DNA analysis system

Abstract

This DNA analysis system comprises: a flow channel device that has a PCR chamber that performs thermal cycles; and a capillary electrophoresis unit that electrophoretically analyzes a PCR reaction liquid. The DNA analysis system stores values of m and n set in advance, the PCR reaction liquid is subjected to m thermal cycles in the PCR chamber to produce a first reaction liquid, part of the first reaction liquid is taken out from the PCR chamber without changing the composition, the part of the first reaction liquid is electrophoretically analyzed in the capillary electrophoresis unit, the first reaction liquid remaining in the PCR chamber is subjected to n-m thermal cycles (provided that n-m is an integer of 2 or more) in the PCR chamber such that the total number of thermal cycles is n to produce a second reaction liquid, at least part of the second reaction liquid is taken out from the PCR chamber without changing the composition, and at least the part of the second reaction liquid is electrophoretically analyzed in the capillary electrophoresis unit.

Description

DNA analysis system

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

[0002] Fragment analysis involves PCR of DNA contained in a sample using primers designed for specific DNA targets, followed by size separation of fluorescently labeled DNA amplification products using capillary electrophoresis (CE). It is used for gene mutation analysis and quantification, cell line authentication, genome editing efficiency assessment, genotyping for amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs), and single nucleotide polymorphisms (SNPs), and for macrosatellite marker analysis. Macrosatellites are repetitive DNA sequences in which specific DNA motifs are repeated multiple times. They are characterized by a higher mutation frequency and genetic diversity than other DNA regions. A typical example of macrosatellite marker analysis is individual identification and identity testing using short tandem repeats (STRs). STR-based DNA analysis is widely used in forensic testing, including paternity testing and matching crime scene DNA to criminals.

[0003] In fragment analysis, CE analysis is sometimes performed by mixing multiple fluorescently labeled DNA fragments of known length (size standards) with the target fluorescently labeled DNA fragment. Using size standards allows the length of each amplified product to be determined. Furthermore, by setting a fixed amount of size standard to be mixed, the amount of amplified product can be calculated from the ratio of the intensity of the DNA fragment of the amplified product obtained by CE analysis to the intensity of the size standard. Furthermore, by mixing a known amount and length of DNA (internal positive control, IPC) with the primers for amplifying it in the PCR reaction solution and amplifying it, and then performing fragment analysis, the amount of target DNA before amplification can be estimated from the ratio of the intensity of the target amplified product to the IPC.

[0004] According to Non-Patent Document 1, DNA testing performed at forensic laboratories involves the following steps: (1) quantifying the concentration of human DNA contained in a sample by DNA quantification using quantitative PCR; (2) preparing the sample so that the human DNA is at an appropriate concentration and performing STR-PCR (PCR containing STR sequences); (3) mixing a portion of the PCR reaction solution with formamide containing a size standard in a certain ratio and heat-denaturing it; (4) measuring the CE of the heat-denatured electrophoresis sample to obtain an electropherogram; and (5) performing DNA testing from the electropherogram.

[0005] In the DNA identification or fragment analysis using the pretreatment-integrated CE analyzer disclosed in Patent Documents 1, 2, 3, 4, and 8, (1) a sample containing DNA is subjected to PCR in a flow path device, (2) a portion of the reaction solution is mixed with formamide containing a size standard in a fixed ratio in the flow path device and heat-denatured, (3) the heat-denatured electrophoresis sample is subjected to CE analysis to obtain an electropherogram, and (4) DNA identification or fragment analysis is performed from the electropherogram. Compared to Non-Patent Document 1, Patent Documents 1, 2, 3, 4, and 8 automate the series of steps, allowing results to be obtained in a shorter time (e.g., 90 minutes).

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

[0007] In the DNA quantification method disclosed in Patent Document 6, (1) a portion of the amplified product at each stage of a plurality of consecutive thermal cycle numbers n (n0 and m are predetermined integers, m is 1 or 2, and n = n0, n0 + m, n0 + 2 m, ...) in PCR of a sample containing DNA is divided by electrophoresis on a flow path device, (2) each divided amplified product is subjected to CE analysis on the flow path device, and (3) DNA quantification (quantification of the original concentration of DNA contained in the sample) is performed based on the relationship between the peak intensity of the amplified product obtained by each CE analysis and the number of thermal cycles n, specifically, the number of thermal cycles at which the peak intensity exceeds a predetermined threshold. In other words, Patent Document 5 is an analytical method for detecting amplified products in real-time PCR using CE.

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

[0009] US Patent Application Publication No. 2022 / 0016632 US Patent No. 9354199 US Patent Application Publication No. 2019 / 0019290 JP 2017-077180 A US Patent No. 7445893 US Patent No. 5494480 A US Patent No. 8715924 US Patent No. 10767225

[0010] John M. Butler, Fundamentals of Forensic DNA Typing (2009), P.29-107 and P.279-336

[0011] In CE analyzers and systems equipped with a CE analysis unit, the ratio of minimum to maximum peak intensity, at which peak intensity and concentration are approximately proportional, is 100 or less, or 1,000 or less, or 10,000 or less, or 100,000 or less. When performing fragment analysis, taking into account variations in CE analyzer intensity, variations in the amount of amplicon injected into the CE, variations in the amplification efficiency and abundance of each allele, variations between dyes, and variations in sample preparation, the ratio of the minimum to maximum measurable DNA concentration is 10, 30, 100, 300, 1,000, 3,000, or 10,000. Meanwhile, the ratio of the minimum to maximum DNA contained in a sample delivered to the analysis system is 30, 300, 3,000, or 30,000. Fragment analysis can fail if the amount of DNA contained in the sample exceeds the range of sample DNA measurable by the analysis system. In this case, the sample and analysis time spent on the analysis are wasted.

[0012] One of the objectives of the present invention is to perform highly accurate and robust DNA identification / fragment analysis or DNA quantification (quantification of the original concentration of an individual's DNA contained in a sample) using PCR and CE analysis on samples containing DNA whose concentration ratios vary over a range of 100-fold or 1000-fold or more (sample concentration range of 100 or 1000 = 2.0 or 3.0 orders of magnitude or more) using a simple, low-cost device. In addition, as in Patent Document 2, the entire process is automated so that results can be obtained in a short time (for example, within 180 minutes, 120 minutes, or 90 minutes).

[0013] In the case of Non-Patent Document 1, the amount of DNA carried over into PCR is quantified using quantitative PCR, and the DNA is diluted to an appropriate concentration before the PCR reaction is performed so that the amount does not exceed the analytical range. When this method is implemented in a flow channel device, an optical system for performing quantitative PCR is required. In addition, a complex flow channel structure is required to determine the dilution concentration according to the quantitative PCR results.

[0014] In Patent Document 1, the solution is divided before PCR, and then STR-PCR and quantitative PCR are performed. Quantitation is performed using quantitative PCR, and the number of cycles for STR-PCR is determined. Because the solution is divided before PCR begins, sensitivity is reduced. In addition, the flow path device for performing two different PCRs is complex, and additional optical systems are required, which inevitably increases costs.

[0015] In Patent Document 2, the solution is divided before PCR to prepare two DNA solutions with different concentrations, and both are subjected to PCR for DNA identification. Since one of the two DNA solutions falls within the analytical concentration range of the analytical system, the analytical range can be expanded. Because the solution is divided before PCR, sensitivity is reduced. Furthermore, since the flow channel device is equipped with a flow channel mechanism for dividing the solution and adjusting the concentration, the flow channel device inevitably becomes more complex.

[0016] In Patent Document 3, the analytical range of DNA testing is expanded by improving the data analysis method. Information on peaks that fall below the detection limit during CE analysis cannot be obtained. Furthermore, if the detection intensity becomes saturated during CE analysis, the correct peak intensity ratio cannot be obtained. If the interpretation of DNA testing is expanded too much, the data obtained by analysis may not reflect the true individual DNA mixture ratio. There is a risk of incorrect profiling.

[0017] In Patent Document 4, a portion of the reaction mixture is removed after PCR, and the presence and amount of amplified product are detected using an optical system. If appropriate amplification is achieved, fragment analysis is performed. If amplification is determined to be incomplete, an additional PCR reaction is performed on the reaction mixture remaining in the PCR section. This method requires the design of additional optical systems and flow path devices suitable for optical detection, which inevitably increases costs. Furthermore, there is a risk of fading of the STR-PCR fluorescent dye during optical detection, or of incorrect optical detection due to overlapping detection wavelength ranges between the STR-PCR fluorescent dye and the optical system.

[0018] In Patent Documents 5 and 6, CE analysis is required for each of the multiple thermal cycles n (multiple consecutive thermal cycles n), which requires a long time to obtain results. High-precision quantification requires three or more CE analyses. While DNA quantification can be performed by replacing PCR with STR-PCR, multiple extractions of the solution require a special flow path structure. Furthermore, DNA analysis is not possible because there is no process for mixing the reaction solution with formamide containing a size standard at a fixed ratio. Furthermore, incorporating a mixing process with formamide containing a size standard (electrophoresis reagent) into the relevant method for DNA analysis requires a new dispensing mechanism or flow path structure for mixing the PCR product of each cycle with the electrophoresis reagent, which increases the complexity and cost of the device. Furthermore, for DNA analysis requiring high-precision and high-resolution CE analysis, CE analysis of the PCR product of each cycle requires a significant analysis time. Furthermore, in Patent Document 6, DNA is extracted from the PCR chamber by applying a voltage, so the composition of the amplified product in the PCR chamber is likely to differ from the composition of the extracted amplified product. In DNA testing, amplicons of different lengths are measured, but there is concern that length-dependent bias may occur when applying voltage to remove the amplified products from the PCR chamber.

[0019] In Patent Document 7, PCR is replaced with STR-PCR to perform DNA quantification, but since microarray analysis is required for each of the many thermal cycle numbers n (multiple consecutive thermal cycle numbers n), it takes a long time to obtain the results. Furthermore, since CE analysis is not performed, DNA identification is not possible.

[0020] On the other hand, Patent Document 7 mentions that microarray analysis may be performed for a small number of thermal cycles n (multiple non-consecutive thermal cycles n), but this is not practical. In microarray analysis, spot intensity for the same DNA concentration generally varies due to variations in the density and number of probes immobilized on each spot, temporal and spatial variations in hybridization efficiency, etc. While the presence or absence of corresponding DNA can be determined from the intensity of the spots, the accuracy of quantifying the corresponding DNA from the spot intensity is low. In other words, to accurately determine the number of thermal cycles n at which the spot intensity exceeds a predetermined threshold, microarray analysis must be performed for each of a large number of thermal cycles n (multiple consecutive thermal cycles n). A method for competitively hybridizing target DNA or target amplification products with reference DNA on the same spot is known as a means for improving the accuracy of DNA quantification by microarray analysis. However, to implement this method, it is necessary to prepare a reference DNA that hybridizes to the probe on the spot with the same efficiency as the target DNA or target amplification product, and further, to label the target DNA or target amplification product and the reference DNA with different fluorophores and measure their respective emitted fluorescence independently. To achieve an expanded dynamic range by preparing a small number of PCR products with different cycle numbers and performing microarray analysis, there are problems with the cost and labor involved.

[0021] Other issues with Patent Document 7 are: (1) in PCR of a sample containing DNA on a flow path device, when a portion of the reaction solution at each stage of multiple thermal cycle numbers n is divided, a fresh PCR solution in the same amount as the divided reaction solution is mixed with each of the remaining reaction solutions that have not been divided, which changes the concentration of DNA contained in the reaction solution and reduces the accuracy of DNA quantification.

[0022] Another problem with Patent Document 7 is that the DNA must be hybridized and dehybridized (washed) repeatedly with the immobilized probe, and each time this occurs, the immobilized probe may peel off or the hybridized DNA may be carried over without being washed away, resulting in low repeatability of microarray analysis and low accuracy of DNA quantification.

[0023] To summarize the challenges of DNA identification / fragment analysis or DNA quantification, there is a need for a DNA analysis method that can be implemented using a simple flow path device, is low cost, highly robust, simple, maintains sensitivity, has a short measurement time, and can expand the range of DNA quantities that can be analyzed.

[0024] An example of a DNA analysis system according to the present invention is a DNA analysis system having: a flow path device having a PCR chamber that performs thermal cycling; and a capillary electrophoresis unit that performs electrophoretic analysis of a PCR reaction solution; wherein the DNA analysis system stores preset values ​​of m and n; in the PCR chamber, performs m thermal cycles on the PCR reaction solution to produce a first reaction solution; removes a portion of the first reaction solution from the PCR chamber without changing its composition; and electrophoretically analyzes the portion of the first reaction solution in the capillary electrophoresis unit; in the PCR chamber, performs n-m thermal cycles (where n-m is an integer of 2 or greater) on the first reaction solution remaining in the PCR chamber so that the total number of thermal cycles is n to produce a second reaction solution; removes at least a portion of the second reaction solution from the PCR chamber without changing its composition; and electrophoretically analyzes the at least a portion of the second reaction solution in the capillary electrophoresis unit.

[0025] an example of a DNA analysis system according to the present invention is a DNA analysis system having: a flow path device having a PCR chamber that performs thermal cycling; and a capillary electrophoresis unit that performs electrophoretic analysis of a PCR reaction solution; wherein the DNA analysis system stores preset values ​​of m and n; performs m thermal cycles on the PCR reaction solution in the PCR chamber to produce a first reaction solution; removes a portion of the first reaction solution from the PCR chamber without changing its composition; performs n-m thermal cycles (where n-m is an integer of 2 or greater) on the first reaction solution remaining in the PCR chamber in the PCR chamber so that the total number of thermal cycles is n to produce a second reaction solution; removes at least a portion of the second reaction solution from the PCR chamber without changing its composition; performs electrophoretic analysis of one of the portion of the first reaction solution and the at least a portion of the second reaction solution in the capillary electrophoresis unit; and controls the execution of electrophoretic analysis of the other of the portion of the first reaction solution or the at least a portion of the second reaction solution based on the results of the electrophoretic analysis of one of the portion of the first reaction solution and the at least a portion of the second reaction solution.

[0026] The effects obtained by the example of the invention disclosed in this application can be briefly explained as follows: That is, according to the example of the present invention, an analytical system equipped with a flow path device and an electrophoresis unit can expand the range of DNA amounts that can be analyzed with high accuracy, high sensitivity, in a short time, at low cost, and using a small-sized device.

[0027] Problems, configurations, and effects other than those described above will become apparent from the following description of the embodiments.

[0028] 1 is a schematic diagram of an analysis system; an example of a method for determining data; a schematic diagram of an analysis system; an example of an analysis system; a schematic diagram and an example of an analysis system; a schematic diagram of an analysis system; a schematic diagram of an analysis system; a schematic diagram of a flow path device; a schematic diagram of a flow path device; a schematic diagram of a flow path device; a schematic diagram of a flow path device; an example of the operation of the flow path device; an example of an analysis system; a schematic diagram of a flow path device; an example of the operation of the flow path device; a schematic diagram of a flow path device; a schematic diagram of a flow path device; an example of the operation of the flow path device; an example of the operation of the flow path device; a result showing the relationship between electrophoresis reagent and peak intensity; a diagram showing the effect of the present invention; a diagram showing an analysis range; an example of a table used as a criterion for setting the number of cycles; a schematic diagram of an obtained electropherogram; an example of a table used as a criterion for setting the number of cycles; an example of an analysis system; an example of a method for determining data; an example of a method for determining data; an example of an analysis system; an example of an analysis system.

[0029] This specification mainly describes procedures and standards for conducting human DNA testing, but the subject of analysis is not limited to human DNA testing.

[0030] In the following, unless otherwise specified, m and n refer to the number of thermal cycles in PCR. m and n are integers, and may be n m ≥ 2. In the following, a thermal cycle may be referred to as a PCR cycle or simply as a cycle.

[0031] Hereinafter, the mixture of PCR reagents and sample-derived DNA will be referred to as the PCR reaction mixture. The DNA from the sample that will be amplified by the PCR reagents will be referred to as the target DNA.

[0032] Hereinafter, the PCR reaction solution obtained after m thermal cycles will be referred to as "PCR reaction solution m," and the PCR reaction solution obtained after n thermal cycles will be referred to as "PCR reaction solution n."

[0033] In the following, the electrophoresis reagent may include deionized formamide, size standards, and pure water. Formamide and pure water may be included to reduce the ionic strength of the electrophoresis sample or to denature DNA. In addition to formamide and pure water, a low-conductivity solution may also be used as the electrophoresis reagent. A low-conductivity solution preferably has a conductivity of 10 mS / cm or less, more preferably 1 mS / cm or less, more preferably 100 μS / cm or less, and even more preferably 10 μS / cm or less. The lower the conductivity of the solution used as the electrophoresis reagent, the greater the amount of DNA injected into CE. A size standard may be added to correlate detected peaks with DNA length or to estimate the amount of DNA contained in the electrophoresis sample from the detected peaks.

[0034] Thus, one example of an analytical system mixes 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) with pure water, formamide, or a solution with a conductivity of 10 mS / cm or less to generate a mixture prior to electrophoretic analysis. Mixing with a solution with low conductivity can increase the CE peak intensity. Mixing with formamide can also denature DNA.

