WO2025009019A1 - Dna analysis system - Google Patents

Abstract

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

Description

DNA Analysis System

The present invention relates to a DNA analysis system.

In fragment analysis, DNA contained in a sample is subjected to PCR using primers designed for specific DNA targets, and fluorescently labeled DNA amplified products are separated by size using capillary electrophoresis (CE). It is used for gene mutation analysis and quantification, cell line authentication, genome editing efficiency evaluation, genotyping for amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs), and single nucleotide polymorphisms (SNPs), and macrosatellite marker analysis. Macrosatellites are repetitive DNA in which a specific DNA motif is repeated multiple times, and are characterized by a higher frequency of mutations and higher genetic diversity than other DNA regions. A representative example of macrosatellite marker analysis is individual identification and identity testing using short tandem repeats (STRs). DNA testing using STRs is widely used in forensic medicine examinations, and is used for applications such as parent-child testing and matching crime scene DNA with criminals.

In fragment analysis, in addition to the fluorescently labeled DNA fragments to be analyzed, multiple types of fluorescently labeled DNA fragments of known lengths (size standards) may be mixed and subjected to CE analysis. The use of size standards allows the fragment length of each amplified product to be identified. Furthermore, by setting the amount of size standard mixed to a fixed amount, the amount of amplified product can be calculated from the ratio of the intensity of the DNA fragments of the amplified product obtained by CE analysis to the intensity of the size standard. Furthermore, by mixing a known amount and length of DNA (internal positive control, IPC) and the primers for amplifying it in the PCR reaction solution and amplifying it, and then performing fragment analysis at the same time, the amount of DNA of the target before amplification can be estimated from the ratio of the intensity of the target amplified product to the IPC.

According to Non-Patent Document 1, DNA analysis at a forensic laboratory involves (1) quantifying the concentration of human DNA in a sample using quantitative PCR, (2) preparing the sample so that the human DNA is at an appropriate concentration and performing STR-PCR (PCR containing STR sequences), (3) mixing a portion of the PCR reaction solution with formamide containing a size standard in a fixed ratio and heat denaturing it, (4) subjecting the heat-denatured electrophoretic sample to CE measurement to obtain an electropherogram, and (5) performing DNA analysis from the electropherogram.

In the DNA identification or fragment analysis using the pretreatment integrated CE analyzer of Patent Document 1, Patent Document 2, Patent Document 3, Patent Document 4, and Patent Document 8, (1) a sample containing DNA is subjected to PCR on a flow path device, (2) a part of the reaction solution is mixed with formamide containing a size standard in a certain ratio on the flow path device and heat denatured, (3) the heat denatured electrophoretic sample is subjected to CE analysis to obtain an electrophoretic pattern, and (4) DNA identification or fragment analysis is performed from the electrophoretic pattern. Compared to Non-Patent Document 1, Patent Documents 1, 2, 3, 4, and 8 automate the series of steps and can obtain results in a short time (e.g., 90 minutes).

In the DNA quantification according to Patent Document 5, (1) in PCR of a sample containing DNA, a portion of the reaction solution is divided at each stage of a series of n thermal cycles (n = n0, n0+1, n0+2, ... where n0 is a preset integer), (2) each divided reaction solution is subjected to CE analysis, and (3) DNA quantification (quantification of the original concentration of DNA contained in the sample) is performed based on the relationship between the peak intensity of the amplified product obtained by CE analysis and the number of thermal cycles n, specifically, the number of thermal cycles at which the peak intensity exceeds a preset threshold. In other words, Patent Document 5 is an analytical method in which CE is used to detect amplified products in real-time PCR.

In the DNA quantification according to Patent Document 6, (1) in a PCR of a sample containing DNA, a portion of the amplified product at each stage of a series of n thermal cycles (n0 and m are preset integers, m is 1 or 2, and n = n0, n0 + m, n0 + 2m, ...) is divided by electrophoresis on a flow path device, (2) each divided amplified product is analyzed by CE on the flow path device, and (3) DNA quantification (quantification of the original concentration of DNA contained in the sample) is performed based on the relationship between the peak intensity of the amplified product from each CE analysis and the number of thermal cycles n, specifically, the number of thermal cycles at which the peak intensity exceeds a predetermined threshold. In other words, Patent Document 5 is an analytical method in which CE is used to detect amplified products in real-time PCR.

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

US Patent Application Publication No. 2022/0016632 U.S. Patent No. 9,354,199 US Patent Application Publication No. 2019/0019290 JP 2017-077180 A U.S. Pat. No. 7,445,893 Patent No. 5494480 U.S. Patent No. 8,715,924 U.S. Pat. No. 1,076,725

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

In CE analyzers or devices equipped with a CE analysis unit, the ratio of the minimum and maximum peak intensities at which peak intensity and concentration are almost proportional is 100 or less, or 1000 or less, or 10000 or less, or 100000 or less. When performing fragment analysis, taking into account the variation in intensity of the CE analyzer, the variation in the amount of amplicon injected into the CE, the variation in the amplification efficiency and the presence rate of each allele, the variation between dyes, and the variation during sample preparation, the ratio of the minimum and maximum amount of measurable DNA concentration is 10 or 30 or 100 or 300 or 1000 or 3000 or 10000. On the other hand, the ratio of the minimum and maximum amount of DNA contained in the sample brought into the analysis system is 30 or 300 or 3000 or 30000. If the amount of DNA contained in the sample exceeds the range of the sample DNA amount that can be measured by the analysis system, fragment analysis may fail. In this case, the sample and analysis time spent on the analysis will be wasted.

One of the objectives of the present invention is to perform highly accurate and robust DNA identification/fragment analysis or DNA quantification (quantification of the original concentration of an individual's DNA contained in a sample) using PCR and CE analysis of samples containing DNA whose concentration ratios vary over a range of 100-fold or 1000-fold or more (sample concentration range is 100 or 1000 = 2.0 or 3.0 orders of magnitude or more) with a simple configuration and low-cost device. In addition, as in Patent Document 2, the series of processes is automated, making it possible to obtain results in a short time (for example, within 180 minutes, within 120 minutes, or within 90 minutes).

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

In Patent Document 1, the solution is divided before PCR, and STR-PCR and quantitative PCR are performed. Quantitation is performed using quantitative PCR, and the number of cycles for STR-PCR is determined. Because the solution is divided before the start of PCR, sensitivity is reduced. In addition, the flow path device for performing two different PCRs is complex, and additional optical systems are required, making high costs unavoidable.

In Patent Document 2, the solution is divided before PCR, two DNA solutions with different concentrations are prepared, and both are subjected to PCR for DNA identification. Since one of the two DNA solutions falls within the analytical concentration range of the analysis system, the analytical range can be expanded. Since the solution is divided before PCR, sensitivity is reduced. In addition, since a flow path mechanism for dividing the solution and adjusting the concentration is installed in the flow path device, it is unavoidable to make the flow path device more complicated.

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

In Patent Document 4, a portion of the reaction solution is taken after PCR, and the presence and amount of amplified products are detected using an optical system, and if appropriate amplification has been achieved, fragment analysis is performed. If it is determined that amplification is incomplete, an additional PCR reaction is performed on the reaction solution remaining in the PCR section. This method requires the design of additional optical systems and flow path devices suitable for optical detection, which inevitably increases costs. In addition, there is a risk that the fluorescent dye of STR-PCR may fade during detection using an optical system, or that optical detection may not be performed correctly due to overlapping detection wavelength ranges between the fluorescent dye of STR-PCR and the optical system.

In Patent Document 5 and Patent Document 6, it takes a long time to obtain results because CE analysis is required for each of the many thermal cycle numbers n (multiple consecutive thermal cycle numbers n). For highly accurate quantification, three or more CE analyses are required. It is possible to replace PCR with STR-PCR and perform DNA quantification, but a special flow path structure is required to extract the solution multiple times. In addition, since there is no process for mixing the reaction solution with formamide containing a size standard at a constant ratio, DNA identification cannot be performed. In addition, when DNA identification is performed by incorporating a mixing process with formamide (electrophoresis reagent) containing a size standard into the corresponding method, a new dispensing mechanism or flow path structure is required to mix the PCR product of each cycle with the electrophoresis reagent, which makes the device more complex and leads to high costs. In addition, for DNA identification that requires CE analysis with high accuracy and high resolution, CE analysis of the PCR product of each cycle requires a huge analysis time. In addition, in the case of Patent Document 6, since DNA is extracted from the PCR chamber by applying voltage, it is assumed that the composition of the amplified product in the PCR chamber is different from the composition of the amplified product extracted. In DNA testing, amplicons of different lengths are measured, but there is concern that length-dependent bias may occur when voltage is applied and the amplified products are removed from the PCR chamber.

In Patent Document 7, PCR can be replaced with STR-PCR to perform DNA quantification, but it takes a long time to obtain results because microarray analysis must be performed for each of a large number of thermal cycles n (multiple consecutive thermal cycles n). In addition, DNA identification cannot be performed because CE analysis is not performed.

On the other hand, Patent Document 7 mentions that microarray analysis may be performed for a small number of thermal cycles n (multiple non-consecutive thermal cycles n), but this is not realistic. In microarray analysis, the spot intensity for the same DNA concentration generally varies due to variations in the density and number of probes immobilized on each spot, and hybridization efficiency varies over time and space, and although the presence or absence of corresponding DNA can be determined from the strength of the spot intensity, the accuracy of quantifying the corresponding DNA from the spot intensity is low. In other words, in order to accurately determine the number of thermal cycles 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 cycles n (multiple consecutive thermal cycles n). As a means for improving the accuracy of DNA quantification by microarray analysis, a method of competitively hybridizing target DNA or target amplification product with reference DNA on the same spot is known. However, to carry out this method, it is necessary to prepare a reference DNA that hybridizes to the probe on the spot with the same efficiency as the target DNA or target amplification product for each target DNA or target amplification product, and further to label the target DNA or target amplification product and the reference DNA with different fluorophores and measure the fluorescence emitted from each independently. If a small number of PCR products with different cycle numbers are prepared and microarray analysis is performed to expand the dynamic range, there is a problem of high cost and labor.

Other issues with Patent Document 7 are: (1) In PCR of a sample containing DNA on a flow path device, when a part of the reaction solution at each stage of multiple thermal cycle numbers n is divided, fresh PCR solution of the same amount as the divided reaction solution is mixed with each of the reaction solutions that remain undivided, which changes the concentration of DNA contained in the reaction solution and reduces the accuracy of DNA quantification.

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

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

An example of a DNA analysis system according to the present invention includes:
A flow path device having a PCR chamber for performing thermal cycling;
a capillary electrophoresis unit for electrophoretic analysis of the PCR reaction solution;
In a DNA analysis system having
The DNA analysis system comprises:
storing the preset values of m and n;
In the PCR chamber, a PCR reaction solution is subjected to a thermal cycle m times to generate a first reaction solution;
removing a portion of the first reaction solution from the PCR chamber without changing its composition;
subjecting the portion of the first reaction solution to electrophoretic analysis in the capillary electrophoresis portion;
In the PCR chamber, the first reaction solution remaining in the PCR chamber is subjected to n-m thermal cycles (where n-m is an integer of 2 or more) so that the total number of thermal cycles is n, thereby generating a second reaction solution;
removing at least a portion of the second reaction solution from the PCR chamber without changing its composition;
At least the portion of the second reaction solution is subjected to electrophoretic analysis in the capillary electrophoresis portion.

An example of a DNA analysis system according to the present invention includes:
A flow path device having a PCR chamber for performing thermal cycling;
a capillary electrophoresis unit for electrophoretic analysis of the PCR reaction solution;
In a DNA analysis system having
The DNA analysis system comprises:
storing the preset values of m and n;
In the PCR chamber, a PCR reaction solution is subjected to a thermal cycle m times to generate a first reaction solution;
removing a portion of the first reaction solution from the PCR chamber without changing its composition;
In the PCR chamber, the first reaction solution remaining in the PCR chamber is subjected to n-m thermal cycles (where n-m is an integer of 2 or more) so that the total number of thermal cycles is n, thereby generating a second reaction solution;
removing at least a portion of the second reaction solution from the PCR chamber without changing its composition;
performing electrophoretic analysis on one of the portion of the first reaction solution and the at least a portion of the second reaction solution in the capillary electrophoresis portion;
Based on the result of the electrophoretic analysis of one of the two, execution of electrophoretic analysis of the other of the portion of the first reaction solution or the at least a portion of the second reaction solution is controlled.

The effects obtained by the example of the invention disclosed in this application can be briefly explained as follows. That is, according to this example of the invention, an analysis system equipped with a flow path device and an electrophoresis unit can expand the range of DNA amounts analyzed with high accuracy, high sensitivity, short time, low cost, and small equipment.

　Other issues, configurations, and advantages will become clear from the description of the embodiments below.

