DNA analysis system

Abstract

A DNA analysis system is provided with: a flow channel device having a PCR chamber in which a thermal cycle is performed; and a capillary electrophoresis unit that performs electrophoretic analysis on a PCR reaction solution, in which the DNA analysis system stores values of m and n that are set in advance, thermal cycles are performed m times on the PCR reaction solution in the PCR chamber to generate a first reaction solution, a part of the first reaction solution is taken out from the PCR chamber without changing the composition, and the first reaction solution is taken out from the PCR chamber without changing the composition of the first reaction solution. Wherein, in the PCR chamber, the first reaction solution remaining in the PCR chamber is subjected to n-m (n-m is an integer of 2 or more) thermal cycles such that the total number of thermal cycles becomes n, and a second reaction solution is generated, and the first reaction solution is subjected to electrophoretic analysis in the capillary electrophoresis unit, and the first reaction solution remaining in the PCR chamber is subjected to n-m (n-m is an integer of 2 or more) thermal cycles in the PCR chamber. At least a portion of the second reaction solution is taken out from the PCR chamber without changing the composition, and the at least a portion of the second reaction solution is subjected to electrophoretic analysis in the capillary electrophoresis unit.

Description

Technical Field

[0001] The present invention relates to a DNA analysis system. Background Art

[0002] In fragment analysis, PCR is performed on the DNA contained in the sample using primers designed for specific DNA targets, and the fluorescently labeled DNA amplification products are separated by size by capillary electrophoresis (CE). This is used for gene mutation analysis or quantification, cell line authentication, determination of genome editing efficiency, genotyping of amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs), single nucleotide polymorphisms (SNPs), and microsatellite marker analysis. Microsatellites refer to repetitive DNA that repeats multiple specific DNA motifs and have a high mutation frequency and high genetic diversity compared to other DNA regions. As a representative example of microsatellite marker analysis, there is individual identification and personal identification using short tandem repeats (STRs). DNA identification using STRs is widely used in forensic testing for paternity testing and for comparing crime scene DNA with criminals.

[0003] In fragment analysis, except the DNA fragments after the fluorescent labeling of the analysis object, sometimes the DNA fragments (molecular weight standards) of known length after mixing multiple fluorescent labels are mixed and CE analysis is performed. By using molecular weight standards, the length of the fragment of each amplified product can be determined. In addition, if the amount of the molecular weight standards mixed is set to a constant amount, the amount of amplified product can be calculated according to the intensity of the DNA fragments of the amplified product obtained by CE analysis and the ratio of the intensity of the molecular weight standards. In addition, by mixing a known amount, a known length of DNA (Internal positive control, IPC) and a primer for amplifying it in the PCR reaction solution and amplifying it, fragment analysis is performed simultaneously, the DNA amount before the amplification of the target can be inferred according to the ratio of the intensity of the target amplified product and IPC.

[0004] According to non-patent document 1, in a DNA identification performed by a forensic medicine research institute, (1) the concentration of human DNA contained in a sample is quantified by DNA quantification using quantitative PCR, (2) the sample is prepared so that the human DNA concentration is appropriate, and STR-PCR (PCR containing STR sequences) is performed, (3) a portion of the PCR reaction solution and formamide containing a molecular weight standard are mixed at a constant ratio and heat denatured, (4) the electrophoretic sample after heat denaturation is measured by CE to obtain an electrophoretogram, and (5) DNA identification is performed based on the electrophoretogram.

[0005] In the DNA identification or fragment analysis performed by pretreatment-integrated CE analysis devices disclosed in Patent Documents 1, 2, 3, 4, and 8, (1) PCR is performed on a sample containing DNA on a flow channel device; (2) a portion of the reaction solution and formamide containing a molecular weight standard are mixed at a constant ratio and heat-denatured on the flow channel device; (3) the heat-denatured elution sample is analyzed by CE to obtain an electrophoretogram; and (4) DNA identification or fragment analysis is performed based on the electrophoretogram. Compared to Non-Patent Document 1, the series of steps in Patent Documents 1, 2, 3, 4, and 8 are automated, enabling results to be obtained in a short time (e.g., 90 minutes).

[0006] In the DNA quantification method disclosed in Patent Document 5, (1) in PCR of a sample containing DNA, a portion of the reaction solution at each of a plurality of successive thermal cycle numbers n (where n0 is a predetermined integer, n=n0, n0+1, n0+2, ...) is divided, (2) each of the divided reaction solutions is subjected to CE analysis, and (3) DNA quantification (the original concentration of the DNA contained in the sample is quantified) is performed based on the relationship between the peak intensity of the amplification product obtained by CE analysis and the thermal cycle number n, specifically, based on the thermal cycle number n at which the peak intensity exceeds a predetermined threshold value. In other words, Patent Document 5 is an analytical method for detecting amplification products in real-time PCR by CE.

[0007] In the DNA quantification method of Patent Document 6, (1) a portion of the amplification product at each of a plurality of successive thermal cycle numbers n (where n0 and m are predetermined integers, m is 1 or 2, n=n0, n0+m, n0+2m, ...) is separated by electrophoresis in a flow channel device during PCR of a sample containing DNA. (2) Each of the separated amplification products is analyzed by CE on the flow channel device. (3) DNA quantification is performed (the original concentration of the DNA contained in the sample is quantified) based on the relationship between the peak intensity of the amplification product obtained by each CE analysis and the thermal cycle number n, specifically, based on the thermal cycle number n at which the peak intensity exceeds a predetermined threshold value. In other words, Patent Document 5 is an analytical method for detecting amplification products in real-time PCR by CE.

[0008] In the DNA quantification method disclosed in Patent Document 7, (1) a portion of a reaction solution at each of a plurality of thermal cycle numbers n is separated on a flow channel device during PCR of a DNA-containing sample; (2) each separated reaction solution is subjected to microarray analysis on the flow channel device; and (3) DNA quantification is performed (the original concentration of the DNA contained in the sample is quantified) based on the relationship between the spot intensity of the amplification product obtained in each microarray analysis and the thermal cycle number n, specifically, based on the thermal cycle number n at which the spot intensity exceeds a predetermined threshold. In other words, Patent Document 7 is an analysis method for analyzing amplification products in real-time PCR using a microarray.

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: U.S. Patent Application Publication No. 2022 / 0016632

[0012] Patent Document 2: U.S. Patent No. 9,354,199

[0013] Patent Document 3: U.S. Patent Application Publication No. 2019 / 0019290

[0014] Patent Document 4: Japanese Patent Application Laid-Open No. 2017-077180

[0015] Patent Document 5: U.S. Patent No. 7,445,893

[0016] Patent Document 6: Patent No. 5494480

[0017] Patent Document 7: U.S. Patent No. 8,715,924

[0018] Patent Document 8: U.S. Patent No. 10,767,225

[0019] Non-patent literature

[0020] Non-patent document 1: John M. Butler, Fundamentals of Forensic DNA Typing (2009), pp. 29-107 and pp. 279-336 Summary of the Invention

[0021] Problems to be solved by the invention

[0022] In a CE analysis device or a device equipped with a CE analysis unit, the ratio of the minimum peak intensity and the maximum peak intensity, in which the peak intensity and concentration are almost in a ratio relationship, is less than 100, or less than 1000, or less than 10,000, or less than 100,000. In the case of performing fragment analysis, if the intensity deviation of the CE analysis device, the deviation of the amount of amplicon injected into CE, the amplification efficiency of different alleles and the deviation of the presence rate of different alleles, the deviation between the pigments, the deviation during sample adjustment, etc., the minimum amount of DNA concentration that can be measured is 10, 30, 100, 300, 1000, 3000, or 10,000. On the other hand, the minimum amount of DNA contained in the sample brought into the analysis system is 30, 300, 3000, or 30,000. Because the DNA amount contained in the sample exceeds the scope of the sample DNA amount that can be measured in the analysis system, fragment analysis failure may be caused. In this case, the sample or analysis time consumed by the analysis can be wasted.

[0023] One of the objectives of the present invention is to use a simple and low-cost device to perform DNA identification / fragment analysis or DNA quantification (quantifying the original concentration of an individual's DNA contained in the sample) with high accuracy and robustness through PCR and CE analysis in samples containing DNA whose concentration ratios vary over a range of 100-fold or 1000-fold or greater (sample concentration range is 100 or 1000 = 2.0 or 3.0 orders of magnitude or greater). Furthermore, similar to Patent Document 2, the series of processes is automated, enabling results to be obtained in a short time (e.g., within 180 minutes, 120 minutes, or 90 minutes).

[0024] In Non-Patent Document 1, quantitative PCR is used to quantify the DNA carried over to the PCR reaction, ensuring that the amount of DNA carried over does not exceed the analytical range. The DNA is diluted to an appropriate concentration before the PCR reaction is performed. Implementing this method using a flow channel device requires an optical system for quantitative PCR. Furthermore, determining the dilution concentration based on the quantitative PCR results requires a complex flow channel structure.

[0025] In Patent Document 1, the solution is split before PCR, and STR-PCR and quantitative PCR are performed. Quantitative PCR is used to determine the number of STR-PCR cycles. Splitting the solution before PCR results in reduced sensitivity. Furthermore, the flow path equipment used for the two different real-time PCR processes is complex and requires an additional optical system, inevitably increasing costs.

[0026] In Patent Document 2, the solution is split before PCR, two DNA solutions of different concentrations are prepared, and PCR is performed on both to perform DNA identification. Either of the two DNA solutions falls within the analytical concentration range of the analysis system, thereby expanding the analytical range. Since the solution is split before PCR, sensitivity is reduced. Furthermore, the flow channel mechanism used to split the solution and adjust the concentration is mounted on the flow channel equipment, which inevitably leads to complexity of the flow channel equipment.

[0027] Patent Document 3 expands the analytical scope of DNA identification by improving the data analysis method. CE analysis fails to provide information on peaks below the detection limit. Furthermore, if the detection intensity saturates during CE analysis, accurate peak intensity ratios cannot be obtained. If the interpretation of DNA identification is overly broadened, the data obtained through analysis may not reflect the original individual DNA mixing ratio, posing the risk of erroneous analysis.

[0028] In Patent Document 4, a portion of the reaction solution is removed after PCR, and the presence or amount of amplification products is detected using an optical system. If appropriate amplification is possible, fragment analysis is performed. If amplification is determined to be incomplete, an additional PCR reaction is performed on the reaction solution remaining in the PCR section. This approach requires an additional optical system or the design of a flow channel device suitable for optical detection, which inevitably leads to increased costs. Furthermore, detection using an optical system may cause the fluorescent dye used in STR-PCR to fade, or the overlap of the fluorescent dye used in STR-PCR with the detection band of the optical system may make accurate optical detection impossible.

[0029] In Patent Documents 5 and 6, CE analysis needs to be performed separately for multiple thermal cycle numbers n (multiple continuous thermal cycle numbers n), so it takes a long time to obtain the results. High-precision quantification requires more than three CE analyses. Although PCR can be replaced with STR-PCR for DNA quantification, a special flow channel structure is required because the solution is taken out multiple times. In addition, there is no process for mixing the reaction solution and formamide containing molecular weight standards at a constant ratio, so DNA identification cannot be performed. In addition, when DNA identification is performed by adding a mixing process with formamide (mobilization reagent) containing molecular weight standards to this method, a dispensing mechanism or flow channel structure for mixing the PCR products of each cycle with the mobile reagent is required again, so the device is complicated and the cost is increased. In addition, especially for DNA identification that requires high-precision and high-resolution CE analysis, it takes a lot of analysis time to perform CE analysis on the PCR products of each cycle. In addition, in Patent Document 6, DNA is taken out of the PCR chamber by applying a voltage, so it is speculated that the composition of the amplified product in the PCR chamber is different from the composition of the amplified product taken out. In the case of DNA identification, although amplicons of different lengths are measured, when a voltage is applied to remove the amplified product from the PCR chamber, there is a possibility that a deviation depending on the length will occur.

[0030] Patent Document 7 proposes that DNA quantification can be performed by replacing PCR with STR-PCR. However, this requires microarray analysis for multiple thermal cycle numbers n (consecutive thermal cycle numbers n), which takes a long time to obtain results. Furthermore, DNA identification is not possible because CE analysis is not performed.

[0031] On the other hand, Patent Document 7 mentions that microarray analysis can be performed for a small number of thermal cycle numbers n (discontinuous multiple thermal cycle numbers n), but this is not realistic. This is because, in microarray analysis, due to the deviation in the density and number of probes immobilized on each spot, or the deviation in hybridization efficiency in time and space, the spot intensity for the same DNA concentration will vary. Although the presence or absence of the corresponding DNA can be determined based on the strength of the spot intensity, the accuracy of quantifying the corresponding DNA based on the spot intensity is low. That is, in order to accurately obtain the thermal cycle number n at which the spot intensity exceeds a pre-set threshold, it is necessary to perform microarray analysis on multiple thermal cycle numbers n (continuous multiple thermal cycle numbers n) respectively. As a method for improving the accuracy of microarray analysis for DNA quantification, a method is known to allow target DNA or target amplification product and reference DNA to compete for hybridization on the same spot. However, in order to implement this method, it is necessary to prepare a reference DNA for each target DNA or target amplification product that hybridizes with the probe on the spot with the same efficiency as the target DNA or target amplification product, then label the target DNA or target amplification product and the reference DNA with different fluorescent substances, and independently measure the luminescent fluorescence of each. When preparing a small number of PCR products with different cycle numbers and performing microarray analysis to expand the dynamic range of analysis, there are problems of increased cost and labor.

[0032] Another problem of Patent Document 7 is that the same amount of fresh PCR solution as that obtained when a portion of the reaction solution of each stage of a plurality of thermal cycle numbers n is divided on a flow channel device in PCR of a sample containing DNA is mixed with each remaining reaction solution without being divided. Therefore, the DNA concentration contained in the reaction solution changes, thereby reducing the accuracy of DNA quantification.

[0033] Another problem of Patent Document 7 is that the hybridization and dehybridization (cleaning) of DNA with the solid-phased probe needs to be repeated. Each time the solid-phased probe is stripped off or the hybridized DNA is not cleaned and remains, so the reproducibility of the microarray analysis is low and the accuracy of DNA quantification is low.

[0034] Summarizing the issues of DNA identification / fragment analysis or DNA quantification, there is a need for a DNA analysis method that can be implemented using simple flow channel equipment, is low-cost, highly robust, simple, maintains sensitivity, shortens measurement time, and can expand the range of DNA amounts analyzed.

[0035] Solutions to Problems

[0036] An example of the DNA analysis system of the present invention includes:

[0037] a flow channel device having a PCR chamber for performing thermal cycling; and

[0038] Capillary electrophoresis unit, which performs electrophoresis analysis on the PCR reaction solution,

[0039] in,

[0040] The DNA analysis system stores the pre-set values ​​of m and n.

[0041] In the PCR chamber, the PCR reaction solution is subjected to m thermal cycles to generate a first reaction solution.

[0042] removing a portion of the first reaction solution from the PCR chamber without changing its composition;

[0043] performing electrophoresis analysis on the portion of the first reaction solution in the capillary electrophoresis unit,

[0044] In the PCR chamber, the first reaction solution remaining in the PCR chamber is subjected to nm thermal cycles so that the total number of thermal cycles becomes n, thereby generating a second reaction solution, wherein nm is an integer greater than or equal to 2.

[0045] removing at least a portion of the second reaction solution from the PCR chamber without changing its composition,

[0046] The at least a portion of the second reaction solution is subjected to electrophoresis analysis in the capillary electrophoresis section.

[0047] An example of the DNA analysis system of the present invention includes:

[0048] a flow channel device having a PCR chamber for performing thermal cycling; and

[0049] Capillary electrophoresis unit, which performs electrophoresis analysis on the PCR reaction solution,

[0050] in,

[0051] The DNA analysis system stores the pre-set values ​​of m and n.

[0052] In the PCR chamber, the PCR reaction solution is subjected to m thermal cycles to generate a first reaction solution.

[0053] removing a portion of the first reaction solution from the PCR chamber without changing its composition;

[0054] In the PCR chamber, the first reaction solution remaining in the PCR chamber is subjected to nm thermal cycles so that the total number of thermal cycles becomes n, thereby generating a second reaction solution, wherein nm is an integer greater than or equal to 2.

[0055] removing at least a portion of the second reaction solution from the PCR chamber without changing its composition,

[0056] performing electrophoresis analysis on one of the portion of the first reaction solution and the at least one portion of the second reaction solution in the capillary electrophoresis section;

[0057] Based on a result of the one electrophoretic analysis, execution of the electrophoretic analysis is controlled for the other of the portion of the first reaction solution and the at least one portion of the second reaction solution.

[0058] Effects of the Invention

[0059] The effects achieved by one example of the invention disclosed in this application are briefly described as follows: Specifically, according to one example of the invention, in an analysis system comprising a flow channel device and an electrophoresis unit, the range of DNA analysis can be expanded with high accuracy, high sensitivity, a short time, and low cost using a compact device.

[0060] Other problems, structures, and effects than those described above will become clear from the following description of the embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 is a schematic diagram of the analysis system.

[0062] Figure 2 This is an example of a data determination method.

[0063] Figure 3 is a schematic diagram of the analysis system.

[0064] Figure 4 is an embodiment of an analysis system.

[0065] Figure 5 is a schematic diagram and embodiment of the analysis system.

[0066] Figure 6 is a schematic diagram of the analysis system.

[0067] Figure 7 is a schematic diagram of the analysis system.

[0068] Figure 8 It is a schematic diagram of the flow channel equipment.

[0069] Figure 9 It is a schematic diagram of the flow channel equipment.

[0070] Figure 10 It is a schematic diagram of the flow channel equipment.

[0071] Figure 11 It is a schematic diagram of the flow channel equipment.