[0035] Hereinafter, the DNA obtained by PCR reaction will be referred to as "amplification product," the amplification product obtained after m cycles will be referred to as "amplification product m," and the amplification product obtained after n cycles will be referred to as "amplification product n."

[0036] In the following, the mixture of PCR reaction solution and electrophoresis reagent (mixture) will be referred to as the "electrophoresis sample." When preparing the electrophoresis sample, it is preferable to include a heating step by heating it to 90°C or higher, as shown in Figure 5(2), which denatures the DNA and makes it more likely to become single-stranded, allowing for more accurate CE analysis. The "electrophoresis sample" refers to the sample either before or after denaturation by heating. The electrophoresis sample prepared from amplification product m will be referred to as "electrophoresis sample m," and the electrophoresis sample prepared from amplification product n will be referred to as "electrophoresis sample n."

[0037] Hereinafter, the PCR reaction procedure in which m cycles of PCR are performed, a portion of the PCR reaction solution m is removed, and nm PCR cycles are performed on the remaining PCR reaction solution m, finally preparing both PCR reaction solutions m and n, will be referred to as "split PCR."

[0038] In the following, locus refers to the location of a gene on a chromosome. A typical kit for STR-PCR contains primers that can amplify each locus specifically.

[0039] In the following, alleles refer to distinct genetic variants at the same locus. When DNA testing is performed, if the DNA comes from a single individual, there can be two alleles at the same locus (heterozygote) or one allele (homozygote).

[0040] In the following, an amplicon refers to an amplification product with a single length. A single allele can produce amplicons of different lengths. For example, multiple amplicons can be generated as by-products (artifacts) during the PCR reaction. In DNA testing, one amplicon peak per allele is often assigned to an individual's DNA. However, in the case of mixed samples, it is difficult to distinguish between artifacts and amplicons derived from alleles, so amplicon peaks that may be artifacts may also be analyzed.

[0041] In the following, CE analysis refers to the process of preparing an electrophoresis sample, performing CE measurement, obtaining an electropherogram, and performing DNA identification or fragment analysis. However, the scope of "CE analysis" does not necessarily include some of the above steps.

[0042] In the following, an electropherogram refers to a diagram obtained by CE measurement, with the horizontal axis representing time, measurement point, or DNA chain length, and the vertical axis representing intensity. The vertical axis may represent wavelength, and the data may be three-dimensional data containing intensity information. Alternatively, the vertical axis may represent intensity, and the data may be three-dimensional data containing dye information. The electropherogram obtained from electrophoresis sample m will be referred to as "electropherogram m," and the electropherogram obtained from electrophoresis sample n will be referred to as "electropherogram n."

[0043] In the following, a DNA profile refers to a DNA type obtained by analyzing an electropherogram, or a two-dimensional sequence including peak intensities and peak lengths, or a data set including the number of DNA repeats assigned to a DNA type and peak intensities.

[0044] In the following, STR-CE refers to the process of preparing the PCR reaction mixture for STR-PCR, performing the PCR reaction, measuring the CE, and analyzing the resulting electropherogram. The data obtained from STR-CE can be an electropherogram or a DNA profile. After STR-CE is complete, some or all of the data may or may not be provided to the user.

[0045] Hereinafter, the analytical range refers to, for example, the range of a given sample amount or biomolecular weight that can be correctly analyzed for a given sample or biomolecule by an analytical system or a part or multiple steps of an implementation procedure included in the analytical system. Here, "correct analysis" can refer to a state in which all required conditions are met, but it does not necessarily have to be met, and it can also refer to a state in which the system can provide the best possible data. For example, if the given sample amount is extremely small, the data obtained will not meet all required conditions, but it will be sufficient to obtain data that comes closest to meeting the required conditions.

[0046] The PCR reaction solution may contain two or more primer sets, and the PCR reaction solution may contain two or more amplified gene regions. Analyzing multiple amplified gene regions improves the ability to identify individuals. In particular, in the case of personal identification, it reduces the risk of mistakenly identifying the same person. By performing PCR reactions on multiple gene regions simultaneously rather than individually, the number of PCR chambers can be minimized, saving reaction reagents. Separating the PCR chambers avoids the risk of reduced sensitivity.

[0047] Embodiment 1.

[0048] [Analysis System] In this embodiment, the analysis system 101 (DNA analysis system) may include a memory for storing program instructions, a control unit including a processor for executing program instructions, a function for receiving and analyzing raw data, optical data, and electropherogram data from the detection unit, a solution transport control mechanism such as a pump and valves, a CE unit (capillary electrophoresis unit) for electrophoretic analysis of PCR reaction solutions, a flow path device, and a heater. The control and analysis unit may be connected to a network to enable uploading, matching, and accessing data to a personal DNA database. For example, it may be connectable to CODIS (Combined DNA Index System). The pump may be a diaphragm pump or a syringe pump. Examples of valves include valves that directly or indirectly transmit motor power to deform a film, or valves that deform using air pressure. The valves may be controlled by the control unit. The valves may be opened and closed by thermal deformation or magnetic force.

[0049] Various parameters related to the analysis protocol may be stored in advance in a database of a computer 102 provided in the analysis system 101. Based on the parameters recorded in the database 103, the computer may be responsible for opening and closing valves in the flow path device 104, the CE unit 105, and their connecting parts, controlling the temperature, and controlling the applied pressure and / or flow rate. The parameters recorded in the computer 102 may include stored functions for setting parameters based on temperature, time, pressure, flow rate, stored parameters, and actual measured values.

[0050] It may be possible to accept a sample containing target DNA and fully automate processes from lysis to purification, PCR, CE measurement, and analysis. It may also be possible to fully automate parts of the process, from lysis to purification to PCR to CE measurement and analysis. For example, the analysis system 101 or the flow path device 104 fully automates the process shown in FIG. 14 (described below), from adjusting the PCR reaction solution to n-m thermal cycles. This increases the efficiency of the process. It also makes it possible for non-experts to perform analysis.

[0051] The integrated device can perform lysis, purification, and PCR reaction mixture preparation. The integrated device can mix and heat PCR and formamide. The integrated device can even perform CE measurement.

[0052] The flow channel device 104 may be disposable, which can prevent contamination between samples.

[0053] The CE unit 105 can be disposable. Making it disposable prevents contamination between samples. In addition, since it can be molded integrally with the device, it becomes easier to store, maintain, and transport. The connection between the pretreatment unit and the CE unit becomes simpler, reducing the frequency of breakdowns and errors.

[0054] While the pretreatment flow path device is disposable, the CE unit can be reused multiple times. Because the CE unit requires precision manufacturing and is expensive, making it reusable can reduce costs.

[0055] FIG. 1 shows a detailed example of an analysis system 101 and a computer 102.

[0056] The computer 102 may be equipped with a user interface 106. Parameters related to the user interface 106 (e.g., time and temperature of each step, pressure, flow rate, procedure, volume of divided liquid, number of PCR cycles, sample information, cartridge information, analysis protocol, etc.) may be received from a user and stored in the database 103. Various parameters may also be stored in the database 103 in advance. The computer 102 may be responsible for opening and closing valves of the flow path device 104, controlling the temperature, and controlling the applied pressure and flow rate, based on the parameters recorded in the database 103.

[0057] The flow path device 104, which is consumed for each measurement, may have an internal tag, and the analysis system 101 may read the information on the tag to set an appropriate analysis protocol.

[0058] The number of PCR cycles (e.g., values ​​of m and n) may be set by the user. The analysis system 101 stores the values ​​of m and n that are set in advance. The user may also input information about the type of sample (e.g., cheek swab / touch sample / casework sample / DVI, etc.) and check it against a database in the computer to determine the appropriate PCR implementation procedure. When electrophoresis is performed multiple times or on multiple electrophoresis samples, the user may select whether to first measure CE for electrophoresis sample m or first measure CE for electrophoresis sample n. The implementation procedure may also be entirely automatically controlled by the computer 102. The user may also assist and implement part of the analysis flow.

[0059] Traditionally, samples were sent to a laboratory or other facility equipped with laboratory equipment, where technicians with specialized knowledge and skills prepared the samples, performed measurements, and then analyzed the data. However, problems with this approach include the time required to transport samples and the significant capital and labor costs required to maintain the laboratory equipment. Furthermore, when batch processing is implemented to increase efficiency, it is difficult to accommodate urgent samples. In recent years, sample-to-answer (StoA) systems, which fully automate the entire process from sample introduction to measurement and data acquisition, have emerged in various fields. StoA systems sometimes use flow-through devices that integrate chambers, flow paths, and reagents. Introducing flow-through devices offers the following advantages: (1) Measurements can be easily performed even by non-experts; (2) Data can be acquired in a short time; (3) Highly portable devices can be designed; (4) Variability due to manual procedures is reduced; and (5) Reagent storage is simplified.

[0060] Potential applications of StoA systems include forensics, DNA testing, in vitro diagnostics, plant and animal species identification, biodefense, medicine, biotechnology, life sciences, defense, public health, and agriculture. StoA systems may be used in laboratories, crime scenes, police stations, hospitals, and automobiles.

[0061] [Flow Channel Device] In this embodiment, the flow channel device 104 refers to a disposable or multiple-use cartridge that contains reagents, chambers, and flow channels. The flow channel device 104 may contain a power source for transporting the solution. Some or all of the reagents may be present within the device. Some of the chambers may be equipped with temperature control functions, molecular capture functions, detection functions, and voltage application functions.

[0062] The materials used for the flow channel device are not particularly limited as long as they are materials commonly used in the relevant technical field. Materials with low DNA adsorption, such as polypropylene, cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polycarbonate, polyethylene terephthalate, and polyurethane, are preferred. It is also desirable to suppress adsorption by modifying the surface to make it negatively charged. Other materials include, for example: - metals such as gold, silver, copper, aluminum, tungsten, molybdenum, chromium, platinum, titanium, and nickel; - alloys such as stainless steel, Hastelloy, Inconel, Monel, and duralumin; - silicon; - glass materials such as glass, quartz glass, fused silica, synthetic quartz, alumina, sapphire, ceramics, forsterite, and photosensitive glass; - 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 vinyl chloride resin; - agarose, dextran, cellulose, polyvinyl alcohol, nitrocellulose, chitin, chitosan, or any combination thereof.

[0063] [Chambers and Reagent Storage Units] A typical chamber or reagent storage unit is a space that can store liquids or solids and allow solutions to react, wait, heat, or change. Chambers may be larger in diameter than the flow channel, but may also be visually indistinguishable from the flow channel. Chambers may have internal membranes or microstructures, or may be made of a different composition than the flow channel, have a different surface treatment, or have a different hydrophilicity. The flow channel device may also have a heater or laser light source on the outside. Reagents may be stored in the chamber, and PCR, lysis, purification, and other processes may be performed in the chamber. The volume of a typical chamber is preferably 0.01 μL to 50 mL.

[0064] A fluidic device may store reagents internally or may be supplied from outside the device or from within the analytical system. For example, a device may contain one or more reagents stored in one or more reagent reservoirs. These reagents include at least one of the following: lysis solution, washing solution, PCR reagents (which may contain polymerase, primers, surfactants, etc.), formamide, pure water, DNA fragments, and oil. Because unintended mixing of these reagents can lead to performance degradation or other unexpected results, it is desirable for them to be separated by a barrier mechanism consisting of valves, film, air, or a flow path narrow enough to prevent spontaneous mixing, or a combination of these, until just before use. Isolating reagents from the outside air also enables long-term storage and device portability. The same reagent may be stored in multiple reagent reservoirs for release in multiple steps. Even when reagents are stored outside the device, they are also desirably stored in an isolated state from the outside air, separated from other purification system components by valves, film, air, etc. Known reagent storage technologies include, for example, a blister reagent reservoir or the reagent reservoirs described in Patent Documents 1 and 2, and similar configurations may be incorporated into this embodiment.

[0065] [Sample Type] The sample to be subjected to the purification system according to this embodiment is not particularly limited as long as it is a biological sample. The biological source from which the sample is derived is also not particularly limited, and samples derived from any biological 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.

[0066] When collecting a sample, a swab, filter paper, cloth, or the like can be used as a carrier, and the carrier may be introduced into the purification system.

[0067] Forensic samples include cheek swabs, bone, muscle tissue, human organs, touch samples containing trace amounts of DNA, bloodstains, skin fragments, hair, bodily fluids, and any artifacts that may contain these. Many forensic samples contain unknown amounts of DNA, ranging from 0.001 ng to 1000 μg of DNA, more frequently from 0.01 ng to 10 μg. Additionally, forensic samples may contain DNA from a single individual, DNA from multiple individuals, or degraded DNA.

[0068] [Solution Delivery Control] The analysis system 101 may include pumps and valves for delivering the solution. The delivery means may be a syringe pump, a diaphragm pump, an electrochemical pump, passive delivery using surface tension, centrifugal force, or a combination thereof.

[0069] The analytical system 101 may be equipped with a valve. The valve is used to specify the solution transport path and to switch the path to which air pressure is applied. The valve may be a diaphragm valve operated by air pressure, a mechanical valve, or a valve using surface tension. The flow path that can be transported may be switched based on the difference in pressure required for transport.

[0070] [PCR reaction] The device may contain PCR reagents. The PCR reagents may be prepared as separate solutions containing polymerase and primers. The PCR reagents may be dry reagents. The sample itself, such as a swab, may be subjected to PCR. DNA purified using silica, Chelex, phenol chloroform, etc. may be mixed with PCR reagents. A membrane (such as a silica membrane) on which DNA is trapped may be mixed with PCR reagents.

[0071] PCR reagents may contain an IPC that is amplified along with the sample DNA and a set of primers for amplifying the IPC. The primers for IPC may be dye-labeled and detectable by CE. The amplicon derived from the IPC can be used for analysis. The amount of DNA in the sample can be estimated by using the intensity ratio of the IPC and sample-derived peaks, as well as the amplification efficiency correction factor and fluorescence intensity correction factor. In addition, checking the intensity of the IPC can be used to estimate whether the PCR reaction is proceeding normally or is inhibited.

[0072] A typical PCR reaction volume is 1 μl to 200 μl, preferably 10 μl to 50 μl. Small volumes offer the advantages of accurate temperature control, high-speed PCR, and low reagent costs. On the other hand, larger volumes allow for the collection of more eluted DNA.

[0073] A typical PCR reaction may consist of an initial denaturing step, an annealing step, an extension step, a denaturing step, and a final extension step, or some of the steps may be missing.

[0074] The initial denaturing step involves heating at 90-99°C for 1 second to 2 minutes at the start of PCR, allowing the PCR reaction to begin. The annealing step involves heating at 50-80°C for 1 second to 2 minutes to allow primers to bind to the template DNA. The extension step involves heating at 50-80°C for 1 second to 2 minutes, raising the temperature to a level where DNA polymerase can function effectively and allowing the DNA to elongate. The denature step involves heating at 80-99°C for 1 second to 2 minutes. The final extension step involves heating at 50-80°C for 1 minute to 60 minutes. The inclusion of a final extension step ensures that the length of the amplified product is consistent. The annealing, extension, and denaturing steps are repeated 10-40 times. The annealing and extension steps can be performed at the same temperature.

[0075] [Detection Method] After amplification, detection is performed by CE. CE may involve injecting the amplified product into a polymer-filled capillary tube using voltage injection. Furthermore, applying a high voltage to both ends of the capillary separates fluorescent DNA fragments by size and detects them using a laser / camera system. While this embodiment only discusses CE analysis, other embodiments may use techniques such as massively parallel sequencing (MPS), pyrosequencing, Sanger sequencing, nanopore sequencing, chromatography, electrical measurements, spectroscopy, NMR, RFLP (Restriction Fragment Length Polymorphisms), and microarrays instead of the CE unit.

[0076] [Analysis and Peak Judgment Criteria] The signal obtained in the CE section is analyzed in the analysis section. Known analysis software includes GeneMapper® ID, GeneMapper ID-X, GeneMarker® HID, i-Cubed™, OSIRIS, and TrueAllele™. During CE analysis, a graph is generated based on the signal intensity vs. time information, with the horizontal axis representing DNA length and the vertical axis representing intensity, based on the size standard peak. Corrections for cosmic rays and pull-up / pull-down effects may be performed. Baseline correction may be performed, or other existing techniques may be used to obtain electropherograms. Peak detection is performed on the resulting electropherogram to examine the intensity and peak position of each amplicon. Analysis may involve partial human intervention or may be fully automated.

[0077] This chapter, Analysis and Peak Criteria, describes peak criteria for DNA analysis. Similar criteria may be used for other fragment analyses.

[0078] Macrosatellites are DNA loci that contain repeated sequences with 2-7 nucleotides per repeat unit. The number of repeats at a particular locus varies between individuals, and can be detected by STR-PCR as differences in the length of the amplified product.

[0079] A typical STR-PCR analysis detects two or more loci. Typically, five or more, ten or more, fifteen or more, twenty or more, or twenty-five or more loci are included. STR-PCR can be performed using commercially available kits such as GlobalFiler™ or PowerPlex®. It is also preferable to include loci designated by various genetic databases, such as CODIS, for forensic or DNA testing purposes. The loci may include loci present on autosomes or genes present only on the Y allele.