FIG. 1 is a schematic diagram of an analysis system. 13 is an example of a method for determining data. FIG. 1 is a schematic diagram of an analysis system. 1 is an example of an analysis system. 1 is a schematic diagram and an embodiment of an analysis system. FIG. 1 is a schematic diagram of an analysis system. FIG. 1 is a schematic diagram of an analysis system. FIG. 2 is a schematic diagram of a flow path device. FIG. 2 is a schematic diagram of a flow path device. FIG. 2 is a schematic diagram of a flow path device. FIG. 2 is a schematic diagram of a flow path device. FIG. 2 is a schematic diagram of a flow path device. 4 is an example of the operation of the flow channel device. 1 is an example of an analysis system. FIG. 2 is a schematic diagram of a flow path device. 4 is an example of the operation of the flow channel device. FIG. 2 is a schematic diagram of a flow path device. FIG. 2 is a schematic diagram of a flow path device. FIG. 2 is a schematic diagram of a flow path device. 4 is an example of the operation of the flow channel device. 4 is an example of the operation of the flow channel device. 4 is an example of the operation of the flow channel device. The results show the relationship between electrophoretic reagent and peak intensity. FIG. 1 is a diagram showing the effect of the present invention. FIG. 1 shows the scope of analysis. 1 is an example of a table used for setting the number of cycles. FIG. 2 is a schematic diagram of the obtained electropherogram. 1 is an example of a table used for setting the number of cycles. 1 is an example of an analysis system. 13 is an example of a method for determining data. 13 is an example of a method for determining data. 1 is an example of an analysis system. 1 is an example of an analysis system.

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

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

In the following, the mixture of PCR reaction reagents and sample-derived DNA will be referred to as the PCR reaction solution. In addition, the DNA from the sample that is amplified by the PCR reaction reagents will be referred to as the target DNA.

In the following, the PCR reaction solution obtained after m thermal cycles will be called "PCR reaction solution m," and the PCR reaction solution obtained after n thermal cycles will be called "PCR reaction solution n."

In the following, the electrophoretic reagent may include deionized formamide, size standards, and pure water. Formamide and pure water may be included to lower the ionic strength of the electrophoretic sample or to denature DNA. The electrophoretic reagent may be a low-conductivity solution as well as formamide and pure water. A low-conductivity solution preferably has a conductivity of 10 mS/cm or less, more preferably 1 mS/cm or less, more preferably 100 μS/cm or less, and more preferably 10 μS/cm or less. The lower the conductivity of the solution used in the electrophoretic reagent, the greater the amount of DNA injected into the CE. A size standard may be mixed in to correspond to the detected peak and DNA length, or to estimate the amount of DNA contained in the electrophoretic sample from the detected peak.

Thus, one example of an analytical system mixes a PCR reaction solution (e.g., at least one of a portion of PCR reaction solution m and at least a portion of PCR reaction solution n) with pure water, formamide, or a solution with a conductivity of 10 mS/cm or less to generate a mixture prior to electrophoretic analysis. By mixing with a solution with low conductivity, it is possible to increase the CE peak intensity. Also, by mixing with formamide, it is possible to denature DNA.

In the following, the DNA obtained by the PCR reaction will be called the "amplification product", the amplification product obtained after m cycles will be called "amplification product m", and the amplification product obtained after n cycles will be called "amplification product n".

In the following, the mixture of the PCR reaction solution and the electrophoresis reagent (mixture) is called the "electrophoresis sample". When preparing the electrophoresis sample, it is preferable to add a heating step by heating to 90°C or higher as shown in Figure 5 (2), which makes it easier for the DNA to denature and become single stranded, allowing for more accurate CE analysis. The "electrophoresis sample" refers to a sample 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".

In the following, the PCR reaction procedure in which m cycles of PCR are performed, a portion of PCR reaction solution m is taken out, and the remaining PCR reaction solution m is subjected to n-m PCR cycles, and finally both PCR reaction solutions m and n are prepared, is referred to as "split PCR."

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

In the following, an allele refers to a genetic variant that can be distinguished at the same locus. When conducting DNA testing, if the DNA comes from one individual, there can be two alleles at the same locus (heterozygote) or one allele (homozygote).

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

In the following, CE analysis refers to the series of steps that involves preparing an electrophoretic sample, performing CE measurement, obtaining an electrophoretic pattern, and performing DNA identification or fragment analysis. However, the scope of "CE analysis" does not necessarily include some of the above steps.

In the following, an electropherogram refers to a diagram obtained by CE measurement, with the horizontal axis representing time, or measurement point, or DNA chain length, and the vertical axis representing intensity. The vertical axis may represent wavelength, and the data may be three-dimensional data including intensity information. The vertical axis may also represent intensity, and the data may be three-dimensional data including dye information. An electropherogram obtained from electrophoretic sample m will be called "electropherogram m", and an electropherogram obtained from electrophoretic sample n will be called "electropherogram n".

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

In the following, STR-CE refers to the series of steps from preparing the PCR reaction solution for STR-PCR, performing the PCR reaction, measuring the CE, and analyzing the resulting electropherogram. The data obtained from STR-CE may be an electropherogram or a DNA profile. Once STR-CE is complete, some or all of the data may or may not be provided to the user.

In the following, analytical range refers to, for example, the range of a given sample amount or biomolecular weight within which an analysis can be performed correctly for a given sample or biomolecule, for an analytical system or for a part or multiple steps of an implementation procedure contained in an analytical system. Here, "capable of analyzing correctly" may refer to a state in which all required conditions are met, but it is not necessary to meet all required conditions, and it may also refer to a state in which the best data possible for the system in question can be provided. For example, if the amount of sample given is extremely small, the data obtained will not be able to meet all required conditions, but it will be sufficient to obtain data that comes closest to meeting the required conditions.

The PCR reaction solution may contain two or more primer sets, and the PCR reaction solution may contain two or more amplified gene regions. Analyzing multiple amplified gene regions increases the ability to identify individuals. In particular, in the case of personal identification, it reduces the risk of mistakenly identifying the same person. By PCR reacting multiple gene regions together at once, rather than individually, it is possible to minimize the number of PCR chambers. This saves on reaction reagents. It is possible to avoid the risk of reduced sensitivity caused by separating the PCR chambers.

Embodiment 1.

[Analysis system]
In this embodiment, the analysis system 101 (DNA analysis system) may include a memory that stores program instructions, a control unit including a processor that executes the program instructions, a function that receives and analyzes raw data, optical data, and electropherogram data from the detection unit, a solution transport control mechanism such as a pump or valve, a CE unit (capillary electrophoresis unit) that performs electrophoretic analysis of the PCR reaction solution, a flow path device, and a heater. The control analysis unit may be connected to a network and may be capable of uploading, collating, 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 magnetic force may be used.

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

It may be possible to accept samples containing target DNA and perform the processes from dissolution to purification, PCR, CE measurement, and analysis in a fully automated manner. It may also be possible to fully automate parts of the processes from dissolution to purification to PCR to CE measurement and analysis. For example, the analysis system 101 or the flow path device 104 fully automates the process shown in FIG. 14 described below, from adjusting the PCR reaction solution to n-m thermal cycles. This makes the process more efficient. It also makes it possible for non-experts to perform the analysis.

In an integrated device, dissolution, purification, and PCR reaction solution preparation can be performed. In an integrated device, PCR and formamide can be mixed and heated. In an integrated device, CE measurement can also be performed.

The flow path device 104 may be disposable. By making it disposable, contamination between samples can be prevented.

The CE unit 105 may be disposable. Making it disposable helps prevent contamination between samples. In addition, since it can be molded integrally with the device, it is easy to store, maintain, and transport. The connection between the pretreatment unit and the CE unit is simplified, which helps reduce the frequency of breakdowns and errors.

While the pretreatment flow path device is disposable, the CE section can be made to be reusable multiple times. The CE section requires precision manufacturing and has a high unit price, so making it reusable can help reduce costs.

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

The computer 102 may be equipped with a user interface 106. Parameters related to the user interface 106 (e.g., time and temperature of each step, pressure, flow rate, procedure, amount of divided liquid, number of PCR cycles, sample information, cartridge information, analysis protocol, etc.) may be accepted from the user and stored in the database 103. In addition, various parameters may be stored in the database 103 in advance. 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 based on the parameters recorded in the database 103.

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

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

　In the past, samples were generally sent to an environment with well-equipped experimental equipment, such as a research institute, where an inspector with specialized knowledge and skills would adjust and measure the samples, and then analyze the data. However, problems include the time it takes to transport samples, and the large equipment and labor costs required to maintain experimental equipment. In addition, when batch processing is introduced to increase efficiency, it is difficult to interrupt urgent samples. In recent years, Sample-to-Answer (StoA) systems, which perform the entire process from sample introduction to measurement and data acquisition in an automated manner, have been appearing in various fields. In some cases, StoA systems use flow path devices that integrate chambers, flow paths, and reagents. The introduction of flow path devices has the following advantages: (1) Measurements can be easily performed even by non-experts, (2) Data can be acquired in a short time, (3) Highly portable equipment can be designed, (4) Variability due to manual techniques is reduced, and (5) Reagents are easy to store.

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

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

The material used for the flow channel device is not particularly limited as long as it is a material commonly used in the technical field. It is preferable to use materials that have a low amount of DNA adsorption, such as polypropylene, cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polycarbonate, polyethylene terephthalate, and polyurethane. It is also preferable to suppress the amount of adsorption by modifying the surface so that it is negatively charged. Other examples of materials include
- metals such as gold, silver, copper, aluminium, tungsten, molybdenum, chromium, platinum, titanium, nickel;
- alloys such as stainless steel, Hastelloy, Inconel, Monel, duralumin, etc.;
-silicon;
- glass materials such as glass, quartz glass, fused silica, synthetic quartz, alumina, sapphire, ceramics, forsterite and photosensitive glass;
Plastics such as polyester resin, polystyrene, polyethylene resin, ABS resin (Acrylonitrile Butadiene Styrene resin), dimethylpolysiloxane (PDMS), nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenolic resin, melamine resin, epoxy resin and polyvinyl chloride resin;
- agarose, dextran, cellulose, polyvinyl alcohol, nitrocellulose, chitin, chitosan,
Or any combination of these.

[Chamber/Reagent storage section]
A typical chamber or reagent reservoir is a space that can contain liquids or solids and where solutions can be reacted, held, heated, or transformed. The chamber may have a larger diameter than the flow channel, but may be indistinguishable from the flow channel by appearance. The chamber may have a membrane or microstructure inside, may be made of a different composition than the flow channel, may have a different surface treatment, or may have a different hydrophilicity. The flow channel device may also have a heater or laser light source on the outside. Reagents may be stored in the chamber, and PCR, lysis, purification, etc. may be performed in the chamber. A typical chamber volume is preferably 0.01 μL to 50 mL.

The flow path device may store reagents within the device, or the reagents may be supplied from outside the flow path device or from inside the analysis system. As an example, the device stores one or more types of reagents in one or more reagent storage sections. The reagents include at least one of the following: [lysis solution, cleaning solution, PCR reagents (which may contain polymerase, primers, surfactants, etc.), formamide, pure water, DNA fragments, and oil].  Since unintended mixing of these can lead to a decrease in performance or other unexpected results, it is desirable that they are separated by a partition mechanism consisting of a valve, film, air, or a flow path thin enough to prevent spontaneous mixing, or a combination of these, until just before use. In addition, by isolating the reagents from the outside air, long-term storage and portability of the device are achieved. The same reagent may be stored in multiple reagent storage sections to be released in multiple steps. Similarly, when reagents are stored outside the device, they are desirably stored in a state isolated from the outside air, and are separated from other purification system components by valves, films, air, etc. Known reagent storage technologies include, for example, a blister reagent storage unit or the reagent storage unit installed in Patent Document 1 and Patent Document 2, and similar configurations may be incorporated into this embodiment.

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

When collecting a sample, a swab, filter paper, cloth, etc. can be used as a carrier, and the carrier itself can be introduced into the purification system.

Forensic samples include cheek swabs, bone, muscle tissue, human organs, touch samples containing very small amounts of DNA, bloodstains, skin fragments, hair, bodily fluids, and items presumed to contain any of these. Many forensic samples contain unknown amounts of DNA, ranging from 0.001ng to 1000μg of DNA, more frequently 0.01ng to 10μg of DNA. Forensic samples may contain only DNA from a single individual, may contain DNA from multiple individuals, and may contain degraded DNA.

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

The analysis system 101 may be equipped with a valve. The valve is used to specify the solution transport path as well as to switch the path to which air pressure is applied. The valve may be a diaphragm valve that operates with air pressure, a mechanical valve, or a valve that uses surface tension. The flow path that can be transported may be switched based on the difference in pressure required for transport.

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

The PCR reagent may include an IPC that is amplified along with the sample DNA and a set of primers for amplifying the IPC. The primers for the IPC may be dyed and detectable by CE. The amplicons derived from the IPC can be used for analysis. The amount of DNA in the sample may be estimated by using the intensity ratio of the IPC and sample-derived peaks, the amplification efficiency correction factor, and the fluorescence intensity correction factor. Also, by checking the intensity of the IPC, it may be estimated whether the PCR reaction is proceeding normally or has been inhibited.

