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

Technical Field

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

Background Art

[0002] In fragment analysis, PCR is performed on the DNA contained in a sample using primers designed for a specific DNA target, and the fluorescence-labeled DNA amplification products are separated by size using capillary electrophoresis (CE). It is used for gene mutation analysis, quantification, cell line authentication, determination of genome editing efficiency, amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR), genotyping of single nucleotide polymorphism (SNP), and microsatellite marker analysis. Microsatellites refer to repetitive DNA in which a specific DNA motif is repeated multiple times, and are characterized by a higher frequency of mutation and higher genetic diversity compared to other DNA regions. A typical example of microsatellite marker analysis is individual identification and personal identification using short tandem repeats (STR). DNA identification using STR is widely used in forensic examinations and is used for applications such as paternity testing and matching DNA at crime scenes with criminals.

[0003] In fragment analysis, in addition to the fluorescence-labeled DNA fragments to be analyzed, multiple types of fluorescence-labeled DNA fragments of known lengths (size standards) may be mixed and analyzed by CE. By using size standards, the length of the fragment length of each amplification product can be specified. Also, if the amount of the mixed size standards is set to a certain amount, the amount of the amplification product can be calculated from the ratio of the intensity of the DNA fragment of the amplification product obtained by CE analysis to the intensity of the size standard. Further, by mixing a known amount and known length of DNA (Internal positive control, IPC) and the primers for amplifying it into the PCR reaction solution, amplifying them, and performing fragment analysis together, the amount of DNA before amplification of the target can be estimated from the ratio of the intensity of the target amplification product to the IPC.

[0004] According to Non-Patent Document 1, DNA analysis performed at a forensic institute involves: (1) quantifying the concentration of human DNA in the sample using quantitative PCR; (2) preparing the sample to an appropriate concentration of human DNA and performing STR-PCR (PCR including STR sequences); (3) mixing a portion of the PCR reaction solution with formamide containing a size standard in a fixed ratio and performing thermal denaturation; (4) obtaining an electrophoresis map by CE measurement of the thermally denatured electrophoretic sample; and (5) performing DNA analysis from the electrophoresis map.

[0005] In the DNA analysis or fragment analysis using the pre-processing integrated CE analyzer described in Patent Documents 1, 2, 3, 4, and 8, (1) a sample containing DNA is subjected to PCR on a fluidic device, (2) a portion of the reaction solution and formamide containing a size standard are mixed in a fixed ratio on the fluidic device for thermal denaturation, (3) the thermally denatured electrophoretic sample is subjected to CE analysis to obtain an electrophoresis map, and (4) DNA analysis or fragment analysis is performed from the electrophoresis map. Compared with Non-Patent Document 1, Patent Documents 1, 2, 3, 4, and 8 automate a series of steps and allow results to be obtained in a short time (e.g., 90 minutes).

[0006] In the DNA quantification method described in Patent Document 5, (1) in PCR of a DNA-containing sample, a portion of the reaction solution at each stage of a series of thermal cycle numbers n (where n0 is a predetermined integer, n=n0, n0+1, n0+2, ...) is divided, (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 from the relationship between the peak intensity of the amplified product obtained by CE analysis and the thermal cycle number n, specifically, from the thermal cycle number n where the peak intensity exceeds a predetermined threshold. In other words, Patent Document 5 is an analytical method that uses CE to detect amplified products in real-time PCR.

[0007] In the DNA quantification method described in Patent Document 6, (1) in PCR of a DNA-containing sample on a flow channel device, a portion of the amplification product at each stage of a series of thermal cycle numbers n (where n0 and m are predetermined integers, m is 1 or 2, and n=n0, n0+m, n0+2m, ...) is separated by electrophoresis; (2) each separated amplification product is subjected to CE analysis on the flow channel device; and (3) DNA quantification (quantification of the original concentration of DNA contained in the sample) is performed from the relationship between the peak intensity of the amplification product obtained by each CE analysis and the thermal cycle number n, specifically from the thermal cycle number n where the peak intensity exceeds a predetermined threshold. In other words, Patent Document 5 is an analytical method that uses CE to detect amplification products in real-time PCR.

[0008] In the DNA quantification method described in Patent Document 7, (1) in PCR of a DNA-containing sample on a flow channel device, a portion of the reaction solution at each stage of multiple thermal cycle numbers n is divided, (2) each divided reaction solution is microarray analyzed on the flow channel device, and (3) DNA quantification (quantification of the original concentration of DNA contained in the sample) is performed from the thermal cycle number n at which the spot intensity of the amplified product obtained from each microarray analysis exceeds a predetermined threshold. In other words, Patent Document 7 is an analytical method that performs the analysis of amplified products in real-time PCR using a microarray. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] U.S. Patent Application Publication No. 2022 / 0016632 [Patent Document 2] U.S. Patent No. 9354199 [Patent Document 3] U.S. Patent Application Publication No. 2019 / 0019290 Specification [Patent Document 4] Japanese Patent Publication No. 2017-077180 [Patent Document 5] U.S. Patent No. 7445893 [Patent Document 6] Patent No. 5494480 [Patent Document 7] U.S. Patent No. 8715924 [Patent Document 8] U.S. Patent No. 10767225 [Non-patent literature]

[0010] [Non-Patent Document 1] John M. Butler, Fundamentals of Forensic DNA Typing (2009), P.29~107 and P.279~336 [Overview of the project] [Problems that the invention aims to solve]

[0011] In CE analyzers and instruments equipped with a CE analysis unit, the ratio of the minimum peak intensity to the maximum peak intensity at which peak intensity and concentration are approximately proportional is 100 or less, or 1000 or less, or 100000 or less. When performing fragment analysis, considering variations in the intensity of the CE analyzer, variations in the amount of amplicon injected into the CE, variations in amplification efficiency and abundance for each allele, variations between dyes, and variations during sample preparation, the ratio of the minimum to maximum measurable DNA concentration is 10, 30, 100, 300, 1000, 3000, or 10000. On the other hand, the ratio of the minimum to maximum amount of DNA contained in the sample brought into the analysis system is 30, 300, 3000, or 30000. Fragment analysis may fail if the amount of DNA contained in the sample exceeds the range of sample DNA that can be measured 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) on samples containing DNA whose concentration ratio changes in 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) by PCR and CE analysis using a simple configuration and low-cost device. In addition, similar to Patent Document 2, the series of processes are automated, and results can be obtained in a short time (for example, within 180 minutes, within 120 minutes, or within 90 minutes).

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

[0014] In Patent Document 1, the solution is divided before PCR, and STR-PCR and quantitative PCR are performed. Quantitative PCR is used to determine the number of cycles for STR-PCR. Because the solution is divided before the start of PCR, sensitivity is reduced. Furthermore, the fluidic device required to perform two different PCRs is complex, and additional optics are necessary, making high costs unavoidable.

[0015] In Patent Document 2, the solution is divided before PCR to prepare two DNA solutions of different concentrations, and both are PCR-treated for DNA analysis. Since one of the two DNA solutions falls within the analytical concentration range of the analysis system, the analytical range can be expanded. Dividing the solution before PCR reduces sensitivity. Also, since a flow channel mechanism for dividing the solution and adjusting the concentration is incorporated into the flow channel device, it is unavoidable that the flow channel device will become more complex.

[0016] Patent Document 3 expands the analytical range of DNA analysis by improving the data analysis method. However, information on peaks that fall below the detection limit during CE analysis cannot be obtained. Also, if the detection intensity saturates during CE analysis, the correct peak intensity ratio cannot be obtained. If the interpretation of DNA analysis is expanded too much, the data obtained from the analysis may not reflect the original individual DNA mixture ratio. There is a risk of incorrect profiling.

[0017] In Patent Document 4, a portion of the reaction solution is removed after PCR, and the presence and amount of amplified products are detected using an optical system. If proper amplification has been achieved, fragment analysis is performed. If it is determined that the amplification is incomplete, an additional PCR reaction is performed on the reaction solution remaining in the PCR section. In this method, the design of additional optical systems and flow channel devices suitable for optical detection is required, making high costs unavoidable. Furthermore, during detection by optical systems, there is a risk that the fluorescent dye of STR-PCR may fade, or that accurate optical detection may not be possible due to overlap between the detection wavelength range of the fluorescent dye of STR-PCR and that of the optical system.

[0018] In Patent Document 5 and Patent Document 6, since it is necessary to perform CE analysis for each of a large number of thermal cycle numbers n (a plurality of consecutive thermal cycle numbers n), it takes a long time to obtain results. For highly accurate quantification, CE analysis three or more times is required. Although PCR can be replaced with STR-PCR and DNA quantification can be performed, a special flow path structure is required because the solution needs to be taken out many times. In addition, since there is no step of mixing the reaction solution and formamide containing the size standard at a certain ratio, DNA identification cannot be performed. Also, when performing DNA identification by incorporating the mixing process with formamide (electrophoresis reagent) containing the size standard into the corresponding method, a new dispensing mechanism or flow path structure for mixing the PCR product of each cycle and the electrophoresis reagent is required, which complicates the device and leads to high costs. Also, for DNA identification that requires CE analysis with particularly high accuracy and high resolution, it takes an enormous analysis time to perform CE analysis on the PCR product of each cycle. Also, in the case of Patent Document 6, since DNA is taken out of the PCR chamber by applying voltage, it is assumed that the composition of the amplification product in the PCR chamber is different from the composition of the taken-out amplification product. In the case of DNA identification, amplicons of different lengths are measured, but when voltage is applied to take out the amplification product from the PCR chamber, there is a concern that a bias depending on the length will occur.

[0019] In Patent Document 7, although PCR can be replaced with STR-PCR and DNA quantification can be performed, since it is necessary to perform microarray analysis for each of a large number of thermal cycle numbers n (a plurality of consecutive thermal cycle numbers n), it takes a long time to obtain results. Also, since CE analysis is not performed, DNA identification cannot be performed.

[0020] On the other hand, Patent Document 7 mentions that microarray analysis may be performed for a small number of thermal cycle counts n (a plurality of non - consecutive thermal cycle counts n), but this is not practical. In microarray analysis, generally, due to variations in the density or number of probes immobilized on each spot, or variations in hybridization efficiency in terms of time and space, etc., the spot intensity for the same DNA concentration fluctuates. Although it is possible to determine the presence or absence of the corresponding DNA from the strength or weakness of the spot intensity, the accuracy of quantifying the corresponding DNA from the spot intensity is low. That is, in order to accurately obtain the thermal cycle count n at which the spot intensity exceeds a predetermined threshold, it is necessary to perform microarray analysis for each of a large number of thermal cycle counts n (a plurality of consecutive thermal cycle counts n). As a means to improve the accuracy of DNA quantification by microarray analysis, a method of competitively hybridizing target DNA or target amplification products with reference DNA to the same spot is known. However, in order to implement this method, for each target DNA or target amplification product, it is necessary to prepare reference DNA that hybridizes to the probe on the spot with the same efficiency as the target DNA or target amplification product, and further, label the target DNA or target amplification product and the reference DNA with different fluorophores and independently measure their emitted fluorescence. When preparing PCR products with a small number of different cycle counts and performing microarray analysis to achieve an analysis with an expanded dynamic range, there are problems that require cost and labor.

[0021] Another problem of Patent Document 7 is that (1) in the PCR of a sample containing DNA on a flow - path device, when a part of the reaction solution at each stage of a plurality of thermal cycle counts n is divided, fresh PCR solution of the same amount as the divided reaction solution is mixed with each of the remaining reaction solutions that are not divided, so that the concentration of DNA contained in the reaction solution changes, reducing the accuracy of DNA quantification.

[0022] Another problem with Patent Document 7 is that it is necessary to repeatedly hybridize and dehybridize (wash) the DNA and the immobilized probe, and each time the immobilized probe peels off or the hybridized DNA is carried over without being washed, resulting in low repeatability of microarray analysis and low accuracy of DNA quantification.

[0023] In summary, the challenges in DNA identification / fragment analysis or DNA quantification require a DNA analysis method that can be implemented with a simple fluidic device, is low-cost, highly robust, simple, maintains sensitivity, has a short measurement time, and can broaden the range of analyzable DNA amounts. [Means for solving the problem]

[0024] An example of a DNA analysis system according to the present invention is: A fluid channel 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 described above is It stores the pre-set values ​​of m and n, In the PCR chamber, the PCR reaction solution is subjected to m thermal cycles to produce the first reaction solution. A portion of the first reaction solution is removed from the PCR chamber without changing its composition. A portion of the first reaction solution is subjected to electrophoretic analysis in the capillary electrophoresis unit. In the PCR chamber, the first reaction solution remaining in the PCR chamber is subjected to a thermal cycle of nm times (where nm is an integer of 2 or more) such that the total number of thermal cycles is n times, thereby generating a second reaction solution. At least a portion of the second reaction solution is removed from the PCR chamber without changing its composition. At least a portion of the second reaction solution is subjected to electrophoretic analysis in the capillary electrophoresis unit.

[0025] An example of a DNA analysis system according to the present invention is: A fluid channel 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 described above is It stores the pre-set values ​​of m and n, In the PCR chamber, the PCR reaction solution is subjected to m thermal cycles to produce the first reaction solution. A portion of the first reaction solution is removed from the PCR chamber without changing its composition. In the PCR chamber, the first reaction solution remaining in the PCR chamber is subjected to a thermal cycle of nm times (where nm is an integer of 2 or more) such that the total number of thermal cycles is n times, thereby generating a second reaction solution. At least a portion of the second reaction solution is removed from the PCR chamber without changing its composition. One of the above-mentioned portion of the first reaction solution and at least a portion of the second reaction solution is subjected to electrophoretic analysis in the capillary electrophoresis unit. Based on the results of the electrophoretic analysis of one of the aforementioned reactions, the execution of the electrophoretic analysis is controlled for the other of the portion of the first reaction solution or at least a portion of the second reaction solution. [Effects of the Invention]

[0026] The effects obtained by an example of the invention disclosed in this application can be briefly explained as follows. That is, according to an example of the present invention, the flow channel device and Capillary An analytical system equipped with an electrophoresis unit allows for high accuracy, high sensitivity, rapid analysis, low cost, and expansion of the range of DNA analysis quantities using a compact device.

[0027] Other issues, configurations, and effects will be clarified by the following description of the embodiments. [Brief explanation of the drawing]

[0028] [Figure 1] This is a schematic diagram of the analysis system. [Figure 2] This is an example of a data classification method. [Figure 3] This is a schematic diagram of the analysis system. [Figure 4] This is an example of an analysis system. [Figure 5] This is a schematic diagram and example of the analysis system. [Figure 6] This is a schematic diagram of the analysis system. [Figure 7] This is a schematic diagram of the analysis system. [Figure 8] This is a schematic diagram of a flow channel device. [Figure 9] This is a schematic diagram of a flow channel device. [Figure 10] This is a schematic diagram of a flow channel device. [Figure 11] This is a schematic diagram of a flow channel device. [Figure 12] This is a schematic diagram of a flow channel device. [Figure 13] This is an example of how a fluidic device operates. [Figure 14] This is an example of an analysis system. [Figure 15] This is a schematic diagram of a flow channel device. [Figure 16] This is an example of how a fluidic device operates. [Figure 17] This is a schematic diagram of a flow channel device. [Figure 18] This is a schematic diagram of a flow channel device. [Figure 19] This is a schematic diagram of a flow channel device. [Figure 20] This is an example of how a fluidic device operates. [Figure 21] This is an example of how a fluidic device operates. [Figure 22] This is an example of how a fluidic device operates. [Figure 23] This result shows the relationship between electrophoresis reagents and peak intensity. [Figure 24] This figure shows the effects of the present invention. [Figure 25] This diagram shows the scope of analysis. [Figure 26] This is an example of a table used as a basis for setting the number of cycles. [Figure 27] This is a schematic diagram of the resulting electrophoretic graph. [Figure 28] This is an example of a table used as a basis for setting the number of cycles. [Figure 29] This is an example of an analysis system. [Figure 30] This is an example of a data classification method. [Figure 31] This is an example of a data classification method. [Figure 32] This is an example of an analysis system. [Figure 33] This is an example of an analysis system. [Modes for carrying out the invention]

[0029] This specification primarily describes procedures and standards for conducting human DNA testing, but the subjects of analysis are 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 nm ≥ 2 may also apply. Note that in the following, thermal cycles may be referred to as PCR cycles or simply cycles.