[0080] When examining DNA types derived from a single individual, which are a mixture of homozygous and heterozygous loci for a particular gene, the number of peaks detected will at least correspond to the number of KIT loci, unless the DNA amount is degraded or insufficient, and at most correspond to a peak corresponding to the sum of twice the number of genes assigned to autosomes and the number of genes assigned to sex chromosomes among the KIT loci.

[0081] In a typical STR-PCR analysis, one fluorescent dye color is assigned to one locus. STR-PCR kits with two, three, four, five, six, seven, or eight dyes may also be used. When detecting DNA types, loci may be assigned based on a combination of length and peak color information. Thresholds for the intensity and position of various peaks may be set for each color, or for each locus or allele.

[0082] In a DNA profile derived from a single individual, one or two alleles are detected per locus. On the other hand, in a DNA profile derived from a mixed sample derived from multiple individuals, one, two, three, or more alleles are detected per locus. When assigning individual DNA profiles from DNA profiles derived from multiple individuals, probabilistic analysis is typically performed using peak intensity ratios. Examples of programs for analyzing mixed samples include Kongho, LikeLTD, LRmix, STRmix, Euroformix, and TrueAllele.

[0083] In DNA testing, artifacts refer to, for example, peaks that do not originate from the DNA type derived from an individual, a balance between peaks that differs from the ideal state, or peak shapes that differ from the ideal state, which cause the electropherogram and DNA profile obtained during actual DNA analysis to differ from the ideal electropherogram and DNA profile that should be obtained from the DNA type derived from an individual.

[0084] Artifacts can arise through multiple mechanisms, and some artifacts are acceptable if the DNA in a sample comes from a single individual.

[0085] On the other hand, if there are many artifacts, the accuracy of mechanical judgment will decrease, making expert review necessary.

[0086] Also, if there are many artifacts, the DNA testing results may be incorrect.

[0087] Furthermore, if the artifact is large, the DNA type that should be obtained may not be detected, resulting in a reduced amount of information being obtained.

[0088] Furthermore, in samples where it is not possible to guarantee that the DNA originates from a single individual, such as forensic samples where the possibility of multiple individuals being involved cannot be ruled out, the presence of artifacts can make electropherogram analysis more complex or difficult. For example, when three or more peaks appear at a single locus, it becomes difficult to determine whether the peaks are due to artifacts or to two or more individuals. Furthermore, while it is common to assign an individual's DNA profile based on the ratio of peak intensities, this becomes more difficult when the possibility of artifacts is taken into account. Therefore, it is desirable to obtain electropherograms with as few artifacts as possible.

[0089] When peaks that are not derived from an individual's DNA occur during CE analysis and are reflected in the analysis results, this state is called "drop in." Similarly, when peaks that should be present in an individual's DNA are not detected during CE analysis and are not reflected in the analysis results, this state is called "drop out." It is preferable to set various thresholds to minimize drop in and drop out.

[0090] <Stutter Peaks> Stutter peaks are by-products of PCR amplification. They arise when one or more repeat sequences are skipped or duplicated during the extension reaction. Stutter peaks typically appear before or after the sample-derived peak, and appear one or two repeats more or less than the sample-derived peak. Typically, stutter peaks have an intensity of about 1-20% of the sample-derived peak.

[0091] Incomplete Adenylation During the extension reaction in STR-PCR, there is a certain probability that an extra adenylate group will be added to amplicons of the correct length (A+ peak). Typical STR-PCR kits add a "final extension" step to the end of the PCR reaction. During this final extension step, amplicons that were not previously adenylated are adenylated. By allowing a sufficient final extension time, almost all amplicons will be adenylated. However, if the final extension time is set too long compared to the amount of amplicon, the proportion of amplicons with extra adenylation (A++ peak) will increase. The A++ peak is detected at a position one base longer than the A+ peak. Furthermore, if the amount of amplicon is excessive, adenylation will not be completed within the final extension time, leaving amplicons that are not adenylated, detected as the A- peak, which is one base shorter. Hereinafter, the A++ peak and the A- peak are collectively referred to as incomplete adenylation peaks (IAPs). Furthermore, when referring specifically to the A- peak, we refer to it as "IAP-," and when referring specifically to the A++ peak or peaks with more adenyl groups, we refer to it as "IAP+." In optimal 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- and A++ peaks are within 50% or less of the A+ peak intensity, preferably 20% or less, and more preferably 10% or less. An IAP intensity threshold or an intensity ratio threshold relative to the main peak (Incomplete Adenylation Peak Ratio Threshold, IAPT) may be set to determine whether optimal STR-CE has been performed by ensuring that the A- or A++ peak intensity falls within the above range. If the intensity ratio of the A- or A++ peak to the A+ peak exceeds a certain level, the intensity of the main peak will no longer reflect the actual gene abundance ratio.Furthermore, in the case of mixed samples or when a peak shifted by one base appears due to genetic polymorphism, accurate assignment becomes impossible, and it may also cause the wrong DNA type to be detected.

[0092] <Peak Intensity Ratio> When sufficient DNA is present, the two peaks originating from heterozygous loci will have approximately the same height. Insufficient DNA increases the likelihood of uneven DNA amounts originating from each gene, resulting in a significant difference in the intensities of the two peaks. Furthermore, excessive amplification results in shorter DNA being amplified preferentially over longer DNA. The heights of peaks originating from the same locus also differ significantly because shorter DNA is amplified preferentially. When the ratio of the two peak intensities becomes large, it becomes impossible to distinguish them from stutter peaks. It also makes it difficult to assign DNA to mixed samples. Therefore, to determine whether a meaningful CE analysis has been performed, the ratio of the intensity of the smaller peak to the larger peak (peak-to-height ratio, PHR) should be 10% or greater, preferably 40% or greater, and more preferably 60% or greater.

[0093] <Ski-slope> When DNA is present in excess, the ratio of dNTPs and polymerase to the amplicon decreases, increasing the tendency for short DNA to be preferentially amplified. In this case, the resulting electropherogram shows a sloped profile, with a small peak for long DNA and a large peak for short DNA. Furthermore, when DNA is degraded, the proportion of short DNA tends to be higher than that of long DNA. This also results in a DNA profile with a slope. Furthermore, the presence of inhibitors tends to reduce the amplification efficiency of long DNA compared to short DNA, which also produces a DNA profile with a slope. Furthermore, when PCR reagents are diluted with DNA solution at a lower ratio than the original mixture ratio, differences in amplification efficiency occur, resulting in preferential amplification of long DNA, resulting in a DNA profile with a reverse slope. Furthermore, when PCR reagents are mixed with a large amount of long DNA primers, creating a reaction system that preferentially amplifies long DNA, a DNA profile with a reverse slope can also be obtained. In DNA testing, sloped profiles and profiles with large peak intensity ratios between loci are undesirable. This is because the difference in peak intensity becomes larger, making it more likely that peaks that saturate CE or fall below the detection limit will appear. It also makes it difficult to assign mixed samples. Typically, PCR reaction parameters should be adjusted so that the inter-locus peak intensity ratio (inter-locus PHR) is such that the smallest peak is at least 1%, more preferably at least 5%, more preferably at least 10%, and even more preferably at least 20% of the largest peak. Small peaks that do not meet the inter-locus PHR threshold can be excluded from peak analysis. To deal with degraded DNA, it is desirable that the PCR amplification amount be within an appropriate range for STR-CE or that the CE dynamic range be designed to be large.

[0094] <CE Saturation> Saturation occurs when an excessive amount of amplicon is introduced into the CE section, causing the fluorescence intensity during CE detection to exceed the upper limit of the detector's detection range. In this case, the ratio of the peak with the highest intensity to the other peaks does not reflect the actual intensity ratio. Furthermore, the intensity ratio of stutter peaks and IAPs relative to the main peak is detected higher than the actual amplicon ratio. This accentuates the artifacts mentioned above. Furthermore, if the sample is a mix, the correct mixing ratio cannot be calculated. Therefore, in preferred DNA analysis, PCR reaction conditions should be set or the PCR amplified product should be diluted to prevent CE detection from oversaturating (OS). The OS threshold can be determined by evaluation on the actual CE device, by the user, or by the user for each measurement, or by computer.

[0095] <CE noise, pull-up> There are also artifacts that occur in the CE detection section.

[0096] Pull-up peaks are peaks that are falsely detected due to other dyes. Pull-up peaks are particularly strong when CE is saturated, but they can also be detected when CE is not saturated.

[0097] If a large amount of DNA is brought into the CE section in the previous measurement, carryover may occur, causing a peak to be detected in the next measurement.

[0098] In addition, air bubbles in the CE section and background noise in the detection section may be reflected in the electropherogram.

[0099] An analytical threshold (AT) may be set during analysis to prevent noise peaks from being erroneously used in the analysis. The analytical threshold may be set by measuring background noise to obtain a sufficient signal-to-noise ratio, or may be set by the user, set by the instrument for each experiment, or may be preset. Furthermore, even if a peak exceeds the AT, a program may be stored and executed to determine whether the peak is due to an amplification product, poor CE migration, or various CE noises.

[0100] The AT may be set in two or more stages, a first standard value and a second standard value. Peaks with a peak intensity greater than the first standard are determined to be true peaks, and peaks with an intensity greater than the second standard but less than the first standard may be determined to be peaks by requiring review by a user or expert, or by satisfying other set conditions. Furthermore, since amplification efficiency may differ depending on the gene locus, and luminescence efficiency and noise intensity may differ depending on the dye, the AT may be set for each gene locus or each dye.

[0101] <Countermeasures for artifacts> We will now explain how to counter the above artifacts. For undegraded DNA and DNA that does not contain PCR inhibitors, inputting an amount of DNA into PCR that falls within the analytical range of STR-CE makes it possible to obtain appropriate peak intensity (above AT, below OS), PHR, and IPA data, while suppressing the appearance of ski slope. Furthermore, even if the DNA is degraded, present in trace amounts, or contains inhibitors, the amount of information obtained can be maximized as long as the appropriate number of cycles and input amount are used. There are parameter sets for the number of PCR cycles and DNA amount that can maximize the amount of information obtained while minimizing the appearance of artifacts.

[0102] <Method of Evaluating CE Analysis Results> When providing CE analysis results to a user, it is also possible to indicate to the user whether the obtained CE data is useful or not, and to what extent it is useful.

[0103] When presenting the CE analysis results to the user, the user may be informed whether the obtained CE data is a complete DNA profile result (Full profile), and the user may be advised whether the obtained CE data requires further analysis.

[0104] Figure 2 shows an evaluation flow for determining whether the obtained DNA profile is a full profile or for informing the user of the data quality. This chart is an example; the order may be reversed, some steps may be omitted, steps not shown may be included, or some or all of the steps may be performed simultaneously so that multiple flags are assigned. Alternatively, a flow like that shown in Figure 2 may be performed for each peak or locus, and a full profile may be determined if all criteria are met for all loci. Even if the profile is not a full profile, information on only the loci that met the criteria may be provided to the user. It is also possible to provide a table or other output showing which criteria were met or not met for each locus.

[0105] 201. None of the peak intensities should saturate the CE detection system (OS). If an OS peak exists, the peak, locus, or analytical result may be flagged as OS.

[0106] 202. All loci must have at least one peak above AT. If there is a locus where no peak is detected, the locus or analysis result may be flagged as Drop out (DO).

[0107] 203. If there is only one peak above AT, that peak must be at least twice as intense as AT (excluding genes that are inherently unique, e.g., sex chromosome loci). If there is no peak at least twice as intense, the peak, locus, or analysis result will be flagged as inconclusive homozygous (IH flag).

[0108] 204. The intensity of the peaks located ±1 base from the main peak must not exceed the IPAT, which is set at a peak ratio of 1%, 5%, 10%, 20%, or 40% of the main peak intensity. If it does exceed the IPAT, the peak, locus, or analytical result will be flagged as having a high IPA intensity (IPA- or IPA++ flag).

[0109] 205. All detected peaks must be at a DNA chain length that can be assigned to a DNA type. If a peak cannot be assigned and does not fall under IPA, it is recognized as an Off Ladder (OL) peak, and the peak, locus, or analytical result is flagged as OL. However, peaks that appear at the position of a stutter peak do not need to be considered for OL judgment.

[0110] 206. Three or more peaks should not be detected per locus (two or more for loci where only one should exist). If detected, the third-highest peak should be 1% or less, 5% or less, 10% or less, 20% or less, or 40% or less in intensity than the second-highest peak. If the ratio of the intensity of the third-highest peak to the second-highest peak exceeds a threshold, the DNA contained in the sample may be determined to be from two or more individuals and a Mix flag may be raised. However, if the third-highest peak occurs at the position where the first or second stutter peak appears, it may be determined to be a stutter peak. If a peak appears at the stutter peak position with an intensity of 1%, 5%, 10%, 15%, 20%, or 40% or less than the main peak, it may be determined to be a stutter peak and no Mix flag may be raised. If a peak exists at a position that could be a stutter peak and its intensity exceeds the threshold for the stutter peak tolerance range, it may be a mix, so a mix flag may be set. Also, if the intensity of the peak with the third highest intensity at a position where a stutter peak should not appear exceeds the threshold, a mix flag may be set. Similar determinations may be made for peaks with the fourth, fifth, and subsequent intensities.

[0111] 207. If two or more peaks are detected and the peak with the second highest intensity is 1% or more, 5% or more, or 10% or more of the intensity of the first peak, and 60% or less, 40% or less, or 20% or less, the PHR is determined to be poor and a PHR flag may be assigned.

[0112] The PHR becomes poor when the amount of DNA sent to PCR is below 0.6 ng, 0.3 ng, 0.15 ng, or 0.075 ng. For example, when the approximate DNA amount is estimated from the IPC intensity or PCR cycle number and peak intensity in CE analysis, if the DNA amount sent to PCR exceeds the aforementioned DNA amount but the PHR does not exceed the threshold, the obtained DNA profile can be determined to be derived from a mixed sample.

[0113] Since peaks that give rise to the IPA flag also give rise to the OL flag, the criteria 205 may also serve as 204, ie the IPA flag may subsume the OL flag.

[0114] If none of the above flags are assigned, a full profile is obtained. If a full profile cannot be obtained, expert review may be requested. This may result in a longer time to complete DNA testing. Therefore, it is important to increase the probability of obtaining a full profile by setting an appropriate number of cycles. Furthermore, even if a full profile cannot be obtained due to a mixed sample, low sample volume, or high PCR inhibitor content, or if not all loci are successfully detected, a DNA profile that detects five or more loci can be useful in criminal investigations. This type of DNA profile is called a partial profile. The more information on loci obtained, the more useful it is for criminal investigations. Therefore, even in situations that result in a partial profile, the analysis protocol, particularly the number of PCR cycles, must be set to detect as many peaks as possible. In particular, for mixed samples or samples with low DNA content, the protocol, particularly the number of PCR cycles, must be set to detect as many peaks as possible while satisfying the 201, 205, and 206 flags on the electropherogram (i.e., without these flags being assigned).

[0115] A threshold can be set to determine whether DO is due to the absence of the corresponding allele in the DNA input to the PCR reaction or to an insufficient number of PCR cycles. For example, if there is a locus with no peaks above AT, but there is a peak in the overall amplification product that is 10 times higher than AT, it can be determined that DO is due to the sample. If not, it can be determined that DO is due to an insufficient number of PCR cycles or is not due to the sample. This determination can be used to determine whether an analysis with an increased number of PCR cycles is necessary.

[0116] [About the scope of analysis] Factors that can cause intensity fluctuations when performing STR-CE include: (1) fluctuations in the concentration of salts contained in the PCR reaction solution and substances that inhibit injection, (2) the mixing ratio of the electrophoresis reagent and the PCR reaction solution, (3) amplification efficiency, (4) deterioration of the electrophoresis reagent or incomplete denaturation, (5) temperature fluctuations in the electrophoresis area, (6) fluctuations during electrolytic injection, (7) fluctuations between capillaries and capillary arrays, and (8) fluctuations in detection intensity at the detection area.

[0117] Hereinafter, the dynamic range of CE may be the dynamic range described in the manual or the like for various CE devices, the ratio of the maximum amplicon amount to the minimum amplicon amount at which a linear signal is obtained for the amount of amplicon actually input into CE, the maximum amplicon amount to the minimum amplicon amount at which the relative ratio of the amplicon amounts input into CE can be assigned from the signal intensity, or 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 vary from measurement to measurement.

[0118] In the following, the analytical range of STR-CE, unless otherwise specified, may simply correspond to the dynamic range of CE. It may also be an experiment conducted by varying the amount of DNA added to PCR to determine the amount of DNA required to correctly detect a specific allele. It may also be an experiment conducted by varying the amount of DNA added to PCR to determine the amount of DNA required to correctly detect a specific set of alleles. Such a range of DNA amounts may be tested in multiple individuals, and the average, minimum, or maximum range may be used as the analytical range. The amount of DNA used for CE measurement is generally proportional to the amount of DNA added to STR-PCR. However, this is not always the case due to the dependence of CE injection efficiency on DNA concentration, and the dependence of PCR amplification efficiency on DNA concentration and cycle number. Furthermore, while the analytical range of STR-CE does not generally change significantly when the PCR cycle number is changed, it may deviate, especially as the amount of DNA is reduced, due to the stochastic effect (a stochastic effect in which the amount of DNA corresponding to the locus to be amplified becomes non-proportional to the amount of DNA added). Therefore, it is desirable to carry out the above examination for each cycle to determine the true STR-CE analysis range, and the analysis range for each cycle determined through such examination is also considered a type of STR-CE analysis range. However, since confirming everything experimentally requires a great deal of effort, some of it may be replaced by calculation.