[0072] Figure 12 It is a schematic diagram of the flow channel equipment.

[0073] Figure 13 This is an example of the operation of the runner equipment.

[0074] Figure 14 is an embodiment of an analysis system.

[0075] Figure 15 It is a schematic diagram of the flow channel equipment.

[0076] Figure 16 This is an example of the operation of the runner equipment.

[0077] Figure 17 It is a schematic diagram of the flow channel equipment.

[0078] Figure 18 It is a schematic diagram of the flow channel equipment.

[0079] Figure 19 It is a schematic diagram of the flow channel equipment.

[0080] Figure 20 This is an example of the operation of the runner equipment.

[0081] Figure 21 This is an example of the operation of the runner equipment.

[0082] Figure 22 This is an example of the operation of the runner equipment.

[0083] Figure 23 The results show the relationship between the mobilization reagent and the peak intensity.

[0084] Figure 24 It is a figure which shows the effect of this invention.

[0085] Figure 25 It is a graph showing the analysis range.

[0086] Figure 26 This is an example of a table for setting standards for the number of cycles.

[0087] Figure 27 is a schematic diagram of the obtained electropherogram.

[0088] Figure 28 This is an example of a table for setting standards for the number of cycles.

[0089] Figure 29 is an embodiment of an analysis system.

[0090] Figure 30 This is an example of a data judgment method.

[0091] Figure 31 This is an example of a data judgment method.

[0092] Figure 32 is an embodiment of an analysis system.

[0093] Figure 33 is an embodiment of an analysis system. DETAILED DESCRIPTION

[0094] In this specification, the procedures and standards for performing human DNA identification are mainly described, but the analysis targets are not limited to human DNA identification.

[0095] Hereinafter, unless otherwise specified, m and n refer to the number of thermal cycles of PCR. M and n are integers, and may be n m ≥ 2. In addition, hereafter, thermal cycles may be referred to as PCR cycles or simply cycles.

[0096] Hereinafter, a mixture of the PCR reaction reagent and the sample-derived DNA is referred to as a PCR reaction solution. Furthermore, the DNA amplified by the PCR reaction reagent in the sample-derived DNA is referred to as target DNA.

[0097] Hereinafter, a PCR reaction solution obtained after m thermal cycles is referred to as “PCR reaction solution m”, and a PCR reaction solution obtained after n thermal cycles is referred to as “PCR reaction solution n”.

[0098] Below, the swimming reagent may also contain deionized formamide or molecular weight standards, pure water. It may also contain formamide or pure water in order to reduce the ionic strength of the swimming sample or denature the DNA. The swimming reagent may also use not only formamide or pure water but also a low-conductivity solution. The low-conductivity solution preferably has a conductivity of 10mS / cm or less, more preferably a conductivity of 1mS / cm or less, more preferably 100μS / cm or less, and further preferably 10μS / cm or less. The lower the conductivity of the solution used for the swimming reagent, the more the amount of DNA injected into CE tends to increase. The mixed molecular weight standards may be mixed in order to correspond the detected peak to the DNA length, or may be mixed in order to infer the amount of DNA contained in the swimming sample based on the detected peak.

[0099] As an example of an analysis system, a PCR reaction solution (e.g., at least one of a portion of PCR reaction solution m and at least a portion of PCR reaction solution n) can be mixed with pure water, formamide, or a solution with a conductivity of 10 mS / cm or less to generate a mixed solution before electrophoresis analysis. Mixing with a solution with low conductivity can increase CE peak intensity. Furthermore, mixing with formamide can denature DNA.

[0100] Hereinafter, the DNA obtained by the PCR reaction is referred to as “amplification product”, the amplification product obtained by m cycles is referred to as “amplification product m”, and the amplification product obtained by n cycles is referred to as “amplification product n”.

[0101] Hereinafter, the liquid (mixed liquid) obtained by mixing the PCR reaction solution and the electrophoresis reagent is referred to as the "electrophoresis sample". Figure 5 When heated to 90°C or higher as shown in (2), DNA is easily denatured and becomes single-stranded, enabling higher-precision CE analysis. Therefore, it is suitable to add a heating step. The "electrophoresis sample" may refer to a sample before or after denaturation by heating. The electrophoresis sample prepared from amplification product m is referred to as "electrophoresis sample m," and the electrophoresis sample prepared from amplification product n is referred to as "electrophoresis sample n."

[0102] Hereinafter, the following process is referred to as "split PCR", that is, performing m cycles of PCR, taking out a portion of the PCR reaction solution m, and performing nm cycles of PCR on the remaining PCR reaction solution m, thereby finally preparing both PCR reaction solutions m and n.

[0103] Hereinafter, a locus refers to the position of a gene on a chromosome. A typical STR-PCR kit includes primers that can uniquely increase each locus.

[0104] Hereinafter, alleles refer to distinguishable gene variants at the same locus. When DNA is identified, if the DNA comes from a single person, there may be both two alleles present at the same locus (heterozygote) and one allele present (homozygote).

[0105] Hereinafter, an amplicon refers to an amplified product having a single length. Amplicons of different lengths may also be generated from a single allele. For example, multiple amplicons are generated by byproducts (artifacts) produced by a PCR reaction. In the case of DNA identification, for a single allele, the peak of an amplicon is often attributed to an individual's DNA. However, in the case of a mixed sample, it is difficult to distinguish between an artifact and an amplicon derived from the allele. Therefore, a peak that may be an amplicon of an artifact may also be the subject of analysis.

[0106] Hereinafter, CE analysis refers to a series of steps including preparing a sample for electrophoresis, performing CE measurement, obtaining an electropherogram, and performing DNA identification or fragment analysis. However, the scope of "CE analysis" may not include some of the above steps.

[0107] Hereinafter, an electropherogram refers to a graph obtained by CE measurement, wherein the horizontal axis is set to time, measurement point, or DNA chain length, and the vertical axis is set to intensity. The vertical axis can also be wavelength, which is three-dimensional data including intensity information. Alternatively, the vertical axis can be intensity, which is three-dimensional data including pigment information. The electropherogram obtained from the electrophoretic sample m is referred to as "electrophoretogram m", and the electropherogram obtained from the electrophoretic sample n is referred to as "electrophoretogram n".

[0108] Hereinafter, the DNA profile refers to DNA types obtained by analyzing electropherograms, or a two-dimensional sequence including peak intensities and peak lengths, or a data set including the number of DNA repeats and peak intensities assigned to DNA types.

[0109] Hereinafter, STR-CE refers to the series of steps involved in preparing the STR-PCR reaction mixture, conducting the PCR reaction, performing CE analysis, and analyzing the resulting electropherogram. The data obtained from STR-CE can be either an electropherogram or a DNA profile. After STR-CE is completed, some, all, or none of the data may be provided to the user.

[0110] Hereinafter, the term "analysis range" refers to the range of sample amounts or biomolecules within which an analytical system, or a portion or multiple processes within the analytical system, can accurately analyze a given sample or biomolecule. Here, "capable of accurate analysis" can mean that all required conditions are met, but it does not necessarily mean that all required conditions are met; it can also mean that the system can provide the best data achievable. For example, if the sample amount is extremely small, even if the data obtained does not meet all required conditions, it is sufficient to obtain data that most closely meets the required conditions.

[0111] The PCR reaction solution can include more than two primer sets, and the PCR reaction solution can include more than two amplified gene regions. By analyzing multiple amplified gene regions, individual / person identification capabilities can be improved. In particular, in the case of personal identification, the risk of misidentification as the same person can be reduced. By performing PCR reactions on multiple gene regions simultaneously, rather than individually, the number of PCR chambers can be minimized, thereby conserving reaction reagents. By separating the PCR chambers, the risk of reduced sensitivity can be avoided.

[0112] Implementation method 1.

[0113] [Analysis System]

[0114] In this embodiment, the analysis system 101 (DNA analysis system) may include: a memory for storing program instructions; a control unit including a processor that executes the program instructions; a functional unit that receives and analyzes raw data, optical data, and electropherogram data from the detection unit; solution delivery control mechanisms such as pumps and valves; a CE unit (capillary electrophoresis) for performing electrophoretic analysis on PCR reaction solutions; a flow channel device; and a heater. The control and analysis unit may also be connected to a network to load, match, and access data from a personal DNA database. For example, it may be connected to CODIS (Combined DNA Index System). A diaphragm pump or a syringe pump may be used as a pump. Examples of valves include those that deform a membrane by directly or indirectly transmitting motor power, or those that deform a membrane using air pressure. The valve may be controlled by the control unit. The valve may be opened and closed by thermal deformation, or by magnetic force.

[0115] Various parameters of the analysis protocol can be pre-stored in a database in computer 102 included in analysis system 101. Based on the parameters stored in database 103, the computer can control the opening and closing of valves, temperature control, applied pressure, and / or flow rate in flow channel device 104, CE unit 105, and their connections. The parameters stored in computer 102 may include functions for setting parameters based on temperature, time, pressure, flow rate, stored parameters, and measured values.

[0116] It is also possible to receive a sample containing target DNA and automatically perform the steps from dissolution to purification, PCR, CE measurement, and analysis. It is also possible to automatically perform a portion of the steps from dissolution to purification to PCR to CE measurement and analysis. For example, the analysis system 101 or the flow channel device 104 will be described later. Figure 14 The process shown here is fully automated, from PCR reaction solution preparation to nanometer thermal cycling. This makes the process more efficient, and even non-experts can perform the analysis.

[0117] The integrated device can dissolve, purify, and adjust the PCR reaction solution. It can also mix and heat PCR and formamide. It can also perform CE analysis.

[0118] The flow channel device 104 may be disposable. By making it disposable, contamination between samples can be prevented.

[0119] The CE unit 105 can also be disposable. This prevents contamination between samples. Furthermore, since it can be integrally formed with the device, storage, maintenance, and transportation are simplified. The connection between the pretreatment unit and the CE unit is simplified, reducing the frequency of malfunctions and errors.

[0120] The pretreatment channel device can be made disposable, while the CE unit can be used multiple times. The CE unit requires precision manufacturing and is expensive, so by making it reusable, costs can be reduced.

[0121] Figure 1 Detailed examples of the analysis system 101 and the computer 102 are shown.

[0122] The computer 102 may include a user interface 106. Parameters (e.g., time or temperature for each step, pressure, flow rate, process flow, splitting solution volume, PCR cycle number, sample information, cartridge information, analysis protocol, etc.) may be received from the user through the user interface 106 and stored in the database 103. Alternatively, various parameters may be pre-stored in the database 103. Based on the parameters stored in the database 103, the computer 102 may be responsible for opening and closing valves, controlling temperature, and controlling applied pressure and flow rate in the flow channel device 104.

[0123] The flow channel device 104 consumed for each measurement may have a tag inside, and the analysis system 101 may read the information on the tag to set an appropriate analysis protocol.

[0124] The number of PCR cycles (e.g., the values ​​of m and n) can be set by the user. The analysis system 101 stores the pre-set values ​​of m and n. Alternatively, the user may input information related to the type of sample (e.g., oral swab / Touch sample / Casework sample / DVI, etc.), match it with the database in the computer, and thus determine the appropriate PCR implementation process. When electrophoresis is performed multiple times or multiple electrophoretic samples are electrophoresed, the user's input can be used to select whether to perform CE determination on electrophoretic sample m first or on electrophoretic sample n first. Alternatively, the entire implementation process may be automatically controlled by the computer 102. Alternatively, the user may assist and implement part of the analysis process.

[0125] In the past, the process was usually as follows: samples were sent to an environment with complete laboratory equipment, such as a research institute, and inspectors with professional knowledge and skills adjusted and measured the samples and analyzed the data. However, there are many problems, such as the time-consuming transportation of samples and the large equipment and labor costs required for the maintenance of laboratory equipment. In addition, when batch processing is introduced to improve efficiency, it is difficult to insert urgent samples. In recent years, Sample-to-answer (StoA) systems that fully automate everything from sample introduction to measurement and data have gradually entered many fields. StoA systems sometimes use flow channel equipment that integrates chambers, flow channels, and reagents. The introduction of flow channel equipment has the following advantages. (1) Measurements can be easily performed even by non-experts; (2) Data can be obtained in a short time; (3) Highly portable devices can be designed; (4) Deviations caused by manual operation are reduced; (5) Reagents can be easily stored.

[0126] Potential applications of the StoA system include forensics, DNA analysis, in vitro diagnostics, plant and animal species identification, biodefense, medicine, biotechnology, life sciences, national defense, public health, and agriculture. The StoA system can also be used in research institutes, crime scenes, police stations, hospitals, and even in vehicles.

[0127] [Runner equipment]

[0128] In this embodiment, the flow channel device 104 refers to a disposable or reusable cartridge that contains reagents, chambers, and flow channels. The flow channel device 104 may also include a power source for transporting solutions. Alternatively, some or all of the reagents may be contained within the device. A portion of the chamber may also include temperature regulation, molecule capture, detection, or voltage application functions.

[0129] The material used in the flow channel device is not particularly limited as long as it is a material commonly used in this technical field. Preferably, as a material with a low DNA adsorption capacity, polypropylene or cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polycarbonate, polyethylene terephthalate, or polyurethane can be used. In addition, it is also preferred to suppress the adsorption capacity by performing negatively charged modification on the surface. As other materials, for example, the following can be mentioned:

[0130] -Metals such as gold, silver, copper, aluminum, tungsten, molybdenum, chromium, platinum, titanium, and nickel;

[0131] - Alloys such as stainless steel, Hastelloy, Inconel, Monel, and Duralumin;

[0132] -silicon;

[0133] - Glass materials such as glass, quartz glass, fused quartz, synthetic quartz, alumina, sapphire, ceramics, forsterite, and photosensitive glass;

[0134] -Plastics such as polyester resin, polystyrene, polyethylene resin, ABS resin (acrylonitrile butadiene styrene resin), dimethylpolysiloxane (PDMS), nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenolic resin, melamine resin, epoxy resin and polyvinyl chloride resin;

[0135] - agarose, dextran, cellulose, polyvinyl alcohol, nitrocellulose, chitin, chitosan;

[0136] Or any combination of them.

[0137] [Chamber, reagent storage]

[0138] A typical chamber or reagent storage portion refers to a space that can store liquids or solids and can react, standby, heat or change solutions. The chamber sometimes has a diameter thicker than the flow channel, and sometimes cannot be distinguished from the flow channel in appearance. The chamber sometimes has a membrane or fine structure inside. In this case, it is sometimes formed with a different composition from the flow channel, or has a different surface treatment, or has a different hydrophilicity. In addition, a heater or laser light source may be provided on the outside of the flow channel device. Reagents can be stored in the chamber, and PCR, dissolution, purification, etc. can also be performed in the chamber. The capacity of a typical chamber is preferably 0.01μL to 50mL.

[0139] The flow channel device can also store reagents in the device, and can also supply reagents from outside the flow channel device and inside the analysis system. As an example, in the device, more than one reagent is stored in one or more reagent storage parts. Reagents include at least one of [lysate, cleaning solution, PCR reagent (can include polymerase, primers, surfactants, etc.), formamide, pure water, DNA fragments, oil]. If these are mixed at an unexpected moment, unexpected results such as performance degradation will result. Therefore, it is preferred that they be separated by a valve, a film, air, or a thin flow channel that is sufficient to prevent active mixing, or a wall mechanism consisting of a combination thereof, just before use. In addition, by isolating the reagent from the external air, long-term storage and the portability of the equipment can be achieved. In order to release the same reagent in multiple steps, it can also be stored in multiple reagent storage parts. In the case where the reagent is stored outside the device, it is also preferably stored in a state isolated from the external air, separated from other purification system components by valves, films, air, etc. As a well-known reagent storage technology, for example, there is a blister reagent storage portion, or a reagent storage portion described in Patent Document 1 or Patent Document 2, and similar forms can also be incorporated into this embodiment.

[0140] [Sample species]

[0141] The sample provided to the purification system of this embodiment is not particularly limited as long as it is of biological origin. The source organism of the sample is also not particularly limited, and samples from any organism, such as vertebrates (e.g., mammals, birds, reptiles, fish, amphibians, etc.), invertebrates (e.g., insects, nematodes, crustaceans, etc.), plants, protozoa, fungi, bacteria, and viruses, can be used.

[0142] When collecting samples, swabs, filter paper, cloth, etc. can be used as carriers, and they can also be introduced into the purification system together with the carrier.

[0143] Forensic samples include buccal swabs, bones, muscle tissue, organs, so-called touch samples (those containing extremely small amounts of DNA), blood, skin flakes, hair, body fluids, and remains suspected of containing these. Most forensic samples contain unknown amounts of DNA, ranging from 0.001ng to 1000μg, and more frequently from 0.01ng to 10μg. Furthermore, forensic samples can contain DNA from only a single individual, multiple individuals, or even degraded DNA.

[0144] [Solution transfer control]

[0145] The analysis system 101 may include a pump or a valve for transporting the solution. As the transport means, a syringe pump, a diaphragm pump, an electrochemical pump, passive transport using surface tension, centrifugal force, or a combination thereof may be used.

[0146] The analysis system 101 may include valves. In addition to specifying the solution transport path, the valves are also used to switch the path to which air pressure is applied. Valves can be diaphragm valves actuated by air pressure, mechanical valves, or valves that utilize surface tension. Alternatively, the pressure difference required for transport can be used to switch the transportable flow path.

[0147] [PCR reaction]

[0148] The device can be equipped with PCR reagents. The PCR reagents can include a solution containing polymerase and a solution containing primers. The PCR reagents can also be dried. Samples such as swabs can also be used for PCR. DNA purified with silica or Chelex, phenol-chloroform, or the like can be mixed with the PCR reagents. A membrane that captures DNA (such as a silica membrane) can also be mixed with the PCR reagents.