[0031] In the following, the mixture of PCR reagent and sample-derived DNA will be referred to as the PCR reaction solution. Furthermore, the DNA from the sample that is amplified by the PCR reagent will be called the target DNA.

[0032] In the following, 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 contain deionized formamide, size standards, or pure water. Formamide and pure water may be included to lower the ionic strength of the electrophoresis sample or to denature the DNA. In addition to formamide and pure water, low conductivity solutions may be used as the electrophoresis reagent. The low conductivity solution is preferably 10 mS / cm or less, more preferably 1 mS / cm or less, more preferably 100 μS / cm or less, and 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 the CE tends to be. Size standards may be mixed in 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 involves mixing the PCR reaction solution (for example, a portion of PCR reaction solution m and at least one portion of PCR reaction solution n) with pure water, formamide, or a solution with a conductivity of 10 mS / cm or less before electrophoresis analysis to produce a mixture. Mixing with a low-conductivity solution makes it possible to increase the peak intensity of CE. Furthermore, mixing with formamide makes it possible to denature DNA.

[0035] In the following, the DNA obtained by the PCR reaction will be referred to as the "amplification product," the amplification product obtained in m cycles will be called "amplification product m," and the amplification product obtained in n cycles will be called "amplification product n."

[0036] In the following, the mixture of the 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, as heating to 90°C or higher, as shown in Figure 5(2), denatures the DNA and makes it more likely to become single-stranded, thus enabling more accurate CE analysis. The sample referred to as the "electrophoresis sample" can be either before or after denaturation by heating. The electrophoresis sample prepared from amplification product m will be called "electrophoresis sample m," and the electrophoresis sample prepared from amplification product n will be called "electrophoresis sample n."

[0037] In the following, the PCR reaction procedure in which m PCR cycles are performed, a portion of the PCR reaction solution m is taken out, and the remaining PCR reaction solution m is subjected to nm PCR cycles, ultimately preparing both PCR reaction solutions m and n, is referred to as "split PCR".

[0038] In the following, a gene locus refers to the location of a gene on a chromosome. Typical STR-PCR kits contain primers that can specifically amplify each gene locus.

[0039] In the following, an allele refers to a gene variant that can be distinguished at the same locus. When performing DNA testing, if the DNA originates from the same person, it is possible to have 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. However, multiple amplicons of different lengths can be generated from a single allele. For example, multiple amplicons can be produced as by-products (artifacts) during the PCR reaction. In DNA analysis, a single amplicon peak is often attributed to an individual's DNA for each allele. However, in mixed samples, it is difficult to distinguish between artifacts and allele-derived amplicons, so peaks of amplicons that may be artifacts may also be included in the analysis.

[0041] In the following, CE analysis refers to the entire process of preparing an electrophoretic sample, performing CE measurement, obtaining an electrophoretic graph, and conducting DNA analysis or fragment analysis. However, the scope of "CE analysis" does not necessarily have to include all of the aforementioned steps.

[0042] In the following, an electrophoresis graph refers to a graph obtained by CE measurement, where the horizontal axis represents time, measurement point, or DNA chain length, and the vertical axis represents intensity. The vertical axis may also represent wavelength and be three-dimensional data including intensity information. Alternatively, the vertical axis may represent intensity and be three-dimensional data including dye information. The electrophoresis graph obtained from electrophoresis sample m will be called "electrophoresis graph m," and the electrophoresis graph obtained from electrophoresis sample n will be called "electrophoresis graph n."

[0043] In the following, a DNA profile refers to a DNA type obtained by analyzing an electrophoresis diagram, or a two-dimensional sequence containing peak intensity and peak length, or a dataset containing the number of repeats and peak intensity of DNA assigned to a DNA type.

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

[0045] In the following, "analytical range" refers to the range of a given sample volume or biomolecular weight that, for example, can be correctly analyzed by an analytical system or some or more of the procedures included within an analytical system for a given sample or biomolecule. Here, "correctly analyzeable" may mean that all requirements are met, but it is not necessary for all requirements to be met; it may mean that the system can provide the best possible data achievable. For example, if the given sample volume is extremely small, the resulting data may not meet all requirements, but it is sufficient to obtain data that comes as close to meeting the requirements as possible.

[0046] The PCR reaction mixture may contain two or more primer sets, and the PCR reaction mixture may contain two or more amplified gene regions. Analyzing multiple amplified gene regions enhances the ability to identify individuals. In particular, in person identification, it can reduce the risk of mistakenly identifying individuals as the same person. By performing PCR reactions on multiple gene regions together at once, rather than individually, the number of PCR chambers can be minimized. This saves on reaction reagents. It also avoids the risk of reduced sensitivity caused by separating PCR chambers.

[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 a detection unit, a solution transport control mechanism such as a pump and valves, a CE unit (capillary electrophoresis unit) for electrophoretic analysis of the PCR reaction solution, a flow channel device, and a heater. The control and analysis unit may be connected to a network and capable of 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. As an example of a valve, a valve that directly / indirectly transmits motor power to deform a film, or a valve that deforms using air pressure may be used. The valve may be controlled by the control unit. The valve may be opened and closed by deformation using heat, or by using magnetism.

[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. The computer may also be responsible for opening and closing valves such as those in the flow path device 104 and the CE section 105 and its connections, as well as temperature control and control of applied pressure and / or flow rate, based on the parameters recorded in the database 103. The parameters recorded in the computer 102 may include temperature, time, pressure, flow rate, and functions for setting parameters based on stored parameters and measured values.

[0050] The system could accept samples containing target DNA and perform the entire process, from dissolution and purification to PCR, CE measurement, and analysis, fully automatically. Alternatively, some steps, such as dissolution, purification, PCR, CE measurement, and analysis, could be fully automated. For example, the analysis system 101 or the fluid device 104 could perform the process shown in Figure 14 (described later) fully automatically, from the preparation of the PCR reaction solution to nanometer thermal cycles. This would streamline the process and enable analysis by non-experts.

[0051] The integrated device may be used for dissolution, purification, and PCR reaction preparation. The integrated device may also be used for mixing PCR and formamide and heating. The integrated device may even be used for CE measurement.

[0052] The flow channel device 104 can be disposable. Disposability prevents contamination between samples.

[0053] The CE section 105 can be disposable. Disposability prevents contamination between samples. Also, because it can be integrally molded with the device, storage, maintenance, and transportation become easier. The connection between the pre-processing section and the CE section is simplified, which reduces the frequency of failures and errors.

[0054] Flow channel While the device itself is disposable, the CE (Computer Emission Control) component can be reused multiple times. Since the CE component requires precise manufacturing and is therefore expensive, making it reusable can help reduce costs.

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

[0056] The computer 102 may be equipped with a user interface 106. The computer 102 may receive parameters related to the user interface 106 (for example, time, temperature, pressure, flow rate, procedure, volume of divided liquid, number of PCR cycles, sample information, cartridge information, analysis protocol, etc.) from the user and store them in the database 103. Alternatively, various parameters may be stored in the database 103 in advance. Based on the parameters recorded in the database 103, the computer 102 may be responsible for opening and closing the valves of the flow path device 104, controlling the temperature, and controlling the applied pressure and flow rate.

[0057] The flow channel device 104, which is consumed with each measurement, has a tag inside, 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 ​​for m and n) may be set by the user. The analysis system 101 stores the pre-set values ​​for m and n. The user may also input information about the sample type (e.g., cheek swab / touch sample / casework sample / DVI, etc.) and compare it with a database in the computer to determine the appropriate PCR procedure. When electrophoresis is performed on multiple samples or multiple electrophoresis samples, the user may select, based on input, whether to perform CE measurement on electrophoresis sample m first or on electrophoresis sample n first. The entire procedure may also be automatically controlled under the computer 102. The user may also assist in and perform parts of the analysis flow.

[0059] Traditionally, samples were sent to well-equipped experimental facilities such as research institutes, where specialized inspectors with expertise and skills prepared and measured the samples, followed by data analysis. However, this process has drawbacks, including the time-consuming nature of sample transport and the significant equipment and personnel costs required to maintain the facilities. Furthermore, if 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, are emerging in various fields. StoA systems sometimes utilize fluidic devices that integrate chambers, fluid channels, and reagents. Introducing fluidic devices offers the following advantages: (1) Easy measurement even for non-experts, (2) Data acquisition in a short time, (3) Design of highly portable devices, (4) Reduced variability due to manual procedures, and (5) Easy storage of reagents.

[0060] Potential applications of the StoA system, including its potential uses, include forensic science, DNA analysis, in vitro diagnostics, identification of plant and animal species, biodefense, medicine, biotechnology, life sciences, defense, public health, and agriculture. The StoA system can be used in laboratories, crime scenes, police stations, hospitals, and automobiles.

[0061] [Flow channel devices] In this embodiment, the fluid channel device 104 refers to a disposable or reusable cartridge that contains reagents, a chamber, and a fluid channel. The fluid channel device 104 may also contain a power source for solution transport. Some or all of the reagents may be present inside the device. Part of the chamber may be equipped with a temperature control function, a molecule capture function, a detection function, or a voltage application function.

[0062] The materials used in fluidic devices are not particularly limited as long as they are materials commonly used in the relevant art. Preferably, materials with low DNA adsorption capacity, such as polypropylene, cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polycarbonate, polyethylene terephthalate, and polyurethane, are used. It is also desirable to reduce adsorption by modifying the surface to be 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 silica, 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 of these.

[0063] [Chamber / Reagent Storage Section] A typical chamber or reagent reservoir refers to a space that can store liquids or solids and allow solutions to react, wait, heat, or be altered. A chamber may have a larger diameter than a channel, but may also be indistinguishable from the channel visually. A chamber may have an internal membrane or microstructure, or it may be made of a different composition, have a different surface treatment, or have a different hydrophilicity than the channel. Heaters or laser light sources may also be attached to the outside of the channel device. Reagents may be stored in the chamber, and PCR, lysis, purification, etc., may be performed within the chamber. A typical chamber volume is preferably between 0.01 μL and 50 mL.

[0064] Fluidized devices may store reagents within the device, or reagents may be supplied from outside the fluidized device or from within the analytical system. For example, a device may contain one or more reagents in one or more reagent reservoirs. The reagents may include at least one of the following: [lysate, washing solution, PCR reagent (which may include polymerase, primer, surfactant, etc.), formamide, pure water, DNA fragments, oil]. Since mixing these reagents at unintended times can lead to performance degradation and other unexpected results, it is desirable that they be separated until immediately before use by a valve, film, air, or a narrow channel that prevents spontaneous mixing, or a partition mechanism consisting of a combination thereof. Furthermore, isolating reagents from the outside air enables long-term storage and portability of the device. The same reagent may be stored in multiple reagent reservoirs to release it in multiple steps. Similarly, if reagents are stored outside the device, it is desirable that they be stored in isolation from the outside air, separated from other purification system components by valves, films, air, etc. Known reagent storage technologies include, for example, blister reagent storage units, or reagent storage units described in Patent Document 1 and Patent Document 2, and similar configurations may be incorporated into this embodiment.

[0065] [Sample type] The sample used in the purification system according to this embodiment is not particularly limited as long as it is a sample of biological origin. The organism from which the sample originates is also not particularly limited, and samples derived from any organism such as vertebrates (e.g., mammals, birds, reptiles, fish, amphibians, etc.), invertebrates (e.g., insects, nematodes, crustaceans, etc.), plants, protists, fungi, bacteria, viruses, etc. can be used.

[0066] When collecting samples, swabs, filter paper, cloth, etc., can be used as carriers, and the entire carrier can be introduced into the purification system.

[0067] Forensic samples include cheek swabs, bones, muscle tissue, human organs, touch samples (samples containing trace amounts of DNA), bloodstains, skin fragments, hair, bodily fluids, and personal belongings to which these are presumed to be attached. Many forensic samples contain unknown amounts of DNA, ranging from 0.001 ng to 1000 μg, and more frequently, from 0.01 ng to 10 μg. Forensic samples may contain DNA from a single individual, DNA from multiple individuals, or degraded DNA.

[0068] [Solution transport control] The analysis system 101 may be equipped with pumps and valves for transporting the solution. The transport means may include syringe pumps, diaphragm pumps, electrochemical pumps, passive transport using surface tension, centrifugal force, and combinations thereof.

[0069] The analysis system 101 may be equipped with valves. Valves are used to specify the solution transport path and to switch the path to which air pressure is applied. The valves may be air-operated diaphragm valves, mechanical valves, or surface tension valves. The transport path may be switched based on the pressure difference required for transport.

[0070] [PCR reaction] The device may be equipped with PCR reagents. The PCR reagents may be provided as separate solutions containing polymerase and primer. The PCR reagents may be dried reagents. The sample itself, such as a swab, may be used for PCR. DNA purified by silica purification, Chelex, phenol chloroform, etc., may be mixed with the PCR reagents. A membrane (such as a silica membrane) in which DNA is trapped may be mixed with the PCR reagents.

[0071] PCR reagents may include a set of IPCs (intracellular protein) that are amplified along with the sample DNA, and a primer for amplifying the IPCs. The IPC primers may be dyed and detectable by CE (Cellular Emission Correction). Amplicons derived from the IPCs can be used for analysis. The amount of DNA in the sample may be estimated by using the intensity ratio of the peaks derived from the IPCs and the sample, as well as amplification efficiency correction factors and fluorescence intensity correction factors. Alternatively, the intensity of the IPCs may be checked to estimate whether the PCR reaction is proceeding normally or is inhibited.

[0072] The typical volume of PCR reaction solution is 1 μl to 200 μl, more preferably 10 μl to 50 μl. Smaller volumes offer advantages such as precise temperature control, faster PCR, and lower reagent costs. Conversely, larger volumes allow for the acquisition 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, although some steps may be omitted.

[0074] The initial denaturing step involves heating at 90°C to 99°C for 1 to 2 minutes at the start of PCR to initiate the reaction. The annealing step involves heating at 50°C to 80°C for 1 to 2 minutes to bind the primer to the template DNA. The extension step involves heating at 50°C to 80°C for 1 to 2 minutes to raise the temperature to a level where DNA polymerase works optimally, allowing for DNA extension. The denature step involves heating at 80°C to 99°C for 1 to 2 minutes. The final extension step involves heating at 50°C to 80°C for 1 to 60 minutes. The inclusion of a final extension step helps to standardize the length of the amplified products. The annealing, extension, and denature steps are repeated 10 to 40 times. The annealing and extension steps may be performed at the same temperature.