[0119] The dynamic range of CE and the analytical range of STR-CE may be evaluated taking into account the variations listed above (1) to (8).

[0120] By changing the threshold value, the analysis range can be broadened or narrowed. However, broadening the analysis range may come at the expense 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 broaden the analysis range.

[0121] In the following, the analytical range of an analytical system refers to the range of DNA content in a sample that can be accurately analyzed for DNA samples input into the analytical system. To expand the analytical range, it is necessary to either expand the analytical range of the STR-CE or control the amount of DNA input into the STR-CE. To expand the analytical range of the STR-CE, it is possible to improve the dynamic range and sensitivity of the CE, reduce variability in the STR-CE, prepare multiple-cycle amplification products, or prepare eluates at different dilutions and subject them to STR-CE. To control the amount of DNA input into the STR-CE, the purification membrane can be reduced in volume or made coarse to cut off the upper limit of the DNA amount that can be processed by the membrane, or the dilution rate can be changed after purification by quantifying the DNA.

[0122] <<Split PCR analysis system and its operation example>>

[0123] [Analysis System] [Configuration Example of a General Analysis System] Fig. 3 shows an example of an analysis system 101. Fig. 4 shows an example of the operation procedure of the analysis system 101. The biomolecule analyzer is equipped with a computer 102 for performing biomolecule analysis and a flow path device 104.

[0124] The flow path device 104 includes a lysis chamber 301 for introducing and lysing a collected sample, a purification membrane chamber 303 containing a purification membrane 302, a PCR chamber 304 (a PCR chamber for performing thermal cycling) for performing DNA amplification and the like, and a waste chamber 305. An external connection port 306 is provided for fluidic connection to the outside of the device. Solutions are transported through the external connection port 306, allowing reagents, amplification products, and the like to be exchanged with the outside of the device. When transporting solutions in the analysis system 101, pumps and valves 307 may be used to control the flow of the solutions. The pumps and valves 307 may all be provided outside the flow path device, or some may be provided within the flow path device 104. Furthermore, a PCR reagent reservoir 308 may contain PCR reagents 309 (polymerase, primer, dNTP, buffer, etc.) required for the PCR reaction, and a migration reagent reservoir 310 may contain a migration reagent 311. The system also includes reagent reservoirs 312, 313, and 314 for storing other reagents required for pretreatment. In a sample loading step 401, before and 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 a lysis step 402. In a purification step 403, the lysis product is transferred from the lysis chamber 301 to the purification membrane chamber 303, where the DNA is bound to the purification membrane 302. A washing solution is then released from the reagent reservoir 313, and purification is performed. After purification, a step of drying the washing solution may be included. An elution solution is released from the reagent reservoir 314, and the DNA eluted from the purification membrane chamber 303 is transferred to the PCR chamber 304. A PCR reagent is transferred from the PCR reagent reservoir 308 to the PCR chamber 304 and mixed with the eluted DNA. In an amplification step 404, the purified DNA in the PCR chamber 304 is mixed with PCR reagent 309 and subjected to a PCR reaction. In the detection step 405 , the amplified DNA is mixed with the migration reagent 311 stored in the migration reagent storage section 310 , and measurement is carried out in the CE section 105 .

[0125] After mixing the electrophoresis reagent and PCR reaction solution, a step of heating to 80-100°C and rapidly cooling to 0-10°C may be added before CE analysis. By adding this step, the DNA is more completely converted into single strands, enabling highly accurate CE analysis.

[0126] [Example of an analysis system used in this embodiment] Figure 5 shows an outline (Figure 5(1)) of the analysis system 101 of this embodiment and an example of the operation procedure (Figure 5(2)). Operation steps 501 to 507 may correspond to the amplification step 404 and the detection step 405.

[0127] As shown in Figure 5(1), the analytical system 101 of this embodiment has a flow path device 104 and a CE unit 105, and the flow path device is equipped with a PCR chamber 304. The analytical system 101 also has a dispensing chamber 320. A PCR reaction solution or an electrophoresis sample is sent to the dispensing chamber 320. However, the dispensing chamber 320 may be omitted. Each element is connected by flow paths 315 and 316.

[0128] 5(2), analysis system 101 prepares a PCR reaction solution in step 501, performs m PCR cycles in PCR chamber 304 in step 502, and then removes PCR reaction solution m from PCR chamber 304 without changing its composition in step 503. In step 504, CE measurement is performed on electrophoresis sample m in CE unit 105. In step 505, the PCR reaction solution m remaining in PCR chamber 304 is thermal cycled nm times to obtain PCR reaction solution n. In step 506, part of PCR reaction solution n is removed from PCR chamber 304 without changing its composition, and in step 507, CE measurement is performed on electrophoresis sample n in CE unit 105.

[0129] The dispensing 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.

[0130] Steps 503 to 505 may be repeated multiple times to increase the number of divisions to three, four, or more.

[0131] Steps 504 and 507 may be performed simultaneously, or 507 may be performed after 504. By performing 507 after 504, the setting of 505 can be changed depending on the result of 504.

[0132] Step 505 may be started immediately after step 503 is completed. Since the time between steps 502 and 505 is short, the occurrence of artifacts can be suppressed. Also, the execution timing of steps 505 and 506 may be set so that step 507 starts at the same time as step 504 is completed.

[0133] [Examples of Derivatives of an Analysis System Used to Implement the Present Invention] As shown in FIG. 6, the analysis system 101 may be equipped with heating units 317 and 318 (temperature control / heating mechanisms) for thermal cycling within the PCR chamber 304. The PCR chamber 304 may be connected to a flow channel 319 and a flow channel 315, which may control the supply of reagents and the pressure. As shown in FIG. 6, the analysis system 101 may also be equipped with a flow channel device and a pump and valve 307 for transporting solutions appropriate for various steps, such as CE measurement. As shown in FIG. 6, the analysis system 101 may also be equipped with a computer 102, which may be equipped with functions such as control of the pump and valve 307, control of CE measurement, temperature control, and functions for analyzing and providing feedback on data obtained from the CE unit 105 and heating units 317 and 318, etc., and providing the data to the user. The flow channel device may also be equipped with a dispensing chamber 320. The dispensing chamber 320 has the function of removing a portion of the amplified product from the PCR chamber 304 without changing its composition upon completion of m thermal cycles. The dispensing chamber 320 may have a measuring function for extracting a specified amount of PCR reaction solution m or n. When the PCR reaction solution is delivered to the dispensing chamber 320, the dispensing chamber 320 may suck up the PCR reaction solution, or the PCR reaction solution may be delivered to the dispensing chamber 320 by pressurization. Alternatively, the dispensing chamber 320 may simply be provided to temporarily store the PCR reaction solution, and may not have a measuring function. The dispensing chamber 320 may be provided outside the flow path device 104.

[0134] The PCR reaction solution m may wait in the dispensing chamber 320 for a period of time before CE measurement. The PCR reaction solution m may be mixed with electrophoresis reagents (formamide and size standards) in the dispensing chamber 320. That is, the analysis system 101 mixes the reaction solution (for example, 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 types of DNA fragments. This allows the concentration to be measured based on the peak height of the size standard.

[0135] As shown in FIG. 6, the analysis system 101 may have a standby unit 321 between the CE unit 105 and the PCR unit. The standby unit 321 may temporarily hold the PCR reaction solution or the electrophoresis sample obtained by mixing the PCR reaction solution with formamide during steps 506 to 507 or steps 503 to 504. The flow channel device in FIG. 6 (and subsequent figures) may be arranged vertically, i.e., so that the main portion of the flow channel or at least a portion of the flow channel is parallel to the direction of gravity. In one form of the flow channel device, the bottom of the drawing faces downward in the direction of gravity. When used vertically, bubbles / entrapped air in each chamber accumulate at the top of the chamber. Therefore, by removing the solution from below when transferring it to the next chamber / flow channel, it is possible to minimize the intrusion of air into the next step.

[0136] The flow channel device can be modified as follows.

[0137] As shown in Fig. 6, one embodiment of the analysis system 101 of this embodiment has a CE unit 105 in the flow path device. Also, as shown in Fig. 7, one embodiment of the analysis system 101 has a pump and valve 307 in the flow path device. Note that the optical system may be located outside the flow path device or inside the analysis system 101.

[0138] 8, the flow channel device 104 may exchange solutions and receive air pressure control with the analysis system 101 via an external connection port 306. Pressure may be applied from the external connection port 306 via a flow channel 322 to send out a PCR product or an electrophoresis sample from a dispensing chamber 320. Valves 323, 324, 325, and 326 are provided within the flow channel device, and are opened and closed depending on the analysis step.

[0139] As shown in FIG. 9 , the flow path device may include a mixing chamber 327 between the dispensing chamber 320 and the external connection port 306. The PCR reaction solution in the dispensing chamber 320 may be mixed with the electrophoresis reagent in the mixing chamber 327. The mixing chamber 327 may be connected to the dispensing chamber 320 by a flow path 328. The mixing chamber 327 may be provided outside the flow path device, inside the analysis system 101, and in some cases, the standby unit 321 may fulfill this role. A flow path 329 is connected to the mixing chamber 327, and by applying pressure to the flow path 329, air bubbles may be sent into the mixing chamber 327 for mixing, or the electrophoresis sample may be sent to the CE unit 105.

[0140] As shown in FIG. 9 , PCR chamber 304 may have three channels 319, 315, and 330. Channel 319 may be connected upstream of the sample, channel 315 to dispensing chamber 320, and channel 330 to mixing chamber 327. PCR reaction solution m removed from PCR chamber 304 may be transported to mixing chamber 327 via dispensing chamber 320, and PCR reaction solution n removed from PCR chamber 304 may be transported to mixing chamber 327 via channel 330 without passing through dispensing chamber 320. Because PCR reaction solution m and PCR reaction solution n pass through separate paths, a decrease in reproducibility due to residual liquid can be suppressed. Furthermore, when reaction solution n is metered but reaction solution m is not metered, and the entire amount is transported to mixing chamber 327 or CE unit 105, there is no need to transport reaction solution m to dispensing chamber 320, making this channel device structure easier to perform split PCR.

[0141] When PCR reaction solution m is also measured, another dispensing chamber may be provided in addition to the dispensing chamber 320 used for PCR reaction solution n.

[0142] As shown in FIG. 10 , the flow channel device 104 may include migration reagent reservoirs 310 and 331 for storing migration reagents (e.g., formamide, DNA fragments, and pure water). The migration reagent reservoirs 310 and 331 may be located on either the flow channel 315 or the flow channel 319. As shown in FIG. 10 , the reagent reservoir may be divided into two or more reservoirs for each type of reagent. By dividing the reservoir into two, a predetermined amount of reagent can be released from each reservoir at the time of division. Alternatively, the amount of liquid released from each reservoir may be controlled so that the reagent is released in two or more separate releases from one reagent reservoir. As shown in FIG. 10 , the flow channel device 104 may include air reservoirs 332 and 333. The air reservoirs 332 and 333 may be located on either the flow channel 315 or the flow channel 319. By releasing a predetermined amount of air from the air reservoir 332 or 333 at the time of division, a predetermined amount of PCR reaction solution can be transported out of the PCR chamber 304. With this flow path device configuration, it is possible to perform split PCR even if the dispensing chamber 320 is omitted. With this flow path device configuration, the mixing chamber 327 may also serve as the dispensing chamber 320. Instead of air, the air reservoir 332 or 333 may contain a liquid that does not affect the PCR reaction, such as oil. The air reservoir 332 or 333 may be filled with PCR reaction liquid, and the PCR reaction liquid may be newly replenished with a volume equivalent to the pushed-out PCR liquid.

[0143] As shown in FIG. 11, the migration reagent reservoirs 310 and 331 may be provided in a pair of flow paths that straddle the PCR chamber 304. As shown in FIG. 11, it is preferable that the migration reagent reservoir 331 be installed closer to the PCR chamber 304 than the air reservoir 333. This is because the migration reagent 311 stored in the flow path 319 or the PCR chamber 304 can be sent in its entirety or in a larger amount to the dispensing chamber 320 or the mixing chamber 327, thereby contributing to improved reproducibility. As shown in FIG. 11, the flow path device 104 may have an air reservoir 334 on the flow path 315 closer to the PCR chamber 304 than the migration reagent reservoir 310. The air reservoir 334 may be used to transport the migration sample m in the dispensing chamber 320 to the mixing chamber 327 or the CE section 105. The air reservoir 334 may be replaced with the flow path 322.

[0144] [Example of Method for Performing Split PCR] This section describes a method for performing split PCR on the flow path device 104. Although a dispensing chamber is mentioned throughout, the solution may be transported directly to the CE section 105 without using a dispensing chamber / mixing chamber.

[0145] Fig. 12 shows an example of a flow channel device 104, and Fig. 13 shows an example of a transport method for performing split PCR on the flow channel device of Fig. 12. For convenience of illustration, some reference numerals are shown in Fig. 12 but omitted in Fig. 13.

[0146] Step I: There is a PCR chamber 304 containing a PCR reaction solution 335, and m cycles of PCR reaction are carried out in the PCR chamber 304 with the valves 326, 323, and 325 closed. This step may correspond to step 502.

[0147] In this way, the analytical system 101 performs m thermal cycles on the PCR reaction solution 335 in the PCR chamber 304 to produce a PCR reaction solution m (first reaction solution).

[0148] Step II: After m cycles of PCR reaction are completed, valves 326 and 323 are opened to transfer a portion of the solution in PCR chamber 304 to dispensing chamber 320. This step may correspond to step 503.

[0149] In this way, the analysis system 101 removes a portion of the PCR reaction solution m without changing its composition from the PCR chamber 304. Here, the analysis system 101 may perform electrophoretic analysis of the portion of the PCR reaction solution m in the CE unit 105.

[0150] In this way, the flow path device 104 has openable and closable valves 326 and 323, and a portion of the PCR reaction solution m is divided and removed by closing the valves 326 and 323 before the start of m thermal cycles and opening the valves 326 and 323 after the completion of m thermal cycles. In this way, the division process can be carried out appropriately.

[0151] Step III: Valves 323 and 326 are closed to push out the migration reagent 311 from the migration reagent reservoir 310, and the PCR reaction solution m stored in the dispensing chamber 320 is transported to the mixing chamber 327. The PCR reaction solution m is mixed with the migration reagent 311 to form the migration sample 336.

[0152] Step IV: The valve 325 is opened, and the electrophoretic sample in the mixing chamber 327 is transported to the outside of the flow path device 104 (to the standby section 321 or CE section 105 of the analysis system). This step may correspond to step 504.

[0153] Step V: Close valve 326 and perform thermal cycling nm times. This step may correspond to step 505.

[0154] In this way, the analysis system 101 performs n-m (where n-m is an integer greater than or equal to 2) thermal cycles on the PCR reaction solution m remaining in the PCR chamber 304 so that the total number of thermal cycles is n, thereby generating PCR reaction solution n (second reaction solution).

[0155] Step VI: After a total of n thermal cycles are completed, valve 325 is closed and valves 326 and 323 are opened to transfer PCR reaction mixture n to mixing chamber 327 .

[0156] In this way, the analytical system 101 removes at least a portion of the PCR reaction solution n from the PCR chamber 304 without changing its composition.

[0157] The migration reagent is pushed out from the migration reagent reservoir 331, and the PCR reaction solution n stored in the dispensing chamber 320 is transported to the mixing chamber 327. The valve 325 is opened, and the migration sample in the mixing chamber is transported outside the flow path device 104 (to the standby section 321 or CE section 105 of the analysis system). This step may correspond to step 507.

[0158] The analysis system 101 may perform electrophoretic analysis of at least a portion of the PCR reaction solution n in the CE section 105.

[0159] [Another example of a method for performing split PCR] Figure 14 shows the analysis process of split PCR, Figure 15 shows an example of a flow path device 104, and Figure 16 shows an example of a transport method for splitting the PCR reaction solution at m=24 cycles and n=30 cycles in Figure 15. For convenience of illustration, some reference numerals are shown in Figure 15 but omitted in Figure 16.

[0160] The flow channel device 104 has a valve 337 on a flow channel 315 connecting the dispensing chamber 320 and the PCR chamber 304. The valve 337 is installed on the PCR chamber 304 side of the branch to the migration reagent reservoir 310. A valve 338 is installed on the flow channel 316. A valve 339 is installed on the flow channel 319 on the flow channel 329 side of the branch to the migration reagent reservoir 331.

[0161] Step I: All valves are closed. There is a PCR chamber 304 containing a μl of PCR reaction mixture 335, and 24 thermal cycles are performed in the PCR chamber 304. After 24 thermal cycles are completed, a final extension is performed for 8 minutes. (Steps 601 to 604 may correspond to steps 501 and 502.)