[0149] PCR reagents can also contain a set of IPCs to be amplified along with the sample DNA and primers for amplifying the IPCs. The primers used for the IPCs are dyed and can be detected by CE. The amplicons from the IPCs can be used for analysis. The amount of DNA in the sample can also be estimated by using the intensity ratio of the IPCs to the peaks from the sample and an amplification efficiency correction factor or a fluorescence intensity correction factor. In addition, by confirming the intensity of the IPCs, it is also possible to infer whether the PCR reaction is proceeding normally or is being hindered.

[0150] The typical PCR reaction volume is 1 μl to 200 μl, more preferably 10 μl to 50 μl. Smaller volumes offer advantages such as precise temperature control, rapid PCR, and low reagent costs. On the other hand, larger volumes allow for greater elution of DNA.

[0151] A typical PCR reaction may consist of an Initial denaturing step, an Annealing step, an Extension step, a Denature step, and a Final Extension step, or may not include some of these steps.

[0152] The Initial denaturing step starts the PCR reaction by heating at 90°C to 99°C for 1 second to 2 minutes. In the Annealing step, the primers are bound to the model DNA by heating at 50°C to 80°C for 1 second to 2 minutes. In the Extension step, the DNA is extended by heating at 50°C to 80°C for 1 second to 2 minutes, raising the temperature to a level at which the DNA polymerase works well. In the Denature step, the DNA is heated at 80°C to 99°C for 1 second to 2 minutes. In the Final Extension step, the DNA is heated at 50°C to 80°C for 1 minute to 60 minutes. By setting up the Final Extension step, the length of the amplified product can be made uniform. The three steps of Annealing, Extension, and Denature can be repeated 10 to 40 times. The Annealing and Extension steps can be performed at the same temperature.

[0153] [Detection method]

[0154] After amplification, detection is performed by CE. In CE, a method of injecting the amplified product into a capillary filled with a polymer by voltage injection can be used. Furthermore, when a high voltage is applied to both ends of the capillary, the fluorescent DNA fragments are separated according to size and detected by a laser / camera system. In this embodiment, only CE analysis is mentioned, but in other embodiments, Massively parallel sequencing (MPS), pyrophosphate sequencing, Sanger sequencing, nanopore sequencing, chromatography, electrical measurement, spectroscopy, NMR, RFLP (Restriction Fragment Length Polymorphisms), microarrays, etc. can also be used to replace the CE part.

[0155] [Analysis and Peak Judgment Criteria]

[0156] The signal obtained by the CE portion is parsed by the analysis unit. As analysis software, known GeneMapper (registered trademark) ID, GeneMapper ID-X, GeneMarker (registered trademark) HID, i-Cubed (trademark), OSIRIS, TrueAllele (trademark) etc. In CE analysis, when parsing, according to the information of signal intensity vs time, based on the molecular weight standard peak, a graph is generated in which the horizontal axis is DNA length and the vertical axis is intensity. It is also possible to correct for cosmic rays or pull-up and pull-down (pullup, pulldown). It is also possible to have baseline correction. In addition, it is also possible to use existing technology to obtain an electropherogram. Peak detection is carried out on the electropherogram obtained to investigate each amplicon intensity and peak position. In addition, when parsing, there can be local human intervention or it can be implemented fully automatically.

[0157] In this chapter, [Analysis and Peak Judgment Criteria] describes peak judgment criteria related to DNA identification. Similar criteria can also be used for analysis of other fragments.

[0158] Microsatellites are DNA loci containing repetitive base sequences with 2 to 7 nucleotides per repeat unit. The number of repeats at a specific locus varies between individuals and can be detected as differences in the length of the amplification product using STR-PCR.

[0159] In a typical STR-PCR analysis, two or more loci are detected. Typically, five or more, ten or more, 15 or more, 20 or more, or 25 or more loci are included. STR-PCR can use kits sold as kits such as GlobalFiler (trademark) and PowerPlex (registered trademark). In addition, it is preferred to include loci for forensic medicine or DNA identification in various countries such as CODIS, or loci specified in various gene databases. The loci may also include loci present on autosomes, or genes present only on the Y gene.

[0160] There are mixed cases of homozygous and heterozygous conditions at a specific locus. Except for cases where the amount of DNA is degraded or insufficient, the number of peaks detected when investigating the DNA type from a single person is a minimum corresponding to the number of loci in the kit, and a maximum corresponding to the peak corresponding to the sum of twice the amount of genes assigned to autosomes and the number of genes assigned to sex chromosomes in the loci in the kit.

[0161] In a typical STR-PCR analysis, a single fluorescent dye is assigned to each locus. STR-PCR kits that utilize two, three, four, five, six, seven, or eight different dye colors are also available. When detecting DNA types, the locus can be assigned based on a combination of peak length and color information. The intensity and position-dependent thresholds for each peak can be set based on color, locus, or allele.

[0162] In a DNA profile from a single individual, one or two alleles are detected for each locus. On the other hand, a DNA profile from a mixed sample of multiple individuals detects one, two, three, or four or more alleles for each locus. When assigning a DNA profile to an individual based on multiple DNA profiles, analysis is typically performed randomly based on peak intensity ratios. Programs for analyzing mixed samples include Kongho, LikeLTD, LRmix, STRmix, Euroformix, and TrueAllele.

[0163] As an example, artifacts in DNA identification refer to peaks that are not from an individual's DNA type or the balance between peaks that are different from the ideal state, or peak shapes that are different from the ideal state, which are the cause of the difference between the electrophoresis pattern or DNA map obtained when the actual DNA analysis is performed and the ideal electrophoresis pattern or DNA map that should be obtained based on the DNA type from the individual.

[0164] Artifacts can be generated by a variety of mechanisms. When a sample contains DNA from a single individual, a certain amount of artifacts is tolerated.

[0165] On the other hand, if there are a large number of artifacts, the accuracy of the machine's determination will decrease, and therefore, expert review will be necessary.

[0166] In addition, when there are many corrections, incorrect DNA identification results may sometimes be obtained.

[0167] Furthermore, when the correction object is large, it may not be possible to detect the desired DNA type, resulting in a reduced amount of information.

[0168] Furthermore, in cases where there's no guarantee that the DNA of a sample originated from a single individual, typically in forensic settings where multiple individuals cannot be excluded, the presence of artifacts complicates or makes analysis of the electropherogram even more difficult. For example, when three or more peaks appear at a single locus, it can be difficult to determine whether these peaks are artifacts or from two or more individuals. Furthermore, while DNA profiles are typically assigned to individuals based on peak intensity ratios, this becomes more difficult if the possibility of artifacts is considered. Therefore, it's preferable to obtain electropherograms with as few artifacts as possible.

[0169] When a peak not derived from an individual's DNA is detected during CE analysis and reflected in the analysis results, this state is called a drop-in. Conversely, when a peak expected to be present in an individual's DNA is not detected during CE analysis and is not reflected in the analysis results, this state is called a drop-out. It is preferable to set various thresholds to minimize the occurrence of drop-in and drop-out.

[0170] <Shadow Peak>

[0171] Ghost peaks are byproducts of PCR amplification. They occur when one or more repeat sequences are skipped or repeated during the extension reaction and amplified, resulting in ghost peaks. Ghost peaks typically appear before or after the sample peak, appearing one or two repeats more or less than the sample peak. Ghost peaks typically have an intensity of approximately 1-20% of the sample peak.

[0172] <Incomplete addition of adenine moiety>

[0173] In STR-PCR, during the extension reaction, excess adenylation (A+ peak) is consistently added to amplicons of the correct length. In typical STR-PCR kits, a reaction step called "Final Extension" is added at the end of the PCR reaction. During this Final Extension step, adenylation is added to amplicons that have not been superfluously added. By setting a sufficient Final Extension time, a state in which adenylation is added to nearly all amplicons is achieved. However, if the Final Extension time is set excessively relative to the amount of amplicons, the proportion of amplicons superfluously added (A++ peak) increases. The A++ peak is detected at a position one base longer than the A+ peak. Furthermore, if the amount of amplicons is excessive, adenylation cannot be completed within the Final Extension time, resulting in residual amplicons that have not been superfluously added, leading to the detection of an A- peak with one base missing. Hereinafter, the A++ and A- peaks are collectively referred to as the Incomplete Adenylation Peak (IAP). Furthermore, when the description is limited to the A-peak, it is described as "IAP-," and when the description is limited to the A++ peak or a peak with a higher number of adenine groups, it is described as "IAP+." In preferred STR-CE, various PCR parameters, such as the number of PCR cycles, final extension time, and input DNA amount, are adjusted so that the A-peak is within a range of 50% or less, more preferably 20% or less, and even more preferably 10% or less relative to the A++ peak. An IAP intensity threshold or an intensity ratio threshold (Incomplete Adenylation Peak Ratio Threshold, IAPT) relative to the main peak can be set to determine whether the intensity of the A- or A++ peak is within the above range and whether preferred STR-CE is performed. If the intensity ratio of the A- or A++ peak relative to the A+ peak exceeds a certain level, the intensity of the main peak may not reflect the original gene abundance ratio. Furthermore, in the case of mixed samples or when peaks shifted by one base due to genetic polymorphism appear, accurate attribution cannot be made. This can also lead to the detection of the wrong DNA type.

[0174] <Peak Intensity Ratio>

[0175] When there is a sufficient amount of DNA, the two peaks from the heterozygous locus show basically the same height. When the amount of DNA is insufficient, the probability of uneven DNA amounts from each gene increases, and the difference in the intensities of the two peaks increases significantly. In addition, in the case of excess amplification, short DNA is amplified in preference to long DNA. The height of the peaks from the same locus is also offset and increased due to the preferential amplification of short DNA. If the ratio of the two peak intensities increases, it cannot be distinguished from the shadow peak. In addition, it is difficult to attribute the DNA of the mixed sample. Therefore, in order to determine whether a meaningful CE analysis has been performed, the benchmark is that the ratio of the intensity of the smaller of the two peaks to the larger peak (Peak to height ratio, PHR) is 10% or more, more preferably 40% or more, and more preferably 60% or more.

[0176] <ski-slope>

[0177] In the case of an excess amount of DNA, the relative presence ratio of dNTP or Polymerase to the amplicon decreases, and the tendency for short DNA to be preferentially amplified is enhanced. In this case, the resulting electropherogram is a sloped electropherogram with a small peak for long DNA and a large peak for short DNA. Additionally, in the case of DNA degradation, there is a tendency for the presence ratio of short DNA to be higher than that of long DNA. At this time, a sloped DNA profile is also obtained. Further, if an inhibitor is contained, there is a tendency for the amplification efficiency of long DNA to be lower than that of short DNA, which also similarly provides a sloped DNA profile. Additionally, if the PCR reagent is diluted to a state thinner than the original mixing ratio by the DNA solution, there is a difference in amplification efficiency, long DNA is preferentially amplified, and an electropherogram with a reverse slope is obtained. Additionally, in the case of a reaction system in which primers for long DNA are mixed in a large amount into the PCR reagent to make long DNA preferentially amplified, an electropherogram with a reverse slope is also obtained. In DNA identification, a sloped profile or a profile with a large peak intensity ratio between loci is not recommended. This is because the difference in peak intensity becomes larger, making it easier for peaks that saturate the CE or fall below the detection limit to appear. Additionally, it also makes it difficult to attribute the mixed sample. Typically, it is preferable to adjust the PCR reaction parameters so that the peak intensity ratio between loci (Inter locus PHR) has a minimum peak to a maximum peak of 1% or more, more preferably 5% or more, more preferably 10% or more, and more preferably 20% or more in intensity. Additionally, small peaks that do not meet the threshold of Inter locus PHR may not be subject to peak analysis. Additionally, in order to cope with degraded DNA, it is preferable that the amplification amount of PCR is within an appropriate range relative to STR-CE, or the dynamic range of CE is designed to be large.

[0178] <Saturation of CE>

[0179] Saturation occurs when the amount of amplicon introduced into the CE section is excessive and the fluorescence intensity at the time of CE detection is higher than the upper limit of the detection range of the detector. At this time, the ratio of the peak showing the maximum intensity to the intensity of other peaks does not reflect the original intensity ratio. Additionally, the intensity ratio relative to the shadow peak or the main peak of IAP is detected as higher than the ratio of the original amplicon. Therefore, the above-mentioned artifacts are meaningless. Additionally, in the case of a Mix sample, the correct mixing ratio cannot be calculated. Thus, in preferred DNA identification, the PCR reaction conditions are set or the PCR amplification product is diluted so that CE detection does not saturate (Oversaturation, saturation, OS). The OS threshold can be set by in-machine evaluation of CE, can be set by the user, can be set by the user each time of measurement, or can be set on a computer.

[0180] <Noise and pull-up of CE>

[0181] There are also artifacts generated by the CE detection unit.

[0182] The pull-up peak refers to the peak erroneously detected from other pigments. Pull-up is significantly prominent when CE is saturated, but it may also be detected in the case of unsaturation.

[0183] When a large amount of DNA was introduced into the CE unit in the previous measurement, it may remain and lead to the detection of a peak in the next measurement.

[0184] In addition, bubbles mixed into the CE unit or background noise of the detection unit may sometimes be reflected in the electropherogram.

[0185] During analysis, Analytical Threshold (AT) can be set so that noise peaks are not erroneously used for analysis. Analytical threshold can be set to obtain a sufficient SN ratio by measuring background noise, can be set by the user, can be set by the device at each experiment, or can be preset. In addition, even for peaks exceeding AT, programs can be stored and executed to determine whether the peak is from an amplification product, from poor migration of CE, or from various noises of CE.

[0186] AT can be set in two stages or more, such as the first standard value and the second standard value. When the peak intensity is greater than the first standard, it is determined as a real peak. Peaks with intensities above the second standard and below the first standard can be set such that user or expert review is required or peak determination is performed when other set conditions are met. In addition, sometimes the amplification efficiency varies according to the locus, and sometimes the luminescence efficiency and noise intensity vary according to the pigment. Therefore, AT can also be set according to the locus or according to the pigment.

[0187] <Measures against artifacts>

[0188] The measures against the above artifacts are described. For undeteriorated DNA and DNA without PCR inhibitors, by introducing the DNA amount within the analysis range of STR-CE into PCR, appropriate peak intensity (above AT and below OS), PHR, and IPA data can be obtained, and the appearance of Skislope can be suppressed. In addition, even in the case of deteriorated, extremely微量, or inhibitor-containing DNA, if the appropriate cycle number and input amount are used, the obtained information amount can be maximized. There is a parameter set of PCR cycle number and DNA amount that can maximize the obtained information amount while minimizing the appearance of artifacts.

[0189] <Evaluation method for CE analysis results>

[0190] When providing the CE analysis results to the user, it is also possible to simultaneously show the user whether the obtained CE data is useful or how useful it is.

[0191] When presenting the CE analysis results to the user, it is also possible to show the user whether the obtained CE data is a complete DNA identification result (Full profile). It is also possible to recommend to the user whether the obtained CE data needs to be analyzed again.

[0192] Figure 2 Represents an evaluation process for determining whether the obtained DNA profile is a Full profile or for notifying the user of the data quality. This chart is an example. The order can be replaced, a part can be omitted, processes not shown here can be included, and one or all of them can be performed simultaneously to assign multiple flags. Additionally, the following processes can be implemented for each peak or locus as Figure 2 shown. When all loci meet all criteria, it is determined as Fullprofile. When it is not a Full profile, it is also possible to only summarize the information of the loci that meet the criteria and provide it to the user. Additionally, for each locus, it is possible to output to a table or the like which criteria are not met or met.

[0193] 201. There is no peak that saturates the CE detection system (OS) among all peak intensities. In the case of a peak with OS, an OS flag can be assigned to the peak, locus, or analysis result.

[0194] 202. There is more than one peak exceeding AT at all loci. In the case of a locus where no peak is detected, a Drop out flag (DO flag) can be assigned to the locus or analysis result.

[0195] 203. In the case where only one peak exceeding AT is detected, the intensity of this peak is more than twice that of AT (excluding genes where only one was originally detected, such as loci on sex chromosomes). In the case where the intensity is not more than twice, an Inconclusive homozygous flag (IH flag) can be assigned to the peak, locus, or analysis result.

[0196] 204. The peak intensity at a position offset by ±1 base amount from the main peak exceeds the IPAT where the peak ratio is set to 1%, 5%, 10%, 20%, or 40% with respect to the intensity of the main peak. In the case of exceeding, a flag indicating a stronger intensity of IPA (IPA- or IPA++ flag) can be assigned to the peak, locus, or analysis result.

[0197] 205. All detected peaks are located within DNA chain lengths that can be assigned to a DNA type. Peaks that cannot be assigned and do not meet the IPA criteria are identified as off-ladder (OL) peaks, and the OL designation is assigned to the peak, locus, or analysis result. However, peaks located at the position of shadow peaks do not need to be considered OL.

[0198] 206. No more than three peaks (or two or more peaks for loci with only one peak) were detected for each locus. If detected, the intensity of the peak with the third highest intensity is less than 1%, less than 5%, less than 10%, less than 20%, or less than 40% of the intensity of the second highest peak. If the intensity ratio of the third highest intensity peak to the second highest peak exceeds a threshold, it can be determined that the DNA contained in the sample comes from more than two people, and the Mix flag is set. However, if the third highest intensity peak is located at a location where the first or second shadow peak appears, it may be determined to be a shadow peak. If a peak with an intensity of less than 1%, less than 5%, less than 10%, less than 15%, less than 20%, or less than 40% of the main peak appears at the shadow peak location, it can be determined to be a shadow peak, and the Mix flag may not be set. If a peak is present at a location that may be a shadow peak and its intensity exceeds the threshold within the allowable range for shadow peaks, it is possible that it is a mix, and the Mix flag may be set. Furthermore, if the intensity of the third highest intensity peak exceeds a threshold at a location where a shadow peak should not appear, the Mix flag may be set. The same determination can be performed on peaks having the fourth and fifth intensities or later.