[0075] [Detection method] After amplification, detection is performed by CE. In CE, a method may be used in which the amplified product is injected into a polymer-filled capillary tube by voltage injection. Furthermore, when a high voltage is applied to both ends of the capillary, fluorescent DNA fragments are separated by size and detected by a laser / camera system. In this embodiment, only CE analysis is mentioned, but in other embodiments, instead of the CE section, Massively Parallel Sequencing (MPS), pyrosequencing, Sanger sequencing, nanopore sequencing, chromatography, electrometric measurements, spectroscopy, NMR, RFLP (Restriction Fragment Length Polymorphisms), microarrays, etc. may be used.

[0076] [Analysis and peak determination criteria] The signals obtained in the CE (Computer Emission) section are analyzed in the analysis section. Known analysis software includes GeneMapper® ID, GeneMapper ID-X, GeneMarker® HID, i-Cubed®, OSIRIS, and TrueAllele®. In CE analysis, a graph is generated based on the signal intensity vs. time information, using size standard peaks, with DNA length on the x-axis and intensity on the y-axis. Correction for cosmic rays and pull-up / pull-down signals is also possible. Baseline correction is also possible, and electrophoresis maps may be obtained using other existing techniques. Peak detection is performed on the obtained electrophoresis map to determine the intensity and peak position of each amplicon. Furthermore, the analysis may involve partial human intervention or be fully automated.

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

[0078] Macrosatellites are DNA loci containing repeating base sequences, each repeating unit having 2 to 7 nucleotides. The number of repeats in a particular locus varies from individual to individual, and this can be detected as differences in the length of amplification products by STR-PCR.

[0079] Typical STR-PCR analysis detects two or more gene loci. Typically, it includes five or more, ten or more, fifteen or more, twenty or more, or twenty-five or more gene loci. STR-PCR can be performed using commercially available kits such as GlobalFiler® or PowerPlex®. It is also preferable to include gene loci designated for forensic or DNA analysis in various national databases such as CODIS, or those specified in various gene databases. Gene loci may include those located on autosomes, or those that exist only on the Y chromosome.

[0080] A mixture of homozygous and heterozygous cases exist for specific gene loci, and the number of peaks detected when examining DNA types derived from a single individual ranges from a minimum of the number of gene loci in the kit to a maximum of the sum of twice the amount of genes assigned to autosomes and the number of genes assigned to sex chromosomes among the gene loci in the kit, unless the amount of DNA is degraded or insufficient.

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

[0082] In DNA profiles derived from a single individual, one or two alleles are detected per locus. In contrast, DNA profiles from mixed samples derived from multiple individuals may show one, two, three, or four or more alleles per locus. When assigning an individual's DNA profile from a multi-individual DNA profile, the analysis is typically performed probabilistically, usually based on peak intensity ratios. Programs for analyzing mixed samples include, for example, Kongho, LikeLTD, LRmix, STRmix, Euroformix, and TrueAllele.

[0083] In DNA analysis, artifacts refer to phenomena such as peaks not derived from the individual's DNA type, differences in the balance between peaks, and differences in peak shape that cause the electrophoresis or DNA profile obtained from DNA analysis to differ from the ideal electrophoresis or DNA profile that should be obtained from the individual's DNA type.

[0084] Multiple mechanisms are involved in the generation of artifacts. If the DNA in the sample originates from a single individual, the presence of some artifacts is acceptable.

[0085] On the other hand, if there are many artifacts, the accuracy of automated judgment decreases, requiring review by experts.

[0086] Furthermore, a high number of artifacts can lead to inaccurate DNA test results.

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

[0088] Furthermore, in the case of samples where it cannot be guaranteed that the DNA originates from a single individual, typically forensic samples where the possibility of involvement from multiple individuals cannot be ruled out, the presence of artifacts makes the analysis of the electrophoresis map more complex or difficult. For example, when three or more peaks appear at a single gene locus, it becomes difficult to interpret whether those peaks are due to artifacts or to two or more individuals. Also, while it is common practice to assign individual DNA profiles based on the ratio of peak intensities, this becomes difficult when the possibility of artifacts is taken into account. Therefore, it is desirable to obtain electrophoresis maps with as few artifacts as possible.

[0089] When peaks not derived from an individual's DNA appear during CE analysis and are reflected in the analysis results, this is called a "drop in." Conversely, 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 is called a "drop out." It is preferable to set various thresholds to minimize the occurrence of drop in and drop out.

[0090] <Starter Peak> A stutter peak is a byproduct of PCR amplification. It occurs 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, 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 adenyl addition> In STR-PCR, during the extension reaction, an extra adenyl group is added to amplicons of the correct length with a certain probability (A+ peak). Typical STR-PCR kits add a reaction step called "Final extension" at the end of the PCR reaction. In this Final extension step, adenyl groups are added to amplicons that did not receive the extra adenyl group. By providing a sufficient Final extension time, it is possible to achieve a state where almost all amplicons receive an adenyl group. However, if the Final extension time is set excessively relative to the amount of amplicon, the proportion of amplicons with an even larger extra adenyl group (A++ peak) will increase. The A++ peak is detected one base longer than the A+ peak. Also, if the amount of amplicon is excessive, the adenyl group addition may not be completed within the Final extension time, leaving some amplicons without adenyl addition, which are detected as an A- peak with one base less. Below, A++ peaks and A- peaks will be collectively referred to as Incomplete adenylation Peaks (IAPs). Furthermore, when describing only the A- peak, it is written as "IAP-", and when describing only the A++ peak or a peak with even more adenyl groups attached, it is written as "IAP+". In a suitable STR-CE, various PCR parameters such as the number of PCR cycles, final extension time, and input DNA amount are adjusted so that the intensity of the A- peak and A++ peak fall within the range of 50% or less, more preferably 20% or less, and more preferably 10% or less, relative to the intensity of the A+ peak. An IAP intensity threshold or an intensity ratio threshold for the main peak (Incomplete adenylation Peak ratio Threshold, IAPT) may be set to determine whether a suitable STR-CE has been performed with the intensity of the A- or A++ peak falling within the aforementioned range. If the intensity ratio of the A- or A++ peak to the A+ peak is above a certain level, the intensity of the main peak will no longer reflect the original gene abundance ratio. Also, in the case of mixed samples or when a peak shifted by one base appears due to gene polymorphism, accurate assignment becomes impossible.Furthermore, this can also lead to the detection of incorrect DNA types.

[0092] <Peak intensity ratio> When the amount of DNA is sufficient, the two peaks originating from heterozygous loci will show approximately the same height. When the amount of DNA is insufficient, the probability of uneven distribution of DNA from each gene increases, and the difference in the intensity of the two peaks becomes significantly larger. Also, in cases of excessive amplification, shorter DNA is amplified preferentially over longer DNA. The height of the peaks originating from the same locus will also shift because shorter DNA is amplified preferentially. When the ratio of the intensities of the two peaks becomes large, it becomes difficult to distinguish them from stunt peaks. Furthermore, it becomes difficult to assign DNA from mixed samples. Therefore, to determine whether a significant CE analysis has been performed, the criterion is that the peak-to-height ratio (PHR) of the two peaks (the ratio of the intensity of the smaller peak to the intensity of the larger peak) should be 10% or more, more preferably 40% or more, and more preferably 60% or more.

[0093] <ski-slope> When there is an excess of DNA, the ratio of dNTPs and polymerases to amplicons decreases, and the tendency for shorter DNA to be preferentially amplified increases. In this case, the resulting electrophoresis map will have a sloped shape, with smaller peaks for long DNA and larger peaks for short DNA. Also, when the DNA is degraded, the proportion of short DNA tends to increase compared to long DNA. In this case as well, a similar sloped DNA profile is obtained. Furthermore, if inhibitors are included, the amplification efficiency of long DNA tends to decrease compared to short DNA, which also results in a similar sloped DNA profile. In addition, if the PCR reagent is diluted in the DNA solution beyond its intended mixing ratio, a difference in amplification efficiency will occur, and long DNA may be preferentially amplified, resulting in a DNA profile with an inverted slope. Also, if the PCR reagent is mixed with a large amount of long DNA primer, creating a reaction system where long DNA is preferentially amplified, a DNA profile with an inverted slope will be obtained. In DNA analysis, a sloped profile or a profile with a large ratio of peak intensity between loci is undesirable. This is because the difference in peak intensity becomes larger, making it more likely for peaks to saturate CE or fall below the detection limit. Furthermore, it becomes difficult to assign mixed samples. Typically, it is desirable to adjust the PCR reaction parameters so that the inter-locus peak intensity ratio (Inter-locus PHR) is such that the smallest peak has an intensity of 1% or more, more preferably 5% or more, more preferably 10% or more, and more preferably 20% or more, compared to the largest peak. Small peaks that do not meet the Inter-locus PHR threshold may be excluded from peak analysis. Additionally, to address degraded DNA, it is desirable that the PCR amplification amount falls within an appropriate range for STR-CE, or that the dynamic range of CE is designed to be large.

[0094] <CEのサチュレーション> Saturation occurs when the amount of amplicon introduced into the CE section is excessive, causing the fluorescence intensity at 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 peak intensities does not reflect the true intensity ratio. Also, the intensity ratio of the stutter peak and IAP to the main peak is detected higher than the true amplicon ratio. Therefore, the artifacts mentioned above are amplified. Furthermore, if the sample is a mix, it is not possible to calculate the correct mixing ratio. For this reason, in desirable DNA analysis, the PCR reaction conditions should be set or the PCR amplification product should be diluted so that CE detection does not oversaturate (OS). The OS threshold may be evaluated and set on the actual CE instrument, set by the user, set by the user for each measurement, or set on a computer.

[0095] <CEのノイズ、プルアップ> Artifacts can also occur in the CE detection unit.

[0096] A pull-up peak refers to the false detection of a peak originating from another pigment. Pull-ups are particularly pronounced when CE is saturated, but they can also be detected when CE is not saturated.

[0097] If a large amount of DNA is introduced into the CE region during the previous measurement, it can carry over, resulting in the detection of a peak in the next measurement.

[0098] Additionally, air bubbles in the CE (Cellular Emission) section and background noise in the detection section may be reflected in the electrophoretic graph.

[0099] An Analytical Threshold (AT) can be set during analysis so that noise peaks are not mistakenly used in the analysis. The analytical threshold can be set by measuring the background noise to obtain a sufficient signal-to-noise ratio, by the user, by the device for each experiment, or it can be preset. Also, even for peaks exceeding the AT, a program for determining whether the peak is derived from an amplification product, from poor CE electrophoresis, or from various noises in CE may be stored and executed.

[0100] The AT may be set in two or more stages of a first reference value and a second reference value. When the peak intensity is greater than the first reference, it is determined as a true peak, and peaks with intensities above the second reference and below the first reference may be set to require review by the user or an expert, or to be peak-determined when other set conditions are met. Also, since the amplification efficiency may vary depending on the locus and the luminescence efficiency and noise intensity may vary for each dye, the AT may be set for each locus or for each dye.

[0101] <Measures against artifacts> The above methods for dealing with artifacts will be described. For non-degraded DNA and DNA without PCR inhibitors, by introducing a DNA amount within the analysis range of STR-CE into PCR, appropriate peak intensity (above AT and below OS), PHR, and IPA data can be obtained, and the appearance of the ski slope can be suppressed. Also, even in the case of degraded, extremely low amounts, or DNA containing inhibitors, if the cycle number and input amount are appropriate, the amount of information obtained can be maximized. There is a parameter set of PCR cycle number and DNA amount that can minimize the appearance of artifacts while maximizing the amount of information obtained.

[0102] <Method for evaluating CE analysis results> When providing users with CE analysis results, it is also acceptable to indicate whether the obtained CE data is useful or not, and to what extent it is useful.

[0103] When presenting CE analysis results to the user, it is acceptable to indicate to the user whether the obtained CE data constitutes a complete DNA profile. It is also acceptable to advise the user whether further analysis of the obtained CE data is necessary.

[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, and the order may be changed, some steps may be omitted, steps not shown here may be included, and some or all of the steps may be performed simultaneously so that multiple flags are assigned. Alternatively, the flow shown in Figure 2 may be performed for each peak or locus, and it may be determined to be a full profile if all loci meet all the criteria. Even if it is not a full profile, only the information of the loci that met the criteria may be provided to the user. Alternatively, a table or similar output may be provided showing which criteria were not met or were met for each locus.

[0105] 201. No peak intensity should saturate (OS) the CE detection system. If an OS peak exists, the OS flag may be assigned to that peak, the gene locus, or the analysis result.

[0106] 202. At least one peak exceeding AT must be present at every locus. If no peaks are detected at any locus, the Drop out (DO) flag may be assigned to that locus or the analysis result.

[0107] 203. If there is only one peak that exceeds the AT, that peak must have at least twice the intensity of the AT (excluding genes that are originally detected only once, such as sex chromosome loci). If there is no peak with at least twice the intensity, the peak, locus, or analysis result shall be assigned the Inconclusive homozygous flag (IH flag).

[0108] 204. The peak intensity located ±1 base pair away from the main peak does not exceed the IPAT set at a peak ratio of 1%, 5%, 10%, 20%, or 40% of the main peak intensity. If it exceeds this, a flag indicating strong IPA intensity will be assigned to that peak or locus, or to the analysis result (IPA- or IPA++ flag).

[0109] 205. All detected peaks must be located within a DNA strand length that can be attributed to a DNA type. If a peak cannot be attributed and does not fall under IPA, it will be identified as an Off-Ladder (OL) peak, and the OL flag will be assigned to that peak, gene locus, or analysis result. However, peaks appearing at the stator peak position do not need to be considered for OL determination.

[0110] 206. No more than three peaks should be detected for a single gene locus (or two or more peaks for a locus that should only have one peak). If peaks are detected, the third highest intensity peak must have an intensity of 1% or less, 5% or less, 10% or less, 20% or less, or 40% or less compared to the second highest intensity peak. If the ratio of the intensity of the third highest intensity peak to the second highest intensity peak exceeds the threshold, the DNA in the sample may be determined to originate from two or more individuals, and the Mix flag may be set. However, if the third highest intensity peak occurs at the position where the first or second stunt peak would appear, it may be determined to be a stunt peak. If a peak appears at the stunt peak position with an intensity of 1% or less, 5% or less, 10% or less, 15% or less, 20% or less, or 40% or less compared to the main peak, it may be determined to be a stunt and the Mix flag may not be set. If a peak exists in a position that could potentially be a stutter peak, and its intensity exceeds the threshold for the acceptable range of a stutter peak, it may be a Mix, and the Mix flag may be set. Also, if the intensity of the third highest peak in a position where a stutter peak should not appear exceeds the threshold, the Mix flag may be set. Similar checks may be performed on the fourth, fifth, and subsequent peaks.

[0111] 207. If two or more peaks are detected, and the second peak has an intensity of 1% or more, 5% or more, or 10% or more of the first peak's intensity, and is 60% or less, 40% or less, or 20% or less, then the PHR is judged to be poor, and the PHR flag may be assigned.

[0112] PHR deteriorates when the amount of DNA subjected to PCR falls below 0.6 ng, 0.3 ng, 0.15 ng, or 0.075 ng. For example, if the amount of DNA subjected to PCR exceeds the aforementioned threshold when estimating the approximate amount of DNA from IPC intensity or the number of PCR cycles and peak intensity using CE analysis, the obtained DNA profile may be determined to originate from a mixed sample.