[0162] Step II: After 24 cycles of PCR reaction are completed, valves 326, 323, and 337 are opened, and a portion of the solution in PCR chamber 304, b μl (where a>b), is transferred to dispensing chamber 320 via flow path 315. (This may correspond to step 605 or step 503.)

[0163] Step III: Valves 323, 326, and 337 are closed, and valves 324 and 325 are opened, so that c μl of migration reagent 311 is pushed out from migration reagent reservoir 310, and PCR reaction solution m stored in dispensing chamber 320 is transported to mixing chamber 327. PCR reaction solution m is mixed with migration reagent 311 to become migration sample 336 (this may correspond to step 606 or step 504). At this time, migration reagent 311 may remain in part of dispensing chamber 320 and flow channel 315.

[0164] Step IV: Close valve 324, open valve 338, and transport the electrophoretic sample in mixing chamber 327 to the outside of the flow path device 104 (to the standby section 321 or CE section 105 of the analytical system). (This may correspond to step 607 or step 504.)

[0165] Step V: Close all valves and perform six thermal cycles on the ab μl PCR reaction mixture 335 remaining in the PCR chamber 304, followed by an eight-minute final extension. (This may correspond to steps 608-610, step 505.)

[0166] Step VI: After a total of 30 thermal cycles are completed, valves 326, 323, and 325 are opened, d μl of migration reagent is pushed out from migration reagent reservoir 331, and PCR reaction solution n is transported to mixing chamber 327. (This may correspond to step 506.)

[0167] Step VII: Valve 325 is closed, valve 339 is opened, and air pressure is applied to send all of the solution remaining in PCR chamber 304 into mixing chamber 327. Alternatively, air may be sent into mixing chamber 327 to agitate and homogenize electrophoresis sample 336. (This step may correspond to step 611 or step 507.)

[0168] Step VIII: The valve 338 is opened, and the electrophoretic sample in the mixing chamber 327 is transported to the outside of the flow path device 104 (to the standby section 321 or CE section 105 of the analytical system). (This may correspond to step 612 or step 507.)

[0169] Table 1 shows examples of the time and temperature for each step of the split PCR process shown in Figure 14.

[0170]

[0171] The basic thermal cycle settings were based on the STR-PCR kit (GlobalFiler™ Express) manual (https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / 4477672_GlobalFilerExpress_UG.pdf). In step 605, the temperature of the PCR chamber 304 during dispensing may be set to any temperature between room temperature and denaturation.

[0172] The solutions may be kept at low temperatures (4° C.) in steps 605, 606, 610, and 611. Keeping the solutions at low temperatures can prevent deterioration of the samples and the progression of unnecessary reactions.

[0173] In step 605, it is preferable to set the temperature during division to the same temperature as that in the denaturation step, since this prevents unnecessary extension reactions and suppresses the occurrence of artifacts.

[0174] In step 605, if the temperature during division is set to the same as the extension temperature, unnecessary extension reactions will occur, but nonspecific amplification can be suppressed. This is also preferable because it avoids inactivation of the polymerase and fluorophores. The impact of unnecessary extension reactions on the analytical accuracy of STR-CE can be ignored.

[0175] In step 605, the temperature during division may be set between the extension temperature and RT. This is convenient because precise temperature control is not required. Since sufficient amplicons exist at the completion of m cycles of PCR, the effects of non-specific amplification can be ignored.

[0176] In step 605, the shorter the time required for division, the better. If the division takes a long time, there is a possibility that artifacts will increase. Furthermore, the longer the division takes, the more likely it is that various biomolecules, such as polymerase, will be inactivated.

[0177] 14, in this embodiment, the analysis system 101 does not perform any analysis other than electrophoretic analysis on each reaction solution (including a portion of PCR reaction solution m and at least a portion of PCR reaction solution n) prior to electrophoretic analysis. This simplifies the configuration of the device, and, for example, eliminates the need for an additional optical system.

[0178] [Volume of PCR reaction solution and electrophoresis reagent] The volume of PCR reaction solution used in this flow channel device is 1 μl to 200 μl, and more preferably 10 μl to 50 μl. A smaller volume of solution allows for more accurate and faster temperature control. On the other hand, a larger volume of solution can accommodate more purified DNA, making it easier to achieve higher sensitivity. Also, a larger volume of solution eliminates the need for highly accurate solution measurement during division.

[0179] An example of the volume of liquid when divided is shown in Table 2. The volume of liquid actually measured does not have to be the exact value in the table; the median value when measuring multiple times can be the value in the table.

[0180]

[0181] Assume that the total volume of PCR reaction solution in step 502 is a μl, the volume of solution removed during cycle m in step 503 is b μl, the migration reagent mixed with PCR reaction solution m in step 504 is c μl, and the migration reagent mixed with PCR reaction solution n in step 507 is d μl. Table 2 above shows examples of suitable relationship equations or set values ​​for a through d. To efficiently introduce the amplified products and size standards into CE, the ionic strength of the migration sample must be sufficiently low. Therefore, it is preferable to mix the migration reagent with a volume that is 2, 5, 10, 20, etc. times larger than the volume of the PCR reaction solution.

[0182] The PCR division can be set so that PCR reaction solution m and PCR reaction solution n are equal. If the amounts of PCR reaction solution m and n are similar, stable liquid transfer can be achieved when transferring the electrophoresis reagent to the CE section.

[0183] The PCR division can be set so that PCR reaction solution m is smaller than PCR reaction solution n. The smaller the volume of solution taken out during division, the smaller the variation in volume when performing PCR nm, allowing for more reproducible preparation of amplified product n.

[0184] Set 1 is an example of the solution volume when the amount of electrophoresis reagent is set to 10 times the amount of PCR reaction solution.

[0185] Set 2 shows an example of the amount of solution when the PCR reagent volume is 15 μl, 5 μl of solution is taken out at the mth cycle, and the entire amount of PCR reaction solution is mixed with the electrophoresis reagent at the nth cycle. In this example, the analysis system 101 transports the entire amount of PCR reaction solution from the PCR chamber to the outside of the PCR chamber at the end of nth thermal cycle. This eliminates the need for a measuring device and simplifies the flow path configuration.

[0186] In the liquid transfer procedure shown in FIG. 16 , migration reagent is left in the dispensing chamber 320. If the amount of migration reagent in the migration reagent reservoir 310 is sufficiently large relative to the dispensing chamber 320, the effect on analytical accuracy and range of the migration reagent left in the dispensing chamber 320 can be ignored. Furthermore, if a flow path 322 is provided as an airline between the valve 337 and the dispensing chamber 320 on the flow path 315, or an air reservoir 334 is provided, the entire amount can be swept into the dispensing chamber 320 and the mixing chamber 327. In the case of a large-volume configuration, almost the entire amount of PCR reaction solution m in the dispensing chamber 320 is sent to the mixing chamber. Furthermore, it is preferable to store extra migration reagent in the reagent reservoir, taking into account the fact that some migration reagent will be left behind in the dispensing chamber 320.

[0187] Set 3 is an example of the amount of solution stored in the migration reagent reservoir 310 in a larger amount than necessary, in anticipation of e μl of migration reagent remaining in the dispensing chamber 320 .

[0188] Set 4 is an example of the amount of solution stored in the migration reagent reservoir 310 when the volume of the dispensing chamber 320 is b μl and it is anticipated that b μl of migration reagent will remain in the dispensing chamber 320.

[0189] [Details of division method] Since variations in the amount of solution during division tend to narrow the effective analysis range of the analysis system, it is desirable to divide the PCR reaction solution with high accuracy. To achieve high-accuracy solution division, it is possible to incorporate functions such as setting an appropriate pressurization pressure and pressurization time in advance, pushing out the PCR reaction solution with a specified volume of liquid or gas, using a liquid level detection sensor, or pushing out the PCR reaction solution into a specified volume of dispensing chamber 320. It is also possible to combine multiple extrusion and metering methods.

[0190] Measurement may be performed when dispensing PCR reaction solution m in step 503 and when dispensing 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 electrophoresis reagent, measurement only needs to be performed in step 503. In other words, the number of measurements may be the number of aliquots minus 1. Similarly, if measurement is performed twice, since there is a specified amount of liquid in the PCR chamber, an additional p PCR thermal cycles may be performed on the reaction solution remaining in the PCR chamber, and the CE of the third aliquot reaction solution after n+p cycles of PCR reaction may be measured.

[0191] When removing the PCR reaction solution from the PCR chamber, pressure can be applied to the PCR chamber with a pump to send the solution to the dispensing chamber. Also, if the valves are closed during PCR heating, internal pressure is applied, so it is possible to set up so that the solution moves to the dispensing chamber by internal pressure without opening valves 326 and 323 in step II.

[0192] After m cycles are completed in step 502, new PCR reagents may be used to push out the PCR reaction solution m, and a specified amount may be transported to the dispensing chamber 320 or the mixing chamber 327 in step 503. This division method may also be implemented by sealing PCR reagents instead of air in the air reservoir 332 in Figure 10. Pushing out with a solution has the advantage of making it easier to specify the volume to be pushed out. Pushing out with a PCR reaction solution also has the advantage of preventing the adverse effects of air bubbles or oil on the reaction. On the other hand, there is a possibility that new PCR reagents may be sent to a later stage, which introduces a new factor in addition to the above-mentioned factors (1) to (8) that contribute to the variability of the analysis range. Furthermore, because the PCR reaction solution is diluted, the PCR reaction solution remaining in the PCR chamber becomes diluted after m cycles, requiring more PCR cycles. Furthermore, adding room-temperature reagents will lower the temperature of the PCR chamber. Furthermore, initial denature must be performed again in step 505. Therefore, pushing out with a PCR reaction solution results in a slightly longer PCR reaction time. In addition, increasing the number of cycles and performing initial denature again contribute to the occurrence of artifacts. Note that it is also possible to solve some of the above problems by performing initial denature of the PCR reaction solution used for extrusion before step 503.

[0193] As shown in Figure 10, air reservoirs 332 and 333 may be installed in the flow path device 104, and when m cycles are completed in step 502, the PCR reagent may be pushed out by the air chamber, and a specified amount may be transported to the dispensing chamber 320 or the mixing chamber 327 in step A3.

[0194] Instead of pushing with air or PCR reagents, the PCR reaction solution can be pushed with oil. This division method can also be implemented by sealing oil instead of air in the air reservoir 332 in Figure 10. Any oil can be used as long as it does not affect the PCR reaction. For example, it can be silicone oil, mineral oil, Fluorinert oil, or a mixture of multiple oils. Pushing with oil has the advantage that, like pushing with reagents, the pushed volume is easily defined and, unlike pushing with PCR reaction solution, it does not mix with the PCR reaction solution. On the other hand, if the PCR reaction product is mixed with oil and flows downstream, it may interfere with CE measurement. When PCR reaction solution m is extracted, it can be separated using a separation membrane or centrifugation to leave the oil in the chamber.

[0195] [Details of the Metering Mechanism] When introducing the PCR reaction solution into the dispensing chamber 320 during division, if air or bubbles from the PCR chamber enter instead of the PCR reaction solution, the measurement accuracy will decrease. Therefore, it is preferable that the dispensing chamber 320 and the channel 315 be designed to prevent air from entering. An example of a structure for preventing air from entering is shown in Figure 17. As shown in Figures 17(a) and 17(b), the channel 315 branches off from the PCR chamber 304 below the liquid level 701 of the PCR reaction solution 335 in the direction of gravity. Because air accumulates above the PCR chamber 304, the amount of air introduced into the channel 315 during division can be minimized. Furthermore, it is desirable to design the liquid volume and chamber shape so that the liquid level 701 is located above the connection between the PCR chamber 304 and the channel 315 in the direction of gravity upon completion of division. As shown in Figure 17(b), the liquid level 701 may be located above the valve 326 or 323 in the direction of gravity. In this case, it is possible to minimize or completely eliminate air bubbles entering the PCR chamber 304. The PCR reaction solution 335 located above the valve 326 or 323 does not need to be subjected to the PCR reaction. However, in this case, unreacted reagents may be mixed into the CE measurement, reducing sensitivity.

[0196] 17(c), the channel 315 may be connected to the bottom of the PCR chamber 304. This configuration has the advantage of preventing air from entering, but the channel 315 is somewhat long and tends to be more susceptible to liquid loss.

[0197] A structure for removing air bubbles may be provided in flow channel 315. Furthermore, a hydrophilic filter that does not allow air to pass through may be provided in the flow channel connecting PCR chamber 304 and dispensing chamber 320 on flow channel 315.

[0198] The dispensing chamber 320 may have a metering function. For example, FIG. 18 shows an example of a dispensing chamber 320 with a metering function. The channels 315 and 328 connected to the dispensing chamber 320 are equipped with valves 324 and 337. Valve 337 may be used not only for metering but also to prevent liquid from spilling into the dispensing chamber 320 during the PCR reaction and to prevent the migration reagent and migration sample from flowing back into the PCR chamber 304 after dispensing. Valve 324 may also be provided not only for metering but also to prevent the migration sample from flowing back after being transported to the mixing chamber. The volume of the dispensing chamber may correspond to the volume of the liquid to be divided and measured from the amplification product m. The dispensing chamber may be spherical or cylindrical, rectangular, elongated, or serpentine, or may have an ellipsoid or elliptical cylindrical shape.

[0199] For example, the analysis system 101 may measure a predetermined amount of PCR reaction solution m within the range of 0.1% to 50% by transporting the PCR reaction solution m to the dispensing chamber 320 (measurement unit) after m thermal cycles have been completed. In this way, an additional measurement process is not required.

[0200] An example of a measurement mechanism is shown in Figure 19. Measurement may be performed by combining multiple mechanisms such as those shown in Figure 19. Measurement mechanisms not shown may also be used. More robust measurement may be achieved by combining multiple measurement mechanisms.

[0201] Figure 19(a) shows an example of a metering mechanism, which is equipped with a vent filter 702 and a flow path 703 that connects to flow path 328. Flow path 703 is connected to flow path 328 on the side of dispensing chamber 320 rather than valve 324. A suitable material for vent filter 702 is a hydrophobic porous filter made of PP, fluorine-based resin, or the like. Steps I to III in Figure 20 show an example of the operation of this metering mechanism.

[0202] Step I: Pressure is applied to the PCR chamber 304, and the PCR reaction mixture is sent into the dispensing chamber 320. Air present in the dispensing chamber 320 and before and after it is released from the vent filter 702 via the flow path 703.

[0203] Step II: Since the amplification products and migration reagents cannot pass through the vent filter 702, the PCR reaction solution is measured to the volume of the dispensing chamber 320 and the flow paths before and after it.

[0204] Step III: The valve 337 is closed and the valve 324 is opened. The PCR reaction mixture in the dispensing chamber 320 is pumped into the mixing chamber 327 by the electrophoresis reagent or air.

[0205] As shown in Figure 19(b), a flow path resistance 704 may be provided on the flow path 328. The flow path resistance 704 may be a hydrophobic filter made of PP, fluororesin, or the like. The hydrophobicity of the flow path surface at the flow path resistance 704 may be higher than that of the flow path 328. The flow path width may be narrowed at the flow path resistance 704, or an obstacle may be installed. The flow path resistance 704 may also have a sudden increase in width. The flow path resistance may be provided using the principle of a capillary stop valve. Air can easily escape until the flow path resistance 704 comes into contact with the liquid. Figures I to III in Figure 21 show an example of the operation of this metering mechanism.

[0206] Step I: Pressure (A kPa) is applied to the PCR chamber 304, and the PCR reaction mixture is sent into the dispensing chamber 320. Air present in the dispensing chamber 320 and before and after it can be smoothly released through the flow path resistance 704.

[0207] Step II: At an applied pressure of A kPa, the PCR reaction solution cannot exceed the flow resistance 704, or it takes a long time for the PCR reaction solution to exceed the flow resistance 704, so that a specified amount of PCR reaction solution can be metered into the dispensing chamber 320.

[0208] Step III: The valve 337 is closed, and B kPa (where B>A) is applied to the dispensing chamber 320 using the migration reagent or air. The PCR reaction mixture in the dispensing chamber 320 can be sent to the mixing chamber 327.

[0209] As shown in FIG. 19(c), a liquid level detection sensor 705 may be attached to flow channel 328 or dispensing chamber 320, and the structure may be such that the transport of liquid is stopped when dispensing chamber 320 is filled with a specified amount of liquid.

[0210] An example of a simple measuring mechanism that does not use a liquid level detection sensor, vent filter, or flow path resistor is shown in Figure 22. However, this method may be combined with a liquid level detection sensor, vent filter, flow path resistor, etc. to provide a robust dispensing mechanism.

[0211] Step I: There is a PCR chamber 304 containing a PCR reaction solution 335, and m cycles of PCR reaction are carried out in the PCR chamber 304 with all valves closed.

[0212] Step II: After m cycles of PCR reaction are completed, the valves 326 and 323 are opened, and the pressure inside the PCR chamber 304 is set to atmospheric pressure (=100 kPa).

[0213] Step III: A pressure of 100 kPa is applied through the flow channel 319 and the flow channel 330 .