[0199] 207. When two or more peaks are detected and the peak with the second intensity is greater than 1%, greater than 5%, or greater than 10% and less than 60%, less than 40%, or less than 20% of the first peak intensity, it is determined to be a PHR difference and a PHR flag can be assigned.

[0200] PHR degradation occurs when the amount of DNA fed to PCR falls below 0.6ng, 0.3ng, 0.15ng, or 0.075ng. For example, in CE analysis, when approximate DNA amount is estimated based on IPC intensity, PCR cycle number, and peak intensity, even if the amount of DNA fed to PCR exceeds the aforementioned amount, if the PHR does not exceed the threshold, the resulting DNA profile can be determined to be from a mixed sample.

[0201] The peak assigned the IPA label is also assigned the OL label, so the judgment standard 205 can also take 204 into consideration, that is, the IPA label can also include the OL label.

[0202] If none of the above-mentioned markers are assigned, a full profile is obtained. If a full profile is not obtained, an expert review may be requested. In this case, the time until the DNA identification is completed may be extended. Therefore, it is necessary to increase the probability of obtaining a full profile by setting an appropriate number of cycles. In addition, if the sample is originally a mixture, the amount is small, or there are many PCR inhibitors, a full profile cannot be obtained. However, if not all loci are detected normally, it can be used in criminal investigations if more than five or ten loci are detected. Such a DNA profile is called a partial profile. The more information about loci obtained, the more helpful it is for criminal investigations. In other words, even if a partial profile is obtained, it is necessary to set an analysis protocol, especially the number of PCR cycles, to detect as many peaks as possible. In particular, in the case of mixed samples or when the amount of DNA is small, it is necessary to set a protocol, especially the number of PCR cycles, to detect as many peaks as possible while ensuring that the electropherogram meets 201, 205, and 206 (i.e., without being assigned these markers).

[0203] A threshold can be set to determine whether DO is caused by the DNA used in the PCR reaction not containing the allele or by a low number of PCR cycles. For example, if a locus lacks any peaks larger than AT, and a peak with a peak size ten times greater than AT is present in all amplified products, DO can be determined to be sample-derived. Otherwise, DO can be determined to be due to a low number of PCR cycles, or to be sample-derived, or not sample-derived. This determination can be used to determine whether an analysis with a higher number of PCR cycles should be performed.

[0204] [About the scope of analysis]

[0205] When implementing STR-CE, factors that may cause intensity fluctuations include: (1) concentration fluctuations of salts or injection-inhibiting substances contained in the PCR reaction solution; (2) the mixing ratio of the moving reagent to the PCR reaction solution; (3) amplification efficiency; (4) degradation of the moving reagent or incomplete denature; (5) temperature deviation of the moving section; (6) deviation during electrolytic injection; (7) deviation between capillaries and between capillary arrays; and (8) deviation in detection intensity in the detection section.

[0206] Hereinafter, the dynamic range of CE may be the dynamic range described in various manuals or the like provided with CE equipment. It may also be the ratio of the maximum amplicon amount to the minimum amplicon amount that linearly produces a signal relative to the amount of amplicon actually fed into CE. It may also be the maximum amplicon amount relative to the minimum amplicon amount that can be attributed to the amount of amplicon fed into CE based on signal intensity. It may also be the ratio of an arbitrary upper analytical limit to an arbitrary lower analytical limit. It may also be the ratio of OT to AT. It may also vary for each measurement.

[0207] The analytical range of STR-CE is not particularly limited. It can simply correspond to the dynamic range of CE. Alternatively, the amount of DNA added to the PCR reaction can be varied in actual experiments to investigate the amount of DNA that can accurately detect a specific allele. Alternatively, the amount of DNA added to the PCR reaction can be varied in actual experiments to investigate the amount of DNA that can accurately detect a specific set of alleles. Alternatively, a range of DNA amounts can be experimentally investigated on multiple individuals, and the average, minimum, or maximum range can be used as the analytical range. The amount of DNA used for CE measurement is generally proportional to the amount of DNA added to the STR-PCR reaction. However, this is not always proportional, as it depends on the DNA concentration of the CE injection efficiency, the DNA concentration of the PCR amplification efficiency, and the number of cycles. Furthermore, the analytical range of STR-CE generally does not change significantly when the number of PCR cycles is varied. However, particularly when the amount of DNA is continuously reduced, a stochastic effect (a random effect in which the amount of DNA corresponding to the locus to be amplified is not proportional to the amount of DNA added) may occur, resulting in a potential shift. Therefore, it is preferable to conduct the above-mentioned research on the true STR-CE analysis range for each cycle. The analysis range set for each cycle based on such research is also a type of STR-CE analysis range. However, full experimental verification requires a lot of labor, so some of it can be replaced by calculation.

[0208] The dynamic range of CE or the analytical range of STR-CE can be evaluated by taking into account the deviations (1) to (8) listed above.

[0209] Changing the threshold value can expand or narrow the analysis range. However, expanding the analysis range may come at the cost of reduced accuracy. If methods such as machine learning can more reliably distinguish between artifacts and true peaks, the threshold value can be changed to expand the analysis range.

[0210] Hereinafter, the analysis range of an analysis system refers to, for example, the range of DNA amounts contained in a sample within which DNA analysis can be accurately performed on a DNA sample fed into the analysis system. To expand the analysis range, it is necessary to expand the analysis range of STR-CE or control the amount of DNA fed into STR-CE. Expanding the analysis range of STR-CE can be accomplished by increasing the dynamic range and sensitivity of CE, reducing STR-CE bias, preparing multiple cycles of amplification products, or preparing eluates at different dilution rates and using each for STR-CE. When controlling the amount of DNA fed into STR-CE, the volume of the refining membrane can be reduced or the mesh size increased to limit the upper limit of the amount of DNA that can be processed by the refining membrane. Alternatively, the dilution rate can be quantitatively altered after refining.

[0211] Analysis System for Split PCR and Its Operation Example

[0212] 〔Analysis System〕

[0213] [Configuration example of a general analysis system]

[0214] Figure 3 An example of the analysis system 101 is shown. Figure 4 FIG. 1 shows an example of an operation flow of the analysis system 101. The biomolecule analysis apparatus includes a computer 102 and a flow channel device 104 for performing biomolecule analysis.

[0215] The flow channel device 104 includes: a dissolution chamber 301 for introducing and dissolving the collected sample; a purification membrane chamber 303 that stores a purification membrane 302; a PCR chamber 304 (a PCR chamber that performs thermal cycling) for performing DNA amplification, etc.; and a waste liquid chamber 305. It has an external connection port 306 that connects to the fluid outside the device. The solution is transported through the external connection port 306, and reagents or amplification products can be exchanged with the outside of the device. When the solution is transported through the analysis system 101, a pump and valve 307 can be used to control the liquid delivery. The pump and valve 307 can be both equipped outside the flow channel device, or some can be equipped inside the flow channel device 104. In addition, the PCR reagent storage unit 308 contains PCR reagents 309 (polymerase, primer, dNTP, buffer, etc.) required for the PCR reaction, and the swimming reagent storage unit 310 can contain swimming reagent 311. In addition, it also includes reagent storage units 312, 313, and 314 for storing reagents required for pretreatment. In the sample introduction step 401, before or after the sample is stored in the lysis chamber 301, a lysis buffer is transferred from the reagent reservoir 312 to the lysis chamber 301. Next, lysis begins in the lysis step 402. In the purification step 403, the lysate is transferred from the lysis chamber 301 to the purification membrane chamber 303, where the DNA is bound to the purification membrane 302. A cleaning solution is then released from the reagent reservoir 313 for purification. After purification, the cleaning solution and other components can be dried. The eluate is released from the reagent reservoir 314, and the dissolved DNA is transferred from the purification membrane chamber 303 to the PCR chamber 304. The PCR reagent is transferred from the PCR reagent reservoir 308 to the PCR chamber 304 and mixed with the dissolved DNA. In the amplification step 404, the DNA purified in the PCR chamber 304 is mixed with the PCR reagent 309 and used for the PCR reaction. In the detection step 405 , the amplified DNA is mixed with the mobilization reagent 311 stored in the mobilization reagent reservoir 310 , and the CE unit 105 performs measurement.

[0216] After mixing the mobilization reagent and PCR reaction solution, and before CE analysis, a step of heating to 80-100°C and rapidly cooling to 0-10°C can be included. This step can more completely single-strand the DNA, enabling high-precision CE analysis.

[0217] [Example of an analysis system used in this embodiment]

[0218] Figure 5 The schematic diagram of the analysis system 101 according to this embodiment is shown ( Figure 5 (1)) and example of action flow ( Figure 5 (2)). Action steps 501 to 507 correspond to the amplification step 404 and the detection step 405.

[0219] like Figure 5 As shown in (1), the analysis system 101 of this embodiment includes a flow channel device 104 and a CE unit 105. The flow channel device includes a PCR chamber 304. In addition, the analysis system 101 includes a dispensing chamber 320. The PCR reaction solution or the electrophoretic sample is fed into the dispensing chamber 320. However, the dispensing chamber 320 can also be omitted. The various elements are connected by flow channels 315 and 316.

[0220] like Figure 5 As shown in (2), the analysis system 101 prepares a PCR reaction solution in step 501, performs m PCR cycles in the PCR chamber 304 in step 502, then removes a portion of the amplified product from the PCR chamber 304 without changing its composition in step 503, performs CE analysis on the electrophoretic sample m by the CE unit 105 in step 504, and performs nm thermal cycles on the PCR reaction solution m remaining in the PCR chamber 304 in step 505 to obtain a PCR reaction solution n. In step 506, a portion of the PCR reaction solution n is removed from the PCR chamber 304 without changing its composition, and in step 507, the electrophoretic sample n is subjected to CE analysis by the CE unit 105.

[0221] Alternatively, the injection step in step 506 may be omitted, and all of the amplification products of n cycles may be mixed with the migration reagent in step 507 .

[0222] Steps 503 to 505 may also be repeated multiple times to increase the number of divisions to three, four, or more.

[0223] Step 504 and step 507 may be performed simultaneously, or step 507 may be performed after step 504. By performing step 507 after step 504, the setting of step 505 can be changed according to the result of step 504.

[0224] Alternatively, step 505 may be started immediately after step 503. Since the time between step 502 and step 505 is short, the occurrence of artifacts can be suppressed. Alternatively, the execution times of step 505 and step 506 may be set so that step 507 can be started based on the time step 504 ends.

[0225] [Derivative Examples of the Analytical System for Implementation of the Present Invention]

[0226] like Figure 6 As shown, the analysis system 101 may include heating units 317 and 318 (temperature adjustment and heating mechanisms) for performing thermal cycling in the PCR chamber 304. Flow channels 319 and 315 may also be connected to the PCR chamber 304 to receive reagent supply or pressure control. Figure 6 As shown in FIG, the analysis system 101 may also include a pump and a valve 307 for carrying out the solution transport suitable for each step of the flow channel device or CE measurement. Figure 6 As shown, the analysis system 101 may also have a computer 102, which may have the functions of controlling the pump and valve 307, controlling the CE measurement, controlling the temperature regulation, analyzing and feeding back the data obtained from the CE unit 105 or the heating units 317, 318, etc., providing functions to the user, etc. The flow channel device may also have a dispensing chamber 320. The dispensing chamber 320 has the function of removing a portion of the amplification product from the PCR chamber 304 without changing the composition at the moment when m thermal cycles are completed. The dispensing chamber 320 may also have a measuring function for removing a predetermined amount of PCR reaction solution m or n. When the PCR reaction solution is fed into the dispensing chamber 320, the dispensing chamber 320 may absorb the PCR reaction solution, or the PCR reaction solution may be fed into the dispensing chamber 320 by pressurization. In addition, the dispensing chamber 320 may also be provided simply for temporarily storing the PCR reaction solution, or may not be given a measuring function. The dispensing chamber 320 may also be provided outside the flow channel device 104.

[0227] The PCR reaction solution m can be kept in standby mode in the dispensing chamber 320 before CE analysis. It is mixed with a mobilization reagent (formamide or a molecular weight standard) in the dispensing chamber 320. Specifically, the analysis system 101 mixes the reaction solution (e.g., at least one of a portion of the PCR reaction solution m and at least a portion of the PCR reaction solution) with a solution containing multiple DNA fragments. This allows concentration to be measured using the peak height of the molecular weight standard as a benchmark.

[0228] like Figure 6 As shown, the analysis system 101 may include a standby unit 321 between the CE unit 105 and the PCR unit. In the standby unit 321, from step 506 to step 507, or from step 503 to step 504, the electrophoresis sample mixed with the PCR reaction solution or the PCR reaction solution and formamide is temporarily stored. Figure 6 The flow channel device (and subsequent figures) is arranged in a longitudinal configuration, that is, with the main portion of the flow channel or at least a portion of the flow channel parallel to the direction of gravity. In one embodiment of the flow channel device, the bottom side of the drawing is used with gravity pointing downward. By using it in a longitudinal configuration, bubbles and air trapped in each chamber remain in the upper portion of the chamber. Therefore, by removing the solution from the bottom during transfer to the next chamber / flow channel, air incorporation into the next step can be minimized.

[0229] The flow channel device can be modified as follows.

[0230] like Figure 6 As shown in FIG. 1 , in one embodiment of the analysis system 101 of the present embodiment, a CE unit 105 is provided in the flow channel device. Figure 7 As shown, in one embodiment of the analysis system 101, a pump and a valve 307 are provided in the flow channel device.

[0231] 〔Runner Equipment〕

[0232] like Figure 8 As shown, the flow channel device 104 can receive solution exchange and air pressure control via the analysis system 101 and the external connection port 306. Pressure is applied from the external connection port 306 through the flow channel 322, dispensing the PCR product or elution sample from the dispensing chamber 320. Valves 323, 324, 325, and 326 are provided within the flow channel device and open and close according to the analysis steps.

[0233] like Figure 9 As shown, the flow channel device may also have a mixing chamber 327 between the dispensing chamber 320 and the external connection port 306. The PCR reaction solution and the swimming reagent in the dispensing chamber 320 are mixed in the mixing chamber 327. The mixing chamber 327 may be connected to the dispensing chamber 320 via a flow channel 328. The mixing chamber 327 may be provided outside the flow channel device and inside the analysis system 101, and depending on the circumstances, the standby unit 321 may also assume this function. The mixing chamber 327 is connected to a flow channel 329, and by applying pressure to the flow channel 329, bubbles may be introduced into the mixing chamber 327 for stirring, or a swimming sample may be introduced into the CE unit 105.

[0234] like Figure 9 As shown, the PCR chamber 304 can have three flow channels 319, 315, and 330. The flow channel 319 can be connected to the upstream side of the sample, the flow channel 315 can be connected to the dispensing chamber 320, and the flow channel 330 can be connected to the mixing chamber 327. The PCR reaction liquid m taken out from the PCR chamber 304 is transported to the mixing chamber 327 through the dispensing chamber 320, and the PCR reaction liquid n taken out from the PCR chamber 304 is transported to the mixing chamber 327 through the flow channel 330 without passing through the dispensing chamber 320. The PCR reaction liquid m and the PCR reaction liquid n pass through different paths, thereby suppressing the reduction in reproducibility caused by liquid residue. In addition, when the reaction liquid n is measured but the reaction liquid m is not measured and the entire amount is transported to the mixing chamber 327 or the CE part 105, there is no need to transport the reaction liquid m to the dispensing chamber 320. Therefore, this flow channel device structure can more easily implement split PCR.

[0235] When the PCR reaction solution m is also measured, another dispensing chamber different from the dispensing chamber 320 for the PCR reaction solution n may be provided.

[0236] like Figure 10 As shown, the flow channel device 104 may include a swimming reagent storage portion 310, 331 for storing swimming reagents (formamide, DNA fragments, pure water, etc.). The swimming reagent storage portion 310, 331 may be located on the flow channel 315 or on the flow channel 319. Figure 10 As shown, the reagent storage unit can be divided into two or more storage units for each reagent. By dividing into two, a predetermined amount of reagent can be released separately when divided. Alternatively, the amount of liquid released from the storage unit can be controlled, and the reagent can be released from one reagent storage unit more than twice. Figure 10 As shown, the flow channel device 104 has air storage parts 332 and 333. The air storage parts 332 and 333 can be located on the flow channel 315 or on the flow channel 319. During the splitting, a predetermined amount of air is released from the air storage part 332 or 333, so that a predetermined amount of PCR reaction liquid can be transported to the outside of the PCR chamber 304. In the case of this flow channel device structure, split PCR can be implemented even if the dispensing chamber 320 is omitted. In the case of this flow channel device structure, the mixing chamber 327 can also serve as the dispensing chamber 320. Instead of air, a liquid such as oil that does not affect the PCR reaction can be added to the air storage part 332 or 333. PCR reaction liquid can also be added to the air storage part 332 or 333, and the same volume of PCR reaction liquid as the squeezed PCR liquid can be newly added.

[0237] like Figure 11 As shown, the swimming reagent storage parts 310 and 331 are respectively provided in the flow channels passing through the PCR chamber 304 and forming a pair. Figure 11 As shown, the swimming reagent storage part 331 is preferably arranged closer to the PCR chamber 304 than the air storage part 333. This is because the swimming reagent 311 remaining in the flow channel 319 or the PCR chamber 304 can be completely sent to the dispensing chamber 320 or the mixing chamber 327, which can help improve reproducibility. Figure 11 As shown, the flow channel device 104 may include an air reservoir 334 on the flow channel 315 closer to the PCR chamber 304 than the elution reagent reservoir 310. The air reservoir 334 can be used to transport the elution sample m in the dispensing chamber 320 to the mixing chamber 327 or the CE unit 105. The air reservoir 334 can be replaced with the flow channel 322.