[0113] Since peaks that give the IPA flag also give the OL flag, criterion 205 may also serve as criterion 204, meaning that the IPA flag may encompass 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 required. In this case, the time required to complete the DNA analysis may be prolonged. Therefore, it is necessary to set an appropriate number of cycles to increase the probability of obtaining a full profile. Also, even if a full profile cannot be obtained, such as when the sample is originally mixed, the quantity is small, or there are many PCR inhibitors, or when not all gene loci are detected properly, if 5 or more, or 10 or more, gene loci are detected, it can be useful in criminal investigations. Such a DNA profile is called a partial profile. The more information about gene loci obtained, the more useful it is in criminal investigations. In other words, even in situations where a partial profile is obtained, the analysis protocol, especially the number of PCR cycles, should be set so that as many peaks as possible can be detected. In particular, in the case of mixed samples or small amounts of DNA, the protocol, especially the number of PCR cycles, should be set so that as many peaks as possible can be detected while satisfying criteria 201, 205, and 206 in the electrophoresis (i.e., these flags are not assigned).

[0115] A threshold can be set to determine whether the DO is caused by the absence of the relevant allele in the DNA introduced into the PCR reaction, or by an insufficient number of PCR cycles. For example, if there is a locus with no peaks exceeding AT, and there is a peak in the overall amplification product that is more than 10 times that of AT, it can be determined that the DO is due to the sample. Otherwise, it can be determined that the DO is due to an insufficient number of PCR cycles, or that it cannot be attributed to the sample. This determination can be used to determine whether an analysis with an increased number of PCR cycles should be performed.

[0116] [Regarding the scope of analysis] When performing STR-CE, the following factors can cause fluctuations in intensity: (1) fluctuations in the concentration of salts or injection-inhibiting substances in the PCR reaction solution, (2) the mixing ratio of the electrophoresis reagent and the PCR reaction solution, (3) amplification efficiency, (4) degradation of the electrophoresis reagent or incomplete denature, (5) temperature variations in the electrophoresis area, (6) variations during electrolytic injection, (7) variations between capillaries and between capillary arrays, and (8) variations in detection intensity in the detection area.

[0117] In the following, the dynamic range of the CE may refer to the dynamic range described in the manuals of various CE devices, the ratio of the maximum amplicon amount to the minimum amplicon amount to which a linear signal can be obtained with respect to the actual amount of amplicon input to the CE, the maximum amplicon to the minimum amplicon amount to which the relative ratio of the amount of amplicon input to the CE can be attributed 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, unless otherwise specified, the STR-CE analysis range may simply correspond to the dynamic range of CE, or it may be determined by actually varying the amount of DNA injected into PCR in experiments and finding the amount of DNA at which a specific allele can be correctly detected, or it may be determined by actually varying the amount of DNA injected into PCR in experiments and finding the amount of DNA at which a specific allele set can be correctly detected, or the range of such DNA amounts may be determined experimentally for multiple different individuals and the average, minimum, or maximum range may be used as the analysis range. The amount of DNA used for CE measurement is generally proportional to the amount of DNA injected into STR-PCR, but it is not always proportional due to the DNA concentration dependence of CE injection efficiency and the DNA concentration and cycle number dependence of PCR amplification efficiency. In addition, while the STR-CE analysis range does not usually change significantly when the number of PCR cycles is changed, it may shift, especially when the amount of DNA is reduced, due to the stochastic effect (a stochastic effect where the amount of DNA corresponding to the gene locus to be amplified no longer corresponds to the amount of DNA injected). Therefore, it is desirable to perform the above considerations for each cycle to determine the true analysis range of STR-CE, and the analysis range for each cycle set through such considerations should also be considered a type of analysis range for STR-CE. However, since verifying everything experimentally would require a great deal of effort, some parts may be replaced by calculations.

[0119] The dynamic range of CE and the analysis range of STR-CE may be evaluated by taking into account the variability mentioned in (1) to (8) above.

[0120] By changing the threshold value, you can broaden or narrow the analysis range. However, broadening the analysis range may come at the cost of decreased accuracy. If methods such as machine learning can more reliably distinguish between artifacts and true peaks, you can change the threshold value to broaden the analysis range.

[0121] In the following, the analytical range of the analysis system refers to the range of DNA amounts in a sample that can be correctly analyzed when a DNA sample is introduced into the analysis 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 introduced into the STR-CE. When expanding the analytical range of the STR-CE, it is possible to improve the dynamic range and sensitivity of the CE, reduce the variability of the STR-CE, prepare amplification products from multiple cycles, or prepare eluents at different dilution ratios and apply each to the STR-CE. When controlling the amount of DNA introduced into the STR-CE, the volume of the purification membrane may be reduced or the mesh size may be made coarser so that the upper limit of the amount of DNA that can be processed by the purification membrane is cut off, or the dilution ratio may be changed after quantification following purification.

[0122] 《Analysis system for segmented PCR and its operation example》

[0123] [Analysis System] [Example of a typical analytical system configuration] Figure 3 shows an example of the analysis system 101. Figure 4 shows an example of the operation procedure of the analysis system 101. The biomolecular analyzer is equipped with a computer 102 for performing biomolecular analysis and a flow channel device 104.

[0124] The fluid channel device 104 includes a lysis chamber 301 for introducing and dissolving collected samples, a purification membrane chamber 303 containing a purification membrane 302, a PCR chamber 304 for DNA amplification (a PCR chamber for performing thermal cycling), and a waste liquid chamber 305. There is an external connection port 306 that is fluidly connected to the outside of the device. Solution transport is performed via the external connection port 306, allowing for the exchange of reagents and amplification products with the outside of the device. When transporting solutions in the analysis system 101, the liquid delivery may be controlled using a pump and valve 307. The pump and valve 307 may all be located outside the fluid channel device, or some may be located inside the fluid channel device 104. In addition, the PCR reagent storage section 308 may contain PCR reagents 309 (polymerase, primer, dNTP, buffer, etc.) necessary for the PCR reaction, and the electrophoresis reagent storage section 310 may contain electrophoresis reagents 311. In addition, reagent storage sections 312, 313, and 314 are included for storing reagents necessary for pretreatment. In the sample loading step 401, before and after the sample is placed in the lysis chamber 301, lysis buffer is transported from the reagent storage section 312 to the lysis chamber 301. Next, lysis begins in the lysis step 402. In the purification step 403, the lysate is sent from the lysis chamber 301 to the purification membrane chamber 303, where the DNA is bound to the purification membrane 302, and washing solution is released from the reagent storage section 313 for purification. After purification, a step for drying the washing solution may be included. Elution solution is released from the reagent storage section 314, and the DNA eluted from the purification membrane chamber 303 is transported to the PCR chamber 304. PCR reagent is transported from the PCR reagent storage section 308 to the PCR chamber 304 and mixed with the eluted DNA. In the amplification step 404, the DNA purified in the PCR chamber 304 is mixed with PCR reagent 309 and subjected to the PCR reaction. In detection step 405, the amplified DNA is mixed with electrophoresis reagent 311 stored in electrophoresis reagent storage section 310, and measurement is performed in CE section 105.

[0125] After mixing the electrophoresis reagent and PCR reaction solution, a step may be added to heat the mixture to 80-100°C and then rapidly cool it to 0-10°C before performing CE analysis. This step helps to more completely unstrand the DNA, enabling highly accurate CE analysis.

[0126] [Example of an analysis system used in this embodiment] Figure 5 shows a schematic (Figure 5(1)) and an example of the operation procedure (Figure 5(2)) of the analysis system 101 of this embodiment. 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 analysis system 101 of this embodiment has a flow channel device 104 and a CE unit 105, and the flow channel device is equipped with a PCR chamber 304. The analysis system 101 is also equipped with a dispensing chamber 320. PCR reaction solution or electrophoresis sample is supplied to the dispensing chamber 320. However, the dispensing chamber 320 may be omitted. Each element is connected by flow channels 315 and 316.

[0128] As shown in Figure 5(2), the analysis system 101 prepares the PCR reaction solution in step 501, performs m PCR cycles in the PCR chamber 304 in step 502, then removes a portion of the amplified product from the PCR chamber 304 without changing its composition, removes PCR reaction solution m in step 503, performs CE measurement on the electrophoretic sample m in the CE section 105 in step 504, and performs n thermal cycles on the remaining PCR reaction solution m in the PCR chamber 304 in step 505 to obtain PCR reaction solution n. In step 506, removes a portion of PCR reaction solution n from the PCR chamber 304 without changing its composition, and performs CE measurement on the electrophoretic sample n in the CE section 105 in step 507.

[0129] The dispensing step in step 506 may be omitted, and all of the amplification products from the n cycles may be mixed with the electrophoresis reagent in step 507.

[0130] You can repeat steps 503 through 505 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. Performing 507 after 504 allows the setting of 505 to be changed according to the result of 504.

[0132] You can start step 505 immediately after step 503 is complete. The short time between 502 and 505 helps to suppress the generation of artifacts. Alternatively, you can set the timing of 505 and 506 so that 507 starts at the same time as 504 finishes.

[0133] [Examples of derivative analytical systems used in the implementation of the present invention] As shown in Figure 6, the analysis system 101 may be equipped with heating units 317 and 318 (temperature control and heating mechanism) for thermal cycling within the PCR chamber 304. Flow channels 319 and 315 are connected to the PCR chamber 304, and reagent supply and pressure control may be provided. As shown in Figure 6, the analysis system 101 may be equipped with a pump and valve 307 for carrying out solution transport suitable for various steps such as flow channel devices and CE measurement. As shown in Figure 6, the analysis system 101 may be equipped with a computer 102, which may have functions for controlling the pump and valve 307, controlling CE measurement, temperature control, analyzing and feeding back data obtained from the CE unit 105 and heating units 317 and 318, etc., and providing this data to the user. The flow channel device may be equipped with a dispensing chamber 320. The dispensing chamber 320 has the function of removing a portion of the amplification product from the PCR chamber 304 without changing its composition when m thermal cycles are completed. The dispensing chamber 320 may be equipped with a measuring function for dispensing a specified amount of PCR reaction solution m or n. When supplying the PCR reaction solution to the dispensing chamber 320, the dispensing chamber 320 may draw up the PCR reaction solution, or the PCR reaction solution may be supplied to the dispensing chamber 320 by pressurization. Alternatively, the dispensing chamber 320 may be provided simply for temporarily storing the PCR reaction solution and may not have a measuring function. The dispensing chamber 320 may be located outside the flow path device 104.

[0134] The PCR reaction solution m may remain in the dispensing chamber 320 for a period of time before CE measurement. In the dispensing chamber 320, it may be mixed with electrophoresis reagents (formamide and size standards). That is, the analysis system 101 may use the reaction solution (for example, a portion of the PCR reaction solution m and the PCR reaction solution) n Mix at least one of at least a portion of the DNA fragments with a solution containing multiple types of DNA fragments. This allows the concentration to be measured based on the peak height of a size standard.

[0135] As shown in Figure 6, the analysis system 101 may have a standby section 321 between the CE section 105 and the PCR section. The standby section 321 may temporarily hold the PCR reaction mixture or the electrophoresis sample mixed with the PCR reaction mixture and formamide during steps 506 to 507 or 503 to 504. The flow channel device in Figure 6 (and subsequent drawings) may be positioned vertically, i.e., the main part of the flow channel or at least part of the flow channel is parallel to the direction of gravity. In one embodiment of the flow channel device, the bottom of the drawing is used facing downwards in the direction of gravity. By using it vertically, bubbles / imported air in each chamber accumulate at the top of the chamber, so when transporting to the next chamber / flow channel, the solution is taken from the bottom, minimizing the introduction of air into the next step.

[0136] The following modifications can be made to the flow channel device.

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

[0138] [Flow channel devices] As shown in Figure 8, the flow channel device 104 may be subject to solution exchange and air pressure control via the analysis system 101 and the external connection port 306. Pressure may be applied from the external connection port 306 through the flow channel 322 to deliver PCR products or electrophoresis samples from the dispensing chamber 320. The flow channel device is equipped with valves 323, 324, 325, and 326, which open and close according to the analysis step.

[0139] As shown in Figure 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 and electrophoresis reagent in the dispensing chamber 320 may be mixed 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 located outside the flow path device, inside the analysis system 101, and in some cases the standby unit 321 may perform 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 introduced into the mixing chamber 327 for agitation, or the electrophoresis sample may be introduced into the CE unit 105.

[0140] As shown in Figure 9, the PCR chamber 304 may have three channels 319, 315, and 330. Channel 319 may be connected to the upstream side of the sample, channel 315 to the dispensing chamber 320, and channel 330 to the mixing chamber 327. Reaction solution m was transported to the mixing chamber 327 via the dispensing chamber 320 and removed from the PCR chamber 304. Reaction solution n may be transported to the mixing chamber 327 via the flow path 330 without passing through the dispensing chamber 320. Reaction solution m and Reaction solution Since n passes through a separate path, the decrease in reproducibility due to residual liquid can be suppressed. Furthermore, if reaction solution n is metered and reaction solution m is not metered but the entire volume is transported to the mixing chamber 327 or CE section 105, there is no need to transport reaction solution m to the dispensing chamber 320, so this flow channel device structure allows for simpler separation PCR.

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

[0142] As shown in Figure 10, the flow channel device 104 may be equipped with electrophoresis reagent storage sections 310 and 331 for storing electrophoresis reagents (formamide, DNA fragments, pure water, etc.). The electrophoresis reagent storage sections 310 and 331 may be located on the flow channel 315 or on the flow channel 319. The reagent storage section may be divided into two or more storage sections for each type of reagent, as shown in Figure 10. By dividing it into two, a predetermined amount of reagent can be released from each section during division. Alternatively, the amount of liquid released from the storage section can be controlled so that the reagent is released from one reagent storage section in two or more separate stages. As shown in Figure 10, the flow channel device 104 may also be equipped with air storage sections 332 and 333. The air storage sections 332 and 333 may be located on the flow channel 315 or on the flow channel 319. During division, a predetermined amount of air can be released from the air storage section 332 or 333 to transport a predetermined amount of PCR reaction solution outside the PCR chamber 304. In this flow channel device configuration, fractional PCR can be performed even if the dispensing chamber 320 is omitted. In this flow channel device configuration, the mixing chamber 327 may also serve as the dispensing chamber 320. Instead of air, a liquid that does not affect the PCR reaction, such as oil, may be placed in the air reservoir 332 or 333. PCR reaction solution may be placed in the air reservoir 332 or 333, and a new volume of PCR reaction solution equivalent to the dispensed PCR solution may be replenished.

[0143] As shown in Figure 11, the electrophoresis reagent storage sections 310 and 331 may be provided in paired channels spanning the PCR chamber 304. Preferably, as shown in Figure 11, the electrophoresis reagent storage section 331 is located closer to the PCR chamber 304 than the air storage section 333. This is because all or more of the electrophoresis reagent 311 accumulated in the channel 319 or PCR chamber 304 can be delivered to the dispensing chamber 320 or mixing chamber 327, contributing to improved reproducibility. As shown in Figure 11, the channel device 104 may also have the air storage section 334 located closer to the PCR chamber 304 than the electrophoresis reagent storage section 310 on the channel 315. The air storage section 334 may be used to transport the electrophoresis sample m in the dispensing chamber 320 to the mixing chamber 327 or CE section 105. The air storage section 334 may be replaced by the channel 322.