[0214] Step IV: Valve 337 is opened, and a portion of the solution in PCR chamber 304 is transferred to dispensing chamber 320. When the air in dispensing chamber 320 is compressed to about half its original volume, the pressures in dispensing chamber 320 and PCR chamber 304 are balanced, and the transfer of the solution stops.

[0215] Step V: The pressure in dispense chamber 320 is returned to atmospheric pressure by closing valve 337 and opening valve 324.

[0216] In this method, when transferring a solution from PCR chamber 304 to dispensing chamber 320, the amount of liquid that enters dispensing chamber 320 can be determined by the pressure applied to dispensing chamber 320. When dispensing chamber 320 has a volume V1 and is filled with air at pressure P1, if a volume V2 of liquid enters the dispensing chamber, the pressure changes according to Boyle's law: P2 = P1 * V1 / V2. When P2 balances with the applied pressure, the solution stops. In step I, if the pressure in the dispensing chamber is 100 kPa, when the volume of dispensing chamber 320 is reduced to half, the pressure in both the dispensing chamber and the PCR chamber becomes 200 kPa, and the forces are balanced. Therefore, PCR reaction solution 335 equivalent to approximately 50% of the volume of the dispensing chamber can be dispensed. Note that the pressure shown here is merely an example; measurements can be performed by appropriately setting the volume of the dispensing chamber, the space in the flow path before and after it, and the amount of liquid to be measured. The disadvantage of this method is that the valve tends to require high pressure resistance. If the volume of the dispensing chamber 320 is set larger than the amount to be measured, the pressure required for dispensing and measuring will decrease, but there is a disadvantage that the amount of residual liquid will increase when it is subsequently pushed out by the migration reagent.

[0217] [About measurement accuracy] In CE analysis, if there is variation in peak intensity when the same sample is measured multiple times, the effective analysis range will narrow. If the introduction of segmented PCR causes large variations in analysis intensity, the effective analysis range will narrow, which is undesirable. If the introduction of segmented PCR causes large variations in the mixing ratio of the electrophoresis reagent and PCR reaction solution, this will be reflected in the variation in peak intensity, narrowing the effective analysis range.

[0218] As shown in Figure 14, we conducted experiments to determine the extent to which changes in the mixing ratio of the PCR reaction solution and the electrophoresis reagent affect the CE peak intensity. Figure 23 shows the CE intensity when the ratio of PCR reaction solution to electrophoresis reagent is changed. The variation in peak intensity between capillaries was normalized by the average intensity of a size standard that did not contain the PCR reaction solution, which was measured separately. The peak intensity of the size standard monotonically decreased as the ratio of the PCR reaction solution increased. On the other hand, the peak intensity of the amplified product monotonically increased, and the variation in peak intensity was limited in response to changes in the liquid volume.

[0219] In CE measurements, when the amplified product is drawn into the capillary, a voltage is applied to the electrophoresis sample and the capillary to perform electric field injection. In electric field injection, the amplified product introduced into the capillary is injected competitively with other ions and nucleic acids. When the voltage and injection time are constant, the relationship shown in Equation 1 holds (β is the current value, and α is a constant). where: k0, C0: mobility and concentration of ions contained in formamide k1, C1: mobility and concentration of size standard DNA k2, C2: mobility and concentration of salts, primers, and dNTPs contained in the PCR reaction solution k3, C3: mobility and concentration of amplified products contained in the PCR reaction solution

[0220] Taking the standard case where 1 μl of PCR reaction solution is mixed with 10 μl of electrophoresis reagent as the standard, if the amount of PCR reaction solution varies by γ μl, Equation 1 can be expressed as follows:

[0221] The injection amount k1*C1 of the size standard can be summarized as follows, and decreases monotonically with increasing γ.

[0222] On the other hand, the injection amount of the amplified product k3*C3 is obtained by rearranging Equation 1 as follows, and increases monotonically with the volume of the PCR reaction solution.

[0223] The PCR reaction solution contains many salts, primers, dNTPs, etc., and the injection volume is nearly saturated. Therefore, even if the ratio of PCR reaction solution to electrophoresis reagent increases slightly, the fluctuation in peak intensity is limited. Even if the PCR reaction solution ratio varies by ±20% from the standard conditions (PCR reaction solution:electrophoresis reagent = 1:10), the fluctuation rate of peak intensity remains within 10%. Furthermore, if the target condition is a slightly more concentrated PCR reaction solution than the standard conditions (PCR reaction solution:electrophoresis reagent = 1.6:10), the fluctuation rate of peak intensity remains within 10% even if the PCR reaction solution ratio varies by ±50% from the target ratio. The results of this experiment suggest that even if the dispensing accuracy of split PCR is somewhat poor, the fluctuation in peak intensity of the amplified product due to variations in the mixing ratio of PCR reaction solution and electrophoresis reagent is limited.

[0224] In some cases, it may be preferable to adopt a simple, low-cost measuring mechanism, even if the accuracy of the measuring mechanism is somewhat low. When using a measuring mechanism with low accuracy, as described above, it is preferable to select a solution composition that minimizes the effect of fluctuations in the mixing ratio of the PCR reaction solution and the electrophoresis reagent on peak intensity. This can be achieved by adding salt to the electrophoresis reagent, or by salts, primers, dNTPs, etc. contained in the PCR reaction solution. Furthermore, by performing measurements under conditions where the mixing ratio of the electrophoresis reagent and the PCR reaction solution is slightly higher than the PCR reaction solution, and measuring at a state closer to saturation, the effect of fluctuations on the ratio is reduced. In this case, it is preferable to mix a slightly larger amount of size standard than the standard mixing ratio in the electrophoresis reagent so that the peak intensity of the size standard contained in the electrophoresis reagent reaches the required level for measurement.

[0225] 《How to set up a split PCR protocol》 [Principle of expanding the analytical range by split PCR]

[0226] In order to analyze target DNAs at various concentrations contained in a sample, PCR reaction solutions m and n containing amplification products at different concentrations are each subjected to electrophoresis analysis. By presetting m and n within an appropriate range according to the concentration range of the target DNA that may be contained in the sample, an appropriate DNA profile can be obtained for a wider range of DNA amounts than when preparing one amplification product for CE analysis without a decrease in sensitivity.

[0227] FIG. 24 shows the principle of expanding the analysis range by split PCR. The horizontal axis in FIG. 24 indicates the amount of DNA input into the PCR, and the vertical axis indicates the concentration of the amplification product or the peak intensity in CE. 801 is the upper limit of the detection intensity of CE or the upper limit of the concentration of the amplification product that can be accurately amplified by PCR, and 802 indicates the lower limit of detection of CE. In the figure, the concentration of the amplification product is shown as increasing linearly with respect to the amount of DNA input, but the actual amplification is sigmoidal, and it is considered that the amplification product approaches a plateau when it reaches a certain amount. Assume that at a certain number of PCR cycles m, the range of DNA amounts that can be correctly analyzed is between a lower limit a and an upper limit b. Let the analysis range of STR-CE that can be analyzed in one cycle number be 803. Also, assume that at a certain number of PCR cycles n, the range of DNA amounts that can be correctly analyzed is between a lower limit c and an upper limit d. At this time, if a < d, the analysis range can be expanded from b / a or d / c to b / c. Let the analysis range of STR-CE when expanded by split PCR be 804. 804 is the range of the amount of DNA in which either m or n falls within the analysis range of STR-CE, and in principle, there is no amount of DNA that spills out of the analysis range within the range. Hereinafter, the analysis range (b / a) at each cycle number is substantially constant and changes by 2 x times with respect to the cycle number difference x. At this time, the interval between m and n is limited to the range of Equation 2 below.

[0228] An example of the case where this is satisfied is shown in FIG. 24(1). For example, when the analysis range b / a with respect to the amount of DNA introduced into the PCR is 80 times, n - m being 6 or less is appropriate, and the expansion rate is 64. The expansion rate is (b / a) y-1Although it can be increased, it is considered that a smaller number of divisions can achieve stable liquid delivery on a simple flow path device. Hereinafter, mainly the case of setting m and n and analyzing amplification products m and n will be described. On the other hand, in a similar setting method, l may be set for three divisions and k may be set for four divisions.

[0229] When c or d is less than 0.2 ng or 0.1 ng, the variation in the intensity balance between peaks becomes large. Therefore, it is preferable to set a cycle number larger than the cycle number set by Equation 2 and make the interval between m and n narrower than the maximum cycle number defined by Equation 2. Hereinafter, unless otherwise specified, the upper and lower limits of STR-CE analysis change by a factor of 2 with respect to the cycle number difference x. x times. <000,0470><000,0471> Fig. 24(2) shows the analysis range when the interval between m and n does not satisfy Equation 2. In this case, the lower limit a that can be analyzed by m exceeds the upper limit d that can be analyzed by n, and correct DNA identification cannot be performed for the DNA concentration located between a and d. If a > d and there is no sample or the frequency is extremely low between a and d, the numerical values of m and n can be set so that the relationship a > d holds. However, since the DNA amount of the actually input sample is often unknown, it is preferable to satisfy Equation 2. <000,0472><000,0473> When covering extremely low-concentration DNA with the cycle number of n, a stochastic effect occurs. Therefore, it is desirable to set the difference to be less than the upper limit of the difference between n and m specified by Equation 2. <000,0474><000,0475> Since there are factors that cause variations in multiple analysis results in STR-CE analysis, it is necessary to have an overlap between the DNA amount range that can be covered by STR-PCR with m cycles and the DNA amount range that can be analyzed by STR-PCR with n cycles, taking into account the variations. If there is no overlap, there will be a DNA amount for which the analysis fails even though it is within the analysis ranges of m and n. That is, it is necessary to set m and n so that a < d always holds for the assumed analysis variations. Hereinafter, it is assumed that m and n are set taking into account the variations of the analysis system. <000,0476>

[0233] [n is set according to the CE analysis result of m] The value of m may be preset in the device.

[0234] In the case of an analytical system in which step 505 is started or ended after step 504, n may be set according to the measurement result of electrophoresis sample m.

[0235] n may be set based on the following conditions: (1) when no peaks of the amplification product m are detected; (2) when peaks are detected and some of the peaks are below the AT; or (3) when peaks are detected and some of the peaks are already saturated or exceed the IAP threshold.

[0236] In the case of (1), if the analytical range of STR-CE is x, an additional nm cycle (nm≦log(x), where x is the analytical range of STR-PCR) may be performed. In this case, it is preferable to use the maximum value of nm that satisfies Equation 2.

[0237] In the case of (2), if the CE saturation intensity is z relative to the maximum intensity y of the detected peak, an additional PCR defined by nm cycles (nm≦log(z / y)) may be performed.

[0238] In case (3), additional PCR is not required.

[0239] [How to set the interval between m and n taking into account the intensity ratio of the sample to be analyzed] If the CE analysis takes a long time, waiting for the completion of m cycles of electrophoresis as described above may result in too long a wait time for the nm PCR reaction, which may increase artifacts or cause the PCR reaction to fail. Therefore, m and n can be set in advance.

[0240] In STR-CE, homozygous and heterozygous loci are mixed together, so even in an ideal analysis system in which all amplification efficiencies, CE injection efficiencies, and fluorescent dye emission efficiencies are equal, there will be a 1:2 difference in intensity between the heterozygous and homozygous peaks.

[0241] In actual STR-CE, there are differences in amplification efficiency depending on the gene locus and DNA length, differences in CE injection efficiency, and differences in luminescence efficiency between dyes, so even if the DNA amount is adjusted appropriately, there will be differences in intensity between peaks of 1:2 to 1:20. Note that AT values ​​may differ between dyes. In such cases, the analytical range must be determined taking into account differences in AT settings.

[0242] When DNA comes from multiple people, the ratio of alleles present varies from 1:2 to 1:1000.

[0243] When DNA is degraded, the allele abundance ratio varies from 1:2 to 1:1000. Typically, DNA from short loci or alleles is amplified more, while the peak intensity of DNA from long loci or alleles tends to decrease.

[0244] If PCR is inhibited, shorter DNA fragments tend to be amplified more, resulting in smaller peak intensities for longer DNA fragments. In such cases, the peak intensity difference can be 1:2 to 1:1000.

[0245] When the input DNA amount is less than 0.1 ng, the variation in intensity between peaks tends to increase. In this case, the effective analytical range becomes even smaller. Therefore, the effective dynamic ranges that can be covered by m and n are not necessarily the same.

[0246] In the following discussion, we will assume that the factors that determine the intensity ratio between peaks are due to differences in the abundance ratio of alleles contained in the DNA input to STR-PCR. Differences in amplification efficiency, CE injection efficiency, and luminescence efficiency also have an effect, so although these differences should be taken into account, they will be omitted and will be included in the discussion of the abundance ratio between alleles.

[0247] The effective analytical range of STR-CE when there is a difference in the abundance ratio of the alleles to be analyzed is explained using Figure 25. The amplification product is assumed to contain allele α and allele β. However, the amounts of allele α and β are assumed to be α>β before or after amplification. Plot 805 is a plot of the amount of allele α in the amplification product or the CE peak intensity against the amount of input DNA. Plot 806 is a plot of the amount of allele β in the amplification product or the CE peak intensity against the amount of input DNA. Plots 805 and 806 are not necessarily linear. Amplicon α can be analyzed in the range of DNA input amounts a to b. Furthermore, amplicon β can be analyzed in the range of DNA input amounts c to d. If PCR amplification is linear in the concentration range a to b, The effective analytical range 803 of STR-CE, where both amplicon α and amplicon β are correctly detected, is given by the following formula 3: b / a (range 807) may correspond to the dynamic range of CE, or may be the analytical range of STR-CE in which peak α can be detected.

[0248] As shown in Equation 3, the effective analytical range becomes smaller as the intensity ratio of the DNA to be analyzed increases.

[0249] Figure 26 shows a table showing the numerical values ​​corresponding to 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 nm setting. For example, if 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 will be analyzed. Conversely, if the intensity ratio of the allele set to be analyzed is greater than the ratio shown in Figure 26, this means that a concentration within the analysis range will appear at which the largest or smallest peak will violate the upper or lower analytical limit. For example, as shown in Figure 27(1), there is no problem if both α and β are detectable at the concentration. However, as shown in Figure 27(2), a situation may occur in which only α can be detected and β cannot.

[0250] Peaks with too large a peak intensity ratio may not be analyzed. Examples are shown below. STR-CE contains stutter peaks, and peaks with a peak intensity ratio of more than 1:20 within a single locus are difficult to distinguish from stutter peaks. Therefore, peaks with an intensity ratio greater than 1:20 or 1:40 within a single locus may not be analyzed. Degraded DNA may also have a reduced total DNA content, so peaks with peak intensities greater than 1:20, 1:40, or 1:100 may not be analyzed. As mentioned above, in electropherograms with extremely large peak intensity ratios, small peaks are insignificant. Therefore, an interlocus PHR threshold may be set, which defines the minimum peak intensity relative to the maximum peak intensity to be analyzed. When setting the m and n intervals using Figure 27, it is preferable to set the abundance ratio of the allele to be analyzed slightly lower than the interlocus PHR threshold.

[0251] [How to set n] If partial profiles are also to be analyzed, 30 to 34 cycles is appropriate.

[0252] A peak derived from one copy of DNA can be detected by setting the cycle number to 36.

[0253] Setting the cycle number to 36 or more is not recommended because, although artifacts (especially stutters and drop-ins) increase when the cycle number exceeds 36, the probability of detecting peaks derived from individuals does not improve.

[0254] If partial profiles are not the subject of analysis, 30 or 29 cycles or less is appropriate.

[0255] For samples containing the smallest amount of DNA, the smallest number of cycles resulting in the fewest number of error calls is preferably selected, which may be determined experimentally.

[0256] The n cycles may be set to the minimum number of PCR cycles at which the peak of the amplicon derived from one copy always exceeds AT.

[0257] The n cycles may be set to the minimum number of PCR cycles that ensures that the amplification product derived from 20 copies of genomic DNA will have a full profile.

[0258] The n cycles may be set to the maximum number of PCR cycles at which the intensity ratio of stutter peaks and other peaks resulting from amplification errors (excluding IAP) does not exceed a threshold value.

[0259] After clarifying the detection limit at a specific cycle number through experiments or calculations, n can be set under the assumption that the detection limit increases or decreases by a factor of 2 with each cycle. However, if the amount of DNA is too small, the stochastic effect will cause greater variability in peak intensity, so it is more preferable to set a cycle number with a slight margin.

[0260] You can set n independently of m using the method described above. However, if the interval between m and n is too large, there is a risk that some DNA will remain unanalyzed.

[0261] If m is set first, it may be set from the nm difference that covers the intensity ratio of the peak to be analyzed and the value of m, as shown in FIG.

[0262] When the analytical range 803 is known, it is preferable to set n with a cycle number difference of log2 (analytical range 803) or less with respect to m.

[0263] The number of cycles may be set with a margin, taking into account fluctuations in various analytical systems and peak intensity ratios.

[0264] [How to set m] In DNA testing, the amount of DNA input to PCR almost never exceeds 1.5 μg, so even if the number of cycles is set to less than 20, the number of loci that can be correctly detected will not increase. Therefore, it is preferable to set the number of cycles to 20 or more.

