fig.13 As shown, when 600bp is determined as the baseline value, in both Example 1 and Example 2, it is confirmed that the CRL clears the baseline value until the 4th run, and the specification is fully met. Assuming that 400bp is determined as the baseline value, it can be seen in both Example 1 and Example 2 that the CRL can clear the baseline value until the 6th run, and the specification is fully met. The baseline value can be changed arbitrarily according to the length of the DNA base sequence to be analyzed. For example, when the DNA base sequence to be analyzed is a PCR amplification product and its length can be expected in advance to be 300bp or 400bp, etc., it can be used as a baseline value for research. For example, when the length of the PCR amplification product is 300bp, according to Fig.13 From the results shown, it is estimated that the specifications can be fully met until the 8th to 10th operation.

[0083] Fig.14 This is a graph comparing the statistical values ​​of the total CRL of Example 1 and Example 2 when the number of sample injections is 1 to 6 (up to Injection 6) (p = 0.04721). Fig.14 As shown, it was confirmed that the specifications were fully satisfied in both Example 1 and Example 2. In addition, it was confirmed that the CRL of Example 2 tended to be higher than that of Example 1. It was concluded that this was because the second pre-operation step S5 was not performed in Example 2, so the decomposition of urea contained in the liquid polymer was suppressed.

[0084] [2] Study 2 In Study 2, the filling 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 following analysis was performed.

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

[0086] <Example 4> In Example 4, the filling frequency of the separation medium 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 in a row as a group, and a total of 16 runs were analyzed. Example 4 was analyzed in the same manner as Example 2 except for this.

[0087] Fig.15 This is a graph showing the transition of CRL in DNA sequence analysis in which the number of sample injections (InjectionID) of Example 4 is 1 to 16, i.e., the number of runs is 1 to 16. In the figure, 1, 2, and 3 on the right side represent the capillary numbers. That is, Fig.15 The line graph shows the transition of CRL of the 1st to 16th runs of the three capillaries. In addition, the "▲" in the figure shows the result of the first run after filling or refilling (changing) the liquid polymer. The "□" in the figure shows the results of the second, third and fourth runs using the liquid polymer used in the first run.

[0088] like Fig.15 As shown, due to the individual differences of capillaries 1 to 3, there are capillaries with good analysis status and capillaries with poor analysis status, and when the number of runs after filling with liquid polymer once increases, there are capillaries whose CRL slightly decreases. However, the DNA base sequence was analyzed without any problems. It can be confirmed that when the CRL benchmark is set to 600bp (when the length of the DNA base sequence to be analyzed is 600bp), if the filling frequency of the separation medium is set to 4, the sufficient specifications are met. From Fig.15 The results shown indicate that, for example, if the CRL standard is 500 bp or less (if the length of the DNA base sequence to be analyzed is 500 bp or less), sufficient specifications can be met even if the filling frequency of the separation medium is increased to 5 or more.

[0089] in addition, Fig.16 This is a graph showing the change of EQ of the maximum base length within the range of single base separation in the fragment analysis of Example 4 with the number of sample injections (InjectionID) being 1 to 12, i.e., the number of runs being the 1st to 12th. In the figure, 1, 2, and 3 on the right side indicate the capillary numbers. That is, Fig.16The line graph shows the transition of EQ of the 1st to 12th runs of the three capillaries. In addition, the "▲" in the figure shows the result of the first run after filling or refilling (changing) the liquid polymer. The "□" in the figure shows the results of the second, third and fourth runs using the liquid polymer used in the first run.

[0090] like Fig.16 As shown, due to the individual differences of capillaries 1 to 3, there are capillaries with good analysis status and capillaries with poor analysis status, and when the number of runs after filling with liquid polymer once increases, there are capillaries with slightly lower EQ. However, the DNA base sequence was analyzed without any problems. From this, it can be confirmed that when the EQ standard is set to 400bp (when the length of the DNA base sequence to be analyzed is 400bp), if the filling frequency of the separation medium is set to 4, the sufficient specifications are met. From Fig.16 The results shown indicate that, for example, if the EQ standard is 200 bp or less (if the length of the DNA base sequence to be analyzed is 200 bp or less), sufficient specifications can be met even if the filling frequency of the separation medium is increased to 5 or more.

[0091] Fig.17 This is an explanatory diagram for explaining the difference in analysis time of the second run in comparison 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 for the first run after filling with a liquid polymer and no pre-run is performed for the second and subsequent runs. Regarding the point that a pre-run is performed for each run, it can be said that Examples 1 and 3 are the same as the conventional method.

[0092] like Fig.17 As shown, since embodiments 1 and 3 (the existing method also) perform a pre-run for each run, in the second run, Fig.17 In the above explanatory diagram, voltage application is performed for the preliminary operation shown in the B portion at the initial stage of the analysis start shown by the two-dot chain line on the left side. In contrast, in Examples 2 and 4, no pre-run was performed in the second and subsequent runs after the liquid polymer was filled. Fig.17 As shown in the following illustration, it is not necessary to Fig.17 The voltage of the pre-run shown in the B part of the above explanatory diagram is applied. Therefore, it is confirmed that Examples 2 and 4 can shorten the analysis time for the second and subsequent runs after filling with liquid polymer. Specifically, Fig.17 The horizontal axis in the above and below illustrations represents the index of analysis time. Fig.17 In the embodiments 1 and 3 shown in the above explanatory diagram, the index from the start to the end of the analysis is about 300, and Fig.17In Examples 2 and 4 shown in the following explanatory diagrams, the index from the start to the end of the analysis was approximately 265. This confirmed that Examples 2 and 4 could shorten the analysis time by approximately 10% compared to Examples 1 and 3 (and the conventional method).