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

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

[0146] Step I: A PCR chamber 304 containing PCR reaction solution 335 is provided. With valves 326, 323, and 325 closed, an m-cycle PCR reaction is performed in the PCR chamber 304. This step may also correspond to Step 502.

[0147] In this manner, the analysis system 101 generates PCR reaction solution m (first reaction solution) by performing m thermal cycles on the PCR reaction solution 335 in the PCR chamber 304.

[0148] Step II: Once the m-cycle PCR reaction is complete, open valves 326 and 323 and transfer a portion of the solution from PCR chamber 304 to dispensing chamber 320. This step may also correspond to step 503.

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

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

[0151] Step III: Close valves 323 and 326 to push the electrophoresis reagent 311 out of the electrophoresis reagent storage section 310, and transfer the PCR reaction mixture m stored in the dispensing chamber 320 to the mixing chamber 327. The PCR reaction mixture m is mixed with the electrophoresis reagent 311 to become the electrophoresis sample 336.

[0152] Step IV: Open valve 325 to transfer the electrophoretic sample in the mixing chamber 327 to the outside of the flow channel device 104 (the standby section 321 or CE section 105 of the analysis system). This step may also correspond to step 504.

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

[0154] Thus, in the PCR chamber 304, the analysis system 101 performs a total of nm thermal cycles (where nm is an integer of 2 or more) 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: Once a total of n thermal cycles are complete, close valve 325 and open valves 326 and 323 to transfer the PCR reaction solution n to the mixing chamber 327.

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

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

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

[0159] [Other examples of split-stage PCR testing methods] Figure 14 shows the analysis process for split PCR, Figure 15 shows an example of the flow channel device 104, and Figure 16 shows an example of a transport method for splitting the PCR reaction solution in m=24 cycles and n=30 cycles as shown in Figure 15. For convenience of illustration, some reference numerals are shown in Figure 15 and omitted in Figure 16.

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

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

[0162] Step II: Once the 24-cycle PCR reaction is complete, open valves 326, 323, and 337 and transfer a portion of the solution from PCR chamber 304, b μl (where a > b), to dispensing chamber 320 via channel 315. (This may also correspond to steps 605 and 503.)

[0163] Step III: Close valves 323, 326, and 337 and open valves 324 and 325 to push c μl of electrophoresis reagent 311 out of the electrophoresis reagent storage section 310 and transfer the PCR reaction mixture m stored in the dispensing chamber 320 to the mixing chamber 327. The PCR reaction mixture m is mixed with the electrophoresis reagent 311 to become the electrophoresis sample 336. (This may correspond to steps 606 and 504.) At this time, some electrophoresis reagent 311 may remain in the dispensing chamber 320 and part of the flow path 315.

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

[0165] Step V: Close all valves and perform six thermal cycles on the ab μl of PCR reaction mixture 335 remaining in PCR chamber 304, followed by an 8-minute final extension. (This may also correspond to steps 608-610 and step 505.)

[0166] Step VI: Once a total of 30 thermal cycles are complete, open valves 326, 323, and 325 to push d μl of electrophoresis reagent out of the electrophoresis reagent reservoir 331 and transfer the PCR reaction mixture n to the mixing chamber 327. (This may correspond to Step 506.)

[0167] Step VII: You may close valve 325, open valve 339, apply air pressure, and transfer all the remaining solution in PCR chamber 304 to mixing chamber 327. Alternatively, you may introduce air into mixing chamber 327 to agitate and homogenize the electrophoresis sample 336. (This may correspond to steps 611 and 507.)

[0168] Step VIII: Open valve 338 to transfer the electrophoretic sample in the mixing chamber 327 to the outside of the flow channel device 104 (the standby section 321 or CE section 105 of the analysis system). (This may correspond to steps 612 and 507.)

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

[0170] [Table 1]

[0171] The basic thermal cycle settings were based on the manual for the STR-PCR kit (GlobalFiler® Express) (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] In steps 605, 606, 610, and 611, the solutions may be kept at a low temperature (4°C). Keeping them at a low temperature prevents sample degradation and prevents unwanted reactions from occurring.

[0173] In step 605, setting the temperature during division to be about the same as the temperature in the denaturation step is preferable because it prevents unnecessary extension reactions and thus suppresses the generation of artifacts.

[0174] In step 605, if the temperature during splitting is the same as the extension temperature, an unnecessary extension reaction occurs, but non-specific amplification can be suppressed. Furthermore, this is preferable because it avoids the inactivation of polymerases and phosphors. The impact of the unnecessary extension reaction on the analytical accuracy of STR-CE is negligible.

[0175] In step 605, the temperature during splitting may be set between the extension temperature and RT. This is simpler as precise temperature control is not required. Since sufficient amplicons are present when m cycles of PCR are completed, the effects of non-specific amplification can be ignored.

[0176] In step 605, a shorter time required for the division is preferable. A longer division time may increase the number of artifacts. Furthermore, a longer division time may lead to increased inactivation of various biomolecules, such as polymerases.

[0177] As shown in Figure 14, in this embodiment, the analysis system 101 does not perform any analysis other than electrophoresis on each reaction solution (including a portion of PCR reaction solution m and at least a portion of PCR reaction solution n) before electrophoresis analysis. This simplifies the configuration of the apparatus and eliminates the need for additional optical systems, for example.

[0178] [Amount of PCR reaction solution and electrophoresis reagent] The volume of PCR reaction solution used in this channel device is 1 μl to 200 μl, more preferably 10 μl to 50 μl. A smaller volume allows for more accurate and faster temperature control. On the other hand, a larger volume allows for the acceptance of more purified DNA, thus facilitating higher sensitivity. Furthermore, a larger volume eliminates the need for highly accurate solution weighing during the splitting process.

[0179] Table 2 shows examples of liquid volumes when divided. The actual liquid volume measured does not have to be the exact value in the table; the median of multiple measurements may be the value in the table.

[0180] [Table 2]

[0181] Assume that in step 502 the total volume of PCR reaction solution is a μl, in step 503 the volume of solution extracted during m cycles is b μl, in step 504 the amount of electrophoresis reagent mixed with PCR reaction solution m is c μl, and in step 507 the amount of electrophoresis reagent mixed with PCR reaction solution n is d μl. Table 2 above shows examples of suitable relationships or set values ​​for a to d. In order to efficiently introduce amplification products and size standards into the CE, it is necessary that the ionic strength of the electrophoresis sample is sufficiently low. Therefore, it is preferable that the amount of electrophoresis reagent be mixed in greater quantities than the volume of PCR reaction solution, such as 2 times, 5 times, 10 times, 20 times, etc.

[0182] The PCR reaction can be divided so that the amounts of PCR reaction solution m and PCR reaction solution n are equal. Having similar amounts of PCR reaction solution m and n allows for more stable delivery of the electrophoresis reagent to the CE section.

[0183] The PCR splitting can be set so that the amount of PCR reaction mixture m is less than the amount of PCR reaction mixture n. Using a smaller volume of solution during splitting reduces the variation in volume when performing nm-level PCR, thus allowing for more reproducible preparation of the amplified product n.

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

[0185] Set 2 is an example of solution volume when the PCR reagent volume is 15 μl, 5 μl of solution is taken out during the splitting of m cycles, and the entire volume of PCR reaction mixture is mixed with the electrophoresis reagent during n cycles. In this example, the analysis system 101 transfers the entire volume of PCR reaction mixture from the PCR chamber to the outside of the PCR chamber at the end of n thermal cycles. This eliminates the need for weighing equipment and simplifies the flow path configuration.

[0186] In the fluid delivery procedure shown in Figure 16, electrophoresis reagent remains in the dispensing chamber 320. If the amount of electrophoresis reagent in the electrophoresis reagent storage unit 310 is sufficiently large relative to the dispensing chamber 320, the impact on analytical accuracy and range can be ignored even if some electrophoresis reagent remains in the dispensing chamber 320. Furthermore, if a flow path 322 is provided as an air line between the valve 337 and the dispensing chamber 320 on the flow path 315, or if an air storage unit 334 is provided, the entire volume can be pushed into the dispensing chamber 320 and the mixing chamber 327. In the case of a large volume configuration, almost the entire volume of PCR reaction solution m in the dispensing chamber 320 is sent to the mixing chamber. In addition, it is preferable to store extra electrophoresis reagent in the reagent storage unit, taking into account that some of the electrophoresis reagent will be left behind in the dispensing chamber 320.

[0187] Set 3 is an example of the solution volume when a larger amount of electrophoresis reagent is stored in the electrophoresis reagent storage unit 310, anticipating that eμl of electrophoresis reagent will remain in the dispensing chamber 320.

[0188] Set 4 is an example of the solution volume when the volume of the dispensing chamber 320 is b μl, and a larger amount of electrophoresis reagent is stored in the electrophoresis reagent storage section 310, anticipating that b μl of electrophoresis reagent will remain in the dispensing chamber 320.

[0189] [Detailed division method] Variations in solution volume during dissolution narrow the effective analytical range of the analysis system; therefore, high precision in dissolution dissolution is desirable when dissolving PCR reaction solutions. To achieve high-precision dissolution dissolution, functions such as pre-setting appropriate pressurizing pressure and pressurizing time, extruding the PCR reaction solution with a liquid or gas of a specified volume, using a liquid level sensor, or extruding the PCR reaction solution into a dispensing chamber 320 of a specified volume may be incorporated. Multiple extrusion and metering methods may also be combined.

[0190] Weighing 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 PCR reaction solution n is mixed with the total electrophoresis reagent, weighing only needs to be performed in step 503. In other words, the number of weighings may be the number of divisions minus 1. Similarly, if weighing is performed twice, since there is a specified amount of liquid in the PCR chamber, an additional p thermal cycles of PCR may be performed on the reaction solution remaining in the PCR chamber, and the CE measurement of the third division reaction solution after n+p cycles of PCR reaction may be performed.

[0191] When removing the PCR reaction solution from the PCR chamber, you can use a pump to pressurize the PCR chamber and send the solution to the dispensing chamber. Alternatively, if the valves are closed during PCR heating, internal pressure is present, so you can set it up so that the solution moves to the dispensing chamber due to the internal pressure without having to open valves 326 and 323 in step II.

[0192] In step 502, after the m cycle is completed, in step 503, the PCR reaction solution m may be extruded with new PCR reagent and transported to the dispensing chamber 320 or mixing chamber 327 in a specified volume. This dispensing method may also be performed by sealing PCR reagent instead of air in the air reservoir 332 shown in Figure 10. Since the solution is extruded, there is an advantage in that the volume extruded is easy to control. Also, when extruding with the PCR reaction solution, there is an advantage in that there is no adverse effect on the reaction due to contamination by air bubbles or oil. On the other hand, there is a possibility that new PCR reagent will be used in a later stage, adding a new factor to the above-mentioned variability factors (1) to (8) in the analytical range. Also, because it is diluted with the PCR reaction solution, the PCR reaction solution remaining in the PCR chamber becomes diluted at the time of the m cycle, requiring more PCR cycles. In addition, if a reagent at room temperature is added, the temperature of the PCR chamber will decrease. Furthermore, it is necessary to perform initial denature again in step 505. For this reason, when extruding with the PCR reaction solution, the PCR reaction time will be slightly longer. Furthermore, increasing the number of cycles or performing initial denature again can contribute to the generation of artifacts. However, performing initial denature of the PCR reaction mixture used for extrusion before step 503 can partially resolve the above issues.

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

[0194] Instead of using air or PCR reagent to extrude the PCR reaction solution, oil may be used. This separation method can also be implemented by sealing oil instead of air in the air reservoir 332 shown in Figure 10. Any oil that does not affect the PCR reaction can be used. For example, silicone oil, mineral oil, fluorinate oil, or a mixture of multiple oils may be used. When extruding with oil, the volume extruded can be easily controlled, similar to when extruding with reagent, and unlike when extruding with the PCR reaction solution, it does not mix with the PCR reaction solution. On the other hand, if oil mixes with the PCR reaction product and flows to the next stage, it may interfere with CE measurement. When the PCR reaction solution m is removed, it may be separated by a separation membrane or centrifugation to leave the oil in the chamber.

[0195] [Details of the weighing mechanism] When introducing the PCR reaction solution into the dispensing chamber 320 during splitting, if air or bubbles from the PCR chamber enter instead of the PCR reaction solution, the weighing accuracy will decrease. Therefore, it is preferable that the dispensing chamber 320 and the flow path 315 are designed to prevent air from entering. An example of a structure to prevent air from entering is shown in Figure 17. As shown in Figures 17(a) and (b), the flow path 315 branches off from the PCR chamber 304 from below the liquid level 701 of the PCR reaction solution 335 in the direction of gravity. Since air accumulates on the upper side of the PCR chamber 304, the amount of air introduced into the flow path 315 during splitting can be minimized. Furthermore, it is desirable to design the liquid volume and chamber shape so that the liquid level 701 is above the connection point between the PCR chamber 304 and the flow path 315 in the direction of gravity when splitting is complete. As shown in Figure 17(b), the liquid level 701 may be above the valve 326 or 323 in the direction of gravity. In this case, the amount of air bubbles entering the PCR chamber 304 can be minimized or completely eliminated. The PCR reaction solution 335, located above valve 326 or 323, does not need to be subjected to the PCR reaction. However, in this case, unreacted reagent may contaminate the CE measurement, potentially reducing its sensitivity.

[0196] As shown in Figure 17(c), the channel 315 may be connected to the bottom of the PCR chamber 304. This configuration has the advantage of making it difficult for air to enter, but the channel 315 becomes somewhat longer, which tends to cause more liquid loss.

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

[0198] The dispensing chamber 320 may have a weighing function. For example, Figure 18 shows an example of a dispensing chamber 320 with a weighing 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 weighing, but also to prevent liquid from splashing out of the dispensing chamber 320 during the PCR reaction, and to prevent electrophoresis reagents and electrophoresis samples from flowing back into the PCR chamber 304 after dispensing. Valve 324 may be provided not only for weighing, but also to prevent electrophoresis samples from flowing back after being transported to the mixing chamber. The volume of the dispensing chamber may correspond to the volume of liquid to be divided and measured out from the amplification product m. The dispensing chamber may be spherical or cylindrical, rectangular, elongated, have a meandering channel, or be ellipsoidal or elliptic.

[0199] For example, the analysis system 101 may weigh a predetermined amount of PCR reaction solution m in the range of 0.1% to 50% by transporting the PCR reaction solution m to the dispensing chamber 320 (weighing unit) after the completion of m thermal cycles. This eliminates the need for additional weighing.

[0200] Figure 19 shows an example of a weighing mechanism. Multiple mechanisms like those shown in Figure 19 can be combined for weighing. Weighing mechanisms not shown can also be used. Combining multiple weighing mechanisms can achieve more robust weighing.

[0201] Figure 19(a) shows an example of a metering mechanism, which includes a vent filter 702 and a flow path 703 connecting to a flow path 328. The flow path 703 is connected to the dispensing chamber 320 side of the valve 324 of the flow path 328. The vent filter 702 is preferably made of a hydrophobic porous filter such as PP or fluororesin. 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 to deliver the PCR reaction mixture into the dispensing chamber 320. Air present in and around the dispensing chamber 320 is released through the flow path 703 and out of the vent filter 702.

[0203] Step II: Since amplification products and electrophoresis reagents cannot pass through the vent filter 702, weigh out the PCR reaction mixture in the volume of the dispensing chamber 320 and the channels before and after it.