The typical volume of PCR reaction solution is 1μl to 200μl, and more preferably 10μl to 50μl. A small volume of solution has the advantages of allowing accurate temperature control, high-speed PCR, and low reagent costs. On the other hand, a larger volume of solution allows for more eluted DNA to be received.

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

In the initial denaturing step, the mixture is heated to 90°C to 99°C for 1s to 2 minutes at the start of PCR, allowing the PCR reaction to begin. In the annealing step, the mixture is heated to 50°C to 80°C for 1s to 2 minutes to allow the primers to bind to the template DNA. In the extension step, the mixture is heated to 50°C to 80°C for 1s to 2 minutes to raise the temperature to a level where DNA polymerase can work well, allowing the DNA to elongate. In the denaturation step, the mixture is heated to 80°C to 99°C for 1s to 2 minutes. In the final extension step, the mixture is heated to 50°C to 80°C for 1min to 60min. By including a final extension step, the length of the amplified products can be made uniform. The annealing, extension, and denaturing steps are repeated 10 to 40 times. The annealing and extension steps may be performed at the same temperature.

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

[Analysis and peak criteria]
The signal obtained in the CE section is analyzed in the analysis section. Known analysis software includes GeneMapper (registered trademark) ID, GeneMapper ID-X, GeneMarker (registered trademark) HID, i-Cubed (trademark), OSIRIS, TrueAllele (trademark), etc. In CE analysis, a graph is generated based on the size standard peak from the signal intensity vs. time information, with the horizontal axis representing DNA length and the vertical axis representing intensity. Cosmic rays and pull-up/pull-down may be corrected. Baseline correction may be performed, or an electropherogram may be obtained using other existing techniques. Peak detection is performed on the obtained electropherogram to examine the intensity and peak position of each amplicon. In addition, the analysis may be performed partially with human intervention or fully automatically.

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

　Macrosatellites are DNA loci that contain repeated base sequences with 2-7 nucleotides per repeat unit. The number of repeats at a particular locus varies from individual to individual, and can be detected as differences in the length of the amplified products by STR-PCR.

A typical STR-PCR analysis detects two or more loci. Typically, five or more, 10 or more, 15 or more, 20 or more, or 25 or more loci are included. STR-PCR may be performed using kits sold by GlobalFiler (trademark) or PowerPlex (registered trademark). It is also preferable to include loci designated by various genetic databases, such as CODIS, for forensic or DNA identification purposes in each country. The loci may include loci present on autosomes, or may include genes present only on the Y gene.

When examining DNA types derived from a single individual, which may be homozygous or heterozygous for a particular locus, the number of peaks detected will, unless the amount of DNA is degraded or insufficient, at a minimum correspond to the number of loci in the kit, and at a maximum correspond to a peak corresponding to the sum of twice the amount of genes assigned to the autosomes and the number of genes assigned to the sex chromosomes among the loci in the kit.

In a typical STR-PCR analysis, one fluorescent dye is assigned to one locus. STR-PCR kits with 2, 3, 4, 5, 6, 7, or 8 dyes may also be used. A combination of length and peak color information may be used to assign loci to detect DNA types. Thresholds for the intensity and position of various peaks may be set for each color, or for each locus or allele.

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

In DNA testing, artifacts refer to, for example, peaks that are not derived from the DNA type derived from an individual, a balance between peaks that is different from the ideal state, or a peak shape that is different from the ideal state, which can cause the electropherogram and DNA profile obtained during actual DNA analysis to differ from the ideal electropherogram and DNA profile that should be obtained from the DNA type derived from an individual.

There are multiple mechanisms involved in the occurrence of artifacts. If the DNA in a sample comes from a single individual, the presence of some artifacts is acceptable.

On the other hand, if there are many artifacts, the accuracy of mechanical assessment decreases, making review by an expert necessary.

In addition, if there are a lot of artifacts, erroneous DNA testing results may occur.

In addition, if the artifacts are large, the DNA type that should be obtained may not be detectable, resulting in a reduced amount of information being obtained.

In addition, for samples where it cannot be guaranteed that the DNA comes from a single individual, typically forensic samples where the possibility that multiple individuals may be involved cannot be ruled out, the presence of artifacts can make analysis of the electropherogram more complex or difficult. For example, when three or more peaks appear at one locus, it becomes difficult to interpret whether those peaks are due to artifacts or to two or more individuals. In addition, while it is common to assign an individual's DNA profile from the ratio of peak intensities, this becomes difficult when the possibility of artifacts is taken into account. Therefore, it is desirable to obtain electropherograms with as few artifacts as possible.

When a peak that does not originate from an individual's DNA occurs during CE analysis and is reflected in the analysis results, this state is called a drop in. When a peak that should be present in an individual's DNA is not detected during CE analysis and is not reflected in the analysis results, this state is called a drop out. It is preferable to set various thresholds to minimize the occurrence of drop in and drop out.

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

<Incomplete adenylation>
In STR-PCR, during the extension reaction, there is a certain probability that an extra adenyl group will be added to amplicons of the correct length (A+ peak). In a typical STR-PCR kit, a reaction step called "final extension" is added to the end of the PCR reaction. In this final extension step, adenyl groups are added to amplicons that have not been given extra adenyl groups. By allowing a sufficient amount of final extension time, it is possible to achieve a state in which almost all amplicons are adenylated. However, if the final extension time is set too long compared to the amount of amplicon, the proportion of amplicons with extra adenyl groups (A++ peak) will increase. The A++ peak is detected at a position one base longer than the A+ peak. Also, if the amount of amplicon is excessive, adenylation is not completed within the final extension time, and amplicons that have not been adenylated remain, and are detected as an A- peak with one base less. In the following, the A++ peak and the A- peak are collectively referred to as incomplete adenylation peaks (IAPs). In addition, when describing only the A-peak, it is written as "IAP-", and when describing only the A++ peak or a peak with more adenyl groups, it is written as "IAP+". In a suitable STR-CE, various PCR parameters such as the number of PCR cycles, the final extension time, and the amount of input DNA are adjusted so that the A-peak and A++ peak are within a range of 50% or less, more preferably 20% or less, and more preferably 10% or less, relative to the A+ peak intensity. An IAP intensity threshold or an intensity ratio threshold to the main peak (Incomplete adenylation Peak ratio Threshold, IAPT) may be set to determine whether the intensity of the A-peak or A++ peak falls within the above range and suitable STR-CE has been performed. If the intensity ratio of the A- or A++ peak to the A+ peak is greater than a certain level, the intensity of the main peak will not reflect the original abundance ratio of the gene. In addition, in the case of a mixed sample or when a peak with one base shift appears due to genetic polymorphism, accurate assignment will not be possible. It may also cause the detection of an incorrect DNA type.

<Peak intensity ratio>
When there is sufficient DNA, the two peaks originating from heterozygous loci show almost the same height. When the amount of DNA is insufficient, the probability that the amount of DNA originating from each gene will be uneven increases, and the difference in the intensity of the two peaks becomes significantly larger. In addition, when amplification is excessive, short DNA is amplified preferentially over long DNA. The height of the peaks originating from the same locus also differs greatly because short DNA is amplified preferentially. When the ratio of the two peak intensities becomes large, it becomes impossible to distinguish from a stutter peak. In addition, it becomes difficult to assign the DNA of a mixed sample. Therefore, to determine whether a significant CE analysis has been performed, the ratio of the intensity of the smaller peak to the larger peak (Peak to height ratio, PHR) of the two peaks is 10% or more, more preferably 40% or more, and more preferably 60% or more.

＜Ski-slope＞
When there is an excess amount of DNA, the ratio of dNTPs and polymerase to the amplicon decreases, and the tendency for short DNA to be preferentially amplified increases. In this case, the electropherogram obtained has a sloped electropherogram with a small peak for long DNA and a large peak for short DNA. Also, when DNA is degraded, the ratio of short DNA to long DNA tends to be higher. In this case, a DNA profile with a slope is also obtained. Also, if an inhibitor is included, the amplification efficiency of long DNA tends to be lower than that of short DNA, which also gives a DNA profile with a slope. Also, when the PCR reagent is diluted with the DNA solution more than the original mixing ratio, the amplification efficiency differs, and long DNA is preferentially amplified, giving a DNA profile with a reverse slope. Also, when a large amount of primers for long DNA is mixed into the PCR reagent, a reaction system in which long DNA is preferentially amplified is obtained, and a DNA profile with a reverse slope is obtained. In DNA identification, a profile with a slope or a profile with a large peak intensity ratio between gene loci is not desirable. This is because the difference in peak intensity becomes larger, and peaks that saturate CE or fall below the detection limit become more likely to appear. Also, 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) has an intensity of 1% or more, more preferably 5% or more, more preferably 10% or more, and more preferably 20% or more relative to the maximum peak. Also, small peaks that do not meet the Inter locus PHR threshold may be excluded from peak analysis. Also, to deal with degraded DNA, it is desirable that the PCR amplification amount is within an appropriate range for STR-CE or that the dynamic range of CE is designed to be large.

<CE Saturation>
If the amount of amplicon introduced into the CE section is excessive and the fluorescence intensity during CE detection exceeds the upper limit of the detection range of the detector, saturation occurs. In this case, the ratio of the peak with the maximum intensity to the other peak intensities does not reflect the original intensity ratio. In addition, the intensity ratio of the stutter peak and the main peak of IAP is detected higher than the original ratio of the amplicon. Therefore, the above-mentioned artifacts are emphasized. In addition, if the sample is a mix, the correct mixing ratio cannot be calculated. Therefore, in a preferred DNA appraisal, the PCR reaction conditions are set or the PCR amplified product is diluted so that the CE detection does not saturate (oversaturation, OS). The OS threshold may be evaluated and set by the actual CE device, may be set by the user, may be set by the user for each measurement, or may be set on the computer.

<CE noise, pull-up>
There are also artifacts that occur in the CE detector.

Pull-up peaks are peaks that are derived from other dyes and are detected incorrectly. Pull-ups are particularly noticeable when CE is saturated, but they can also be detected when CE is not saturated.

If a large amount of DNA is brought into the CE section during the previous measurement, carryover may occur and a peak may be detected during the next measurement.

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

An analytical threshold (AT) may be set during analysis to prevent noise peaks from being mistakenly used in the analysis. The analytical threshold may be set by measuring background noise to obtain a sufficient signal-to-noise ratio, or may be set by the user, set by the device for each experiment, or may be preset. In addition, a program may be stored and executed to determine whether a peak that exceeds the AT is due to an amplification product, poor CE migration, or various types of CE noise.

The AT may be set in two or more stages, a first and a second reference value. If the peak has a peak intensity greater than the first reference, it is determined to be a true peak, and if the peak has an intensity greater than the second reference and less than the first reference, review by the user or an expert is required, or it may be set to be determined to be a peak when other set conditions are met. In addition, since amplification efficiency may differ depending on the gene locus, and luminescence efficiency and noise intensity may differ depending on the dye, AT may be set for each gene locus or each dye.

<Countermeasures against artifacts>
We will now discuss countermeasures for the above artifacts. For DNA that is not degraded or does not contain PCR inhibitors, inputting an amount of DNA into PCR that falls within the analytical range of STR-CE makes it possible to obtain appropriate peak intensity (above AT, below OS), PHR, and IPA data, and to suppress the appearance of the ski slope. Even if the DNA is degraded, in extremely small amounts, or contains inhibitors, the amount of information obtained can be maximized if the number of cycles and input amount are appropriate. There is a parameter set of the number of PCR cycles and the amount of DNA that can maximize the amount of information obtained while minimizing the appearance of artifacts.

<Evaluation method of CE analysis results>
When providing the CE analysis results to a user, it may also be possible to indicate to the user whether the obtained CE data is useful or not, and to what extent it is useful.

When presenting the CE analysis results to the user, it is also possible to show the user whether the obtained CE data is a complete DNA profile result (full profile). It is also possible to advise the user whether the obtained CE data requires further analysis.

Figure 2 shows an evaluation flow for determining whether the obtained DNA profile is a Full profile or for informing the user of the quality of the data. This chart is one example, and the order may be reversed, 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. Also, a flow like that of Figure 2 may be performed for each peak or locus, and it may be determined to be a Full profile if all criteria are met for all loci. Even if it is not a Full profile, information on only the loci that met the criteria may be provided to the user. Also, it may be possible to provide a table or other output showing which criteria were or were not met for each locus.

201. None of the peak intensities should saturate (OS) the CE detection system. If any peaks are OS, the peak or locus or analytical results may be flagged as OS.

202. All loci should have at least one peak above AT. If there are loci where no peaks are detected, the loci or analysis results may be flagged as Drop out (DO).