[0093] Fig.18 This is a table showing the operation time and the like of the 1st to 10th operations of the four capillaries (Cap1 to Cap4) in Example 4. Fig.18 The table shown shows these running time [minutes], average running time (Average run time) [minutes], specification of average running time (Average run time spec) [minutes], judgment on the specification of average running time (Average run time spec judge), uniformity of migration time (Migration Time) (MT Uniformity: MT uniformity), specification of migration time uniformity (Spec), judgment on the specification of migration time uniformity (MTUniformity judge: MT uniformity judgment).

[0094] like Fig.18 As shown in the table, the running time of Cap1 to Cap4 in the second run is longer, so the average running time is also longer. Therefore, the second run cannot meet the average running time specification of less than 30 minutes, and the average running time specification is judged as unqualified (Fail). The other 1st, 3rd to 10th runs are qualified (Pass). In addition, Fig.18 As shown in the table, in general, the running time is 25 minutes to 30 minutes, with some deviations. On the other hand, the uniformity of the swimming time all satisfied the standard of the uniformity of the swimming time of 5.0% or less, and all the 1st to 10th runs were qualified.

[0095] Above, the continuous use method of the separation medium of the present invention is described in detail by the embodiments and examples, but the present invention is not limited to the above-mentioned embodiments, and includes various modifications. For example, in the above-mentioned embodiments, the present invention is described in detail for easy understanding, but is not necessarily limited to including all structures described. In addition, a part of the structure of a certain embodiment can be replaced with the structure of other embodiments, and in addition, the structure of other embodiments can also be added to the structure of a certain embodiment. In addition, other structures can also be added, deleted, and replaced to a part of the structure of each embodiment. Description of symbols

[0096] 1 Electrophoresis device (this device) 10 Capillary array 20 Separation medium container 30 Anode side buffer container 31 Anode side cleaning tank 32 Anode side electrophoresis buffer tank 33 Buffer tank for sample introduction 40 Cathode side buffer container 41 Wastewater tank 42 Cathode side cleaning tank 43 Cathode side electrophoresis buffer tank 50 Sample container 60 Liquid delivery mechanism 61 Plunger 80 Sampler Base 85 Y-axis drive body 90 Z-axis drive body 95 X-axis drive body 100 Sample tray 101 Guide 110 Constant temperature bath unit 115 Electrode 120 Constant temperature bath door 121 First Ammeter 122 High Voltage Power Supply 123 Second Ammeter 124 Hollow Electrode 125 Capillary 125a Front End 126 Loading head 127 Capillary head 128 Testing Department 129a, 129b GND 130 Irradiation detection unit 141 Microcomputer 142 Controller 150 Autosampler Unit 160 irradiation detection / constant temperature chamber unit S1 Separation medium filling process S2 First Pre-running Process S3 First sample injection step S4 First electrophoresis step S5 Second pre-operation process S6 Second sample injection step S7: second electrophoresis step.

Claims

1. A method for continuous use of a separation medium, characterized in that: have: A separation medium filling step of filling the capillary with a separation medium; A first pre-operation step of applying voltage to a capillary filled with the separation medium to perform a pre-operation; a first sample injection step of injecting a first sample into the capillary that has undergone the pre-operation; a first electrophoresis step of applying voltage to the capillary into which the first sample is injected to perform first electrophoresis; A second pre-running step of applying voltage to the capillary after the first electrophoresis step to perform a pre-running; a second sample injection step of injecting a next amount of sample into the capillary having undergone the second pre-operation step; as well as A voltage is applied to the capillary into which the next amount of sample is injected to perform a second electrophoresis step of electrophoresis of the next amount of sample, The second pre-operation step, the second sample injection step, and the second electrophoresis step are repeatedly performed a preset number of times.

2. A method for continuous use of a separation medium, characterized in that: have: A separation medium filling step of filling the capillary with a separation medium; A first pre-operation step of applying voltage to a capillary filled with the separation medium to perform a pre-operation; a first sample injection step of injecting a first sample into the capillary that has undergone the pre-operation; a first electrophoresis step of applying voltage to the capillary into which the first sample is injected to perform first electrophoresis; A second sample injection step of injecting a next amount of sample into the capillary after the first electrophoresis step; as well as A voltage is applied to the capillary into which the next amount of sample is injected to perform a second electrophoresis step of electrophoresis of the next amount of sample, The second sample injection step and the second electrophoresis step are repeatedly performed a preset number of times.

3. The method for continuous use of a separation medium according to claim 1 or 2, characterized in that: The separation medium is a liquid polymer.

4. The method for continuous use of a separation medium according to claim 1 or 2, characterized in that: The filling frequency of the separation medium in the separation medium filling step is set by a graphical user interface.

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