[0204] Step III: Close valve 337 and open valve 324. Dispense the PCR reaction mixture from dispensing chamber 320 into mixing chamber 327 using electrophoresis reagent or air.

[0205] As shown in Figure 19(b), a flow resistance 704 may be provided on the flow path 328. The flow resistance 704 may be a hydrophobic filter such as PP or fluororesin. Also, the hydrophobicity of the flow path surface in the flow resistance 704 may be higher than that of the flow path 328 portion. Furthermore, the flow path width may be narrowed by the flow resistance 704, and obstacles may be installed. Also, the flow path width may widen abruptly in the flow resistance 704. The flow resistance 704 may be provided using the principle of a capillary stop valve. Air can easily escape until the flow resistance 704 comes into contact with the liquid. Figures 21 I to III show examples of the operation of this metering mechanism.

[0206] Step I: Pressure (A kPa) is applied to the PCR chamber 304 and the PCR reaction solution is delivered to the dispensing chamber 320. Air present in and around the dispensing chamber 320 can escape smoothly against the flow resistance 704.

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

[0208] Step III: Close valve 337 and apply B kPa (where B > A) to dispensing chamber 320 with electrophoresis reagent or air. The PCR reaction mixture in dispensing chamber 320 can then be transferred to mixing chamber 327.

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

[0210] Figure 22 shows an example of a simple metering mechanism that does not use a liquid level sensor, vent filter, or flow resistance element. However, this method may be modified to incorporate a robust dispensing mechanism using a liquid level sensor, vent filter, flow resistance element, etc.

[0211] Step I: A PCR chamber 304 containing PCR reaction solution 335 is provided. With all valves closed, the m-cycle PCR reaction is performed in the PCR chamber 304.

[0212] Step II: Once the m-cycle PCR reaction is complete, open valves 326 and 323 to bring the pressure inside the PCR chamber 304 to atmospheric pressure (=100kPa).

[0213] Step III: Apply a pressure of 100 kPa from channels 319 and 330.

[0214] Step IV: Open valve 337 to transfer a portion of the solution from PCR chamber 304 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 balance out, and the transfer of solution stops.

[0215] Step V: Close valve 337 and open valve 324 to return the pressure inside the dispensing chamber 320 to atmospheric pressure.

[0216] In this method, when transferring the solution from the PCR chamber 304 to the dispensing chamber 320, the amount of liquid that enters the dispensing chamber 320 can be controlled by the pressure applied to the dispensing chamber 320. When the volume of the dispensing chamber 320 is V1 and it is filled with air at pressure P1, when a volume V2 of liquid enters the dispensing chamber, according to Boyle's Law, the pressure changes to P2 = P1 * V1 / V2. When P2 balances the applied pressure, the solution stops flowing. In step I, if the pressure inside the dispensing chamber is 100 kPa, when the volume of the dispensing chamber 320 decreases to half, the pressure inside both the dispensing chamber and the PCR chamber becomes 200 kPa, and the forces balance. Therefore, a 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 just an example, and the volume of the dispensing chamber, the space in the flow path before and after it, and the amount of liquid to be measured can be appropriately set for measurement. A disadvantage of this method is that it tends to require valves with 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 this will result in a larger amount of residual liquid when the electrophoresis reagent is pushed out afterward.

[0217] [Regarding measurement accuracy] In CE analysis, if there is variability in peak intensity when the same sample is measured multiple times, the effective analytical range becomes narrower. If the analytical intensity variates significantly due to the introduction of segmented PCR, the effective analytical range becomes narrower, which is undesirable. If the mixing ratio of electrophoresis reagent and PCR reaction solution becomes large due to the introduction of segmented PCR, this will be reflected in the variability of peak intensity, narrowing the effective analytical range.

[0218] As shown in Figure 14, experiments were conducted to confirm how much variation in the mixing ratio of the PCR reaction solution and electrophoresis reagent affects the peak intensity of the CE. Figure 23 shows the CE intensity when the ratio of PCR reaction solution to electrophoresis reagent is varied. The variation in peak intensity between capillaries was normalized by the average intensity of size standards without PCR reaction solution, which were measured separately. The peak intensity of the size standards decreased monotonically with increasing ratio of PCR reaction solution. On the other hand, the peak intensity of the amplification product increased monotonically, and the variation in peak intensity was limited with respect to the change in liquid volume.

[0219] In CE measurements, when the amplified product is drawn into the capillary, a voltage is applied to both the electrophoretic sample and the capillary to perform electric field implantation. In electric field implantation, the amplified product introduced into the capillary is implanted competitively with other ions and nucleic acids. When the voltage and implantation time are constant, the relationship shown in Equation 1 holds (where β is the current value and α is a constant).

number

[0220] Assuming that the standard is a mixture of 1 μl of PCR reaction solution to 10 μl of electrophoresis reagent, when the amount of PCR reaction solution varies by γ μl, Equation 1 can be expressed as follows:

number

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

number

[0222] On the other hand, the injection volume of the amplification product k3*C3, obtained by rearranging Equation 1, is monotonically increasing with respect to the volume of the PCR reaction solution.

number

[0223] PCR reaction solutions contain many salts, primers, dNTPs, etc., and the injection volume is almost saturated. Therefore, even if the proportion of PCR reaction solution relative to the electrophoresis reagent increases slightly, the fluctuation in peak intensity is limited. Even if the ratio of PCR reaction solution to electrophoresis reagent 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 higher concentration of PCR reaction solution than the standard conditions (PCR reaction solution: electrophoresis reagent = 1.6:10), even if the ratio of PCR reaction solution varies by ±50% from the target ratio, the fluctuation rate of peak intensity remains below 10%. These experimental results suggest that even if the dispensing accuracy of segmented PCR is somewhat poor, the fluctuation in peak intensity derived from the amplification product due to fluctuations in the mixing ratio of PCR reaction solution to electrophoresis reagent is limited.

[0224] In some cases, it is preferable to adopt a simple and low-cost weighing mechanism, even if its accuracy is somewhat poor. When using a low-accuracy weighing mechanism, as described above, it is preferable to select a solution composition in which fluctuations in the mixing ratio of the PCR reaction solution and electrophoresis reagent do not significantly affect the peak intensity. This can be achieved by adding salt to the electrophoresis reagent, or by using salts, primers, dNTPs, etc., contained in the PCR reaction solution. Furthermore, by performing the measurement under conditions where the mixing ratio of the electrophoresis reagent and the PCR reaction solution is slightly higher in the PCR reaction solution, and measuring when the mixture is closer to saturation, the influence of fluctuations in the ratio can be reduced. In this case, it is preferable to mix in a slightly larger amount of size standards than the standard mixing ratio so that the peak intensity of the size standards contained in the electrophoresis reagent reaches the level required for measurement.

[0225] "Method for Setting Split PCR Protocol" [Principle of Expanding Analysis Range by Split PCR]

[0226] In order to enable the analysis of various concentrations of target DNA contained in a sample, the PCR reaction solutions m and n containing amplification products of different concentrations are each subjected to electrophoresis analysis. Depending on the concentration range of the target DNA that may be contained in the sample, by setting m and n in advance within an appropriate range, 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] Figure 24 shows the principle of expanding the analysis range by split PCR. The horizontal axis in Figure 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 CE detection. In the figure, the concentration of the amplification product is shown as increasing linearly with respect to the DNA input amount, 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 the lower limit a and the 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 the lower limit c and the upper limit d. At this time, if a < d, the analysis range can be expanded from b / a or d / c to b / c. Let the analysis range of STR-CE when expanded by split PCR be 804. 804 is the range of DNA amounts within which either m or n falls within the analysis range of STR-CE, and in principle, there is no DNA amount that spills out of the analysis range within the range. Hereinafter, the analysis range (b / a) at each cycle number is approximately constant and doubles with respect to the cycle number difference x. , , , times the change. At this time, the interval between m and n is limited to the range of the following formula 2.

Equation

[0228] An example satisfying this is shown in Fig. 24(1). For example, when the analysis range b / a with respect to the amount of DNA introduced into PCR is 80 times, n - m is preferably 6 or less, and the magnification factor is 64. The magnification factor can be increased by (b / a) with respect to the number of divisions y, but it is considered that stable liquid feeding can be realized on a simpler flow path device with a smaller number of divisions. Hereinafter, m and n are set, and the case of analyzing amplified products m and n will be mainly described. On the other hand, in the same setting method, l may be set for three divisions and k may be set for four divisions. y-1 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.

[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 to change by a factor of two.

[0230] 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 analyzable at m exceeds the upper limit d analyzable at n, and correct DNA identification cannot be performed at 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 may be set so that the relationship a > d holds. However, since the amount of DNA in the actually input sample is often unknown, it is preferable to satisfy Equation 2.

[0231] When covering extremely low-concentration DNA at 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.

[0232] Since there are factors that cause variations in multiple analysis results in STR-CE analysis, it is necessary to take into account the variations and make the analysis DNA amount ranges that can be covered by STR-PCR with m cycles and the DNA amount ranges that can be analyzed by STR-PCR with n cycles overlap. If there is no overlap, even though the DNA amount is within the analysis ranges of m and n, there will be a DNA amount for which the analysis fails. That is, it is necessary to set m and n such 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.

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

[0234] In the case of an analysis system that starts or ends step 505 after step 504, n may be set according to the measurement result of the electrophoretic sample m.

[0235] n may be set by branching to the following conditions: (1) When no peak of the amplification product m is detected (2) When peaks are detected and some of the peaks are below AT (3) When peaks are detected and some of the peaks are already saturated or exceed the IAP threshold.

[0236] In the case of (1), when the analysis range of STR-CE is x, additional n - m cycles (n - m ≤ log(x), where x is the analysis range of STR-PCR) may be performed. In this case, it is preferable to use the maximum value such that n - m satisfies Equation 2.

[0237] In the case of (2), when the saturation intensity of CE is z with respect to the maximum intensity y of the detected peaks, additional PCR defined by n - m cycles (n - m ≤ log(z / y)) may be performed.

[0238] In the case of (3), additional PCR may not be performed.

[0239] [How to set the interval between m and n, taking into account the intensity ratio of the sample to be analyzed] Especially when CE analysis takes a long time, waiting until the m-cycle electrophoresis is complete, as described above, can lead to excessive waiting time for the nm-cycle PCR reaction, potentially increasing artifacts or even causing the PCR reaction to fail. Therefore, m and n can be set in advance.

[0240] In STR-CE, homozygous and heterozygous gene loci are present together. Therefore, even in an ideal analytical system where all amplification efficiencies, CE injection efficiencies, and fluorescent dye emission efficiencies are equal, there will be a 1:2 difference in intensity between heterozygous and homozygous peaks.

[0241] In actual STR-CE analysis, differences in amplification efficiency, CE injection efficiency, and luminescence efficiency between dyes exist depending on the gene locus and DNA length. Therefore, even when the amount of DNA is properly adjusted, there are differences in intensity between peaks ranging from 1:2 to 1:20. Furthermore, AT values ​​may differ between dyes. In such cases, it is necessary to consider the differences in AT settings when determining the analysis range.

[0242] When DNA originates from multiple individuals, the allele ratio can vary from 1:2 to 1:1000.

[0243] When DNA is degraded, there is a difference in the allele ratio of 1:2 to 1:1000. Typically, DNA from shorter loci or alleles is amplified more, while the peak intensity of DNA from longer loci or alleles tends to decrease.

[0244] When PCR is inhibited, shorter DNA tends to be amplified more, while the peak intensity of longer DNA tends to decrease. In such cases, the difference in peak intensity can range from 1:2 to 1:1000.

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

[0246] In what follows, the factors determining the intensity ratio between peaks will be discussed on the assumption that they are derived from the difference in the abundance ratio of alleles contained in the DNA input into STR-PCR. Although differences in amplification efficiency, CE injection efficiency, and luminescence efficiency also have an impact, these differences will be omitted for the sake of simplicity and considered to be subsumed in the discussion of the abundance ratio between alleles.

[0247] The effective STR-CE analysis range in the case where there are differences in the abundance ratio of the alleles to be analyzed will be explained using Fig. 25. Assume that the amplification products contain allele α and allele β. However, assume that the amounts of allele α and β are such that α > β before or after amplification. Let plot 805 be the plot of the amount in the amplification product or the CE peak intensity against the input DNA amount of allele α. Let plot 806 be the plot of the amount in the amplification product or the CE peak intensity against the input DNA amount of allele β. Plots 805 and 806 are not necessarily straight lines. Assume that amplicon α can be analyzed in the range from DNA input amount a to b. Also assume that amplicon β can be analyzed in the range from DNA input amount c to d. If the PCR amplification is linear in the concentration range of a to b,

Equation

Equation

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

[0249] Figure 26 shows a correspondence table between the numerical values ​​corresponding to range 807 and the nm setting, specifically the intensity ratio α / β of the largest amplicon to the smallest amplicon that can be analyzed by split-resolved PCR. For example, if range 807 is 120 and nm is set to 5, the allele with an abundance ratio of 1 / 3.75 to the allele with the highest abundance ratio will be the target of analysis. Conversely, if the intensity ratio of the allele set to be analyzed is greater than the ratio shown in Figure 26, it means that concentrations where the largest or smallest peak exceeds the upper or lower limit of analysis will appear within the analysis range. For example, as shown in Figure 27(1), there is no problem if both α and β are detectable at the concentration, but as shown in Figure 27(2), a situation may occur where only α is detected and β is not.

[0250] Samples with excessively high peak intensity ratios do not need to be included in the analysis. An example is shown below. STR-CE contains stutter peaks, and since it is difficult to distinguish peaks with an intensity ratio of 1:20 or more within a single locus from stutter peaks, it is not necessary to analyze peaks with an intensity ratio greater than 1:20 or 1:40 within a single locus. • Since degraded DNA may have a reduced overall DNA volume, it is not necessary to analyze peaks whose peak intensities are separated by more than 1:20, 1:40, or 1:100. As described above, in electrophoresis diagrams where the intensity ratio between peaks is extremely large, smaller peaks are not significant, so an Inter-locus PHR threshold may be set that defines the minimum peak intensity relative to the maximum peak intensity to be analyzed. When setting the interval between m and n using Figure 27, it is preferable to set the abundance ratio of the allele to be analyzed to slightly less than the Inter-locus PHR threshold.

[0251] [How to configure n] If partial profiles are also included in the analysis, 30-34 cycles is appropriate.

[0252] Peaks originating from a single copy of DNA can be detected by setting the cycle count to 36.

[0253] Setting the number of cycles to 36 or more is not advisable, because while artifacts (particularly stutters and drop-ins) increase significantly when the cycle count exceeds 36, the probability of detecting individual-derived peaks does not improve.

[0254] If partial profiles are not included in the analysis, 30 or 29 cycles or fewer is appropriate.

[0255] For samples containing the minimum amount of DNA, it is preferable to select the minimum number of cycles that results in the fewest error judgments, and this can be determined experimentally.

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

[0257] n cycles can be set to the minimum number of PCR cycles required to ensure that the amplification product derived from 20 copies of genomic DNA always produces a full profile.

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

[0259] After experimentally or computationally determining the detection limit at a specific number of cycles, you can set n under the assumption that the detection limit doubles or decreases with each cycle. However, if the amount of DNA is too small, the stochastic effect will increase the variability of the peak intensity, so it is preferable to set a number of cycles with some margin.