203. If there is only one peak above AT, that peak should have an intensity at least twice that of AT (excluding genes that are uniquely detected, e.g. sex chromosome loci). If there is no intensity at least twice that of AT, the peak, locus, or analysis result will be flagged as inconclusive homozygous (IH flag).

204. The intensity of the peaks present at ±1 base offset from the main peak must not exceed the IPAT, which is set at a peak ratio of 1%, 5%, 10%, 20%, or 40% of the intensity of the main peak. If it does exceed the IPAT, a flag indicating that the IPA intensity is high for that peak, locus, or analytical result (IPA- or IPA++ flag) will be assigned.

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

206. Three or more peaks should not be detected per locus (two or more for loci where only one should exist). If detected, the third-highest peak should be 1% or less, 5% or less, 10% or less, 20% or less, or 40% or less intense than the second-highest peak. If the ratio of the intensity of the third-highest peak to the intensity of the second-highest peak exceeds a threshold, the DNA contained in the sample may be determined to be from two or more individuals and a Mix flag may be set. However, if the third-highest peak occurs at the position where the first or second stutter peak appears, it may be determined to be a stutter peak. If a peak appears at the stutter peak position with an intensity of 1% or less, 5% or less, 10% or less, 15% or less, 20% or less, or 40% or less intense than the main peak, it may be determined to be a stutter and a Mix flag may not be set. If a peak exists at a position that could be a stutter peak, and its intensity exceeds the threshold of the acceptable range for stutter peaks, it may be a Mix, so a Mix flag may be set. Also, if the intensity of the peak with the third highest intensity in a position where a stutter peak should not appear exceeds the threshold, a Mix flag may be set. A similar determination may be made for peaks with the fourth, fifth, and subsequent intensities.

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

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

A peak that gives an IPA flag also gives an OL flag, so the criterion 205 may also serve as 204, i.e., the IPA flag may include the OL flag.

If none of the above flags are given, a full profile will be obtained. If a full profile cannot be obtained, an expert review may be requested. In this case, it may take longer to complete the DNA analysis. Therefore, it is necessary to increase the probability of obtaining a full profile by setting an appropriate number of cycles. Also, if the sample is originally mixed, the amount is small, there is a lot of PCR inhibitors, a full profile cannot be obtained but not all loci are detected normally, etc., it can be useful in criminal investigations if 5 or more or 10 or more loci are detected. Such a DNA profile is called a partial profile. The more information on loci obtained, the more useful it is for criminal investigations. In other words, even in a situation where a partial profile is obtained, the analysis protocol, especially the number of PCR cycles, must be set so that as many peaks as possible can be detected. In particular, in the case of a mixed sample or a small amount of DNA, the protocol, especially the number of PCR cycles, must be set so that as many peaks as possible can be detected while the electropherogram satisfies 201, 205, and 206 (i.e., these flags are not given).

A threshold may be set to determine whether DO is due to the absence of the relevant allele in the DNA input to the PCR reaction, or due to a low number of PCR cycles. For example, if there is a locus with no peaks above AT, and there is a peak in the entire amplified product that is 10 times higher than AT, it may be determined that the DO is due to the sample; if not, it may be determined that the DO is due to a low number of PCR cycles, or that it cannot be due to the sample. This determination can be used to determine whether an analysis with an increased number of PCR cycles should be performed.

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

In the following, the dynamic range of CE may be the dynamic range described in the manuals set for various CE devices, or it may be the ratio of the maximum amount of amplicon to the minimum amount of amplicon that gives a linear signal relative to the amount of amplicon actually input into CE, or it may be the maximum amount of amplicon to the minimum amount of amplicon that allows the relative ratio of the amount of amplicon input into CE to be assigned from the signal intensity, or it may be the ratio of an arbitrary upper analytical limit to an arbitrary lower analytical limit. It may also be the ratio of OT to AT. It may vary for each measurement.

In the following, unless otherwise specified, the analytical range of STR-CE may simply correspond to the dynamic range of CE, or may be an experiment in which the amount of DNA put into PCR is changed to examine the amount of DNA at which a specific allele can be correctly detected, or may be an experiment in which the amount of DNA put into PCR is changed to examine the amount of DNA at which a specific allele set can be correctly detected, or such a range of DNA amounts may be examined in experiments for multiple different individuals, and the average, minimum, or maximum range may be used as the analytical range. The amount of DNA used for CE measurement is generally proportional to the amount of DNA put into STR-PCR, but this is not always the case because the CE injection efficiency depends on the DNA concentration, and the PCR amplification efficiency depends on the DNA concentration and the number of cycles. In addition, when the number of PCR cycles is changed, the analytical range of STR-CE does not change significantly in most cases, but it may deviate, especially when the amount of DNA is reduced, due to the occurrence of a stochastic effect (a stochastic effect in which the amount of DNA corresponding to the locus to be amplified is no longer proportional to the amount of DNA input). Therefore, it is desirable to carry out the above examination for each cycle to determine the true STR-CE analysis range, and the analysis range for each cycle determined through such examination is also considered to be a type of STR-CE analysis range. However, since it would require a great deal of effort to confirm everything through experiments, some of it may be replaced by calculation.

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

By changing the threshold you set, you can widen or narrow the analysis range. However, trying to widen the analysis range can come at the expense of reduced accuracy. If you can more reliably distinguish between artifacts and true peaks using methods such as machine learning, you can change the threshold to widen the analysis range.

In the following, the analytical range of an analytical system refers to, for example, the range of DNA contained in a sample that allows DNA analysis to be performed correctly for a DNA sample input into the analytical system. To expand the analytical range, it is necessary to either expand the analytical range of STR-CE or control the amount of DNA input into STR-CE. When expanding the analytical range of STR-CE, it is possible to improve the dynamic range and sensitivity of CE, reduce the variability of STR-CE, prepare multiple cycles of amplification products, prepare eluates with different dilution rates and subject each to STR-CE, etc. When controlling the amount of DNA input into STR-CE, the volume or mesh of the purification membrane can be reduced so that the upper limit of the amount of DNA that can be processed by the purification membrane can be cut off, or the dilution rate can be changed by quantifying after purification.

《Analysis system for split PCR and its operation example》

[Analysis System]
[Example of a typical analysis system configuration]
Fig. 3 shows an example of the analysis system 101. Fig. 4 shows an example of the operation procedure of the analysis system 101. The biomolecule analyzer includes a computer 102 for performing biomolecule analysis, and a flow path device 104.

The flow path device 104 includes a dissolution chamber 301 for introducing and dissolving the collected sample, a purification membrane chamber 303 containing a purification membrane 302, a PCR chamber 304 (a PCR chamber for performing a thermal cycle) for amplifying DNA, and a waste liquid chamber 305. An external connection port 306 is provided that is fluidically connected to the outside of the device. The solution is transported through the external connection port 306, and reagents, amplified products, etc. can be exchanged with the outside of the device. When transporting the solution in the analysis system 101, the liquid transfer may be controlled using a pump and valve 307. The pump and valve 307 may all be provided outside the flow path device, or some of them may be provided inside the flow path device 104. In addition, the PCR reagent storage section 308 may be provided with PCR reagents 309 (polymerase, primer, dNTP, buffer, etc.) required for the PCR reaction, and the migration reagent storage section 310 may be provided with migration reagent 311. In addition, the chamber includes reagent reservoirs 312, 313, and 314 for storing reagents necessary for pretreatment. In a sample input step 401, before and after the sample is stored in the lysis chamber 301, a lysis buffer is transported from the reagent reservoir 312 to the lysis chamber 301. Next, lysis begins in a lysis step 402. In a purification step 403, the lysis product is transported from the lysis chamber 301 to the purification membrane chamber 303, where the DNA is bound to the purification membrane 302, and a cleaning solution is released from the reagent reservoir 313, and purification is performed. After purification, a step of drying the cleaning solution and the like may be included. An elution solution is released from the reagent reservoir 314, and the DNA eluted from the purification membrane chamber 303 is transported to the PCR chamber 304. A PCR reagent is transported from the PCR reagent reservoir 308 to the PCR chamber 304 and mixed with the eluted DNA. In an amplification step 404, the purified DNA in the PCR chamber 304 is mixed with a PCR reagent 309 and subjected to a PCR reaction. In the detection step 405, the amplified DNA is mixed with the migration reagent 311 stored in the migration reagent storage section 310, and measurement is performed in the CE section 105.

After mixing the electrophoresis reagent and the PCR reaction solution, a step can be added in which the mixture is heated to 80-100°C and then rapidly cooled to 0-10°C before CE analysis. By adding this step, the DNA is more completely converted into single strands, enabling highly accurate CE analysis.

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

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

As shown in FIG. 5(2), the analysis system 101 prepares a PCR reaction solution in step 501, performs m PCR cycles in the PCR chamber 304 in step 502, and then removes a portion of the amplified product from the PCR chamber 304 as PCR reaction solution m without changing its composition in step 503, performs CE measurement on the electrophoresis sample m in the CE unit 105 in step 504, and performs thermal cycling n-m times on the PCR reaction solution m remaining in the PCR chamber 304 in step 505 to obtain a PCR reaction solution n. In step 506, a portion of the PCR reaction solution n is removed from the PCR chamber 304 without changing its composition, and performs CE measurement on the electrophoresis sample n in the CE unit 105 in step 507.

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

Steps 503 to 505 can be repeated multiple times to increase the number of divisions to three, four, or more.

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

You can start step 505 immediately after step 503 is finished. Because the time between 502 and 505 is short, the occurrence of artifacts can be suppressed. You can also set the execution timing of 505 and 506 so that 507 starts at the same time as 504 ends.

[Examples of derivatives of the analysis system used in the practice of the present invention]
As shown in FIG. 6, the analysis system 101 may be equipped with heating units 317 and 318 (temperature control/heating mechanism) for performing thermal cycling in the PCR chamber 304. A flow path 319 and a flow path 315 are connected to the PCR chamber 304, and the supply of reagents and pressure may be controlled. As shown in FIG. 6, the analysis system 101 may be equipped with a pump and valve 307 for performing solution transport suitable for various steps such as a flow path device and CE measurement. As shown in FIG. 6, the analysis system 101 may be equipped with a computer 102, which may be equipped with a function of controlling the pump and valve 307, controlling the CE measurement, controlling temperature, analyzing data obtained from the CE unit 105 and the heating units 317 and 318, feeding back the data, providing the data to the user, and the like. The flow path device may be equipped with a dispensing chamber 320. The dispensing chamber 320 has a function of taking out a part of the amplified product from the PCR chamber 304 without changing the composition at the timing when m thermal cycles are completed. The dispensing chamber 320 may have a measuring function for extracting a specified amount of the PCR reaction liquid m or n. When the PCR reaction liquid is sent to the dispensing chamber 320, the dispensing chamber 320 may suck up the PCR reaction liquid, or the PCR reaction liquid may be sent to the dispensing chamber 320 by pressurization. In addition, the dispensing chamber 320 may be provided simply to temporarily store the PCR reaction liquid, and may not have a measuring function. The dispensing chamber 320 may be provided outside the flow path device 104.

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

As shown in FIG. 6, the analysis system 101 may have a waiting section 321 between the CE section 105 and the PCR section. The waiting section 321 may temporarily hold the PCR reaction solution or the electrophoretic sample in which the PCR reaction solution and formamide are mixed from step 506 to 507 or from 503 to 504. The flow path device in FIG. 6 (and the following figures) may be arranged vertically, that is, so that the main part of the flow path or at least a part of the flow path is parallel to the direction of gravity. In one form of the flow path device, the bottom side of the figure is used facing downward in the direction of gravity. By using it vertically, air bubbles/mixed air in each chamber accumulate at the top of the chamber, so that when transporting to the next chamber/flow path, the solution is removed from the bottom, minimizing the mixing of air into the next step.

The flow path device can be modified as follows:

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

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

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

As shown in FIG. 9, the PCR chamber 304 may have three flow paths 319, 315, and 330. The flow path 319 may be connected to the upstream side of the sample, the flow path 315 to the dispensing chamber 320, and the flow path 330 to the mixing chamber 327. The PCR reaction liquid m taken out from the PCR chamber 304 may be transported to the mixing chamber 327 via the dispensing chamber 320, and the PCR reaction liquid n taken out from the PCR chamber 304 may be transported to the mixing chamber 327 via the flow path 330 without passing through the dispensing chamber 320. Since the PCR reaction liquid m and the PCR reaction liquid n pass through different paths, it is possible to suppress the decrease in reproducibility caused by the remaining liquid. In addition, when the reaction liquid n is measured and the reaction liquid m is not measured and the entire amount is transported to the mixing chamber 327 or the CE unit 105, there is no need to transport the reaction liquid m to the dispensing chamber 320, so this flow path device structure makes it easier to perform split PCR.

If PCR reaction liquid m is also to be measured, another dispensing chamber may be provided in addition to the dispensing chamber 320 used for PCR reaction liquid n.