[0265] It is preferable to evaluate the amount of DNA input to PCR from a sample containing the maximum amount of DNA that can be brought into the analysis system, and set the maximum PCR cycle that does not produce an OS or IPA flag when the DNA is subjected to STR-CE analysis.

[0266] If the maximum amount of DNA to be input into STR-CE is determined, it is more preferable to set the maximum number of cycles that can satisfy 201 to 207 in Figure 2 when that amount of DNA is input.

[0267] The number of cycles may be set with a margin, taking into account fluctuations in various analytical systems and peak intensity ratios.

[0268] The lower limit of detection, the amount of DNA input to PCR, and the upper limit of detection at a specific number of cycles can be determined, and m can be set under the assumption that the lower and upper limits increase or decrease by a factor of two with each cycle.

[0269] You can set m independently of n using the method described above. However, if the interval between m and n is too large, there is a risk that some DNA will remain unanalyzed.

[0270] When n is set first, it may be set from the difference in nm that can cover the intensity ratio of the peak to be analyzed and the value of n, as shown in FIG.

[0271] When the analytical range 803 is known, it is preferable to set m with a cycle number difference of log2 (analytical range 803) or less.

[0272] Even if the interval between m and n is 2 or 3, the expansion rate of the analytical range is only 4 or 8 times. To perform highly robust DNA testing, it is desirable to expand the analytical range by at least one order of magnitude. Therefore, it is preferable to set the interval between m and n to 4 or more. As mentioned above, in a suitable split PCR, the maximum setting value for n is 36 and the minimum setting value for m is 20, so the minimum setting value for n is 24 and the maximum setting value for m is 32.

[0273] [Setting m and n from a table] Figure 28(1) shows the analytical range expansion ratio when m and n are set. You can use this table to determine the interval between m and n to obtain the required expansion ratio. However, this table does not take into account changes in the analytical range that depend on the DNA concentration range, such as the Stochastic effect. Also, if the original analytical range is larger than the expansion ratio, it is inappropriate because it will result in a range of DNA that cannot be analyzed, as shown in Figure 24(2). Furthermore, when analyzing DNA with large differences in abundance ratios between alleles, setting the expansion ratio to the limit of the original analytical range will result in alleles that cannot be analyzed.

[0274] Figure 28(2) shows the analytical range (number of digits) expanded by split PCR. In a typical STR-CE, 0.75 ng to 48 ng (1.8 digits) of genomic DNA can be analyzed with 25 PCR cycles. However, this range varies depending on the individual's DNA, the quality of the DNA, and the CE measurement system. Therefore, this range must be evaluated for each measurement system. Variation must also be taken into account. Based on these results, the number of digits in the analytical range when m and n are changed, as well as the lower analytical limit for n cycles and the upper analytical limit for m cycles are shown here. The stochastic effect when the amount of DNA is reduced is not taken into account. All are 2 for the number of cycles. n The table was created assuming that m and n change depending on the analysis range. You can create a table like this and determine m and n from the appropriate upper and lower limits and analysis range. However, if the expansion rate of the analysis range exceeds 1.8 digits in this case, a range of DNA amounts that falls outside the analysis range will appear in the section considered to be the analysis range, as shown in Figure 24(2).

[0275] The dynamic range of a typical CE is 2000 or less. Even in ideal DNA testing, the peak intensity ratio is more than double, so it is desirable that the difference between n and m be 9 or less.

[0276] The dynamic range of more powerful CE is 4000 or less. In many DNA testing, the peak intensity ratio is 4 times or more, so it is desirable that the difference between n and m is 9 or less.

[0277] As described above, in one preferred range, m is equal to or greater than 20 and equal to or less than 32. In another preferred range, n is equal to or greater than 24 and equal to or less than 36. In another preferred range, n is 4 to 9 more than m.

[0278] [Timing of Division] As shown in Figure 14, a final extension step can be performed after m thermal cycles, followed by division, and then final extension can be performed again after n thermal cycles. In this case, for example, the analysis system 101 holds PCR reaction solution m at a constant temperature between 50°C and 80°C for 1 to 20 minutes, and then removes a portion. With this configuration, only one heater is required around the PCR, simplifying the structure of the equipment and flow path device.

[0279] After m thermal cycles, the mixture can be divided and then the final extension step can be performed.

[0280] Figure 29 shows an excerpt of how to use the analysis system 101. Explanations of parts common to Figure 14 may be omitted. Steps 601 to 612 shown in Figure 29 may correspond to an example of detailed steps from step 404 (amplification of the sample) to step 405, and steps 601 to 612 shown in Figure 29 may be a process independent of steps 404 to 405.

[0281] The operation shown in FIG. 29 can be performed by the flow channel device 104 shown in FIG. 6. In FIG. 6, a heating unit 318 is installed in contact with the dispensing chamber 320. After m cycles are completed in step 603, the mixture is divided in the dispensing chamber 320 in step 605, and a heater final extension step is performed in step 604. The PCR reaction solution remaining in the PCR chamber may be thermal cycled nm times in step 608 in parallel with step 604, or may be performed at different times. After n thermal cycles, a final extension step may be performed on product n in a holding chamber. That is, in the example shown in FIG. 29, the analysis system 101 holds a portion of the PCR reaction solution m at a constant temperature between 50°C and 80°C for 1 to 20 minutes.

[0282] The advantage of performing this step after division is that product n does not undergo the final extension step twice, which reduces artifacts and does not increase the overall time.

[0283] [Method of providing CE analysis results to users] When CE is performed more than once using split PCR, more than one electropherogram will be generated. Either the two electropherograms or the DNA analysis results may be provided to the user. Two or more electropherograms may be scored to determine which is more suitable for DNA analysis and provided together with the CE analysis results.

[0284] For example, the analysis system 101 may determine which of the results of electrophoretic analysis of a portion of PCR reaction solution m and the results of electrophoretic analysis of at least a portion of PCR reaction solution n is better, and output the better result. Alternatively, it may output information that allows for determining which result is better. Furthermore, it may notify whether or not a full profile is achieved using a portion of the flowchart shown in FIG. 2. Scoring may be performed using the number of loci that meet the criteria shown in FIG. 2, the number of loci that do not meet the criteria, the number of flags that indicate that the criteria are not met, or a comprehensive calculation using an algorithm based on the criteria. Data provided to the user may be intermediate data from the DNA analysis. Only the analysis results for the full profile or the one determined to have the better electropherogram may be provided to the user. This allows for efficient comparison of results. Furthermore, it makes it easier for non-experts to select the appropriate results when receiving data from the system.

[0285] If both analyses are unsuccessful, you can request a peer review.

[0286] The two data sets may be combined for DNA analysis. In particular, in the case of DNA analysis with a large peak intensity ratio, it is possible that the allele showing the minimum intensity at m is less than AT, and the allele showing the maximum intensity at n is oversaturated. In this case, significant peaks or DNA analysis results can be extracted from m and n, and then combined and provided. In other words, this embodiment can be used to expand the dynamic range of CE.

[0287] The number of times that CE is measured using the analytical system may be two, one, or even three or more times.

[0288] Depending on the condition of the sample, it may be possible to analyze it once, twice, or even more times. Compared to preparing only one PCR product and performing only one CE analysis each time, this method increases the chances of obtaining accurate DNA identification results, and also eliminates the need to perform multiple measurements each time, resulting in lower costs, shorter analysis times, and improved throughput.

[0289] When analyzing only once, the amplification product m or the amplification product n may be measured.

[0290] When measuring twice, the product of cycle m may be measured twice, the product of cycle n may be measured twice, the product of cycle n may be measured after cycle m, the analysis of cycle m may be performed after cycle n, the analysis of cycle n may be started regardless of the status of the data of cycle m, the analysis of cycle m may be started regardless of the status of the data of cycle n, or the analyses of cycle m and n may be performed completely simultaneously.

[0291] If the first run results in poor migration, you can analyze the amplified products again using the same number of cycles. Also, if the first run results in poor migration, you can analyze the products using the other number of cycles. A poor migration here refers to a situation where some or all of the size standard peaks are not detected, or where the peaks get stuck somewhere during the transport of the solution.

[0292] After obtaining the analytical results of either amplification product m or n, the obtained DNA identification results can be compared with the database, and based on the obtained feedback, analysis of the other amplification product can be initiated or continued.

[0293] After obtaining the analysis results for either amplification product m or n, the user may decide whether to analyze the other amplification product. The user may make the decision based on the first data or analysis score, or may decide to perform a second measurement at any time regardless of the data. Product m or n may be held inside or outside the device, and after the measurement is completed, it may be removed from the cartridge or analysis system 101 and measured outside the device. Alternatively, it may be stored in the cartridge for a certain period of time and then re-measured later in the device. During this time, it is desirable that the amplification product be stored refrigerated or frozen. The amplification product may be mixed with the electrophoresis reagent and stored in the form of an electrophoresis sample, or it may be stored in a state before being mixed with the electrophoresis reagent.

[0294] <When analyzing amplification product m first> When analyzing amplification product m first, it may be decided whether to analyze amplification product n according to the flowchart shown in Figure 30. However, the flowchart shown in Figure 30 is just one example, and judgment criteria and branching conditions not shown here may be included. For example, the judgment criteria may change depending on the number of peaks obtained, the number of loci, whether the data is mixed, etc. Furthermore, the judgment criteria shown in Figure 30 may be omitted in part or in whole, or may be replaced with other criteria.

[0295] The electrophoresis results of m may be provided to the user, who may then decide whether to analyze the product n and begin.

[0296] Furthermore, when n are being analyzed simultaneously or in parallel, the analysis may be interrupted midway based on the determination result of the user or the flowchart of FIG.

[0297] Decision set 1: Is there a peak that saturates the CE detection system? If so, do not perform or discontinue analysis of amplification product n.

[0298] Decision set 2: Is a full profile obtained? If so, do not perform or discontinue analysis of amplification product n.

[0299] Decision Set 3: Is the IAP+ flag present? If so, start or continue analysis of amplicon n.

[0300] Decision set 4: Are all peaks at half the OS intensity or less? Or are all peaks at an intensity equal to or less than the OS divided by the amplification factor expected by split PCR? Or are they at an intensity that does not cause saturation when additional PCR cycles are performed nm times? Or are any peak intensities not reached? If so, do not analyze amplification product n or discontinue analysis.

[0301] When the interval between m and n is 2, if almost no peaks are detected in the CE analysis of electrophoretic product m, significant data cannot be obtained from electrophoretic product n, and therefore it is meaningless to analyze n after obtaining the analysis results of m. Similarly, when the interval between m and n is narrow, if the analysis results of m indicate that the DNA amount is too low, there is no need to analyze n. When determining whether the DNA amount is too low, the number of detected peaks or the peak intensity can be used.

[0302] The various judgment sets may use different judgment criteria sets depending on whether each locus is heterozygous, homozygous, mixed, or single.

[0303] If the IAP peak exceeds the threshold, you can perform n-cycle analysis. The IAP peak occurs when the amount of input DNA is too small for the PCR cycles, so it can be reduced by increasing the number of cycles.

[0304] In addition, various thresholds and judgment algorithms may be provided so that if it is determined from the CE analysis results of m that significant CE analysis results will clearly not be obtained even if analysis of n is performed, a decision can be made to discontinue or not perform analysis of n.

[0305] <When analyzing amplification product n first> When analyzing amplification product n first, it may be decided whether to analyze amplification product m according to the flowchart shown in Figure 31. However, the flowchart shown in Figure 31 is just one example, and judgment criteria and branching conditions not shown here may be included. For example, judgment criteria that change depending on the number of peaks obtained, the number of loci, whether the data is mixed, etc. may be envisioned. Furthermore, the judgment criteria shown in Figure 31 may be omitted in part or in whole, or replaced with other criteria.

[0306] The electrophoresis results of n may be provided to the user to decide whether to analyze the product m and then start.

[0307] Furthermore, when m is being analyzed simultaneously or in parallel, the analysis may be interrupted midway based on the determination result of the user or the flowchart of FIG.

[0308] Decision Set 1: Is there a peak that is saturating the CE detection system? If so, start or continue analysis of amplification product m.

[0309] Decision set 2: Is the IAP+ flag present? If not, do not analyze the amplification product m or discontinue analysis.

[0310] In addition, various thresholds and judgment algorithms may be provided so that if it is determined from the CE analysis results of n that significant CE analysis results will clearly not be obtained even if analysis of m is performed, a decision can be made to discontinue or not perform analysis of m.

[0311] In this way, the analysis system 101 performs electrophoretic analysis on one of a portion of the PCR reaction solution m and at least a portion of the PCR reaction solution n in the CE unit 105, and controls the execution of the other electrophoretic analysis based on the results of the electrophoretic analysis of the other. For example, it may be possible to determine whether to start the electrophoretic analysis of one of the PCR reaction solutions based on the results of the other electrophoretic analysis, or, after the other electrophoretic analysis has started, it may be possible to determine whether to continue the other electrophoretic analysis based on the results of the one electrophoretic analysis. In this way, unnecessary or inefficient electrophoretic analyses are omitted, improving the efficiency of the overall process.

[0312] [Both n and m are preset] Two different cycle numbers, n and m, may be preset in the analysis system 101. As mentioned above, it is preferable that m and n are appropriately set depending on the CE analysis range and the amplifiable amount of DNA.

[0313] Depending on the type of sample to be measured, different sets of n and m and analysis protocols may be set, as shown in Table 3.

[0314]

[0315] For example, if a buccal swab contains a relatively large amount of stable DNA, select "buccal swab mode" and perform STR-CE analysis in 26 cycles without splitting (or splitting, but only analyzing one side of the sample, and only if the other amplicon fails). A "DVI sample mode" may also be available for DVI samples (disaster victim identification, DNA testing for identifying bodies from disasters or large-scale terrorist attacks). Because DVI samples are likely to contain a relatively large amount of DNA, for example, the m cycles can be set to [m=25, n=31]. Because DVI samples often contain a relatively large amount of DNA, analysis of m cycles can be initiated first, and then analysis of amplicon n can be initiated or continued using the judgment flow shown in Figure 30. Furthermore, when analyzing DNA obtained at a crime scene, the DNA amount is often low, so the "Casework sample mode" with [m=26, n=31] can be selected. In the case of casework samples, the probability that the amount of DNA contained is extremely small is high, so analysis of n can be started first, and then a decision can be made to proceed with analysis of m or continue using the decision flow shown in Figure 31. In addition, in the case of touch samples, the DNA contained is likely to be very small, or it may have been degraded, resulting in large variations in peak intensity, or it may be mixed DNA. For this reason, it is preferable to set the interval between m and n narrower in "touch sample mode" than in other modes. Furthermore, because there is a high probability that the DNA contained is small, it is preferable to start analysis with sample n.

[0316] If two or more CEs exist in a sample, the analyses for m and n may be performed simultaneously. The preset m and n values ​​and the measurement order may be changed by the user. The values ​​for m and n shown in Table 3 are examples; the preset m and n may be set during development through validation testing to maximize the probability of successful DNA analysis.

[0317] [Key Points of the Present Embodiment] In the above embodiment, the case where both m and n are analyzed each time and the case where only one of m and n is analyzed are described.

[0318] If one of the amplification products prepared using two cycles does not provide data of the required quality, the other amplification product is more likely to provide data of the required quality than if it were prepared using a single cycle.

[0319] Even if one of the amplification products prepared using two cycles fails to provide data of the required quality, another amplification product is prepared, reducing the probability of sample waste. Furthermore, because the probability of sample waste is reduced, the effort of collecting and pretreating samples again is eliminated. Consequently, preparing two samples using this embodiment and analyzing them both shortens the average data acquisition time compared to not using this embodiment.

[0320] If the m-cycle PCR product is divided and the remaining PCR product is not cycled, but only the m-cycle PCR product is subjected to CE analysis, and if the CE analysis results of the m-cycle PCR product do not meet the required quality, nm PCR can be performed on the remaining PCR product to prepare n-cycle PCR product. However, if the PCR product is left to stand for the waiting time for CE analysis of electrophoresis sample m, the activity of the polymerase will decrease, and depending on the standing temperature, a large number of artifacts will increase, so the CE analysis results of the n-cycle PCR product will also not meet the required quality.

[0321] If only n-cycle PCR products are prepared and only n-cycle PCR products are subjected to CE analysis, and the CE analysis results of the n-cycle PCR products do not meet the required quality, the sample will be wasted unless m-cycle PCR products are prepared.

[0322] If the DNA solution used for PCR is divided before PCR, the amount of DNA input to each reaction is reduced, resulting in a decrease in sensitivity. However, once the PCR reaction has progressed to a certain extent (e.g., after four or more cycles), each allele contains 10 or more amplicons, so division does not affect sensitivity.

[0323] It is desirable to divide the mixture after m cycles have been completed. If the mixture is divided before m cycles have been completed and the remaining PCR reaction is performed in each chamber after division so that a total of m cycles have been completed in each chamber, a PCR temperature controller must be installed in each chamber, which increases the size of the apparatus. Furthermore, because there is a possibility of artifacts occurring during division, it is preferable to divide the mixture after the number of cycles has reached as high as possible.