[0260] n can be set independently of m using the method described above. However, if the interval between m and n is too wide, there is a possibility that some DNA may remain unanalyzed.

[0261] If m is set first, you can also set it using the difference in nm that covers the intensity ratio of the peaks you want to analyze, as shown in Figure 26, and the value of m.

[0262] If the analysis range 803 is known, it is preferable to set n to have a cycle count difference of log2(analysis range 803) or less from m.

[0263] You may want to set a cycle count with some margin, taking into account fluctuations and peak intensity ratios in various analysis systems.

[0264] [How to set m] In DNA analysis, the amount of DNA introduced into PCR rarely exceeds 1.5 μg. Therefore, reducing the number of cycles to less than 20 will not increase the number of loci that can be correctly detected. For this reason, it is preferable to set the number of cycles to 20 or more.

[0265] It is preferable to evaluate the amount of DNA introduced into PCR from a sample containing the maximum amount of DNA that can be brought into the analysis system, and then set the PCR cycle to the longest possible cycle in which OS or IPA flags are not detected when that DNA is analyzed using STR-CE.

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

[0267] You may want to set a cycle count with some margin, taking into account fluctuations and peak intensity ratios in various analysis systems.

[0268] You can determine m by examining the lower limit of detection, the amount of DNA to be injected into PCR, and the upper limit of detection DNA for a specific number of cycles, and assuming that the lower and upper limits increase or decrease by a factor of two with each cycle.

[0269] m can be set independently of n using the method described above. However, if the interval between m and n is too wide, there is a possibility that some DNA may remain unanalyzed.

[0270] If you set n first, you can set it based on the difference in nm that covers the intensity ratio of the peaks you want to analyze, as shown in Figure 26, and the value of n.

[0271] If the analysis range 803 is known, it is preferable to set m to have a difference in the number of cycles of log2(analysis range 803) or less.

[0272] Even if the interval between m and n is 2 or 3, the expansion of the analysis range is limited to 4 or 8 times. To perform robust DNA analysis, it is desirable to expand the analysis range by more than an order of magnitude. Therefore, it is preferable to set the interval between m and n to 4 or greater. 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] [Set m and n from the table] Figure 28(1) shows the analysis range expansion rate when m and n are set. Based on this table, you may determine the interval between m and n to obtain the required expansion rate. However, this table does not take into account changes in the analysis range that depend on the DNA concentration range, such as the stochastic effect. Also, if the original analysis range is larger than the expansion rate, it is unsuitable because it will result in a range of DNA amounts that cannot be analyzed, as shown in Figure 24(2). Furthermore, when analyzing DNA with large differences in the abundance ratio between alleles, setting the expansion rate to the absolute limit relative to the original analysis range will result in alleles that cannot be analyzed.

[0274] Figure 28(2) shows the expanded analytical range (in terms of orders of magnitude) by split PCR. In a typical STR-CE, 0.75 ng to 48 ng (1.8 orders of magnitude) of Genomic DNA can be analyzed after 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 needs to be evaluated for each measurement system. Variability also needs to be taken into account. Based on these results, the orders of magnitude of the analytical range, the lower limit of analysis for n cycles, and the upper limit of analysis for m cycles are shown here. The stochastic effect when reducing the amount of DNA is not considered. All values ​​are 2 for the number of cycles. n The table was created to show the values ​​that change accordingly. You can create a table like this one and determine m and n from the appropriate lower and upper limits and the analysis range. However, if the expansion rate of the analysis range exceeds 1.8 orders of magnitude in this case, a range of DNA amounts that falls outside the analysis range will occur within the interval designated as the analysis range, as shown in Figure 24(2).

[0275] The typical dynamic range for CE is less than 2000. Even in ideal DNA analysis, the peak intensity ratio will be more than 2, so it is desirable that the difference between n and m be 9 or less.

[0276] Higher-performance CEs have a dynamic range of 4000 or less. In many DNA analyses, the peak intensity ratio is 4:1 or more, so it is desirable that the difference between n and m be 9 or less.

[0277] As mentioned above, one example of a suitable range is when m is between 20 and 32. Another example of a suitable range is when n is between 24 and 36. Yet another example of a suitable range is when n is 4 to 9 more than m.

[0278] [Timing of splitting] As shown in Figure 14, a Final Extension step may be performed after m thermal cycles to divide the mixture, and then another Final Extension may be performed after n thermal cycles. In this case, for example, the analysis system 101 holds the PCR reaction mixture m at a constant temperature in the range of 50°C to 80°C for 1 to 20 minutes, and then takes out a portion. With this configuration, only one heater is needed around the PCR, so the structure of the apparatus and flow path devices becomes simpler.

[0279] Alternatively, you can split the process after m thermal cycles are complete and then perform the Final extension step.

[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 601-612 shown in Figure 29 may correspond to detailed examples of steps 404 (sample amplification) through 405, and steps 601-612 shown in Figure 29 may be processes independent of steps 404 through 405.

[0281] The operation shown in Figure 29 can be performed with the flow channel device 104 in Figure 6. In Figure 6, the heating unit 318 is positioned 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 the heater final extension step is performed in step 604. The PCR reaction solution remaining in the PCR chamber may be thermally cycled n times in step 608 in parallel with step 604, or this may be done with a time delay. After n thermal cycles, amplification The final extension step may be performed on product n in a holding chamber. That is, in the example in Figure 29, for example, the analysis system 101 holds a portion of the PCR reaction solution m at a constant temperature in the range of 50°C to 80°C for 1 to 20 minutes.

[0282] The advantages of doing it after the division are, amplification Since product n does not experience the Final extension step twice, artifacts are reduced and the overall time does not increase.

[0283] [Method of providing CE analysis results to users] When performing CE two or more times in split-stage PCR, two or more electrophoresis maps will be generated. Either the two electrophoresis maps or the DNA analysis results may be provided to the user. Alternatively, the two or more electrophoresis maps may be scored to determine which is more suitable for DNA analysis, and this score may be provided along with the CE analysis results.

[0284] For example, the analysis system 101 may determine which of the electrophoretic results—the result of electrophoretic analysis on a portion of PCR reaction solution m or the result of electrophoretic analysis on at least a portion of PCR reaction solution n—is better, and output the better result. Alternatively, it may output information that allows for the determination of which is better. It may also use part of the flowchart shown in Figure 2 to indicate whether it is a Full profile or not. Scoring may use the number of gene loci that met the criteria shown in Figure 2, the number of gene loci that did not meet the criteria, the number of flags that were determined not to meet the criteria, or it may be calculated comprehensively by an algorithm based on the criteria. The user may be provided with data from an intermediate stage of DNA analysis. Only the analysis result that yielded a Full profile or was determined to be a better electrophoretic figure may be provided to the user. This allows for efficient comparison of results. It also makes it easier for non-experts to select the appropriate result when they receive data from the system.

[0285] If both analysis results are poor, you may request a review by an expert.

[0286] It is also possible to synthesize two sets of data for DNA analysis. In particular, in DNA analysis where the peak intensity ratio is large, it is conceivable that the allele showing the minimum intensity in m will be below AT, and the allele showing the maximum intensity in n will be oversaturated. In this case, significant peaks or DNA analysis results can be extracted from m and n respectively and provided as a synthesized result. In other words, this embodiment can be used to expand the dynamic range of CE.

[0287] The number of CE measurements performed in the analysis system can be two, one, or three or more times.

[0288] Depending on the sample's condition, analysis may be performed 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 likelihood of obtaining accurate DNA identification results and eliminates the need for multiple measurements each time, resulting in lower costs, shorter analysis times, and improved throughput.

[0289] When performing a single analysis, it is acceptable to measure either the amplified product m or the amplified product n.

[0290] When two measurements are taken, the product of the m cycle may be measured twice, the product of the n cycle may be measured twice, the product of the n cycle may be measured after the m cycle, the analysis of the m cycle may be performed after the n cycle, the analysis of the n cycle may be started regardless of the state of the data of the m cycle, the analysis of the m cycle may be started regardless of the state of the data of the n cycle, and the analyses of m and n may be performed completely simultaneously.

[0291] If the electrophoresis result for the first run is poor, you may analyze the amplification product from the same number of cycles again. Alternatively, if the electrophoresis result for the first run is poor, you may analyze the product from the other number of cycles. Poor electrophoresis here refers to a situation where some or all of the size standard peaks are not detected, or where the solution gets stuck somewhere during transport.

[0292] After obtaining the analysis results for either amplification product m or n, the obtained DNA analysis results can be compared with a database, and based on the feedback received, the analysis of the other amplification product may be started 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 decide based on the first data or analysis score, or they may decide whether to measure a second time 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, may be removed from the cartridge or analysis system 101 and measured outside the instrument. Alternatively, it may be stored in the cartridge for a certain period and later remeasured inside the instrument. During that time, it is desirable that the amplification product be stored refrigerated or frozen. The amplification product may be stored in the form of an electrophoretic sample mixed with the electrophoretic reagent, or it may be stored in its state before being mixed with the electrophoretic reagent.

[0294] <When analyzing the amplification product m first> If the amplification product of m is analyzed first, the decision of whether or not to analyze the amplification product n may be made according to the flowchart shown in Figure 30. However, the flowchart shown in Figure 30 is just one example, and other decision criteria and branching conditions may be included. For example, the decision criteria may change depending on the number of peaks obtained, the number of loci, whether the data is mixed or not, etc. Also, the decision criteria shown in Figure 30 may be partially or completely omitted, or replaced with other criteria.

[0295] The electrophoresis results for m can be provided to the user, and the analysis of the product of n can be initiated after determining whether to proceed.

[0296] Furthermore, if n is being analyzed simultaneously or in parallel, the analysis may be interrupted midway based on the user's judgment or the flowchart in Figure 30.

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

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

[0299] Decision Set 3: Check if the IAP+ flag is displayed. If so, start or continue the analysis of amplification product n.

[0300] Judgment Set 4: Are all peaks less than or equal to half the OS intensity? Or are all peaks less than or equal to the intensity obtained by dividing the OS by the expected amplification factor from split PCR? Or are they less than or equal to the intensity that does not cause saturation when an additional nm PCR cycle is performed? Or have they not reached any peak intensity? If they have, do not perform or interrupt the analysis of the amplified product n.

[0301] If the interval between m and n is 2, amplification If almost no peaks are detected in the CE analysis of product m, amplification Since significant data cannot be obtained from product n, analyzing n after obtaining the analysis results for m is not meaningful. Similarly, if the interval between m and n is narrow, if the analysis results for m indicate that the amount of DNA is too low, it is not necessary to perform the analysis of n. When determining whether the amount of DNA is too low, either the number of detected peaks or the peak intensity may be used.

[0302] Different sets of criteria may be used for each determination set, depending on whether each gene locus is heterozygous, homozygous, mixed, or single.

[0303] If the IAP peak exceeds the threshold, an n-cycle analysis may be performed. The IAP peak occurs when the amount of input DNA is insufficient for the PCR cycle, so it is thought that increasing the number of cycles can mitigate it.

[0304] Furthermore, if the CE analysis results for m indicate that a significant CE analysis result cannot be obtained by performing the analysis on n, various thresholds and decision algorithms may be established to allow for the decision to interrupt or not perform the analysis on n.

[0305] <When analyzing the amplification product n first> If the amplification product of n is analyzed first, the decision of whether or not to analyze the amplification product m may be made according to the flowchart shown in Figure 31. However, the flowchart shown in Figure 31 is just one example, and other decision criteria and branching conditions may be included. For example, decision criteria that change depending on the number of peaks obtained, the number of gene loci, whether the data is mixed or not, etc. may be conceivable. Also, the decision criteria shown in Figure 31 may be partially or completely omitted, or replaced with other criteria.

[0306] The electrophoresis results for n may be provided to the user, and the analysis of the product of m may be initiated after determining whether to proceed.

[0307] Furthermore, if m is being analyzed simultaneously or in parallel, the analysis may be interrupted midway based on the user's judgment or the flowchart in Figure 30.

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

[0309] Decision Set 2: Check if the IAP+ flag is displayed. If not, do not perform or interrupt the analysis of the amplification product m.

[0310] Furthermore, if the CE analysis results for n indicate that a significant CE analysis result cannot be obtained by performing the analysis on m, various thresholds and decision algorithms may be established to allow for the decision to interrupt or not perform the analysis on m.

[0311] In this manner, the analysis system 101 performs electrophoretic analysis on one of the two parts of PCR reaction solution m and at least one of the two parts of PCR reaction solution n in the CE unit 105, and controls the execution of electrophoretic analysis on the other based on the results of the electrophoretic analysis of the one part. For example, it may be decided whether or not to start the electrophoretic analysis of the other based on the results of the electrophoretic analysis of the one part, or after the electrophoretic analysis of the other part has started, it may be decided whether or not to continue the electrophoretic analysis of the other based on the results of the electrophoretic analysis of the one part. In this way, unnecessary or inefficient electrophoretic analysis is omitted, and the overall process is made more efficient.

[0312] [Both n and m are preset] The analysis system 101 may have two different cycle counts, n and m, pre-set. It is preferable that m and n are appropriately set according to the CE analysis range and the amount of DNA that can be amplified, as mentioned earlier.

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

[0314] [Table 3]

[0315] For example, if a large amount of DNA is present in a relatively stable manner, such as in a cheek swab, you can select "cheek swab mode" and perform STR-CE analysis in 26 cycles without splitting (or if split, analyze only one CE analysis, and only analyze the amplification product of the other if that fails). Additionally, a "DVI sample mode" may be provided for DVI samples (disaster victim identification, DNA analysis to identify bodies resulting from disasters or large-scale terrorist attacks). Since DVI samples are likely to contain a relatively large amount of DNA, you can set it to, for example, [m=25,n=31]. In many cases, DVI samples contain a relatively large amount of DNA, so you can start the m-cycle analysis first and then start or continue the analysis of the amplification product n using the judgment flow shown in Figure 30. Furthermore, when analyzing DNA obtained from a crime scene, where the amount of DNA is often small, you can select "Casework sample mode" with [m=26,n=31] set. In the case of a casework sample, since the probability of containing only a trace amount of DNA is higher, it is acceptable to start the analysis of sample n first and then determine whether to proceed with or continue the analysis of sample m using the decision flow shown in Figure 31. Furthermore, in the case of a touch sample, the amount of DNA contained is likely to be very small, or it may be degraded with large variations in peak intensity, or it may be mixed DNA. Therefore, in "Touch sample mode," it is preferable to set a narrower interval between m and n compared to other modes. Also, since the probability of containing a small amount of DNA is higher, it is preferable to start the analysis with sample n.

[0316] If there are two or more CE samples per sample, m and n analysis may be performed simultaneously. Furthermore, the preset m and n values ​​and measurement order may be changed by the user each time. The m and n values ​​shown in Table 3 are examples; the preset m and n values ​​may be set during development after validation testing to maximize the probability of successful DNA analysis.

[0317] [Key points of this embodiment] The embodiments described above include cases where both m and n are analyzed each time, and cases where only one of m or n is analyzed each time.

[0318] If one of the amplification products prepared with two different cycle counts fails to provide data of the required quality, the probability of obtaining data of the required quality from the other amplification product is higher than when prepared with only one cycle count.

[0319] If one of the two amplification products prepared with two cycle counts fails to provide data of the required quality, the probability of sample waste is reduced because the other amplification product is still available. Furthermore, the reduced probability of sample waste eliminates the need to re-collect and pre-process samples. Consequently, preparing and analyzing two samples using this embodiment results in a shorter average data acquisition time compared to not using this embodiment.