As shown in FIG. 10, the flow path device 104 may have electrophoretic reagent storage sections 310 and 331 for storing electrophoretic reagents (formamide, DNA fragments, pure water, etc.). The electrophoretic reagent storage sections 310 and 331 may be located on the flow path 315 or on the flow path 319. The reagent storage section may be divided into two or more storage sections for one type of reagent as shown in FIG. 10. By dividing into two, a specified amount of reagent can be released from each storage section at the time of division. Alternatively, the amount of liquid released from the storage section may be controlled so that the reagent can be released in two or more separate times from one reagent storage section. As shown in FIG. 10, the flow path device 104 may have air storage sections 332 and 333. The air storage sections 332 and 333 may be located on the flow path 315 or on the flow path 319. By releasing a specified amount of air from the air storage section 332 or 333 at the time of division, a specified amount of PCR reaction liquid can be transported outside the PCR chamber 304. In the case of this flow path device configuration, it is possible to perform split PCR even if dispensing chamber 320 is omitted. In the case of this flow path device configuration, mixing chamber 327 may also serve as dispensing chamber 320. Air reservoir 332 or 333 may contain a liquid that does not affect the PCR reaction, such as oil, instead of air. Air reservoir 332 or 333 may be filled with PCR reaction liquid, and the PCR reaction liquid may be newly replenished with a volume equivalent to the pushed-out PCR liquid.

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

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

FIG. 12 shows an example of a flow path device 104, and FIG. 13 shows an example of a transport method for performing split PCR on the flow path device of FIG. 12. For ease of illustration, some reference numerals are shown in FIG. 12 but omitted in FIG. 13.

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

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

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

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

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

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

Step IV: Open the valve 325 and transport the electrophoretic sample in the mixing chamber 327 to the outside of the flow path device 104 (to the standby section 321 or CE section 105 of the analysis system). This step may correspond to step 504.

Step V: Close valve 326 and perform thermal cycle n-m times. This step may correspond to step 505.

In this way, the analysis system 101 performs n-m thermal cycles (where n-m is an integer of 2 or greater) on the PCR reaction liquid m remaining in the PCR chamber 304 so that the total number of thermal cycles is n, to generate PCR reaction liquid n (second reaction liquid).

Step VI: After a total of n thermal cycles are completed, close valve 325, open valves 326 and 323, and transport PCR reaction solution n to mixing chamber 327.

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.

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

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

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

The flow path device 104 has a valve 337 on the flow path 315 that connects the dispensing chamber 320 and the PCR chamber 304. The valve 337 is installed on the PCR chamber 304 side of the branch with the migration reagent storage section 310. A valve 338 is also installed in the flow path 316. A valve 339 is also installed on the flow path 319, on the flow path 329 side of the branch with the migration reagent storage section 331.

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

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

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

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

Step V: Close all valves and perform 6 thermal cycles on the a-b μl PCR reaction solution 335 remaining in the PCR chamber 304, then perform a final extension for 8 minutes. (This may correspond to steps 608 to 610 or step 505.)

Step VI: After a total of 30 thermal cycles are completed, open valves 326, 323, and 325 to push out d μl of electrophoretic reagent from electrophoretic reagent reservoir 331, and transport PCR reaction solution n to mixing chamber 327. (This may correspond to step 506.)

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

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

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

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

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

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

In step 605, if the temperature during division is set to be equal to the extension temperature, an unnecessary extension reaction occurs, but non-specific amplification can be suppressed. This is also preferable because it avoids inactivation of the polymerase and fluorophore. The impact of the unnecessary extension reaction on the analytical accuracy of STR-CE can be ignored.

In step 605, the temperature during division may be between the extension temperature and RT. In this case, precise temperature control is not required, which is convenient. Since there are sufficient amplicons at the time when m cycles of PCR are completed, the effect of non-specific amplification can be ignored.

In step 605, the shorter the time required for division, the better. If the division takes a long time, there is a possibility that artifacts will increase. In addition, the longer the division takes, the greater the risk of inactivation of various biomolecules, such as polymerase.

As shown in FIG. 14, in this embodiment, the analysis system 101 does not perform analysis by a method other than electrophoretic analysis on each reaction solution (including a part of PCR reaction solution m and at least a part of PCR reaction solution n) before electrophoretic analysis. This simplifies the configuration of the device, and, for example, does not require an additional optical system.

[Amount of PCR reaction solution and electrophoresis reagent]
The volume of PCR reaction solution used in this flow path device is 1μl to 200μl, and more preferably 10μl to 50μl. The smaller the volume of solution, the more accurate and faster the temperature control can be. On the other hand, the larger the volume of solution, the more purified DNA can be accommodated, making it easier to achieve high sensitivity. Also, the larger the volume of solution, the less precise the measurement of the solution becomes when dividing.

Table 2 shows an example of the amount of liquid when divided. The amount of liquid actually measured does not have to be the value in the table, and the median value when multiple measurements are taken can be the value in the table.

In step 502, the total amount of PCR reaction solution is a μl, the amount of solution removed during cycle m in step 503 is b μl, the amount of migration reagent mixed with PCR reaction solution m in step 504 is c μl, and the amount of migration reagent mixed with PCR reaction solution n in step 507 is d μl. Table 2 above shows an example of a suitable relationship or set value for a-d. In order to efficiently introduce the amplified products and size standards into CE, it is necessary that the ionic strength of the migration sample is sufficiently low. Therefore, it is preferable to mix the amount of migration reagent in a volume ratio of 2, 5, 10, 20, etc., greater than the amount of PCR reaction solution.

The PCR division can be set so that PCR reaction solution m and PCR reaction solution n are equal. If the amounts of PCR reaction solution m and n are close, stable delivery of the electrophoresis reagent to the CE section can be achieved.

The PCR division can be set so that PCR reaction solution m is smaller than PCR reaction solution n. The smaller the volume of liquid taken out during division, the smaller the variation in volume of liquid when performing PCR n-m, and the more reproducible the amplified product n can be prepared.

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

Set 2 is an example of the amount of solution when the amount of PCR reagent is 15 μl, 5 μl of solution is taken out at the time of division into m cycles, and the entire amount of PCR reaction solution is mixed with the electrophoresis reagent at the time of n cycles. In this example, the analysis system 101 transports the entire amount of PCR reaction solution in the PCR chamber out of the PCR chamber at the end of n thermal cycles. In this way, the device required for measurement can be omitted, and the configuration of the flow path can be simplified.

In the liquid transfer procedure shown in FIG. 16, the migration reagent is left in the dispensing chamber 320. If the amount of migration reagent in the migration reagent storage section 310 is sufficiently large compared to the dispensing chamber 320, the influence on the analytical accuracy and range can be ignored even if the migration reagent is left in the dispensing chamber 320. In addition, 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 an air storage section 334 is provided, the entire amount can be pushed into the dispensing chamber 320 and the mixing chamber 327. In the case of a large volume configuration, almost the entire amount of the PCR reaction liquid m in the dispensing chamber 320 is sent to the mixing chamber. In addition, it is preferable to store an extra migration reagent in the reagent storage section, taking into account that a part of the migration reagent is left behind in the dispensing chamber 320.

Set 3 is an example of the amount of solution stored in the migration reagent reservoir 310 when it is anticipated that eμl of migration reagent will remain in the dispensing chamber 320.

Set 4 is an example of the amount of solution stored in the electrophoretic reagent storage section 310 when the volume of the dispensing chamber 320 is b μl and it is expected that b μl of electrophoretic reagent will remain in the dispensing chamber 320.

[Detailed division method]
Since variation in the amount of solution during division tends to narrow the effective analysis range of the analysis system, it is desirable to divide the PCR reaction solution with high accuracy. To achieve high-accuracy solution division, functions such as setting an appropriate pressurizing pressure and pressurizing time in advance, pushing out the PCR reaction solution with a liquid or gas of a specified volume, using a liquid level detection sensor, and pushing out the PCR reaction solution into a dispensing chamber 320 of a specified volume may be incorporated. Multiple pushing and measuring methods may be combined.

Measurement may be performed when dispensing PCR reaction solution m in step 503 and when dispensing PCR reaction solution n in step 506. If step 506 is omitted and the entire amount of PCR reaction solution n is mixed with the electrophoresis reagent, measurement may be performed only in step 503. In other words, the number of measurements may be the number of divisions minus 1. Similarly, if measurement is performed twice, since there is a specified amount of liquid in the PCR chamber, an additional p PCR thermal cycles may be performed on the reaction solution remaining in the PCR chamber, and the CE of the third division reaction solution after n+p cycles of PCR reaction may be measured.

When removing the PCR reaction solution from the PCR chamber, you can apply pressure to the PCR chamber with a pump to send the solution to the dispensing chamber. Also, if the valve is closed during PCR heating, internal pressure is applied, so you can set it so that the solution moves to the dispensing chamber using internal pressure without opening valves 326 and 323 in step II.

After m cycles are completed in step 502, new PCR reagent may be used to push out the PCR reaction solution m in step 503, and a specified amount may be transported to the dispensing chamber 320 or the mixing chamber 327. This division method may be implemented by sealing PCR reagent instead of air in the air reservoir 332 in FIG. 10. Since it is pushed out with a solution, it has the advantage that the pushed-out volume is easy to specify. In addition, when pushing out with PCR reaction solution, it has the advantage that there is no adverse effect on the reaction due to the inclusion of air bubbles or oil. On the other hand, there is a possibility that new PCR reagent may be sent to the latter stage, which adds a new factor in addition to the above-mentioned factors of variation in the analysis range (1) to (8). In addition, since it is diluted with PCR reaction solution, the PCR reaction solution remaining in the PCR chamber is diluted at the point of m cycles, and more PCR cycles are required. In addition, when reagent at room temperature is added, the temperature of the PCR chamber drops. In addition, it is necessary to perform the initial denaturation again in step 505. Therefore, when pushing out with PCR reaction solution, the PCR reaction time becomes slightly longer. Furthermore, increasing the number of cycles and performing the initial denaturation again can contribute to the occurrence of artifacts. Note that it is also possible to solve some of the above problems by performing the initial denaturation of the PCR reaction solution used for extrusion prior to step 503.

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

Instead of pushing with air or PCR reagent, the PCR reaction liquid may be pushed with oil. This division method may be implemented by sealing oil instead of air in the air reservoir 332 in FIG. 10. The oil may be any oil that does not affect the PCR reaction. For example, it may be silicone oil, mineral oil, Fluorinert oil, or a mixture of multiple oils. When pushing with oil, as with pushing with reagent, the pushed volume is easy to define, and unlike pushing with PCR reaction liquid, it has the advantage that it does not mix with the PCR reaction liquid. On the other hand, if the PCR reaction product is mixed with oil and flows to the rear stage, it may interfere with the CE measurement. When the PCR reaction liquid m is taken out, it may be separated by a separation membrane or centrifugation to leave the oil in the chamber.

[Weighting mechanism details]
When the PCR reaction solution is introduced into the dispensing chamber 320 during division, if air or air bubbles in the PCR chamber enter instead of the PCR reaction solution, the measurement accuracy will decrease. Therefore, it is preferable that the dispensing chamber 320 and the flow path 315 are designed to prevent air from entering. An example of a structure for preventing air from entering is shown in FIG. 17. As shown in FIGS. 17(a) and 17(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 division can be minimized. In addition, it is preferable to design the liquid volume and the chamber shape so that the liquid level 701 is located above the connection part of the PCR chamber 304 and the flow path 315 in the direction of gravity when division is completed. As shown in FIG. 17(b), the liquid level 701 may be located above the valve 326 or 323 in the direction of gravity. In this case, it is possible to minimize or completely eliminate air bubbles entering the PCR chamber 304. The PCR reaction solution 335 located above the valve 326 or 323 does not need to be subjected to the PCR reaction. However, in this case, there is a possibility that unreacted reagents may be mixed into the CE measurement, decreasing the sensitivity.

As shown in FIG. 17(c), the flow channel 315 may be connected to the bottom of the PCR chamber 304. This configuration has the advantage that air is less likely to enter, but the flow channel 315 is somewhat long, which tends to cause liquid loss.

The flow path 315 may be provided with a structure for removing air bubbles. In addition, a hydrophilic filter that does not allow air to pass through may be placed on the flow path 315 in the flow path connecting the PCR chamber 304 and the dispensing chamber 320.

The dispensing chamber 320 may have a measuring function. For example, FIG. 18 shows an example of a dispensing chamber 320 with a measuring function. The flow paths 315 and 328 connected to the dispensing chamber 320 are provided with valves 324 and 337. The valve 337 may be used not only for measuring, but also to prevent liquid from splashing out into the dispensing chamber 320 during the PCR reaction, and to prevent the migration reagent and migration sample from flowing back into the PCR chamber 304 after dispensing. The valve 324 may be provided not only for measuring, but also to prevent the migration sample from flowing back after being transported to the mixing chamber. The volume of the dispensing chamber may correspond to the amount of liquid to be measured by dividing the amplification product m. The dispensing chamber may be spherical or cylindrical, rectangular, elongated, or serpentine, or may be ellipsoidal or elliptical cylindrical.