[0238] [Example of split PCR implementation method]

[0239] This section describes a method for performing split PCR on the flow channel device 104. Although the aliquot chamber is mentioned throughout, the solution may be directly transferred to the CE unit 105 without using an aliquot chamber or a mixing chamber.

[0240] Figure 12 An example of the flow channel device 104 is shown. Figure 13 Indicates that it is used in Figure 12 An example of a method for carrying out split PCR on a flow channel device. In addition, for the convenience of illustration, some reference symbols are shown in Figure 12 , and in Figure 13 omitted.

[0241] Step I: The PCR chamber 304 is provided with a PCR reaction solution 335 . With valves 326 , 323 , and 325 closed, an m-cycle PCR reaction is performed in the PCR chamber 304 . This step may correspond to step 502 .

[0242] In this manner, the analysis system 101 performs m thermal cycles on the PCR reaction solution 335 in the PCR chamber 304 to generate the PCR reaction solution m (first reaction solution).

[0243] Step II: After the m-cycle PCR reaction is completed, valves 326 and 323 are opened to transfer a portion of the solution in the PCR chamber 304 to the dispensing chamber 320 . This step may correspond to step 503 .

[0244] In this way, the analysis system 101 removes a portion of the PCR reaction solution m from the PCR chamber 304 without changing its composition. Here, the analysis system 101 can perform electrophoresis analysis on the portion of the PCR reaction solution m in the CE unit 105 .

[0245] Thus, the flow channel device 104 includes openable and closable valves 326 and 323. By closing valves 326 and 323 before the start of m thermal cycles and opening valves 326 and 323 after the completion of m thermal cycles, a portion of the PCR reaction solution m can be split and removed. This allows for appropriate splitting processing.

[0246] Step III: Close valves 323 and 326, squeeze out the running reagent 311 from the running reagent reservoir 310, and transfer the PCR reaction solution m stored in the dispensing chamber 320 to the mixing chamber 327. The PCR reaction solution m is mixed with the running reagent 311 to form a running sample 336.

[0247] Step IV: Open the valve 325 to transfer the electrophoretic sample in the mixing chamber 327 to the outside of the flow channel device 104 (the standby unit 321 or the CE unit 105 of the analysis system). This step may correspond to step 504 .

[0248] Step V: Close valve 326 and perform nm thermal cycles. This step may correspond to step 505.

[0249] In this manner, the analysis system 101 performs nm thermal cycles (where nm is an integer greater than or equal to 2) on the PCR reaction solution m remaining in the PCR chamber 304 so that the total number of thermal cycles becomes n, thereby generating a PCR reaction solution n (second reaction solution).

[0250] Step VI: After a total of n thermal cycles are completed, valve 325 is closed, valves 326 and 323 are opened, and the PCR reaction solution n is transferred to the mixing chamber 327 .

[0251] In this way, the analysis system 101 removes at least a portion of the PCR reaction solution n from the PCR chamber 304 without changing its composition.

[0252] The mobilization reagent is squeezed out of the mobilization reagent reservoir 331, transferring the PCR reaction solution n stored in the dispensing chamber 320 to the mixing chamber 327. Valve 325 is opened to transfer the mobilization sample in the mixing chamber to the outside of the flow channel device 104 (to the standby section 321 or CE section 105 of the analysis system). This step may correspond to step 507.

[0253] The analysis system 101 can perform electrophoresis analysis on the at least a portion of the PCR reaction solution n in the CE unit 105 .

[0254] [Other Examples of Split PCR Methods]

[0255] Figure 14 Indicates the analysis process of split PCR, Figure 15 An example of the flow channel device 104 is shown. Figure 16 Indicates Figure 15 An example of a method for transporting a PCR reaction solution with m=24 cycles and n=30 cycles. Figure 15 In the Figure 16 omitted.

[0256] The flow channel device 104 includes a valve 337 on the flow channel 315 connecting the dispensing chamber 320 and the PCR chamber 304. Valve 337 is located on the PCR chamber 304 side of the branch with the mobilization reagent reservoir 310. Furthermore, a valve 338 is provided on the flow channel 316. Furthermore, a valve 339 is provided on the flow channel 319 on the flow channel 329 side of the branch with the mobilization reagent reservoir 331.

[0257] Step I: Close all valves. A PCR chamber 304 containing a μl of PCR reaction solution 335 is provided. 24 thermal cycles are performed in the PCR chamber 304. After the 24 thermal cycles are completed, a final extension is performed for 8 minutes. (Steps 601 to 604 may correspond to steps 501 and 502.)

[0258] Step II: After the 24-cycle PCR reaction is completed, valves 326, 323 and 337 are opened to transfer a portion bμl (where a>b) of the solution in the PCR chamber 304 to the dispensing chamber 320 via the flow channel 315 (which may correspond to step 605 and step 503).

[0259] Step III: Close valves 323, 326, and 337, open valves 324 and 325, and squeeze c μl of running reagent 311 from the running reagent reservoir 310. The PCR reaction solution m stored in the dispensing chamber 320 is transferred to the mixing chamber 327. The PCR reaction solution m is mixed with the running reagent 311 to form the running sample 336 (which may correspond to steps 606 and 504). At this point, the running reagent 311 may remain in the dispensing chamber 320 and a portion of the flow channel 315.

[0260] Step IV: Close valve 324, open valve 338, and transfer the electrophoretic sample in the mixing chamber 327 to the outside of the flow channel device 104 (the standby part 321 or the CE part 105 of the analysis system) (which may correspond to step 607 or step 504).

[0261] Step V: Close all valves, perform six thermal cycles on the a-b μl PCR reaction solution 335 remaining in the PCR chamber 304, and then perform an 8-minute final extension (which may correspond to steps 608 to 610 and step 505).

[0262] Step VI: After a total of 30 thermal cycles are completed, valves 326, 323, and 325 are opened to squeeze out d μl of the running reagent from the running reagent reservoir 331 and transfer the PCR reaction solution n to the mixing chamber 327 (which may correspond to step 506).

[0263] Step VII: Valve 325 can be closed, valve 339 can be opened, and air pressure can be applied to move all the solution remaining in PCR chamber 304 into mixing chamber 327. Alternatively, air can be introduced into mixing chamber 327 to agitate and homogenize the eluted sample 336 (which can correspond to steps 611 and 507).

[0264] Step VIII: Open the valve 338 to transfer the electrophoretic sample in the mixing chamber 327 to the outside of the flow channel device 104 (the standby unit 321 or the CE unit 105 of the analysis system) (which may correspond to step 612 or step 507 ).

[0265] Will Figure 14 An example of the time and temperature for each step of the split PCR process is shown in Table 1.

[0266] [Table 1]

[0267] Table 1 Split PCR scheme

[0268]

[0269] For basic thermal cycle settings, refer to the manual of the STR-PCR kit (GlobalFiler (trademark) Express) (https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / 4477672_GlobalFilerExpress_UG.pdf). In step 605, the temperature of the PCR chamber 304 during aliquoting can be set to any temperature between room temperature and denaturation.

[0270] In steps 605, 606, 610, and 611, the solution may be kept at a low temperature (4°C). By keeping the solution at a low temperature, it is possible to prevent the sample from being degraded or an unnecessary reaction from occurring.

[0271] In step 605, it is preferable to set the temperature during the division to be substantially the same as the temperature in the denaturation step, because this can prevent excessive elongation reactions and thus suppress the generation of artifacts.

[0272] In step 605, setting the temperature for segmentation to the same as the extension temperature will cause an unnecessary extension reaction, but it can suppress nonspecific amplification. Furthermore, this is preferable because it avoids inactivation of the polymerase or fluorophore. The effect of the unnecessary extension reaction on STR-CE analysis accuracy can be minimized.

[0273] In step 605, the temperature during the splitting can also be set between the Extension temperature and RT. In this case, accurate temperature adjustment is not required, which is simple. When the m-cycle PCR is completed, there are sufficient amplicons, so the influence of non-specific amplification can be ignored.

[0274] In step 605, the shorter the time required for segmentation, the better. If the time required for segmentation is long, it is likely to lead to an increase in artifacts. In addition, the longer the time required for segmentation, the more likely it is that various biomolecules such as polymerase will be inactivated.

[0275] In addition, if Figure 14 As shown, in this embodiment, analysis system 101 does not perform any analysis other than electrophoresis on each reaction solution (including a portion of PCR reaction solution m and at least a portion of PCR reaction solution n) prior to electrophoresis analysis. This simplifies the device structure, eliminating the need for an additional optical system, for example.

[0276] [Amount of PCR reaction solution and electrophoresis reagent]

[0277] The PCR reaction solution used in this flow channel device has a volume of 1μl to 200μl, preferably 10μl to 50μl. Smaller volumes allow for accurate and rapid temperature regulation. On the other hand, larger volumes allow for the collection of more purified DNA, facilitating high sensitivity. Furthermore, larger volumes eliminate the need for highly precise solution measurements during fractionation.

[0278] Table 2 shows an example of the liquid volume when divided. The liquid volume actually measured may not be the value in the table itself, and the median value of multiple measurements may be used as the value in the table.

[0279] [Table 2]

[0280] Table 2 Liquid volume for split PCR

[0281] a[μl] b[μl] c[μl] d[μl] Episode 1 a b 10a 10(ba) Episode 2 15 5 50 100 Episode 3 a b 10a+e 10(ba) Episode 4 a b 11a 10(ba)

[0282] The total amount of PCR reaction solution in step 502 is set to a μl, the amount of solution removed during the mth cycle in step 503 is set to b μl, the amount of running reagent mixed with PCR reaction solution m in step 504 is set to c μl, and the amount of running reagent mixed with PCR reaction solution n in step 507 is set to d μl. Table 2 above shows examples of preferred relationship equations or setting values ​​for a to d. To efficiently introduce amplification products or molecular weight standards into CE, the ionic strength of the running sample must be sufficiently low. Therefore, it is preferable to mix the running reagent in a volumetric ratio of 2, 5, 10, or 20 times the amount of PCR reaction solution.

[0283] The PCR division can also be set so that the PCR reaction solution m is equal to the PCR reaction solution n. When the amounts of the PCR reaction solutions m and n are close, more stable liquid delivery can be achieved when the moving reagent is delivered to the CE part.

[0284] PCR splitting can be set so that the PCR reaction solution m is smaller than the PCR reaction solution n. When the amount of liquid removed during splitting is smaller, the variation in the liquid amount during the PCR of nm is smaller, and thus the amplification product n can be prepared with higher reproducibility.

[0285] Set 1 is an example of the solution amount when the amount of the running reagent is set to 10 times that of the PCR reaction solution.

[0286] Set 2 shows an example of the solution volume when the PCR reagent volume is 15 μl. 5 μl of the solution is removed during the mth cycle, and the entire PCR reaction solution is mixed with the running reagent during the nth cycle. In this example, analysis system 101 transfers the entire PCR reaction solution from the PCR chamber to the outside of the chamber at the end of the nth thermal cycle. This eliminates the need for measurement equipment and simplifies the flow path structure.

[0287] exist Figure 16 In the shown liquid delivery process, there is residual swimming reagent in the dispensing chamber 320. When the amount of swimming reagent in the swimming reagent storage part 310 is sufficient relative to the dispensing chamber 320, even if the swimming reagent remains in the dispensing chamber 320, the influence on the analysis accuracy or range can be ignored. In addition, if a flow channel 322 is provided between the valve 337 and the dispensing chamber 320 on the flow channel 315 or an air storage part 334 is provided as an air line, the full amount can be flowed into the dispensing chamber 320 and the mixing chamber 327. Under the wide structure of the large volume, the PCR reaction solution m in the dispensing chamber 320 is almost fully sent to the mixing chamber. In addition, considering that a part of the swimming reagent remains in the dispensing chamber 320, it is preferred to keep an excessive amount of swimming reagent in the reagent storage part.

[0288] Set 3 shows an example of the solution amount when e μl of the moving reagent is expected to remain in the dispensing chamber 320 and an excessive amount of the moving reagent is stored in the moving reagent reservoir 310 .

[0289] Set 4 is an example of the solution amount when the volume of the dispensing chamber 320 is b μl and b μl of the moving reagent is expected to remain in the dispensing chamber 320 and an excess of the moving reagent is stored in the moving reagent reservoir 310 .

[0290] [Detailed segmentation method]

[0291] Variations in the solution volume during splitting can easily narrow the effective analytical range of the analysis system. Therefore, high precision is desirable when splitting PCR reaction solutions. To achieve high-precision solution splitting, the following functions can be incorporated: presetting an appropriate pressurization pressure and time; extruding the PCR reaction solution with a predetermined volume of liquid or gas; employing a liquid level sensor; and extruding the PCR reaction solution into the dispensing chamber 320 to a predetermined volume. Multiple extrusion and measurement methods can also be combined.

[0292] Measurements can be performed during the dispensing of PCR reaction solution m in step 503 and during the dispensing of PCR reaction solution n in step 506. If step 506 is omitted and the entire amount of PCR reaction solution n is mixed with the mobilization reagent, measurement can be performed only in step 503. In other words, the number of measurements can be the number obtained by subtracting 1 from the number of divisions. Similarly, when performing two measurements, since a predetermined amount of liquid remains in the PCR chamber, the reaction solution remaining in the PCR chamber can be subjected to an additional p PCR thermal cycle, and CE analysis can be performed on the third division of the PCR reaction solution after n+p cycles of PCR.

[0293] When removing the PCR reaction solution from the PCR chamber, a pump can be used to apply pressure to the PCR chamber to deliver the solution to the dispensing chamber. Furthermore, if the valves are closed during PCR heating, internal pressure is applied. Therefore, even if valves 326 and 323 are not opened in step II, the internal pressure can still be used to move the solution to the dispensing chamber.

[0294] In step 502, after the m cycles are completed, the PCR reaction solution m can be squeezed out using new PCR reagents in step 503, and a predetermined amount can be transferred to the dispensing chamber 320 or the mixing chamber 327. Figure 10 The PCR reagent is sealed in the air storage section 332 instead of air. Since the solution is squeezed out, it has the advantage of being easy to determine the volume of the squeezed out. In addition, when the PCR reaction solution is squeezed out, there is the advantage that there is no adverse effect of bubbles or oil mixed in on the reaction. On the contrary, new PCR reagent may enter the later stage, resulting in the addition of new factors in addition to the above-mentioned deviation factors (1) to (8) of the analysis range. In addition, since the PCR reaction solution is diluted, the PCR reaction solution remaining in the PCR chamber is diluted at the time of m cycles, and more PCR cycles are required. In addition, when a reagent at room temperature is added, the temperature of the PCR chamber will decrease. In addition, it is necessary to perform initial denaturation again in step 505. Therefore, when the PCR reaction solution is squeezed out, the PCR reaction time is slightly longer. In addition, increasing the number of cycles and performing initial denaturation again will help to produce artifacts. In addition, by performing initial denaturation of the PCR reaction solution used for extrusion before step 503, part of the above-mentioned problem can also be solved.

[0295] like Figure 10 As shown, air storage parts 332 and 333 are set in the flow channel device 104. When the m cycle ends in step 502, the PCR reagent can also be squeezed out through the air chamber and transported to the dispensing chamber 320 or the mixing chamber 327 in step A3.

[0296] Alternatively, oil can be used to squeeze out the PCR reaction solution instead of using air or PCR reagents to squeeze out the PCR reaction solution. Figure 10 The air storage portion 332 is sealed with oil instead of air. Any oil can be used as long as it does not affect the PCR reaction. For example, it can be silicone oil, mineral oil, fluorine-inert oil, or a mixture of multiple oils. In the case of extrusion with oil, there are the following advantages: as with extrusion with reagents, it is easy to specify the extruded volume; unlike the case of extrusion with PCR reaction liquid, it will not mix with the PCR reaction liquid. On the other hand, if oil mixes with the PCR reaction product and flows into the latter stage, it may cause obstacles to the CE determination. When taking out the PCR reaction liquid m, it can also be separated by a separation membrane or centrifugal separation to leave the oil in the chamber.

[0297] [Details of measurement organization]

[0298] When the PCR reaction solution is introduced into the dispensing chamber 320 during the splitting process, if air or bubbles in the PCR chamber enter instead of the PCR reaction solution, the measurement accuracy will be reduced. Therefore, it is preferable to implement an improvement to prevent air from entering the dispensing chamber 320 and the flow channel 315. Figure 17 An example of a structure for preventing air from entering is shown. Figure 17 As shown in (a) and (b), the flow channel 315 branches from the PCR chamber 304 at a position lower than the liquid level 701 of the PCR reaction solution 335 in the direction of gravity. Air is retained on the upper side of the PCR chamber 304, so the amount of air introduced into the flow channel 315 during the splitting can be minimized. In addition, it is preferable to design the liquid volume or the chamber shape so that the liquid level 701 is located above the connection between the PCR chamber 304 and the flow channel 315 in the direction of gravity when the splitting is completed. Figure 17 As shown in (b), liquid level 701 may also be located above valve 326 or 323 in the direction of gravity. In this case, the ingress of bubbles into PCR chamber 304 can be minimized or completely eliminated. PCR reaction solution 335 located above valve 326 or 323 may not be used for the PCR reaction. However, in this case, unreacted reagent may enter the CE assay, resulting in reduced sensitivity.

[0299] like Figure 17 As shown in (c), the flow channel 315 can also be connected to the lower part of the PCR chamber 304. In this structure, there is an advantage that air is difficult to enter, but the flow channel 315 is slightly long, and there is a tendency to easily cause liquid loss.