[0320] If the m-cycle PCR product is split and only the m-cycle PCR product is subjected to CE analysis without further PCR cycling, if the CE analysis results for the m-cycle PCR product do not meet the required quality, it is possible to perform nm PCR on the remaining PCR product to prepare the n-cycle PCR product. However, if the PCR product is left standing during the CE analysis of the m-cycle electrophoresis sample, polymerase activity may decrease, and depending on the storage temperature, a large amount of artifacts may increase, so the CE analysis results for the n-cycle PCR product may 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, then the samples will be wasted if m-cycle PCR products are not prepared.

[0322] If the DNA solution used in the PCR reaction is split before PCR, the amount of DNA added to each part decreases, thus lowering the sensitivity. On the other hand, if the PCR reaction has progressed to a certain extent (for example, 4 cycles or more), each allele contains 10 or more amplicons, so splitting does not affect the sensitivity.

[0323] Splitting is preferably performed when m cycles are completed. If splitting is performed before m cycles are completed, and the remaining PCR reaction is carried out in each chamber after splitting so that the total number of cycles is m, a PCR temperature controller must be installed in each chamber, which increases the size of the apparatus. Also, since artifacts may occur during splitting, it is preferable to split after the number of cycles has been maximized.

[0324] By preparing multiple electrophoresis samples at different dilution ratios after PCR, the effective analytical range of CE can be expanded. This means that the occurrence of CE oversaturation and the frequency of peak intensity falling below AT can be suppressed. However, problems such as imbalances in peak intensity and peak splitting due to excessive amplification products cannot be solved by dilution after PCR; these issues must be addressed before or during PCR. Therefore, segmented PCR is appropriate.

[0325] Because the number of cycles for splitting is appropriately set for the analytical sample, the maximum analytical range expansion is achieved with the minimum number of splits. In other words, the measurement time during analytical range expansion is minimized.

[0326] By controlling the amount of DNA before it is introduced into PCR, the effective analytical range can be expanded. For example, the upper limit of the amount of DNA adsorbed onto the purification membrane can be reduced by changing the volume of the purification membrane or the purification protocol. However, with typical purification membranes, reducing the volume to lower the adsorption limit can lead to poor solution flow and a decrease in DNA yield (especially short, degraded DNA). Therefore, there are limitations to controlling the amount of DNA at the purification stage. Alternatively, quantifying the amount of DNA before PCR can be used to control the number of PCR cycles or change the dilution ratio, thereby expanding the effective analytical range. However, the quantification step requires an additional detection system and inevitably increases the complexity of the fluidic device. There is also a possibility of analytical failure due to quantification errors, so segmented PCR is preferable.

[0327] This embodiment and the control of dilution and purified DNA volume after PCR may be carried out in combination. Combining them allows for more reliable or broader range of DNA analysis.

[0328] By taking out the PCR reaction solution after each cycle, or skipping a cycle, and performing CE measurement each time, DNA analysis suitable for all DNA concentration ranges can be achieved. However, in a fluid channel device, it is difficult to provide a mechanism that allows for taking out the solution three or more times, four or more times, or five or more times, and then mixing it with the electrophoresis reagent. Furthermore, even if two, three or four amplification products are measured by splitting the solution after each cycle, or skipping a cycle, the expansion of the analysis range is limited to 2x, 4x, or 8x, respectively, and is not necessarily suitable for analyzing samples containing various amounts of DNA. In addition, in STR-PCR, when the final extension is performed in the same chamber as the PCR chamber, if the interval between m and n is narrow (e.g., 1 cycle or 2 cycles), depending on the amount of DNA introduced into PCR, the final extension time may be excessive, potentially resulting in many A++ peaks. In particular, in analytical systems without a heating unit 318, and in the case of a split PCR device that performs final extension in the same chamber as shown in Figure 14, a narrow interval between n and m will result in many A++ peaks, so a interval of 2 or more is preferable.

[0329] [How to flush the capillaries according to the number of capillaries] i) When only one capillary tube can be used per sample Figure 32 shows a typical example of the operation timing of the analysis system 101. In this operation timing, first, the first sample A is introduced in step 401, the sample is processed in the pretreatment cartridge in steps 402 to 404, and the electrophoretic sample m or n is analyzed by CE in step 405.

[0330] The operating procedure shown in Figure 32 is suitable when there is one CE unit for each sample preprocessing unit. This configuration makes the device compact and easy to carry.

[0331] The user may be provided with the data at the end of the first CE analysis in step 405. The user may decide whether to start or continue the analysis of the second electrophoresis sample. Alternatively, they may decide whether to start or continue the second analysis using the method described above. This is because measuring twice each time doubles the CE measurement time, thus not increasing throughput.

[0332] The pre-processing of sample B (steps 401-404) may begin at the same time that the pre-processing of sample A is completed. In this case, since the cartridge containing sample A is removed, electrophoresis samples that have not yet been analyzed for CE may be held in the standby unit 321, and CE samples may be in the process of electrophoresis.

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

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

[0335] As shown in Figure 33(1), electrophoresis sample m and electrophoresis sample n may be measured in step 405 as soon as they are ready, or as shown in Figure 33(2), both may be prepared and then analyzed simultaneously in step 405. Also, if the number of CE units is the same as the number of sample processing units and there are no available units, electrophoresis sample m or electrophoresis sample n may be measured in order.

[0336] [Example 1] The analysis system 101 is equipped with one flow channel device 104 and one CE unit 105.

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

[0338] After performing m-cycle PCR in step 502, the m-cycle amplification product is extracted in step 503. The sample is then sent to the CE unit 105, and CE measurement is started in step 504. Step 504 In parallel with this, in step 505, n cycles of PCR are performed on the PCR reaction mixture remaining in the PCR chamber 304. Once the CE measurement in step 504 is complete and the next CE measurement can begin, the product from the n cycles is sent to the CE section 105 and CE measurement is performed in step 507.

[0339] [Example 2] The analysis system 101 is equipped with one flow channel device 104 and two CE units 105.

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

[0341] After performing m-cycle PCR in step 502, the amplified product m is removed from the PCR chamber 304 in step 503. The sample is then fed into one of the CE units 105. In parallel with step 503, n-cycle PCR is performed on the PCR reaction mixture remaining in the PCR chamber in step 505. In step 506, the product from the n-cycle is fed into the CE unit 105, and with electrophoretic samples m and n respectively stored in the two capillaries, steps 504 and 507 are started simultaneously. [Explanation of Symbols]

[0342] 101 Analysis System 102 Computer 103 Databases 104 Flow Channel Devices 105 CE Department 106 User Interface 201~207 Judgment criteria 301 Dissolution Chamber 302 Purification membrane 303 Purification membrane chamber 304 PCR Chambers 305 Waste liquid chamber 306 External connection port 307 Pumps and Valves 308 PCR Reagent Storage Section 309 PCR reagents 310 Electrophoresis reagent storage section 311 Electrophoresis reagent 312-314 Reagent storage section 315 channel 316 Flow channels 317 Heating section 318 Heating section 319 Channels 320 dispensing chamber 321 Waiting section 322 Flow Channels 323-326 Valves 327 Mixing Chamber 328~330 Flow path 331 Electrophoresis reagent storage section 332-334 Air storage section 335 PCR reaction solution 336 electrophoresis samples 337-339 Valves 401 Sample loading step 402 Dissolution step 403 Purification Step 404 Amplification Step 405 Detection Step 701 Liquid level 702 Vent Filter 703 Flow channel 704 Flow Resistor 705 Liquid level detection sensor 801 CE detection limit or PCR amplification product concentration limit 802 CE detection limit 803 STR-CE analysis range Analysis range of STR-CE by 804-fold PCR 805,806 plots Dynamic range of the 807 CE

Claims

1. A fluid channel 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 described above is It stores the pre-set values ​​of m and n, In the PCR chamber, the PCR reaction solution is subjected to m thermal cycles to produce the first reaction solution. A portion of the first reaction solution is removed from the PCR chamber without changing its composition. A portion of the first reaction solution is subjected 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 between 20 and 36, and n-m is an integer between 2 and 9) to produce a second reaction solution, such that the total number of thermal cycles is n. At least a portion of the second reaction solution is removed from the PCR chamber without changing its composition. At least a portion of the second reaction solution is subjected to electrophoretic analysis in the capillary electrophoresis unit. DNA analysis system.

2. The DNA analysis system according to claim 1, wherein, prior to the electrophoretic analysis, a portion of the first reaction solution and at least a portion of the second reaction solution are mixed with pure water, formamide, or a solution with a conductivity of 10 mS / cm or less to produce a mixture, and the mixture is subjected to electrophoresis.

3. A fluid channel device having a PCR chamber for performing thermal cycling, A capillary electrophoresis unit for electrophoretic analysis of the PCR reaction solution, Storage section and In a DNA analysis system having, The DNA analysis system described above is It stores the pre-set values ​​of m and n, In the PCR chamber, the PCR reaction solution is subjected to m thermal cycles to produce the 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) to produce a second reaction solution, such that the total number of thermal cycles is n. Remove at least a portion of the second reaction solution from the PCR chamber. At least a portion of the second reaction solution is subjected to electrophoretic analysis in the capillary electrophoresis unit. Based on the results of the electrophoretic analysis, the execution of the electrophoretic analysis of a portion of the first reaction solution is controlled. DNA analysis system.

4. In claim 2, The DNA analysis system is a DNA analysis system that 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 the n-m ratio is between 3 and 6.

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

8. A fluid channel device having a PCR chamber for performing thermal cycling, A DNA analysis system having a capillary electrophoresis unit for electrophoretic analysis of a PCR reaction solution, and performing reaction A and reaction B in sequence, The aforementioned reaction A consists of a temperature-controlled condition including a thermal denaturation step in PCR, and annealing and extension reactions. The above reaction B consists of temperature-controlled conditions that do not include the heat denaturation step, The DNA analysis system described above is It stores the pre-set values ​​of m and n, In the PCR chamber, reaction A is performed m times on the PCR reaction solution, and reaction B is performed once to produce the first reaction solution. A portion of the first reaction solution is removed from the PCR chamber without changing its composition. A portion of the first reaction solution is subjected 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 cycles of reaction A (where n and m are integers satisfying n > m + 2) so that the total number of thermal cycles is n, and then reaction B is performed once to produce the second reaction solution. At least a portion of the second reaction solution is removed from the PCR chamber without changing its composition. At least a portion of the second reaction solution is subjected to electrophoretic analysis in the capillary electrophoresis unit. DNA analysis system.

9. In claim 8, The DNA analysis system comprises mixing a portion of the first reaction solution and at least a portion of the second reaction solution with pure water, formamide, or a solution with a conductivity of 10 mS / cm or less, respectively, before electrophoretic analysis to produce a mixture, and then performing electrophoresis on the mixture.

10. In claim 9, Reaction B is a DNA analysis system in which the PCR reaction solution is held for 1 to 20 minutes under the temperature conditions at which the extension reaction in the PCR reaction occurs.

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

12. In claim 11, A DNA analysis system in which m is an integer between 20 and 36, and n - m is between 3 and 9 (inclusive).

13. A fluid channel device having a PCR chamber for performing thermal cycling, A DNA analysis system having a capillary electrophoresis unit for electrophoretic analysis of a PCR reaction solution, and performing reaction A and reaction B in sequence, The aforementioned reaction A consists of a temperature-controlled condition including a thermal denaturation step in PCR, and annealing and extension reactions. The above reaction B consists of temperature-controlled conditions that do not include the heat denaturation step, The DNA analysis system described above is It stores the pre-set values ​​of m and n, In the PCR chamber, the above reaction A is carried out m times with respect to the PCR reaction solution to produce the first reaction solution. A portion of the first reaction solution is removed from the PCR chamber without changing its composition, and reaction B is carried out once. A portion of the first reaction solution is subjected 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 cycles of reaction A (where n and m are integers satisfying n > m) so that the total number of thermal cycles is n, and then reaction B is performed once to produce the second reaction solution. At least a portion of the second reaction solution is removed from the PCR chamber without changing its composition. At least a portion of the second reaction solution is subjected to electrophoretic analysis in the capillary electrophoresis unit. DNA analysis system.

14. In claim 13, Reaction B is a DNA analysis system in which the PCR reaction solution is held for 1 to 20 minutes under the temperature conditions at which the extension reaction in the PCR reaction occurs.

15. In claim 14, The DNA analysis system comprises mixing a portion of the first reaction solution and at least a portion of the second reaction solution with pure water, formamide, or a solution with a conductivity of 10 mS / cm or less, respectively, before electrophoretic analysis to produce a mixture, and then performing electrophoresis on the mixture.

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

17. In claim 16, A DNA analysis system in which m is an integer between 20 and 36, and n - m is between 2 and 9 (inclusive).

18. In claim 17, The DNA analysis system further comprises a storage unit, A DNA analysis system in which the storage section is installed in contact with a heating section, and the reaction B in the first reaction solution is carried out by heating the storage section with the heating section.

19. A fluid channel 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 described above is It stores the pre-set values ​​of m and n, In the PCR chamber, the PCR reaction solution is subjected to m thermal cycles to produce the first reaction solution. A portion of the first reaction solution is removed from the PCR chamber without changing its composition. A portion of the first reaction solution is subjected 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) to produce a second reaction solution, such that the total number of thermal cycles is n. The entire volume of the second reaction solution is removed from the PCR chamber. At least a portion of the second reaction solution is subjected to electrophoretic analysis in the capillary electrophoresis unit. Determine which of the results of the electrophoretic analysis on a portion of the first reaction solution and the results of the electrophoretic analysis on at least a portion of the second reaction solution is better. Provide users with information that allows them to determine which outcome is better. DNA analysis system.

20. In claim 19, The DNA analysis system comprises mixing a portion of the first reaction solution and at least a portion of the second reaction solution with pure water, formamide, or a solution with a conductivity of 10 mS / cm or less, respectively, before electrophoretic analysis to produce a mixture, and then performing electrophoresis on the mixture.

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

22. In claim 21, A DNA analysis system in which m is an integer between 20 and 36, and n - m is between 2 and 9 (inclusive).

23. A fluid channel 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 described above is It stores the pre-set values ​​of m and n, In the PCR chamber, the PCR reaction solution is subjected to m thermal cycles to produce the first reaction solution. A portion of the first reaction solution is removed from the PCR chamber without changing its composition. A portion of the first reaction solution is subjected 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) to produce a second reaction solution, such that the total number of thermal cycles is n. The entire volume of the second reaction solution is removed from the PCR chamber. At least a portion of the second reaction solution is subjected to electrophoretic analysis in the capillary electrophoresis unit. Determine which of the results of the electrophoretic analysis on a portion of the first reaction solution and the results of the electrophoretic analysis on at least a 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 comprises mixing a portion of the first reaction solution and at least a portion of the second reaction solution with pure water, formamide, or a solution with a conductivity of 10 mS / cm or less, respectively, before electrophoretic analysis to produce a mixture, and then performing electrophoresis on the mixture.

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

26. In claim 25, A DNA analysis system in which m is an integer between 20 and 36, and n - m is between 2 and 9 (inclusive).

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

28. In claim 27, The target sample is a forensic sample, and the system is designed for DNA analysis.

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

30. In claim 29, A DNA analysis system in which m is an integer between 20 and 36, and n - m is between 2 and 9 (inclusive).

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