For example, the analysis system 101 may measure a predetermined amount of PCR reaction liquid m within the range of 0.1% to 50% by transporting the PCR reaction liquid m to the dispensing chamber 320 (measurement unit) after m thermal cycles have been completed. In this way, no additional measurement process is required.

Figure 19 shows an example of a weighing mechanism. Multiple mechanisms like those shown in Figure 19 may be combined for weighing. A weighing mechanism not shown may also be used. More robust weighing may be achieved by combining multiple weighing mechanisms.

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

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

Step II: Since the amplified products and electrophoretic reagents cannot pass through the vent filter 702, the PCR reaction liquid is weighed to the volume of the dispensing chamber 320 and the flow paths before and after it.

Step III: Close valve 337 and open valve 324. The PCR reaction mixture in dispensing chamber 320 is pumped to mixing chamber 327 using electrophoretic reagent or air.

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

Step I: Pressure (A kPa) is applied to the PCR chamber 304, and the PCR reaction liquid is sent to the dispensing chamber 320. The air present in the dispensing chamber 320 and before and after it can be smoothly removed through the flow path resistance 704.

Step II: At an applied pressure of A kPa, the PCR reaction liquid cannot exceed the flow resistance 704 or can only exceed it for a long time, so that a specified amount of PCR reaction liquid can be measured out in the dispensing chamber 320.

Step III: Close valve 337 and apply B kPa (where B>A) to dispensing chamber 320 using electrophoretic reagent or air. The PCR reaction solution in dispensing chamber 320 can be sent to mixing chamber 327.

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

Figure 22 shows an example of a simple metering mechanism that does not use a liquid level sensor, vent filter, or flow path resistor. However, this method can also be combined with a liquid level sensor, vent filter, flow path resistor, etc. to provide a robust dispensing mechanism.

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

Step II: After m cycles of PCR reaction are completed, open valves 326 and 323 and set the pressure in PCR chamber 304 to atmospheric pressure (=100 kPa).

Step III: Apply a pressure of 100 kPa through flow paths 319 and 330.

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

Step V: Close valve 337 and open valve 324 to return the pressure in dispensing chamber 320 to atmospheric pressure.

In this method, when the solution is transported from the PCR chamber 304 to the dispensing chamber 320, the amount of liquid that has entered the dispensing chamber 320 can be determined by the pressure applied to the dispensing chamber 320. When the volume of the dispensing chamber 320 is V1 and the inside is filled with air at pressure P1, if the liquid enters the dispensing chamber by volume V2, the pressure changes to P2 = P1 * V1 / V2 according to Boyle's law. When P2 is balanced with the applied pressure, the solution stops. In step I, if the pressure in the dispensing chamber is 100 kPa, when the volume of the dispensing chamber 320 is reduced to half, the pressure in both the dispensing chamber and the PCR chamber becomes 200 kPa and the forces are balanced. Therefore, it is possible to dispense PCR reaction solution 335 equivalent to about 50% of the volume of the dispensing chamber. Note that the pressure shown here is just one 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 may be appropriately set and measured. The disadvantage of this method is that the valve tends to require high pressure resistance. If the volume of the dispensing chamber 320 is set to a value larger than the amount to be measured, the pressure required for dispensing and measuring will be reduced, but the disadvantage is that the amount of residual liquid will be large when it is pushed out with the migration reagent afterwards.

[About weighing accuracy]
In CE analysis, if there is variation in peak intensity when the same sample is measured multiple times, the effective analysis range will be narrowed. If the analysis intensity varies greatly due to the introduction of split PCR, the effective analysis range will be narrowed, which is undesirable. If the introduction of split PCR increases the variation in the mixing ratio of the electrophoresis reagent and the PCR reaction solution, this will be reflected in the variation in peak intensity, narrowing the effective analysis range.

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

　ただし：
　k₀,C₀：ホルムアミドに含まれるイオンの移動度と濃度
　k₁,C₁：サイズスタンダードのDNAの移動度と濃度
　k₂,C₂：PCR反応液に含まれる塩やプライマ、dNTPの移動度と濃度
　k₃,C₃：PCR反応液に含まれる増幅産物の移動度と濃度
である。 In CE measurements, when the amplified products are drawn into the capillary, a voltage is applied to the electrophoretic sample and the capillary to perform electric field injection. In electric field injection, the amplified products introduced into the capillary are competitively injected with other ions and nucleic acids. When the voltage and injection time are constant, the relationship shown in Equation 1 holds (β is the current value, and α is a constant).

however:
_k0 , _C0 : mobility and concentration of ions contained in formamide _{; k1} , _C1 : mobility and concentration of size standard DNA _{; k2} , _C2 : mobility and concentration of salts, primers, and dNTPs contained in the PCR reaction solution; _k3 , _C3 : mobility and concentration of amplified products contained in the PCR reaction solution.

Taking the standard case where 1 μl of PCR reaction solution is mixed with 10 μl of electrophoresis reagent as the standard, when the amount of PCR reaction solution varies by γ μl, Equation 1 is expressed as follows.

The injection amount k ₁ *C ₁ of the size standard can be rearranged as follows, and it decreases monotonically with increasing γ.

On the other hand, the injection amount of the amplified product k ₃ *C ₃ is obtained by transforming Equation 1 as follows, and increases monotonically with the volume of the PCR reaction solution.

The PCR reaction solution contains many salts, primers, dNTPs, etc., and the injection amount is nearly saturated. Therefore, even if the ratio of the PCR reaction solution to the electrophoretic reagent increases slightly, the fluctuation in peak intensity is limited. Even if the ratio of the PCR reaction solution varies by ±20% from the standard conditions (PCR reaction solution: electrophoretic reagent = 1:10), the fluctuation rate of the peak intensity remains within 10%. In addition, if the target conditions are slightly more concentrated PCR reaction solution than the standard conditions (PCR reaction solution: electrophoretic reagent = 1.6:10), the fluctuation rate of the peak intensity will be less than 10% even if the ratio of the PCR reaction solution varies by ±50% from the target ratio. The results of this experiment suggest that even if the dispensing accuracy of the split PCR is somewhat poor, the fluctuation in peak intensity from the amplified product resulting from the fluctuation in the mixing ratio of the PCR reaction solution and the electrophoretic reagent is limited.

There are cases where it is preferable to adopt a simple, low-cost measuring mechanism, even if the accuracy of the measuring mechanism is somewhat poor. When using a measuring mechanism with low accuracy, as shown above, it is preferable to select a solution composition in which fluctuations in the mixing ratio of the PCR reaction solution and the electrophoretic reagent are unlikely to affect the peak intensity. This can be done by adding salt to the electrophoretic reagent, or the salt, primers, dNTPs, etc. contained in the PCR reaction solution can play this role. In addition, the mixing ratio of the electrophoretic reagent and the PCR reaction solution should be performed under conditions where the PCR reaction solution is slightly higher, and measurements are performed in a state closer to saturation, making it less susceptible to the effects of fluctuations in the ratio. In this case, it is preferable to mix a slightly larger amount of size standard than the standard mixing ratio so that the peak intensity of the size standard contained in the electrophoretic reagent reaches the level required for measurement.

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

In order to be able to analyze target DNA of various concentrations contained in a sample, PCR reaction solution m and PCR reaction solution n, each containing an amplified product of different concentrations, are subjected to electrophoretic analysis. By setting m and n in advance within an appropriate range according to the range of concentrations of the target DNA that may be contained in the sample, it is possible to obtain an appropriate DNA profile for a wider range of DNA amounts without a decrease in sensitivity than when a single amplified product is prepared and analyzed by CE.

Figure 24 shows the principle of expanding the analytical range by split PCR. The horizontal axis of Figure 24 shows the amount of DNA input to PCR, and the vertical axis shows the concentration of the amplified product or the peak intensity in CE. 801 is the upper detection limit intensity of CE or the upper limit of the concentration of the amplified product that can be amplified accurately by PCR, and 802 is the lower detection limit of CE. In the figure, the concentration of the amplified product is shown to increase linearly with the amount of DNA input, but the actual amplification is sigmoidal, and it is considered that it approaches a plateau when the amount of amplified product reaches a certain amount. It is assumed that the range of DNA amount that can be correctly analyzed at a certain PCR cycle number m is between the lower limit a and the upper limit b. The analytical range of STR-CE that can be analyzed with one cycle number is 803. Also, it is assumed that the range of DNA amount that can be correctly analyzed at a certain PCR cycle number n is between the lower limit c and the upper limit d. In this case, if a<d, the analytical range can be expanded from b/a or d/c to b/c. The analytical range of STR-CE when expanded by split PCR is 804. 804 is the range of DNA amount where either m or n falls within the analytical range of STR-CE, and in principle, there is no DNA amount falling outside the analytical range. In the following, the analytical range (b/a) at each cycle number is assumed to be almost constant and to change by ^2x times with respect to the cycle number difference x. In this case, the interval between m and n is limited to the range of the following formula 2.

An example of a case where this requirement is met is shown in Figure 24(1). For example, when the analysis range b/a for the amount of DNA brought into the PCR is 80 times, an appropriate nm is 6 or less, and the magnification ratio is 64. The magnification ratio can be increased by (b/a) ^y-1 for the number of divisions y, but it is thought that a smaller number of divisions will enable stable liquid delivery on a simple flow path device. Below, we will mainly describe the case where m and n are set and the amplified products m and n are analyzed. On the other hand, it is also possible to set l to divide into 3 and k to divide into 4 using a similar setting method.

When c or d is less than 0.2 ng or 0.1 ng, the variation in the intensity balance between peaks increases, so it is preferable to set a number of cycles greater than that set in formula 2 and to set the interval between m and n narrower than the maximum number of cycles defined in formula 2. In the following, unless otherwise specified, the upper and lower analytical limits of STR-CE change by a factor of ^2x with respect to the difference in cycle number x.

Figure 24(2) shows the analysis range when the interval between m and n does not satisfy formula 2. In this case, the lower limit a that can be analyzed with m exceeds the upper limit d that can be analyzed with n, and correct DNA identification cannot be performed with a DNA concentration between a and d. If a>d and there are no samples between a and d, or if the frequency is extremely low, it is acceptable to set the values of m and n so that the relationship a>d holds, but since the amount of DNA in the sample actually input is often unknown, it is preferable to satisfy formula 2.

When covering extremely low concentrations of DNA with the number of cycles n, a stochastic effect will occur, so it is desirable to set the difference between n and m to a value smaller than the upper limit of the difference specified in Equation 2.

Since there are multiple factors that can cause variability in the analytical results of STR-CE analysis, it is necessary to allow for overlap between the range of DNA amounts that can be analyzed by m cycles of STR-PCR and the range of DNA amounts that can be analyzed by n cycles of STR-PCR, taking variability into account. If there is no overlap, there will be an amount of DNA that will cause the analysis to fail, even if it falls within the analytical range of m and n. In other words, m and n must be set so that a<d is always true for the expected analytical variability. In the following, it is assumed that m and n are set taking into account the variability of the analysis system.

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

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

n may be set according to the following conditions:
(1) No peaks of amplification product m are detected at all. (2) Peaks are detected and some peaks are below the AT. (3) Peaks are detected and some peaks are already saturated or exceed the IAP threshold.

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

In the case of (2), if the CE saturation intensity is z for the maximum intensity y of the detected peak, an additional PCR may be performed with n-m cycles (n-m≦log(z/y)).

In case (3), there is no need to perform an additional PCR test.

[How to set the interval between m and n taking into account the intensity ratio of the sample you want to analyze]
In particular, if the time required for CE analysis is long, waiting for the completion of m cycles of electrophoresis as described above may result in an excessively long waiting time for the nm PCR reaction, which may increase artifacts or cause the PCR reaction to fail. Therefore, m and n may be set in advance.

STR-CE contains a mixture of homozygous and heterozygous loci, so even in an ideal analysis 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 the heterozygous and homozygous peaks.

In actual STR-CE, there are differences in amplification efficiency depending on the gene locus and DNA length, differences in CE injection efficiency, and differences in luminescence efficiency between dyes, so even if the amount of DNA is appropriately adjusted, there is a difference in intensity between peaks of 1:2 to 1:20. Note that AT values may differ between dyes. In such cases, it is necessary to consider the analysis range taking into account the difference in AT setting value.

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

When DNA is degraded, the ratio of alleles present varies from 1:2 to 1:1000. Typically, DNA from short loci or alleles is amplified in large amounts, while the peak intensity of DNA from long loci or alleles tends to be reduced.

If PCR is inhibited, shorter DNA will tend to be amplified more and longer DNA will tend to have smaller peak intensities. In such cases, the difference in peak intensities will be 1:2 to 1:1000.

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

In the following, we will proceed with the discussion assuming that the factor that determines the intensity ratio between peaks is due to the difference in the abundance ratio of alleles contained in the DNA input to STR-PCR. Differences in amplification efficiency, CE injection efficiency, and luminescence efficiency also have an effect, so although these differences should be taken into consideration, they will be omitted and will be included in the discussion of the abundance ratio between alleles.