[0300] A structure for removing bubbles may also be provided in the flow channel 315. In addition, a hydrophilic filter may be placed in the flow channel 315 to prevent air from passing through the flow channel connecting the PCR chamber 304 and the dispensing chamber 320.

[0301] The dispensing chamber 320 may have a measuring function. For example, Figure 18 The example of the dispensing chamber 320 with a measuring function is shown. The flow channels 315 and 328 connected to the dispensing chamber 320 have valves 324 and 337. The valve 337 can be used not only for measurement, but also for preventing liquid from splashing into the dispensing chamber 320 during the PCR reaction, or for preventing the swimming reagent and swimming sample from flowing back to the PCR chamber 304 after the dispensing. In addition, the valve 324 can be used not only for measurement, but also for preventing the swimming sample from flowing back after being transported to the mixing chamber. The volume of the dispensing chamber can be consistent with the liquid volume of the amplification product m that you want to divide and measure. The dispensing chamber can be spherical or cylindrical, or it can be a cube, or it can be elongated, or it can be a winding flow channel, or it can be an ellipsoid or an elliptical cylinder.

[0302] For example, after m thermal cycles, the analysis system 101 can measure a predetermined amount of PCR reaction solution m within a range of 0.1% to 50% by transferring the PCR reaction solution m to the dispensing chamber 320 (measurement unit).

[0303] Figure 19 Indicates an example of a measuring mechanism. Figure 19 The illustrated mechanisms may be combined to perform measurement. A measurement mechanism not shown may also be used. By combining multiple measurement mechanisms, robust measurement can be achieved.

[0304] Figure 19 (a) is an example of a measuring mechanism, comprising a ventilation filter 702 and a flow channel 703 connected to the flow channel 328. The flow channel 703 is connected to the flow channel 328 on the side closer to the dispensing chamber 320 than the valve 324. The ventilation filter 702 is preferably a hydrophobic porous filter formed of PP or a fluorine-based resin. Figure 20 Steps I to III show an example of the operation of this measuring mechanism.

[0305] Step I: Apply pressure to the PCR chamber 304 to deliver the PCR reaction solution into the dispensing chamber 320. The air in the dispensing chamber 320 or in front of and behind it is released from the ventilation filter 702 via the flow channel 703.

[0306] Step II: The amplified product or the mobile reagent cannot pass through the ventilation filter 702 , so the volume of the PCR reaction solution in the dispensing chamber 320 and the flow channels before and after it is measured.

[0307] Step III: Close valve 337 and open valve 324. The PCR reaction solution in the dispensing chamber 320 is delivered to the mixing chamber 327 through the mobilization reagent or air.

[0308] like Figure 19 As shown in (b), a flow channel resistance body 704 can be provided on the flow channel 328. The flow channel resistance body 704 can be a hydrophobic filter such as PP or fluorine-based resin. In addition, the hydrophobicity of the flow channel surface in the flow channel resistance body 704 can be higher than that of the flow channel 328 portion. In addition, the flow channel width can be narrowed at the flow channel resistance body 704, or an obstacle can be provided. In addition, the flow channel resistance body 704 can quickly widen the flow channel width. In the flow channel resistance body 704, the flow channel resistance body can be provided using the principle of a capillary stop valve. Air can be easily removed before the flow channel resistance body 704 comes into contact with the liquid. Figure 21 I to III show operation examples of this measuring mechanism.

[0309] Step I: Apply pressure (AkPa) to the PCR chamber 304 to deliver the PCR reaction solution into the dispensing chamber 320. The air in the dispensing chamber 320 or before and after the dispensing chamber 320 can be smoothly released from the flow path resistor 704.

[0310] Step II: Under the applied pressure of AkPa, the PCR reaction solution cannot pass through the flow channel resistance body 704 or cannot pass through without taking a long time, so a predetermined amount of PCR reaction solution can be measured in the dispensing chamber 320 .

[0311] Step III: Close the valve 337 and apply B kPa (where B>A) to the dispensing chamber 320 using a mobilization reagent or air, so that the PCR reaction solution in the dispensing chamber 320 can be transferred to the mixing chamber 327 .

[0312] like Figure 19 As shown in (c), the structure can be such that a liquid level detection sensor 705 is installed on the flow channel 328 or the dispensing chamber 320, and the liquid transportation is stopped after the dispensing chamber 320 is filled with a predetermined amount of liquid.

[0313] Figure 22 This example shows a simple measurement mechanism that does not use a liquid level detection sensor, a ventilation filter, or a flow path resistor. However, in this embodiment, a mechanism that combines a liquid level detection sensor, a ventilation filter, a flow path resistor, etc. to perform stable dispensing can also be provided.

[0314] Step I: A PCR chamber 304 containing a PCR reaction solution 335 is provided. With all valves closed, an m-cycle PCR reaction is performed in the PCR chamber 304 .

[0315] Step II: After the m-cycle PCR reaction is completed, valves 326 and 323 are opened to make the pressure in the PCR chamber 304 become atmospheric pressure (=100 kPa).

[0316] Step III: Apply a pressure of 100 kPa from flow channel 319 and flow channel 330 .

[0317] Step IV: Open valve 337 to transfer a portion of the solution in PCR chamber 304 to dispensing chamber 320. When the pressure in dispensing chamber 320 is reduced to about half, the pressures in dispensing chamber 320 and PCR chamber 304 reach equilibrium, and the solution transfer stops.

[0318] Step V: Close valve 337 and open valve 324 to restore the pressure in the dispensing chamber 320 to atmospheric pressure.

[0319] In this method, when transferring solution from the PCR chamber 304 to the dispensing chamber 320, the amount of liquid entering the dispensing chamber 320 can be regulated by the pressure applied to the dispensing chamber 320. When the volume of the dispensing chamber 320 is V1 and the interior is filled with air at a pressure of P1, when liquid of a volume of V2 enters the dispensing chamber, according to Boyle's law, the pressure changes to P2 = P1 * V1 / V2. When P2 balances the applied pressure, the solution stops. In step I, when the pressure in the dispensing chamber is 100 kPa, when the volume of the dispensing chamber 320 is reduced to half, the pressure in both the dispensing chamber and the PCR chamber is 200 kPa, achieving force equilibrium. Therefore, a PCR reaction solution 335 equivalent to approximately 50% of the volume of the dispensing chamber can be dispensed. The pressures shown here are merely examples; the volume of the dispensing chamber, the space in the flow channel before and after the dispensing chamber, and the desired amount of liquid can also be appropriately set and measured. The disadvantage of this method is that it tends to require a high pressure resistance of the valve. If the volume of the dispensing chamber 320 is set larger than the amount to be measured, the pressure required for dispensing and measurement will be reduced, but there will be a disadvantage that the amount of residual liquid will increase when the reagent is squeezed out later.

[0320] [About measurement accuracy]

[0321] In CE analysis, if peak intensities vary when measuring the same sample multiple times, the effective analysis range will be narrowed. If the introduction of split PCR causes significant variation in analysis intensity, the effective analysis range will be narrowed, making this technique not recommended. If the introduction of split PCR causes significant variation in the mixing ratio of the mobilization reagent to the PCR reaction solution, this will be reflected in variations in peak intensity, narrowing the effective analysis range.

[0322] like Figure 14 As shown, it was experimentally confirmed how much the change in the mixing ratio affects the CE peak intensity when a PCR reaction solution and a running reagent are mixed. Figure 23 This value represents the CE intensity when the ratio of the PCR reaction solution to the running reagent is varied. The variation in peak intensity between capillaries is normalized by the average intensity of a separately measured molecular weight standard that does not contain the PCR reaction solution. The peak intensity of the molecular weight standard decreases monotonically with increasing ratios of the PCR reaction solution. On the other hand, the peak intensity of the amplified product increases monotonically, and the variation in peak intensity is limited by changes in the liquid volume.

[0323] In CE, a voltage is applied to the moving sample and capillary tube as the amplification product is drawn into the capillary, creating an electric field injection. During electric field injection, the amplification product introduced into the capillary competes with other ions or nucleic acids for injection. For a constant voltage and injection time, the relationship shown in Equation 1 holds true (β is the current value, α is a constant).

[0324] [Mathematical formula 1]

[0325] α(k0*C0+k1*C1+k2*C2+k3*C3)=β…(Formula 1)

[0326] in,

[0327] k0 and C0 are the mobility and concentration of ions contained in formamide,

[0328] k1 and C1 are the mobility and concentration of the molecular weight standard DNA,

[0329] k2 and C2 are the mobility and concentration of salts, primers, and dNTPs contained in the PCR reaction solution.

[0330] k3 and C3 are the mobility and concentration of the amplification product contained in the PCR reaction solution.

[0331] Assuming that 1 μl of the PCR reaction solution is mixed with 10 μl of the running reagent as a standard, when the amount of the PCR reaction solution is varied by γ μl, the following expression 1 is obtained.

[0332] [Mathematical formula 2]

[0333]

[0334] The injection amount k1*C1 of the molecular weight standard is adjusted as follows, and it decreases monotonically with the increase of γ.

[0335] [Mathematical formula 3]

[0336]

[0337] On the other hand, by modifying Formula 1, the injection amount k3*C3 of the amplification product increases monotonically with respect to the amount of the PCR reaction solution.

[0338] [Formula 4]

[0339]

[0340] PCR reaction mixtures contain large amounts of salts, primers, dNTPs, and other substances, and the injection volume is essentially saturated. Therefore, even if the ratio of PCR reaction mixture to running reagent is slightly increased, the fluctuation in peak intensity is limited. Even if the ratio of PCR reaction mixture fluctuates by ±20% relative to standard conditions (PCR reaction mixture: running reagent = 1:10), the fluctuation in peak intensity remains within 10%. Furthermore, if the target PCR reaction mixture is slightly more concentrated than the standard conditions (PCR reaction mixture: running reagent = 1.6:10), the fluctuation in peak intensity remains below 10%, even if the ratio of PCR reaction mixture fluctuates by ±50% from the target ratio. These experimental results suggest that even if the distribution accuracy of split PCR is slightly reduced, the fluctuation in peak intensity of the amplified product caused by changes in the mixing ratio of PCR reaction mixture and running reagent is limited.

[0341] Even if the precision of measuring mechanism is slightly reduced sometimes, still preferably adopt simple and low-cost measuring mechanism.In the case of adopting the measuring mechanism with low precision, as shown above, preferably select to make the variation of the mixing ratio of PCR reaction solution and swimming reagent difficult for the solution composition that has an impact on peak intensity.This can add salt to swimming reagent, can make the salt, primer, dNTP etc. contained by PCR reaction solution assume this function.In addition, by implementing under the condition that the mixing ratio of swimming reagent and PCR reaction solution is slightly more than PCR reaction solution, measure under the state closer to saturation, can be difficult to be influenced by the variation ratio.In this case, preferably slightly more than the mixing ratio of standard mixed molecular weight standard, so that the peak intensity of the molecular weight standard contained by swimming reagent reaches the required level of mensuration.

[0342] How to set up a split PCR protocol

[0343] [Principle of expanded analytical range by split PCR]

[0344] To analyze target DNA concentrations of varying sizes within a sample, separate electrophoresis analyses are performed on PCR reaction solutions m and n containing different concentrations of amplified product. By presetting m and n within appropriate ranges based on the potential target DNA concentration range within the sample, appropriate DNA profiles can be generated for a wider range of DNA amounts compared to preparing a single amplified product for CE analysis without compromising sensitivity.

[0345] Figure 24 This figure shows the principle of expansion of the analytical range by split PCR. Figure 24 The horizontal axis represents the amount of DNA input into the PCR, and the vertical axis represents the concentration of the amplification product or the peak intensity in CE. 801 refers to the upper limit of the detection intensity in CE or the upper limit of the concentration of the amplification product that can be accurately amplified by PCR. 802 refers to the lower limit of the detection in CE. In the figure, the concentration of the amplification product is recorded as linearly increasing with respect to the DNA input amount, but the actual amplification is curvilinear, and it can be considered that when the amplification product reaches a certain amount, it approaches a plateau. In a certain number of PCR cycles m, the range of the amount of DNA that can be accurately analyzed is between the lower limit a and the upper limit b. The analysis range of STR-CE that can be analyzed by one cycle number is set as 803. Additionally, in a certain number of PCR cycles n, the range of the amount of DNA that can be accurately analyzed is between the lower limit c and the upper limit d. At this time, if a < d, the analysis range can be expanded from b / a or d / c to b / c. The analysis range of STR-CE when expanded by split PCR is set as 804. 804 is the range of the amount of DNA within the analysis range of STR-CE in either m or n, and in principle, there is no amount of DNA outside the analysis range within this range. Hereinafter, it is assumed that the analysis range (b / a) for each cycle number is basically constant, and it changes by 2 x , y-1 times with respect to the cycle number difference x. At this time, the interval between m and n is limited to the range of the following formula 2.

[0346] [Mathematical formula 5]

[0347]

[0348] Figure 24 (1) of represents an example that satisfies the above formula. For example, when the analysis range b / a with respect to the amount of DNA introduced into the PCR is 80 times, n - m is preferably 6 or less, and the expansion rate is 64. The expansion rate can increase with respect to the number of splits y by (b / a) y-1 but it is considered that the fewer the number of splits, the more stable liquid feeding can be achieved on a simple flow channel device. Hereinafter, mainly the cases of setting m and n and analyzing the amplification products m and n will be described. On the other hand, according to the same setting method, l can be set for three splits and k can be set for four splits.

[0349] When c or d is less than 0.2 ng or 0.1 ng, the deviation of the intensity balance between the peaks becomes large. Therefore, it is preferable to set a cycle number larger than the cycle number set by formula 2, and make the interval between m and n narrower than the maximum cycle number specified by formula 2. Hereinafter, unless otherwise specified, the upper and lower limits of the analysis of STR-CE change by 2 x times with respect to the cycle number difference x.

[0350] Figure 24 The (2) indicates the analysis range when the interval between m and n does not satisfy Formula 2. In this case, the lower limit a that can be analyzed by m is greater than the upper limit d that can be analyzed by n, and accurate DNA identification cannot be performed at the DNA concentration between a and d. In the case where a > d and there is no sample between a and d, or in the case of extremely low frequency, the values of m and n that make the relationship a > d hold can be勉强 set, but in fact, the DNA amount of the input sample is mostly unknown, so it is preferable to satisfy Formula 2.

[0351] When the n cycle number covers extremely low-concentration DNA, a Stochastic effect will occur. Therefore, it is preferable to set a difference smaller than the upper limit of the difference between n and m specified by Formula 2.

[0352] There are multiple factors causing analysis result deviation in STR-CE analysis. Therefore, considering the deviation, it is necessary to make the analysis DNA amount range that can be covered by the STR-PCR of the m cycle and the DNA amount range that can be analyzed by the STR-PCR of the n cycle overlap. In the case where there is no overlap, there is a DNA amount for which the analysis fails whether it is within the analysis ranges of m and n. That is, it is necessary to set m and n so that a < d is surely established for the assumed analysis deviation. Hereinafter, m and n are set considering the deviation of the analysis system.

[0353] [Set n according to the CE analysis result of m]

[0354] The value of m can also be preset in the device.

[0355] In the case of the analysis system that ends or starts step 505 after step 504, n can be set according to the measurement result of the electrophoresed sample m.

[0356] n can be set according to the following conditions:

[0357] (1) When no peak of the amplification product m is detected at all;

[0358] (2) When a peak is detected and some peaks are smaller than AT; and

[0359] (3) When a peak is detected and some peaks are saturated or exceed the IAP threshold.

[0360] In the case of (1), when the analysis range of STR-CE is x, the n - m cycle can be additionally implemented (n - m ≤ log(x), where x is the analysis range of STR-PCR). In this case, n - m is preferably used with the maximum value in a manner that satisfies Formula 2.

[0361] In the case of (2), when the saturation intensity of CE is z relative to the maximum intensity y of the detected peak, PCR defined by nm cycles (nm≤log(z / y)) can be additionally performed.

[0362] In case (3), additional PCR may not be performed.

[0363] [How to set the interval between m and n in consideration of the intensity ratio of the sample to be analyzed]

[0364] Especially when CE analysis takes a long time, waiting until the m cycles of electrophoresis are complete will increase the waiting time for the nm PCR reaction, potentially increasing artifacts or preventing the PCR reaction from proceeding smoothly. Therefore, m and n should be set in advance.

[0365] In STR-CE, since homozygous and heterozygous loci are mixed, even if an ideal analysis system is assumed in which all amplification efficiencies, CE injection efficiencies, and fluorescent dye luminescence efficiencies are equal, there will still be an intensity difference of 1:2 between the heterozygous peak and the homozygous peak.

[0366] In actual STR-CE, there are differences in amplification efficiency depending on the locus and DNA length, differences in CE injection efficiency, and differences in luminescence efficiency between dyes. Therefore, even when the DNA amount is adjusted appropriately, peak intensity differences of 1:2 to 1:20 may exist. Furthermore, AT values ​​may differ between dyes. In such cases, the analytical range must be considered to account for these differences in AT settings.

[0367] When DNA comes from multiple people, the allele abundance ratio varies from 1:2 to 1:1000.

[0368] When DNA is degraded, the allele abundance ratio differs by 1:2 to 1:1000. Typically, DNA with short loci or alleles is amplified in large quantities, while the peak intensity of DNA with long loci or alleles tends to decrease.

[0369] When PCR is inhibited, there is a tendency for shorter DNA to be amplified more and for the peak intensity of longer DNA to decrease. In this case, the difference in peak intensity is 1:2 to 1:1000.

[0370] If the amount of DNA input is less than 0.1 ng, the intensity variation between peaks tends to increase. In this case, the effective analysis range is further reduced. Therefore, the effective dynamic range that can be covered by m and n does not necessarily have to be the same.