[0324] By preparing multiple electrophoretic samples with different dilution rates after PCR, the effective analytical range of CE can be expanded. This means that the occurrence of CE oversaturation and the frequency with which peak intensities fall below the AT can be reduced. However, problems such as unbalanced peak intensities and peak splitting caused by excessive amplification products cannot be solved by dilution after PCR; these must be addressed before or during PCR. In other words, split PCR is appropriate.

[0325] The number of cycles for dividing is set appropriately for the analytical sample, so the maximum expansion rate of the analytical range can be obtained with the minimum number of divisions. In other words, the measurement time when the analytical range is expanded can be minimized.

[0326] Controlling the amount of DNA before PCR can also expand the effective analytical range. For example, the upper limit of DNA adsorption to a purification membrane can be reduced by adjusting the membrane's capacity or volume or by modifying the purification protocol. However, with typical purification membranes, reducing the volume to lower the upper limit of adsorption can impair solution passage and reduce DNA yield (especially for short, degraded DNA). This limits the control of DNA quantity during the purification stage. Quantifying the DNA amount before PCR can also expand the effective analytical range by controlling the PCR cycle number or changing the dilution ratio. However, the quantification step requires an additional detection system and inevitably increases the complexity of the flow path device. Because of the risk of analytical failure due to quantification errors, split PCR is preferable.

[0327] This embodiment may be combined with post-PCR dilution and control of the amount of purified DNA, which allows for more reliable DNA analysis or a wider range of DNA amounts.

[0328] By removing the PCR reaction solution after each cycle or every other cycle and measuring the CE, DNA analysis can be performed over a wide range of DNA concentrations. However, it is difficult to implement a flow path device that allows for three, four, or five times more extraction and subsequent mixing with electrophoresis reagents. Furthermore, even if two, three, or four amplification products are measured by splitting the PCR reaction solution after each cycle or every other cycle, the expansion ratio of the analytical range is limited to 2x, 4x, or 8x, which is not necessarily suitable for analyzing samples containing various amounts of DNA. Furthermore, when final extension is performed in the same chamber as the PCR chamber in STR-PCR, if the interval between m and n is narrow (e.g., one or two cycles), the final extension time may be excessive depending on the amount of DNA input, resulting in excessive A++ peaks. In particular, in analytical systems without a heating unit 318 and in segmented PCR devices that perform final extension in the same chamber as shown in Figure 14, a narrow interval between n and m is preferable because it results in excessive A++ peaks.

[0329] [Flow Method According to the Number of Capillaries] i) When Only One Capillary Can Be Used Per Sample Figure 32 shows a typical example of the operation timing of the analysis system 101. In this operation timing, the first sample A is loaded in step 401, the sample is processed in the pretreatment cartridge in steps 402 to 404, and the electrophoresis sample m or n is analyzed by CE in step 405.

[0330] The operating procedure shown in Figure 32 is suitable for a case where one CE unit is used for one sample pretreatment unit. This configuration makes the device compact and easy to carry.

[0331] Data may be provided to the user when the first CE analysis is completed in step 405. The user may decide whether to start or continue the analysis of the second electrophoresis sample. Alternatively, the user may decide whether to start or continue the second analysis using the determination method described above. This is because performing two measurements each time doubles the CE measurement time, which does not increase throughput.

[0332] Pretreatment of sample B (steps 401 to 404) may be started at the same time as the pretreatment of sample A is completed. In this case, the cartridge containing sample A is removed, so the electrophoretic sample for which CE analysis has not yet begun may be held in the standby section 321, and the CE sample may be in the middle of electrophoresis.

[0333] ii) When two capillaries can be used for one sample Figure 33 shows a typical example of the operation timing of an analytical system.

[0334] Two CE units may be provided for one sample analysis unit. In this case, it is preferable to provide twice as many CE units as there are sample processing units. Alternatively, the number of CE units may be equal to or greater than the number of sample analysis units.

[0335] As shown in Figure 33(1), CE measurement of electrophoresis sample m and electrophoresis sample n may be performed in step 405 as soon as they are prepared, or as shown in Figure 33(2), analysis may be started simultaneously in step 405 after both are prepared. Also, when there are only as many CE units as sample processing units and no free space, measurement may be performed starting with electrophoresis sample m or electrophoresis sample n.

[0336] Example 1 An analysis system 101 is provided with one flow path device 104 and one CE unit 105 .

[0337] A forensic sample containing an unknown amount of DNA is placed in the lysis chamber 301 (sample inlet) of the flow channel device 104. The processes of steps 401 to 403 are automatically performed within the flow channel device 104.

[0338] After m cycles of PCR are performed in step 502, the m cycles of amplification product is removed in step 503. The sample is sent to CE unit 105, and CE measurement is started in step 504. In parallel with step 320, n cycles of PCR are performed on the PCR reaction solution remaining in PCR chamber 304 in step 505. When the CE measurement in step 504 is completed and the next CE measurement can be started, the n cycles of product are sent to CE unit 105, and CE measurement is performed in step 507.

[0339] Example 2 An analytical system 101 is provided with one flow path device 104 and two CE units 105 .

[0340] A forensic sample containing an unknown amount of DNA is placed in the lysis chamber 301 (sample inlet) of the flow channel device 104. The processes of steps 401 to 403 are automatically performed within the flow channel device 104.

[0341] After m cycles of PCR are performed in step 502, amplification product m is removed from PCR chamber 304 in step 503. The sample is sent to one side of CE section 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 n cycles is sent to CE section 105, and with electrophoresis samples m and n stored in the two capillaries, steps 504 and 507 are started simultaneously.

[0342] 101 Analysis system 102 Computer 103 Database 104 Flow path device 105 CE section 106 User interface 201-207 Judgment criteria 301 Lysis chamber 302 Purification membrane 303 Purification membrane chamber 304 PCR chamber 305 Waste chamber 306 External connection port 307 Pump and valve 308 PCR reagent storage section 309 PCR reagent 310 Running reagent storage section 311 Running reagent 312-314 Reagent storage section 315 Flow path 316 Flow path 317 Heating section 318 Heating section 319 Flow path 320 Dispensing chamber 321 Waiting section 322 Flow path 323-326 Valve 327 Mixing chamber 328-330 Flow path 331 Running reagent storage section 332-334 Air reservoir 335 PCR reaction solution 336 Electrophoresis sample 337-339 Valve 401 Sample input step 402 Dissolution step 403 Purification step 404 Amplification step 405 Detection step 701 Liquid level 702 Vent filter 703 Flow path 704 Flow path resistor 705 Liquid level detection sensor 801 Upper limit of CE detection or upper limit of PCR amplification product concentration 802 Lower limit of CE detection 803 Analysis range of STR-CE 804 Analysis range of STR-CE by split PCR 805,806 Plot 807 Dynamic range of CE

Claims

1. a flow path device having a PCR chamber for performing thermal cycling; a capillary electrophoresis unit for electrophoretic analysis of the PCR reaction solution; In a DNA analysis system having The DNA analysis system includes: memorizing the preset values ​​of m and n; In the PCR chamber, the PCR reaction solution is subjected to m thermal cycles to produce a first reaction solution; removing a portion of the first reaction solution from the PCR chamber without changing its composition; subjecting the portion of the first reaction solution to electrophoretic analysis in the capillary electrophoresis unit; In the PCR chamber, the first reaction solution remaining in the PCR chamber is subjected to n-m thermal cycles (where m is an integer that satisfies the conditions of 20 or more and n is an integer that is 36 or less, and n-m is an integer that is 2 or more and 9 or less) so that the total number of thermal cycles is n, thereby producing a second reaction solution; removing at least a portion of the second reaction solution from the PCR chamber without changing its composition; subjecting at least a portion of the second reaction solution to electrophoretic analysis in the capillary electrophoresis portion; DNA analysis system.

2. 2. The DNA analysis system according to claim 1, wherein the DNA analysis system mixes the portion of the first reaction solution and at least the portion of the second reaction solution with pure water, formamide, or a solution having a conductivity of 10 mS / cm or less, respectively, before the electrophoretic analysis to generate a mixed solution, and then performs the electrophoresis on the mixed solution.

3. a flow path device having a PCR chamber for performing thermal cycling; a capillary electrophoresis unit for electrophoretic analysis of the PCR reaction solution; a reservoir; In a DNA analysis system having The DNA analysis system includes: memorizing the preset values ​​of m and n; In the PCR chamber, the PCR reaction solution is subjected to m thermal cycles to produce a first reaction solution; A portion of the first reaction solution is removed from the PCR chamber without changing its composition and stored in the storage section; In the PCR chamber, the first reaction solution remaining in the PCR chamber is subjected to n-m thermal cycles (where n and m are integers satisfying n>m) so that the total number of thermal cycles is n, thereby producing a second reaction solution; removing at least a portion of the second reaction solution from the PCR chamber; subjecting at least a portion of the second reaction solution to electrophoretic analysis in the capillary electrophoresis portion; controlling the execution of electrophoretic analysis of the portion of the first reaction solution based on the result of the electrophoretic analysis; DNA analysis system.

4. In claim 2, The DNA analysis system performs STR-CE analysis by electrophoresis.

5. In claim 1, A DNA analysis system where n is 34 or less.

6. In claim 5, A DNA analysis system in which n-m is between 3 and 6.

7. In claim 1, A DNA analysis system, wherein the entire amount of the second reaction solution is removed from the PCR chamber.

8. a flow path device having a PCR chamber for performing thermal cycling; a capillary electrophoresis unit for electrophoretically analyzing a PCR reaction solution, and a DNA analysis system for sequentially carrying out reaction A and reaction B, Reaction A is composed of temperature control conditions including a thermal denaturation step in PCR, and an annealing and extension reaction, Reaction B is performed under temperature control conditions that do not include the thermal denaturation step, The DNA analysis system includes: memorizing the preset values ​​of m and n; In the PCR chamber, the reaction A is carried out m times and the reaction B is carried out once on the PCR reaction solution to produce a first reaction solution; removing a portion of the first reaction solution from the PCR chamber without changing its composition; subjecting the portion of the first reaction solution to electrophoretic analysis in the capillary electrophoresis unit; In the PCR chamber, the first reaction solution remaining in the PCR chamber is subjected to reaction A n-m times (where n and m are integers satisfying n>m+2) so that the total number of thermal cycles is n, and reaction B is performed once to produce a second reaction solution; removing at least a portion of the second reaction solution from the PCR chamber without changing its composition; subjecting at least a portion of the second reaction solution to electrophoretic analysis in the capillary electrophoresis portion; DNA analysis system.

9. In claim 8, The DNA analysis system mixes the portion of the first reaction solution and at least the portion of the second reaction solution with pure water, formamide, or a solution having a conductivity of 10 mS / cm or less, respectively, before the electrophoretic analysis to generate a mixed solution, and then performs the electrophoresis on the mixed solution.

10. In claim 9, In the DNA analysis system, the reaction B is a reaction in which the PCR reaction solution is maintained for 1 to 20 minutes under temperature conditions that allow an extension reaction in the PCR reaction.

11. In claim 10, The DNA analysis system performs STR-CE analysis by electrophoresis.

12. In claim 11, A DNA analysis system in which m is an integer greater than or equal to 20, n is an integer less than or equal to 36, and n-m is an integer greater than or equal to 3 and less than or equal to 9.

13. a flow path device having a PCR chamber for performing thermal cycling; a capillary electrophoresis unit for electrophoretically analyzing a PCR reaction solution, and a DNA analysis system for sequentially carrying out reaction A and reaction B, Reaction A is composed of temperature control conditions including a thermal denaturation step in PCR, and an annealing and extension reaction, Reaction B is performed under temperature control conditions that do not include the thermal denaturation step, The DNA analysis system includes: memorizing the preset values ​​of m and n; In the PCR chamber, the reaction A is carried out m times on the PCR reaction solution to produce a first reaction solution; A portion of the first reaction solution is removed from the PCR chamber without changing the composition, and one reaction B is carried out thereon; subjecting the portion of the first reaction solution to electrophoretic analysis in the capillary electrophoresis unit; In the PCR chamber, the first reaction solution remaining in the PCR chamber is subjected to reaction A n-m times (where n and m are integers satisfying n>m) so that the total number of thermal cycles is n, and reaction B is performed once to produce a second reaction solution; removing at least a portion of the second reaction solution from the PCR chamber without changing its composition; subjecting at least a portion of the second reaction solution to electrophoretic analysis in the capillary electrophoresis portion; DNA analysis system.

14. In claim 13, In the DNA analysis system, the reaction B is a reaction in which the PCR reaction solution is maintained for 1 to 20 minutes under temperature conditions that allow an extension reaction in the PCR reaction.

15. In claim 14, The DNA analysis system mixes the portion of the first reaction solution and at least the portion of the second reaction solution with pure water, formamide, or a solution having a conductivity of 10 mS / cm or less, respectively, before the electrophoretic analysis to generate a mixed solution, and then performs the electrophoresis on the mixed solution.

16. In claim 15, The DNA analysis system performs STR-CE analysis by electrophoresis.

17. In claim 16, A DNA analysis system in which m is an integer greater than or equal to 20, n is an integer less than or equal to 36, and n-m is an integer greater than or equal to 2 and less than or equal to 9.

18. In claim 17, The DNA analysis system further comprises a storage unit, A DNA analysis system, wherein the storage section is placed so as to be in contact with a heating section, and the reaction B on the first reaction solution is carried out by heating the storage section with the heating section.

19. a flow path device having a PCR chamber for performing thermal cycling; a capillary electrophoresis unit for electrophoretic analysis of the PCR reaction solution; In a DNA analysis system having The DNA analysis system includes: memorizing the preset values ​​of m and n; In the PCR chamber, the PCR reaction solution is subjected to m thermal cycles to produce a first reaction solution; removing a portion of the first reaction solution from the PCR chamber without changing its composition; subjecting the portion of the first reaction solution to electrophoretic analysis in the capillary electrophoresis unit; In the PCR chamber, the first reaction solution remaining in the PCR chamber is subjected to n-m thermal cycles (where n and m are integers satisfying n>m) so that the total number of thermal cycles is n, thereby producing a second reaction solution; Remove the entire amount of the second reaction solution from the PCR chamber; subjecting at least a portion of the second reaction solution to electrophoretic analysis in the capillary electrophoresis portion; determining which of the results of the electrophoretic analysis of the portion of the first reaction solution and the results of the electrophoretic analysis of the at least portion of the second reaction solution is better; Providing users with information that allows them to determine which is the better outcome; DNA analysis system.

20. In claim 19, The DNA analysis system mixes the portion of the first reaction solution and at least the portion of the second reaction solution with pure water, formamide, or a solution having a conductivity of 10 mS / cm or less, respectively, before the electrophoretic analysis to generate a mixed solution, and then performs the electrophoresis on the mixed solution.

21. In claim 20, The DNA analysis system performs STR-CE analysis by electrophoresis.

22. In claim 21, A DNA analysis system in which m is an integer greater than or equal to 20, n is an integer less than or equal to 36, and n-m is an integer greater than or equal to 2 and less than or equal to 9.

23. a flow path device having a PCR chamber for performing thermal cycling; a capillary electrophoresis unit for electrophoretic analysis of the PCR reaction solution; In a DNA analysis system having The DNA analysis system includes: memorizing the preset values ​​of m and n; In the PCR chamber, the PCR reaction solution is subjected to m thermal cycles to produce a first reaction solution; removing a portion of the first reaction solution from the PCR chamber without changing its composition; subjecting the portion of the first reaction solution to electrophoretic analysis in the capillary electrophoresis unit; In the PCR chamber, the first reaction solution remaining in the PCR chamber is subjected to n-m thermal cycles (where n and m are integers satisfying n>m) so that the total number of thermal cycles is n, thereby producing a second reaction solution; Remove the entire amount of the second reaction solution from the PCR chamber; subjecting at least a portion of the second reaction solution to electrophoretic analysis in the capillary electrophoresis portion; determining which of the results of the electrophoretic analysis of the portion of the first reaction solution and the results of the electrophoretic analysis of the at least portion of the second reaction solution is better; Outputting better results than the above DNA analysis system.

24. In claim 23, The DNA analysis system mixes the portion of the first reaction solution and at least the portion of the second reaction solution with pure water, formamide, or a solution having a conductivity of 10 mS / cm or less, respectively, before the electrophoretic analysis to generate a mixed solution, and then performs the electrophoresis on the mixed solution.

25. 25. In claim 24, The DNA analysis system performs STR-CE analysis by electrophoresis.

26. 26. In claim 25, A DNA analysis system in which m is an integer greater than or equal to 20, n is an integer less than or equal to 36, and n-m is an integer greater than or equal to 2 and less than or equal to 9.

27. In claim 3, The DNA analysis system performs STR-CE analysis by electrophoresis.

28. In claim 27, The target samples are forensic samples,DNA analysis system.

29. 29. In claim 28, A DNA analysis system, wherein the entire amount of the second reaction solution is removed from the PCR chamber.

30. 30. In claim 29, A DNA analysis system in which m is an integer greater than or equal to 20, n is an integer less than or equal to 36, and n-m is an integer greater than or equal to 2 and less than or equal to 9.