が成り立つ。アンプリコンαとアンプリコンβの両方が正しく検出される実効的なSTR-CEの分析範囲803は、以下の式3

により表すことができる。b/a(範囲807)はCEのダイナミックレンジに対応していてもよく、ピークαを検出できるSTR-CEの分析範囲でもいい。 The effective analytical range of STR-CE when there is a difference in the abundance ratio of the alleles to be analyzed is explained using Figure 25. It is assumed that the amplification product contains allele α and allele β. However, it is assumed that the amounts of allele α and β are α>β before or after amplification. Plot 805 is a plot of the amount of allele α in the amplification product or the CE peak intensity against the amount of input DNA. Plot 806 is a plot of the amount of allele β in the amplification product or the CE peak intensity against the amount of input DNA. Plots 805 and 806 are not necessarily linear. It is assumed that amplicon α can be analyzed with DNA input amounts ranging from a to b. It is also assumed that amplicon β can be analyzed with DNA input amounts ranging from c to d. If PCR amplification is linear in the concentration range from a to b,

The effective analytical range of STR-CE 803 where both amplicon α and amplicon β are correctly detected is expressed by the following formula 3:

b/a (range 807) may correspond to the dynamic range of CE, or may be the analytical range of STR-CE in which peak α can be detected.

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

Figure 26 shows a table showing the numerical values corresponding to the range 807 and the intensity ratio α/β of the maximum amplicon to the minimum amplicon that can be analyzed by split PCR for each set value of n-m. For example, if the range 807 is 120 and n-m is set to 5, the allele with an abundance ratio of 1/3.75 relative to the allele with the highest abundance ratio will be the analysis target. Conversely, if the intensity ratio of the allele set to be analyzed is greater than the ratio shown in Figure 26, it means that a concentration where the maximum or minimum peak hits the upper or lower analytical limit will appear within the analytical range. For example, as in Figure 27(1), there is no problem if the concentration is such that both α and β can be detected, but as in Figure 27(2), a situation may occur where only α can be detected and β cannot be detected.

If the peak intensity ratio is too large, it is not necessary to analyze it. An example is shown below.
- STR-CE includes stutter peaks, and peaks with peak intensities more than 1:20 apart within a single locus are difficult to distinguish from stutter peaks, so there is no need to analyze peaks with intensity ratios greater than 1:20 or 1:40 within a single locus.
- Degraded DNA may also have a reduced total DNA amount, so there is no need to analyze peaks whose peak intensities are separated by more than 1:20, 1:40, or 1:100.
As described above, in an electropherogram in which the intensity ratio between peaks is extremely large, small peaks are not significant, so an inter locus PHR threshold that specifies the minimum peak intensity relative to the maximum peak intensity to be analyzed may be set. 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 be slightly lower than the inter locus PHR threshold.

[How to set n]
When a partial profile is also to be analyzed, 30 to 34 cycles is appropriate.

The peak resulting from one copy of DNA can be detected by setting the cycle number to 36.

　When the number of cycles exceeds 36, the probability of detecting individual peaks does not improve, although the number of artifacts (especially stutters and drop-ins) increases. Therefore, it is not advisable to set the number of cycles to 36 or more.

If partial profiles are not of interest for analysis, 30 or 29 cycles or less is appropriate.

For the sample containing the smallest amount of DNA, it is preferable to select the smallest number of cycles that results in the fewest number of errors, which can be determined experimentally.

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

n cycles may be set to the minimum number of PCR cycles that ensures that the amplification products derived from 20 copies of genomic DNA will be full profile.

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

After clarifying the detection limit at a specific cycle number through experiments or calculations, n can be set under the assumption that the detection limit increases or decreases by a factor of two with each cycle. However, if the amount of DNA is too small, the stochastic effect will cause greater variance in peak intensity, so it is more appropriate to set a cycle number with some leeway.

You can set n as described above, independent of m. However, if the interval between m and n is too large, there is a possibility that some DNA will remain in between them and cannot be analyzed.

If m is set first, it can be set from the difference of n-m that covers the intensity ratio of the peak to be analyzed, and the value of m, as shown in Figure 26.

When the analytical range 803 is known, it is preferable to set n having a cycle number difference of log ₂ (analytical range 803) or less with respect to m.

The number of cycles can be set with some leeway, taking into account the fluctuations of various analysis systems and peak intensity ratios.

[How to set m]
In DNA testing, the amount of DNA input to PCR rarely exceeds 1.5 μg, so setting the number of cycles below 20 will not increase the number of loci that can be correctly detected. Therefore, it is preferable to set the number of cycles to 20 or more.

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

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

The number of cycles can be set with some leeway, taking into account the fluctuations of various analysis systems and peak intensity ratios.

You can set m by examining the lower limit of detection, the amount of DNA input to PCR, and the upper limit of detection at a specific cycle number, and assuming that the lower and upper limits increase or decrease by a factor of two with each cycle.

You can set m as described above, independent of n. However, if the interval between m and n is too large, there is a possibility that some DNA will remain in between them and cannot be analyzed.

If you set n first, you can set it from the difference between n-m that covers the intensity ratio of the peak you want to analyze, as shown in Figure 26, and the value of n.

When the analytical range 803 is known, it is preferable to set m with a cycle number difference of log ₂ (analytical range 803) or less.

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

[Set m and n from the table]
Figure 28(1) shows the analysis range expansion ratio when m and n are set. The interval between m and n can be determined based on this table to obtain the required expansion ratio. 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. In addition, if the original analysis range is larger than the expansion ratio, it is inappropriate because it will result in a range of DNA amounts that cannot be analyzed, as shown in Figure 24(2). In addition, when analyzing DNA with a large difference in the abundance ratio between alleles, setting the expansion ratio to the limit of the original analysis range will result in alleles that cannot be analyzed.

Figure 28(2) shows the analytical range (number of digits) expanded by split PCR. In a typical STR-CE, genomic DNA 0.75 ng to 48 ng (1.8 digits) can be analyzed at 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. Variation must also be taken into account. Based on this result, the number of digits of the analytical range when m and n are changed, the analytical lower limit for n cycles, and the analytical upper limit for m cycles are shown here. The stochastic effect when the amount of DNA is reduced is not taken into account. The table was created assuming that everything changes by ²ⁿ with respect to the number of cycles. You can create a table like this and determine m and n from the appropriate lower and upper limits and analytical range. However, if the expansion rate of the analytical range exceeds 1.8 digits in this case, a range of DNA amount that falls outside the analytical range will be generated in the section considered to be the analytical range, as shown in Figure 24(2).

The typical dynamic range of CE is 2000 or less. Even in ideal DNA testing, the peak intensity ratio will be more than twice as high, so it is desirable for the difference between n and m to be 9 or less.

The dynamic range of higher performance CE is 4000 or less. In many DNA tests, the peak intensity ratio is 4 times or more, so it is desirable for the difference between n and m to be 9 or less.

As mentioned above, one example of a suitable range is m equal to or greater than 20 and equal to or less than 32. Another example of a suitable range is n equal to or greater than 24 and equal to or less than 36. Another example of a suitable range is n being 4 to 9 more than m.

[Timing of division]
As shown in Figure 14, a final extension step may be performed after m thermal cycles, and the mixture may be divided, and then final extension may be performed again after n thermal cycles. In this case, for example, the analysis system 101 may hold PCR reaction solution m at a constant temperature in the range of 50°C to 80°C for 1 to 20 minutes, and then remove a portion of it. With this configuration, only one heater is required around the PCR, making the apparatus and flow path device structure simple.

After m thermal cycles are completed, you can split it up and then perform the final extension step.

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

The operation shown in FIG. 29 can be performed by the flow path device 104 in FIG. 6. In FIG. 6, the heating unit 318 is installed so as to be in contact with the dispensing chamber 320. After m cycles are completed in step 603, the mixture is divided in the dispensing chamber 320 in step 605, and a heater final extension step is performed in step 604. The PCR reaction solution remaining in the PCR chamber may be thermal cycled n-m times in step 608 in parallel with step 604, or may be performed with a time lag. After n thermal cycles, a final extension step may be performed on product n in a holding chamber. That is, in the example of FIG. 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.

The advantage of performing this after division is that product n does not go through the Final extension step twice, reducing artifacts and not lengthening the overall time.

[How CE analysis results are provided to users]
If CE is performed more than once using split PCR, more than one electropherogram will be generated. Either the two electropherograms or the DNA analysis results may be provided to the user. The two or more electropherograms may be scored as to which is more suitable for DNA analysis and provided together with the CE analysis results.

For example, the analysis system 101 may determine which of the results of electrophoretic analysis of a portion of the PCR reaction solution m and the results of electrophoretic analysis of at least a portion of the PCR reaction solution n is the better result, and output the better result. Alternatively, it may output information that allows for determining which result is the better result. Also, it may notify whether or not it is a full profile using a part of the flowchart shown in FIG. 2. For scoring, the number of loci that meet the judgment criteria shown in FIG. 2 may be used, the number of loci that do not meet the judgment criteria may be used, the number of flags that are judged not to meet the judgment criteria may be used, or a comprehensive calculation may be performed using an algorithm based on the judgment criteria. Data from an intermediate stage of DNA identification analysis may be provided to the user. Only the analysis results for the side that has obtained a full profile or is judged to have a better electropherogram may be provided to the user. In this way, the results can be compared efficiently. Also, it is easy for non-experts to select the appropriate result when they receive data from the system.

If both analyses come up with poor results, you may request an expert review.

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

The number of times CE is measured using an analytical system may be two, one, or even three or more times.

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

If you are analyzing only once, you can measure either amplification product m or amplification product n.

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

If the first electrophoresis result is poor, you can analyze the amplified products with the same number of cycles again. Also, if the first electrophoresis result is poor, you can analyze the products with the other number of cycles. Poor electrophoresis here refers to a situation where some or all of the size standard peaks cannot be detected, or where the solution gets stuck somewhere during transport.

After obtaining analytical results for either amplification product m or n, the DNA identification results obtained can be compared against the database, and based on the feedback obtained, analysis of the other amplification product can be started or continued.

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

<When analyzing amplification product m first>
When the amplification product m is analyzed first, it may be determined whether or not to analyze the amplification product n according to a flowchart as shown in Figure 30. However, the flowchart shown in Figure 30 is only an example, and judgment criteria and branching conditions not shown here may be included. For example, the judgment criteria may change depending on the number of peaks obtained, the number of loci, whether the data is mixed, etc. Furthermore, the judgment criteria shown in Figure 30 may be omitted partially or entirely, or may be replaced with other criteria.

The electrophoresis results of m can be provided to the user to decide whether to analyze the product of n and then start.

In addition, if n is being analyzed simultaneously or in parallel, the analysis may be interrupted midway based on the judgment results of the user or the flowchart in Figure 30.

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

Criteria set 2: Has a full profile been obtained? If so, do not perform or discontinue analysis of amplification product n.

Case Set 3: Is the IAP+ flag present? If so, begin or continue analysis of amplicon n.

Criteria set 4: Are all peaks at half or less than the OS intensity? Or are all peaks at or less than the intensity calculated by dividing the OS by the amplification rate expected by split PCR? Or are they at or below the intensity that would not cause saturation when additional PCR cycles n-m times are performed? Or have any peak intensities been reached? If so, do not perform or discontinue analysis of amplification product n.

When the interval between m and n is 2, if almost no peaks are detected in the CE analysis of electrophoretic product m, no significant data can be obtained from electrophoretic product n, so it is meaningless to analyze n after obtaining the analysis results of m. Similarly, when the interval between m and n is narrow, if the analysis results of m indicate that the amount of DNA is too low, there is no need to analyze n. When determining whether the amount of DNA is too low, the number of detected peaks or the peak intensity can be used.

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

If the IAP peak exceeds the threshold, you can perform n cycles of analysis. IAP peaks occur when an insufficient amount of DNA is input compared to the PCR cycles, so they can be reduced by increasing the number of cycles.

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

<When analyzing amplification product n first>
When the amplification product n is analyzed first, it may be determined whether or not to analyze the amplification product m according to a flowchart as shown in FIG. 31. However, the flowchart shown in FIG. 31 is only an example, and judgment criteria and branching conditions not shown here may be included. For example, judgment criteria that change depending on the number of peaks obtained, the number of loci, whether the data is mixed, etc. may be conceivable. Furthermore, the judgment criteria shown in FIG. 31 may be omitted in part or in whole, or replaced with other criteria.

The electrophoretic results of n can be provided to the user to decide whether to analyze the product of m and then start.

In addition, if m is being analyzed simultaneously or in parallel, the analysis may be interrupted midway based on the judgment results of the user or the flowchart in Figure 30.

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

Criteria set 2: Is the IAP+ flag present? If not, do not perform or discontinue analysis of amplification product m.