[0371] The factors that determine the intensity ratio between peaks will be discussed below, assuming that they arise from differences in the abundance ratios of alleles contained in the DNA used for STR-PCR. Differences in amplification efficiency, CE injection efficiency, and luminescence efficiency also have an impact, and while these differences should be considered, they are omitted here and included in the discussion of the abundance ratio between alleles.

[0372] use Figure 25 , the effective STR-CE analysis range when the presence ratio of the analyzed alleles is poor is described. The amplification product contains allele α and allele β. However, the amount of alleles α and β before or after amplification is α>β. The plot of the amount of allele α in the amplification product relative to the input DNA amount or the peak intensity of CE is set as plot 805. The plot of the amount of allele β in the amplification product relative to the input DNA amount or the peak intensity of CE is set as plot 806. Plots 805 and 806 are not limited to being straight lines. Amplicon α can be analyzed within the range of DNA input amount a to b. In addition, amplicon β can be analyzed within the range of DNA input amount c to d. Assuming that the PCR amplification is linear in the concentration range of a to b, the following formula holds:

[0373] [Formula 6]

[0374]

[0375] The effective STR-CE analysis range 803 for accurately detecting both amplicon α and amplicon β can be expressed by the following formula 3:

[0376] [Formula 7]

[0377]

[0378] b / a (range 807) may correspond to the dynamic range of CE, or may be the analytical range of STR-CE capable of detecting peak α.

[0379] As shown in Formula 3, the larger the intensity ratio of the DNA to be analyzed, the smaller the effective analysis range.

[0380] Figure 26 The table shows the correspondence between the values ​​of range 807 and the intensity ratio α / β of the largest amplicon to the smallest amplicon that can be analyzed by split PCR relative to the set value of nm. For example, when range 807 is 120 and nm is set to 5, the allele with an abundance ratio of 1 / 3.75 relative to the allele with the highest abundance ratio is the target of analysis. Conversely, when the intensity ratio of the allele set to be analyzed is 1 / 3.75, the allele with the highest abundance ratio is 1 / 3.75. Figure 26 If the ratio shown is large, it means that the concentration of the largest peak or smallest peak that conflicts with the upper or lower limit of analysis appears within the analysis range. For example, if Figure 27 If the concentrations of both α and β can be detected as shown in (1), there is no problem, but the following may occur: Figure 27 As shown in (2), only α can be detected but β cannot be detected.

[0381] Targets with excessively large peak intensity ratios may not be analyzed. An example is shown below.

[0382] STR-CE contains ghost peaks. Peaks with intensity differences of more than 1:20 within a locus are difficult to distinguish from ghost peaks. Therefore, peaks with intensity ratios greater than 1:20 or 1:40 within a locus may not be analyzed.

[0383] Degraded DNA may also reduce the overall DNA amount. Therefore, peaks with intensities that differ by 1:20, 1:40, or 1:100 or more may not be analyzed.

[0384] As described above, in an electropherogram where the inter-peak intensity ratio becomes extremely large, small peaks are meaningless. Therefore, a threshold value of Inter-locus PHR can be set to define the minimum peak intensity relative to the maximum peak intensity to be analyzed. Figure 27 When setting the interval between m and n, it is preferable that the abundance ratio of the allele to be analyzed be set slightly smaller than the threshold value of the Inter-locus PHR.

[0385] [How to set n]

[0386] When a partial profile is also analyzed, 30 to 34 cycles are preferred.

[0387] By setting the cycle number to 36, a peak derived from one copy of DNA can be detected.

[0388] When the number of cycles exceeds 36, even if the artifacts (particularly, the frequency of occurrence of shadows or drop-ins increases significantly) increase, the probability of detecting the peak from an individual does not increase. Therefore, setting the number of cycles to 36 or more is not appropriate.

[0389] When the partial profile is not the subject of analysis, it is preferably 30 or 29 cycles or less.

[0390] For samples containing the minimum amount of DNA, it is preferable to select the minimum cycle number that minimizes the number of false positives, which can also be investigated experimentally.

[0391] n cycles can be set to the minimum number of PCR cycles at which the peak from one copy of the amplicon must be greater than AT.

[0392] n cycles can be set as the minimum number of PCR cycles that ensures that the amplified product from 20 copies of genomic DNA has a full profile.

[0393] The n cycle can be set as the maximum number of PCR cycles at which the intensity ratio of ghost peaks and other peaks resulting from amplification errors (except IAP) does not exceed a threshold value.

[0394] Alternatively, n can be set based on the assumption that the lower limit of detection at a specific number of cycles has been determined experimentally or by calculation, and then the lower limit of detection is doubled or decreased with each cycle. However, if the amount of DNA is too small, the stochastic effect can lead to greater peak intensity variation, so setting a cycle number with a slight margin is more preferable.

[0395] n can also be set by the above method independently of m. However, if the interval between m and n is too large, an amount of DNA that cannot be analyzed may remain between them.

[0396] When m is preset, the Figure 26 The difference in nm and the numerical value of m that can cover the intensity ratio of the peak to be analyzed are set as shown.

[0397] When the analysis range 803 is known, it is preferable to set n so as to have a cycle number difference of log2(analysis range 803) or less with respect to m.

[0398] The number of cycles may be set with a margin in consideration of fluctuations in various analysis systems or peak intensity ratios.

[0399] [How to set m]

[0400] In DNA identification, the amount of DNA put into PCR rarely exceeds 1.5 μg, so the number of loci that can be accurately detected does not increase even if the cycle number is set to less than 20. Therefore, it is preferable to set the cycle number to 20 or more.

[0401] It is preferred to evaluate the amount of DNA input to PCR based on a sample containing the maximum amount of DNA that can be introduced into the analysis system, and set the maximum PCR cycle at which no OS or IPA markers appear when the DNA is analyzed by STR-CE.

[0402] When the maximum amount of DNA to be fed to STR-CE is determined, it is more preferable to set the amount of DNA to be fed to meet the requirement. Figure 2 The maximum number of cycles is 201 to 207.

[0403] The number of cycles can be set with a margin in consideration of fluctuations in various analysis systems or peak intensity ratios.

[0404] Alternatively, m may be set under the assumption that the lower limit of detection, the amount of DNA input for PCR, and the upper limit of detection are investigated at a specific number of cycles, and the lower and upper limits are doubled or reduced with each cycle.

[0405] By the above method, m can also be set independently of n. However, if the interval between m and n is too large, there is a possibility that an amount of DNA that cannot be analyzed will remain in the interval.

[0406] When n is preset, the Figure 26 The difference in nm and the numerical value of m that can cover the intensity ratio of the peak to be analyzed are set as shown.

[0407] When the analysis range 803 is known, it is preferable to set m so as to have a cycle number difference of less than log2(analysis range 803).

[0408] Even if the interval between m and n is 2 or 3, the expansion rate of the analysis range remains at 4 or 8 times. To achieve highly robust DNA identification, it is preferable to expand the analysis range by at least one order of magnitude. Therefore, the interval between m and n is preferably set to 4 or more. As mentioned above, in a suitable split PCR, the maximum setting value of n is 36, and the minimum setting value of m is 20. Therefore, the minimum setting value of n is 24, and the maximum setting value of m is 32.

[0409] [Set m and n according to the table]

[0410] Figure 28 (1) shows the expansion rate of the analysis range when m and n are set. Based on this table, the interval between m and n can be determined to obtain the desired expansion rate. However, this table does not take into account the changes in the analysis range that depend on the DNA concentration range, such as the stochastic effect. In addition, if the original analysis range is larger than the expansion rate, Figure 24 (2) is not suitable because it produces a range of DNA amounts that cannot be analyzed. In addition, when DNA with a large difference in the presence ratio between alleles is used as the analysis object, if the expansion rate is set to a limit relative to the original analysis range, alleles that cannot be analyzed will appear.

[0411] Figure 28 (2) represents the analysis range (order of magnitude) after expansion by split PCR. In a typical STR-CE, 0.75ng to 48ng (1.8 orders of magnitude) of Genomic DNA can be analyzed at PCR 25 cycles. However, this range varies depending on the DNA of the individual, and also varies depending on the quality of the DNA and the CE measurement system. Therefore, it is necessary to evaluate this range for each measurement system. In addition, deviations need to be considered. Here, the order of magnitude of the analysis range when m and n are changed based on this result, as well as the analysis lower limit of n cycles and the analysis upper limit of m cycles are shown. Stochastic effect when the amount of DNA is reduced is not considered. Set the total number of cycles to 2 n A table is created based on the changes. It is also possible to create a file like this table and determine m and n based on the appropriate lower and upper limits and analysis range. However, in this case, when the expansion rate of the analysis range exceeds 1.8 orders of magnitude, as Figure 24 As shown in (2), a DNA amount range exceeding the analysis range may occur within the interval set as the analysis range.

[0412] The dynamic range of typical CE is less than 2000. Even for ideal DNA identification, the peak intensity ratio is more than 2 times, so the difference between n and m is preferably 9 or less.

[0413] The dynamic range of higher performance CE is less than 4000. In many DNA identifications, the peak intensity ratio is 4 times or more, so the difference between n and m is preferably 9 or less.

[0414] As described above, in one example of a suitable range, m is 20 or more and 32 or less. In another example of a suitable range, n is 24 or more and 36 or less. In another example of a suitable range, n is 4 to 9 more than m.

[0415] [The moment of division]

[0416] like Figure 14 As shown, the final extension step can be performed after m thermal cycles, and then the final extension can be performed again after n thermal cycles. In this case, for example, the analysis system 101 maintains the PCR reaction solution m at a constant temperature within the range of 50°C to 80°C for 1 to 20 minutes, then removes a portion. With this configuration, only one heater is required around the PCR, simplifying the device and flow path configuration.

[0417] Alternatively, segmentation may be performed after m thermal cycles are completed, followed by a Final extension step.

[0418] Figure 29 An excerpt showing how to use the analysis system 101 is provided. Figure 14 The description of the same parts is sometimes omitted. Figure 29 Steps 601 to 612 shown in the figure may correspond to an example of detailed steps from step 404 (amplification of the sample) to step 405. Figure 29 The steps shown in steps 601 to 612 may be a process independent of the steps 404 to 405 .

[0419] Figure 29 The actions shown can be performed by Figure 6 The flow channel device 104 is implemented. Figure 6 In the embodiment, the heating unit 318 is provided to be connected to the dispensing chamber 320. After the m cycles are completed in step 603, the dispensing chamber 320 is divided in step 605, and the heater final extension step is performed in step 604. The PCR reaction liquid remaining in the PCR chamber can be subjected to nm thermal cycles in step 608 in parallel with step 604, or it can be performed with a time difference. After n thermal cycles, the final extension step can be performed on the product n in the holding chamber. That is, in step 608, the PCR reaction liquid remaining in the PCR chamber can be subjected to nm thermal cycles in parallel with step 604, or it can be performed with a time difference. Figure 29 In the example, for example, the analysis system 101 maintains a portion of the PCR reaction solution m at a constant temperature within a range of 50° C. to 80° C. for 1 to 20 minutes.

[0420] Advantages of performing the process after splitting include: product n does not need to undergo two final extension steps, thus reducing artifacts; and the overall time is not extended.

[0421] [Method for providing CE analysis results to users]

[0422] When CE is performed two or more times using split PCR, two or more electropherograms are generated. Both electropherograms or DNA identification results can be provided to the user. For each of the two or more electropherograms, a score can be assigned to determine which is more suitable for DNA identification and provided to the user along with the CE analysis results.

[0423] For example, the analysis system 101 may determine which of the results of the electrophoresis analysis of a portion of the PCR reaction solution m and the results of the electrophoresis analysis of at least a portion of the PCR reaction solution n is a better result, and output the better result. Alternatively, information that can be used to determine which result is better may be output. Figure 2 The part of the flowchart shown in the figure is used to inform whether it is a Full profile. Figure 2 The number of the loci of the criterion that appears in the determination criteria can also be used, the number of the loci that do not meet the determination criteria can also be used, the number of the marks that are judged to not meet the determination criteria can also be used, or the algorithm based on the determination criteria can be used to calculate comprehensively. The data provided to the user can also be the data of the midway stage of DNA identification analysis. It is also possible to only provide the user with the analysis result of the Full profile or be judged to be the analysis result of a better electrophoretogram. Like this, the comparison of the result can be carried out efficiently. In addition, when non-professionals receive data from the system, they can easily select suitable results.

[0424] In the event that the analysis results are all poor, an expert review may also be requested.

[0425] Alternatively, two data sets can be synthesized for DNA identification. In particular, in the case of DNA identification with relatively large peak intensities, it can be inferred that at m, the allele with the minimum intensity is less than AT, while at n, the allele with the maximum intensity is oversaturated. In this case, meaningful peaks or DNA identification results can be extracted from m and n, respectively, and synthesized and provided. This embodiment can be utilized to expand the dynamic range of CE.

[0426] The number of times CE measurement is performed by the analysis system may be two, one, or three or more times.

[0427] Depending on the sample's condition, analysis may be performed only once, twice, or more times. Compared to preparing only one PCR product and performing CE analysis once, accurate DNA identification results can be obtained without requiring multiple measurements, thus reducing costs, shortening analysis time, and increasing throughput.

[0428] In the case of only one analysis, either amplification product m or amplification product n can be measured.

[0429] In the case of two measurements, the product of the m cycle can be measured twice, the product of the n cycle can be measured twice, the product of the n cycle can be measured after the m cycle, the analysis of the m cycle can be performed after the n cycle, the analysis of the n cycle can be started regardless of the status of the data of the m cycle, the analysis of the m cycle can be started regardless of the status of the data of the n cycle, or the analysis of m and n can be performed completely and simultaneously.

[0430] If the first run indicates poor run, the amplification products of the same number of cycles may be analyzed again. Alternatively, if the first run indicates poor run, the products of a different number of cycles may be analyzed. Poor run, as used herein, refers to a situation where part or all of the molecular weight standard peak is not detected, or where there is a blockage somewhere in the solution transport.

[0431] After obtaining the analysis result of either amplification product m or n, the obtained DNA identification result may be matched with the database, and based on the obtained feedback, the analysis of another amplification product may be continued or started.

[0432] Alternatively, after obtaining the analysis result for either amplification product m or n, the user can decide whether to analyze the other amplification product. The user can make a decision after viewing the first data or analysis score, or can decide at any time whether to perform a second measurement regardless of the data. Alternatively, product m or n can be retained inside or outside the device, and once the measurement is completed, it can be removed from the cassette or analysis system 101 and measured outside the device. Alternatively, it can be stored in the cassette for a certain period of time and then re-measured in the device. During this period, the amplification product is preferably stored refrigerated or frozen. The amplification product can be mixed with a migration reagent and stored as a migration sample, or it can be stored in a state before mixing with the migration reagent.

[0433] <Analysis of amplification product m first>

[0434] In the case of analyzing the amplified product of m first, Figure 30 The flowchart shown in the figure can be used to decide whether to analyze amplification product n. However, Figure 30 The flowchart shown is an example and may include criteria and branch conditions not shown here. The criteria may vary depending on, for example, the number of peaks obtained, the number of loci, whether the data is mixed, etc. Figure 30 The judgment criteria shown may be omitted in part or in whole, or may be replaced by other criteria.

[0435] The electrophoresis results of m can also be provided to the user to determine whether to analyze the product of n and then start.

[0436] In addition, when analyzing n simultaneously or in parallel, it is possible to analyze n based on the user or Figure 30 The judgment result of the flowchart is interrupted midway.

[0437] Decision Set 1: Is there a peak that saturates the CE detection system? If so, do not perform or interrupt the analysis of amplification product n.

[0438] Judgment set 2: Whether a full profile is obtained. If a full profile is obtained, the analysis of amplification product n is not performed or is interrupted.

[0439] Decision set 3: Whether the IAP+ flag appears. If it does, continue or start the analysis of amplification product n.

[0440] Judgment Set 4: Are all peaks less than 1 / 2 the intensity of OS? Alternatively, are all peaks less than the intensity obtained by dividing OS by the expected amplification rate from fractional PCR? Alternatively, are they less than the intensity that would not cause saturation after additional nm cycles of PCR? Alternatively, are any peak intensities not reached? If they are reached, analysis of amplification product n is not performed or is interrupted.

[0441] If the interval between m and n is 2, and if almost no peaks are detected in the CE analysis of the migrating product m, no meaningful data can be obtained even for migrating product n. Therefore, it is meaningful to analyze n after obtaining the analysis results for m. Similarly, if the interval between m and n is narrow, if the analysis results for m indicate that the DNA amount is too low, analysis for n can be omitted. The determination of whether the DNA amount is too low can be based on either the number of detected peaks or the peak intensity.

[0442] Various decision sets can use different decision criteria sets depending on whether each locus is heterozygous, homozygous, mixed, or single.

[0443] If the IAP peak exceeds the threshold, analysis can be performed for n cycles. IAP peaks occur when the amount of input DNA is insufficient for the number of PCR cycles, so increasing the number of cycles may be considered to alleviate the problem.

[0444] Furthermore, various thresholds or determination algorithms may be provided to allow for interruption or non-implementation of analysis n when it is determined from the CE analysis results of m that no meaningful CE analysis results can be obtained even if analysis n is performed.

[0445] <Analysis of amplification product n first>

[0446] In the case of analyzing the amplification product of n first, the following Figure 31 The flowchart shown determines whether to analyze the amplification product m. However, Figure 31 The flowchart shown is an example and may include criteria and branch conditions not shown here. It is conceivable that the criteria may vary depending on, for example, the number of peaks obtained, the number of loci, whether the data is mixed, etc. Figure 31 The judgment criteria shown may be omitted in part or in whole, or replaced by other criteria.

[0447] The electrophoresis results of n can also be provided to the user to determine whether to analyze the product of m and then start.

[0448] In addition, when analyzing m simultaneously or in parallel, it is possible to analyze m based on the user or Figure 30 The judgment result of the flowchart is interrupted midway.

[0449] Decision Set 1: Is there a peak that saturates the CE detection system? If so, continue or start the analysis of amplification product m.

[0450] Decision set 2: Whether the IAP+ marker appears. If it does, the analysis of the amplification product m is not performed or is interrupted.

[0451] Furthermore, various thresholds and determination algorithms may be provided so that when it is determined from the CE analysis results of n that a meaningful CE analysis result cannot be clearly obtained even if analysis of m is performed, it may be determined that analysis of m is interrupted or not performed.

[0452] In this manner, analysis system 101 performs electrophoretic analysis on either a portion of PCR reaction solution m or at least a portion of PCR reaction solution n in CE unit 105. Based on the results of the electrophoretic analysis of one, the execution of the electrophoretic analysis of the other is controlled. For example, the results of the electrophoretic analysis of one can be used to determine whether to initiate the electrophoretic analysis of the other. Alternatively, after initiating the electrophoretic analysis of the other, the results of the electrophoretic analysis of one can be used to determine whether to continue the electrophoretic analysis of the other. This eliminates unnecessary or inefficient electrophoretic analysis, improving overall processing efficiency.

[0453] [Both n and m are assumed]

[0454] Two different cycle numbers, n and m, may also be preset in the analysis system 101. As described above, m and n are preferably appropriately set according to the CE analysis range or the amplifiable amount of DNA.

[0455] As shown in Table 3, different sets of n and m and analysis schemes can be set according to the type of sample to be measured.

[0456] [Table 3]

[0457] Table 3 Example of preset mode

[0458] m n Measure first Oral swab mode 26 - - DVI Mode 25 31 Start from m Casework 26 31 Start with n Touch sample 28 32 Start with n

[0459] For example, in the case of oral swabs that contain a large amount of DNA stably to a certain extent, the "oral swab mode" can be selected, and STR-CE analysis can be performed with 26 cycles without segmentation (or even if segmented, only CE analysis of one side is performed, and analysis of the amplified product of the other side is performed only in the event of failure). In addition, a "DVI sample mode" can be provided for the case of DVI samples (disaster victim identification, a DNA identification method for identifying remains resulting from disasters or large-scale terrorist attacks). Since DVI samples are likely to contain more DNA, they are set to, for example, [m=25, n=31]. In most cases, DVI samples contain more DNA, so the analysis can be started with m cycles first, and then the analysis can be completed with 10 cycles. Figure 30 The judgment flow shown above continues or starts the analysis of amplification product n. In addition, when analyzing DNA obtained at a crime scene, the probability of a small amount of DNA is high, so the "Casework sample mode" with the settings [m=26, n=31] can be selected. In the case of a casework sample, the probability of containing an extremely small amount of DNA is high, so the analysis of n can be started first, and then the analysis of the product n can be started. Figure 31 The determination flow shown above determines whether to continue or proceed with analysis of sample m. Furthermore, in the case of touch samples, there is a high probability that the sample contains minimal amounts of DNA, or that it is degraded, causing large variations in peak intensity, or that it is a mixed DNA sample. Therefore, in "Touch Sample Mode," it is preferable to set the interval between m and n narrower than in other modes. Furthermore, since the probability of containing low amounts of DNA is high, it is preferable to start analysis with sample n.

[0460] When two or more CEs are present per sample, both m and n can be analyzed simultaneously. Furthermore, the preset m, n, and measurement order described above can be modified by the user as appropriate. The m and n values ​​shown in Table 3 are examples. The preset m and n values ​​can be set during validation testing during development to maximize the probability of successful DNA identification.

[0461] [Key Points of This Embodiment]

[0462] In the above embodiment, description has been given of the case where both m and n are analyzed at a time and the case where only one of m and n is analyzed.

[0463] Even if one of the amplification products prepared by two cycles fails to provide data of the required quality, the possibility of obtaining data of the required quality from the other amplification product is higher than that of the case of preparation by one cycle.

[0464] Even if either of the amplification products prepared through two cycles fails to provide data of the required quality, the other amplification product is still available, thus reducing the probability of sample waste. Furthermore, this reduced probability of sample waste eliminates the need for recollection and sample pretreatment. Consequently, using this embodiment to prepare two samples and perform two analyses shortens the average data acquisition time compared to a method not using this embodiment.

[0465] Even if the m-cycle PCR product is split and only the m-cycle PCR product is subjected to CE analysis without further PCR cycles, if the CE analysis results of the m-cycle PCR product do not meet the required quality, the remaining PCR product can be subjected to nm PCR to prepare n-cycle PCR products. However, if the PCR product is left for the waiting time for m-cycle CE analysis of the PCR sample, the polymerase activity decreases or the correction factor increases significantly depending on the storage temperature. Therefore, the CE analysis results of the n-cycle PCR product may not meet the required quality.

[0466] When only n-cycle PCR products are prepared and CE analysis is performed on the n-cycle PCR products, if the CE analysis results of the n-cycle PCR products do not meet the required quality, the samples are wasted if m-cycle PCR products are not prepared.

[0467] When the DNA solution used for PCR is split before PCR, the amount of DNA added to each reaction decreases, thereby reducing sensitivity. On the other hand, if the PCR reaction proceeds to a certain point (e.g., four or more cycles), each allele contains more than 10 amplicons, so splitting does not affect sensitivity.

[0468] It is preferable to perform splitting at the end of the cycle. If splitting is performed before the end of m cycles and the remaining PCR reactions are performed in each chamber after the splitting, a PCR temperature control device must be installed in each chamber, increasing the size of the apparatus. Furthermore, artifacts may be generated during splitting, so it is preferable to perform splitting after the number of cycles is as large as possible.

[0469] Preparing multiple elution samples at different dilution ratios after PCR can expand the effective CE analysis range. This can reduce the occurrence of CE oversaturation or the frequency of peak intensities lower than those observed in AT. However, peak intensity imbalance or peak splitting caused by excessive amplification product cannot be resolved by post-PCR dilution and must be addressed before or during PCR. Specifically, split PCR is suitable.

[0470] Since the number of cycles for splitting is appropriately set for the analysis sample, the maximum analysis range expansion rate is achieved with the minimum number of splits. In other words, the measurement time is minimized when the analysis range is expanded.

[0471] By controlling the amount of DNA before it is added to the PCR, the effective analysis range can also be expanded. For example, the upper limit of the amount of DNA adsorbed on the refining membrane can be reduced by adjusting the capacity or volume of the refining membrane, changing the refining scheme, etc. However, in a typical refining membrane, when the volume is reduced in order to lower the adsorption upper limit, the solution flow rate will deteriorate, or the DNA yield (especially short, degraded DNA) will decrease. Therefore, there are limitations on the control of the amount of DNA in the refining stage. In addition, the effective analysis range can be expanded by quantifying the amount of DNA before PCR, controlling the number of PCR cycles, or changing the dilution rate. However, in addition to requiring an additional detection system, the quantitative step will inevitably lead to complex flow channel equipment. It is also possible that quantitative errors will lead to analysis failure, so split PCR is appropriate.

[0472] This embodiment can be combined with post-PCR dilution and purified DNA quantity control to enable DNA analysis to be performed more reliably or over a wide range of DNA quantities.

[0473] The PCR reaction solution is taken out for CE determination in each cycle or every other cycle, so that appropriate DNA analysis can be performed on all DNA concentration ranges. However, in the flow channel device, it is difficult to set up a mechanism that can take out the solution three or more times, or four or five times, and then mix it with the swimming reagent. In addition, even if two, three or four amplification products are divided and measured in each cycle or every other cycle, the expansion rate of the analysis range remains at 2 times, 4 times, or 8 times, which is not necessarily suitable for the analysis of samples containing various DNA amounts. Moreover, in the case where Final extension is implemented in the same chamber as the PCR chamber in STR-PCR, when the interval between m and n is narrow (for example, 1 cycle or 2 cycles), depending on the amount of DNA input to PCR, there is a possibility that the Final extension time is excessive, resulting in the appearance of a large number of A++ peaks. In particular, in an analysis system where a heating unit 318 is not provided, in a case such as Figure 14 In the case of the split PCR device that performs Final extension in the same chamber as shown, when the interval between n and m is narrow, a large number of A++ peaks appear, so two or more are preferably used.

[0474] [Flow method according to the number of capillaries]

[0475] i) When only one capillary can be used for each sample

[0476] Figure 32 This shows a typical example of the operation timing of the analysis system 101. In this operation timing, first, the first sample A is added in step 401, the sample is processed by the pre-processing cartridge in steps 402 to 404, and the eluted sample m or n is analyzed by CE in step 405.

[0477] like Figure 32 The operation flow is suitable for the case where there is one CE unit for one sample pretreatment unit. Such a structure makes the device small and easy to carry.

[0478] Alternatively, data may be provided to the user at the time the first CE analysis is completed in step 405. The user can then determine whether to continue or start the second analysis of the eluted sample. Alternatively, the determination method described above may be used to determine whether to continue or start the second analysis. This is because performing two measurements each time doubles the CE measurement time, thus preventing an increase in throughput.

[0479] The pretreatment of sample B (steps 401 to 404 ) can be started after the pretreatment of sample A is completed. In this case, the cartridge containing sample A is removed, so the eluted sample that has not yet been subjected to CE analysis can be held in the standby unit 321 , and the CE sample can be in the elution process.

[0480] ii) Two capillaries can be used for each sample

[0481] Figure 33 This section shows a typical example of the operating timing of the analysis system.

[0482] Two CE units can be prepared for one sample analysis unit. In this case, the number of CE units is preferably twice that of the sample processing units. Alternatively, the number of CE units may be the same as or greater than the number of sample analysis units.

[0483] You can Figure 33 As shown in (1), after the electrophoresis sample m and the electrophoresis sample n are prepared, CE measurement is performed immediately in step 405, or as shown in Figure 33 After both are prepared as shown in (2), analysis is started simultaneously in step 405. In addition, when the number of CE units is the same as the number of sample processing units and there are no vacancies, measurement can be performed sequentially starting from the electrophoretic sample m or the electrophoretic sample n.

[0484] [Example 1]

[0485] The analysis system 101 includes a flow channel device 104 and a CE unit 105 .

[0486] A forensic sample containing an unknown amount of DNA is introduced into the lysis chamber 301 (sample inlet) of the flow channel device 104. The processes of steps 401 to 403 are automatically performed in the flow channel device 104.

[0487] After performing m cycles of PCR in step 502, the amplified products of the m cycles are removed in step 503. The sample is then transferred to the CE unit 105, and CE measurement begins in step 504. In parallel with step 320, n cycles of PCR are performed on the PCR reaction solution remaining in the PCR chamber 304 in step 505. After the CE measurement in step 504 is completed and the next CE measurement is ready to begin, the products of the n cycles are transferred to the CE unit 105, and CE measurement is performed in step 507.

[0488] [Example 2]

[0489] The analysis system 101 includes one flow channel device 104 and two CE units 105 .

[0490] A forensic sample containing an unknown amount of DNA is introduced into the lysis chamber 301 (sample inlet) of the flow channel device 104 . The processes of steps 401 to 403 are automatically performed in the flow channel device 104 .

[0491] After performing m cycles of PCR in step 502, amplification product m is removed from PCR chamber 304 in step 503. The sample is transferred to one side of CE unit 105. In parallel with step 503, n cycles of PCR are performed on the PCR reaction solution remaining in the PCR chamber in step 505. In step 506, the product of cycle n is transferred to CE unit 105. With the electrophoretic samples m and n stored in two capillaries, respectively, steps 504 and 507 are simultaneously initiated.

[0492] Explanation of symbols

[0493] 101—Analysis system, 102—Computer, 103—Database, 104—Flow channel device, 105—CE unit, 106—User interface, 201-207—Judgment criteria, 301—Dissolution chamber, 302—Refined membrane, 303—Refined membrane chamber, 304—PCR chamber, 305—Waste liquid chamber, 306—External connection port, 307—Pump and valve, 308—PCR reagent storage unit, 309—PCR reagent, 310—Mobilization reagent storage unit, 311—Mobilization reagent, 312-314—Reagent storage unit, 315—Flow channel, 316—Flow channel, 317—Heating unit, 318—Heating unit, 319—Flow channel, 320—Dispensing chamber, 321—Standby unit, 322—Flow channel, 323-326—Valve, 327—Mixer Chamber, 328-330—flow channel, 331—mobilization reagent storage portion, 332-334—air storage portion, 335—PCR reaction solution, 336—mobilization sample, 337-339—valve, 401—sample input step, 402—dissolution step, 403—purification step, 404—amplification step, 405—detection step, 701—liquid level, 702—ventilation filter, 703—flow channel, 704—flow channel resistance body, 705—liquid level detection sensor, 801—CE detection upper limit or PCR amplification product concentration upper limit, 802—CE detection lower limit, 803—STR-CE analysis range, 804—STR-CE analysis range using split PCR, 805, 806—plotting, 807—CE dynamic range.

Claims

1. A DNA analysis system comprising: a flow channel device having a PCR chamber for performing thermal cycling; and Capillary electrophoresis unit, which performs electrophoresis analysis on the PCR reaction solution, The DNA analysis system is characterized by: The DNA analysis system stores the pre-set values ​​of m and n. In the PCR chamber, the PCR reaction solution is subjected to m thermal cycles to generate a first reaction solution. removing a portion of the first reaction solution from the PCR chamber without changing its composition, performing electrophoresis analysis on the portion of the first reaction solution in the capillary electrophoresis unit, In the PCR chamber, the first reaction solution remaining in the PCR chamber is subjected to nm thermal cycles so that the total number of thermal cycles becomes n, thereby generating a second reaction solution, wherein: nm is an integer greater than or equal to 2, removing at least a portion of the second reaction solution from the PCR chamber without changing its composition, The at least a portion of the second reaction solution is subjected to electrophoresis analysis in the capillary electrophoresis section.

2. The DNA analysis system according to claim 1, characterized in that m is 20 or more and 32 or less.

3. The DNA analysis system according to claim 1, wherein n is 24 or more and 36 or less.

4. The DNA analysis system according to claim 1, wherein n is 4 to 9 more than m.

5. The DNA analysis system according to claim 1, characterized in that The DNA analysis system does not perform analysis by a method other than electrophoresis analysis on the portion of the first reaction solution and the at least portion of the second reaction solution before the electrophoresis analysis.

6. The DNA analysis system according to claim 1, wherein: The DNA analysis system mixes at least one of the portion of the first reaction solution and the at least portion of the second reaction solution with a solution containing a plurality of DNA fragments.

7. The DNA analysis system according to claim 1, characterized in that The DNA analysis system mixes at least one of the portion of the first reaction solution and the at least one portion of the second reaction solution with pure water, formamide, or a solution having a conductivity of 10 mS / cm or less to generate a mixed solution before electrophoresis analysis.

8. The DNA analysis system according to claim 7, characterized in that The DNA analysis system heats the mixed solution to above 90°C.

9. The DNA analysis system according to claim 1, wherein: The flow channel device fully automatically implements the process from adjusting the PCR reaction solution to the nm thermal cycle.

10. The DNA analysis system according to claim 1, wherein The flow channel device has a measuring part, The DNA analysis system transfers the first reaction solution to the measurement unit after the m thermal cycles are completed, thereby measuring a predetermined amount of the first reaction solution within a range of 0.1% to 50%.

11. The DNA analysis system according to claim 1, wherein The flow channel device has a valve that can be opened and closed, The valve is closed before the m thermal cycles are started, and the valve is opened after the m thermal cycles are completed, thereby dividing and taking out the portion of the first reaction solution.

12. The DNA analysis system according to claim 1, wherein: The DNA analysis system maintains the portion of the first reaction solution at a constant temperature within a range of 50° C. to 80° C. for 1 to 20 minutes.

13. The DNA analysis system according to claim 1, wherein The DNA analysis system maintains the first reaction solution at a constant temperature within a range of 50° C. to 80° C. for 1 to 20 minutes, and then takes out the portion.

14. The DNA analysis system according to claim 1, wherein The DNA analysis system transports all the PCR reaction liquid in the PCR chamber to the outside of the PCR chamber at the end of n thermal cycles.

15. The DNA analysis system according to claim 1, wherein The PCR reaction solution contains two or more primer sets, The PCR reaction solution contains DNA including two or more amplified gene regions.

16. The DNA analysis system according to claim 1, wherein The DNA analysis system determines which of the results of the electrophoresis analysis on the portion of the first reaction solution and the results of the electrophoresis analysis on the at least portion of the second reaction solution is a better result, The better result is output, or information that can determine which one is the better result is output.

17. A DNA analysis system comprising: a flow channel device having a PCR chamber for performing thermal cycling; and Capillary electrophoresis unit, which performs electrophoresis analysis on the PCR reaction solution, The DNA analysis system is characterized by: The DNA analysis system stores the pre-set values ​​of m and n. In the PCR chamber, the PCR reaction solution is subjected to m thermal cycles to generate a first reaction solution. removing a portion of the first reaction solution from the PCR chamber without changing its composition, In the PCR chamber, the first reaction solution remaining in the PCR chamber is subjected to nm thermal cycles so that the total number of thermal cycles becomes n to generate a second reaction solution, wherein: nm is an integer greater than or equal to 2, removing at least a portion of the second reaction solution from the PCR chamber without changing its composition, performing electrophoresis analysis on one of the portion of the first reaction solution and the at least one portion of the second reaction solution in the capillary electrophoresis unit; Based on a result of the one electrophoretic analysis, execution of the electrophoretic analysis is controlled for the other of the portion of the first reaction solution and the at least a portion of the second reaction solution.

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