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

In this way, the analysis system 101 performs electrophoretic analysis on one of a portion of the PCR reaction solution m and at least a portion of the PCR reaction solution n in the CE unit 105, and controls the execution of the electrophoretic analysis of the other based on the results of the electrophoretic analysis of the one. For example, it may be determined whether or not to start the electrophoretic analysis of the other based on the results of the electrophoretic analysis of one, or, after the electrophoretic analysis of the other has started, it may be determined whether or not to continue the electrophoretic analysis of the one based on the results of the electrophoretic analysis of the other. In this way, unnecessary or inefficient electrophoretic analyses are omitted, making the overall processing more efficient.

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

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

For example, if a buccal swab contains a relatively large amount of stable DNA, the buccal swab mode can be selected and STR-CE analysis can be performed in 26 cycles without splitting (or splitting and analyzing only one side of the sample for CE analysis, and only analyzing the other amplification product if that fails). A DVI sample mode can also be provided for DVI samples (disaster victim identification, DNA analysis for identifying bodies from disasters or large-scale terrorist attacks). DVI samples are likely to contain a relatively large amount of DNA, so for example, [m=25,n=31] can be set. In many cases, DVI samples contain a relatively large amount of DNA, so analysis of m cycles can be started first, and analysis of amplification product n can be started or continued using the judgment flow shown in Figure 30. When analyzing DNA obtained at a crime scene, the amount of DNA is often low, so the casework sample mode with [m=26,n=31] can be selected. In the case of a casework sample, there is a high probability that the amount of DNA contained will be extremely small, so analysis of n can be started first, and a decision can be made to proceed with or continue analysis of m using the decision flow shown in Figure 31. In addition, in the case of a touch sample, there is a high possibility that the DNA contained will be extremely small, will have been degraded and the peak intensity will vary greatly, or will be mixed DNA. For this reason, it is preferable to set the interval between m and n narrower in the "touch sample mode" compared to other modes. Also, since there is a high probability that the DNA contained will be small, it is preferable to start analysis with sample n.

If there are two or more CEs per sample, the analyses of m and n may be performed simultaneously. The above preset m and n and the measurement order may be changed by the user on each occasion. The values of m and n shown in Table 3 are examples, and the preset m and n may be set during development through validation testing to maximize the probability of successful DNA identification.

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

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

Even if one of the amplification products prepared using two cycle numbers cannot provide data of the required quality, the other amplification product is prepared, reducing the probability of the sample going to waste. In addition, because the probability of the sample going to waste is reduced, the effort of collecting and pretreating the sample again is eliminated. As a result, the average data acquisition time is shorter when two samples are prepared using this embodiment and two samples are analyzed compared to when this embodiment is not used.

If the m cycle PCR product is divided and the remaining PCR product is not PCR cycled, and only the m cycle PCR product is CE analyzed, if the CE analysis result of the m cycle PCR product does not meet the required quality, it is possible to perform n-m PCR on the remaining PCR product to prepare the n cycle PCR product. However, if the PCR product is left unattended during the waiting time for CE analysis of electrophoresis sample m, the activity of the polymerase will decrease, and depending on the leaving temperature, a large amount of artifacts will increase, so the CE analysis result of the n cycle PCR product will not meet the required quality either.

If only n-cycle PCR products are prepared and only n-cycle PCR products are subjected to CE analysis, if the CE analysis results of the n-cycle PCR products do not meet the required quality and no m-cycle PCR products are prepared, the sample will be wasted.

If the DNA solution used in the PCR reaction is divided before PCR, the amount of DNA added to each part is reduced, resulting in a decrease in sensitivity. On the other hand, if the PCR reaction has progressed to a certain extent (for example, after 4 cycles or more), each allele contains 10 or more amplicons, so division does not affect sensitivity.

It is desirable to divide the sample when m cycles have been completed. If the sample is divided before m cycles have been completed and the remaining PCR reactions are carried out in each chamber after the division so that a total of m cycles are achieved, a PCR temperature controller must be installed in each chamber, which increases the size of the device. Also, since there is a possibility of artifacts occurring during division, it is preferable to divide the sample after the number of cycles has reached as large as possible.

By preparing multiple electrophoretic samples with different dilution rates after PCR, the effective analytical range of CE can be expanded. In other words, the occurrence of CE oversaturation and the frequency with which peak intensities fall below the AT can be suppressed. However, problems with peak intensity balance and peak splitting caused by too many amplified products cannot be solved by dilution after PCR, and must be addressed before or during PCR. In other words, split PCR is appropriate.

The number of cycles for division is set appropriately for the analytical sample, so the maximum expansion rate of the analytical range can be obtained with the minimum number of divisions. In other words, the measurement time is kept to a minimum when the analytical range is expanded.

　By controlling the amount of DNA before inputting it into PCR, the effective analytical range can be expanded. For example, the upper limit of the amount of DNA adsorbed to the purification membrane can be reduced by changing the capacity or volume of the purification membrane or the purification protocol. However, with a typical purification membrane, reducing the volume to lower the upper limit of adsorption can result in poor passage of the solution and a decrease in the DNA yield (especially for short, degraded DNA). Therefore, there is a limit to how much DNA can be controlled at the purification stage. In addition, by quantifying the amount of DNA before PCR, it is possible to control the number of PCR cycles or change the dilution rate to expand the effective analytical range. However, the quantification step requires an additional detection system and inevitably complicates the flow path device. Since there is a possibility of analysis failure due to quantification errors, split PCR is more preferable.

This embodiment may be combined with dilution after PCR and control of the amount of purified DNA. By combining them, DNA analysis can be performed more reliably or over a wide range of DNA amounts.

By taking out the PCR reaction solution after each cycle or after every cycle and measuring the CE, DNA analysis suitable for all DNA concentration ranges can be performed. However, it is difficult to provide a mechanism for taking out the solution three or more times, four or more times, or five or more times in a flow path device and then mixing it with the electrophoresis reagent. In addition, even if two, three, or four amplification products are measured after each cycle or after every cycle, the expansion rate of the analysis range is only 2x, 4x, or 8x, which is not necessarily suitable for analyzing samples containing various amounts of DNA. In addition, when performing final extension in STR-PCR in the same chamber as the PCR chamber, if the interval between m and n is narrow (for example, 1 cycle or 2 cycles), the final extension time may be excessive depending on the amount of DNA input to the PCR, and many A++ peaks may be obtained. In particular, in the case of an analysis system that does not have a heating unit 318 and a split PCR device that performs final extension in the same chamber as shown in Figure 14, if the interval between n and m is narrow, many A++ peaks will appear, so a value of 2 or more is preferable.

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

The operating procedure shown in Figure 32 is suitable when there is one CE section for one sample pretreatment section. This type of configuration makes the device small and easy to carry.

The data may be provided to the user when the first CE analysis is completed in step 405. The user may decide to start or continue the analysis of the second electrophoretic sample. The user may also decide to start or continue the second analysis using the determination method described above. This is because performing two measurements each time would double the CE measurement time, which would not increase throughput.

When the pretreatment of sample A is completed, the pretreatment of sample B (steps 401 to 404) may be started. In this case, the cartridge containing sample A is removed, so the electrophoretic sample for which CE analysis has not yet begun may be held in the waiting section 321, and the CE sample may be in the middle of electrophoresis.

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

Two CE units may be provided for one sample analysis unit. In this case, it is preferable that the number of CE units is twice as many as the number of sample processing units. Also, the same number or more CE units as multiple sample analysis units may be provided.

As shown in Figure 33(1), electrophoretic sample m and electrophoretic sample n may be subjected to CE measurement in step 405 as soon as they are prepared, or as shown in Figure 33(2), analysis may start simultaneously in step 405 after both are prepared. Also, when there are only as many CE sections as sample processing sections and no free space, electrophoretic sample m or electrophoretic sample n may be measured in that order.

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

A forensic sample containing an unknown amount of DNA is placed into the dissolution chamber 301 (sample inlet) of the flow path device 104. The processes of steps 401 to 403 are automatically performed within the flow path device 104.

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

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

A forensic sample containing an unknown amount of DNA is placed into the dissolution chamber 301 (sample inlet) of the flow path device 104. The processes of steps 401 to 403 are automatically performed within the flow path device 104.

After performing m cycles of PCR in step 502, amplified product m is extracted from PCR chamber 304 in step 503. The sample is sent to one side of CE section 105. In parallel with step 503, n cycles of PCR are performed on the PCR reaction solution remaining in the PCR chamber in step 505. In step 506, the product of n cycles is sent to CE section 105, and with electrophoretic samples m and n stored in the two capillaries, steps 504 and 507 are started simultaneously.

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

Claims (17)

A flow path device having a PCR chamber for performing thermal cycling;
a capillary electrophoresis unit for electrophoretic analysis of the PCR reaction solution;
In a DNA analysis system having
The DNA analysis system comprises:
storing the preset values of m and n;
In the PCR chamber, a PCR reaction solution is subjected to a thermal cycle m times to generate a first reaction solution;
removing a portion of the first reaction solution from the PCR chamber without changing its composition;
subjecting the portion of the first reaction solution to electrophoretic analysis in the capillary electrophoresis portion;
In the PCR chamber, the first reaction solution remaining in the PCR chamber is subjected to n-m thermal cycles (where n-m is an integer of 2 or more) so that the total number of thermal cycles is n, thereby generating a second reaction solution;
removing at least a portion of the second reaction solution from the PCR chamber without changing its composition;
subjecting at least a portion of the second reaction solution to electrophoretic analysis in the capillary electrophoresis portion;
DNA analysis system. The DNA analysis system of claim 1, wherein m is 20 or more and 32 or less. The DNA analysis system of claim 1, wherein n is 24 or more and 36 or less. The DNA analysis system according to claim 1, wherein n is 4 to 9 more than m. The DNA analysis system according to claim 1, wherein the DNA analysis system does not perform analysis by a method other than electrophoretic analysis on the portion of the first reaction solution and at least the portion of the second reaction solution prior to the electrophoretic analysis. The DNA analysis system according to claim 1, wherein at least one of the portion of the first reaction solution and the at least a portion of the second reaction solution is mixed with a solution containing multiple types of DNA fragments. 　A DNA analysis system according to claim 1, which mixes at least one of the part of the first reaction solution and at least the part of the second reaction solution with pure water, formamide, or a solution having a conductivity of 10 mS/cm or less prior to electrophoretic analysis to generate a mixed solution. The DNA analysis system according to claim 7, wherein the mixed solution is heated to 90°C or higher. In claim 1, the flow path device is a DNA analysis system that performs all steps from preparation of the PCR reaction solution to the n-m thermal cycles in a fully automated manner. In claim 1,
The flow path device has a metering portion,
After the m number of thermal cycles are completed, the DNA analysis system conveys the first reaction solution to the measuring unit, thereby measuring a predetermined amount of the first reaction solution within a range of 0.1% to 50%.
DNA analysis system. A DNA analysis system according to claim 1, wherein the flow path device has a valve that can be opened and closed, and the valve is closed before the start of the m number of thermal cycles and opened after the m number of thermal cycles are completed, thereby dividing and extracting the portion of the first reaction solution. The DNA analysis system according to claim 1, wherein the portion of the first reaction solution is maintained at a constant temperature within a range of 50°C to 80°C for 1 to 20 minutes. The DNA analysis system according to claim 1 holds the first reaction solution at a constant temperature within the range of 50°C to 80°C for 1 to 20 minutes, and then removes the portion. In claim 1, the DNA analysis system transports the entire PCR reaction solution in the PCR chamber out of the PCR chamber when n thermal cycles are completed. In claim 1,
The PCR reaction solution contains two or more primer sets,
The PCR reaction solution contains DNA containing two or more amplified gene regions.
DNA analysis system. The DNA analysis system according to claim 1,
determining which of the results of the electrophoretic analysis of the portion of the first reaction solution and the results of the electrophoretic analysis of the at least a portion of the second reaction solution is better;
Outputting the better result, or outputting information that allows one to determine which result is better;
DNA analysis system. A flow path device having a PCR chamber for performing thermal cycling;
a capillary electrophoresis unit for electrophoretic analysis of the PCR reaction solution;
In a DNA analysis system having
The DNA analysis system comprises:
storing the preset values of m and n;
In the PCR chamber, a PCR reaction solution is subjected to a thermal cycle m times to generate a first reaction solution;
removing a portion of the first reaction solution from the PCR chamber without changing its composition;
In the PCR chamber, the first reaction solution remaining in the PCR chamber is subjected to n-m thermal cycles (where n-m is an integer of 2 or more) so that the total number of thermal cycles is n, thereby generating a second reaction solution;
removing at least a portion of the second reaction solution from the PCR chamber without changing its composition;
performing electrophoretic analysis on one of the portion of the first reaction solution and the at least a portion of the second reaction solution in the capillary electrophoresis portion;
controlling the execution of electrophoretic analysis of the other of the part of the first reaction solution or the at least a part of the second reaction solution based on a result of the electrophoretic analysis of the other.
DNA analysis system.

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