DNA analysis device and DNA analysis method

Abstract

Provided are a DNA analysis device and a DNA analysis method which have reduced cost. A DNA analysis device (100) according to the present invention amplifies DNA in a sample to be measured, and analyzes the DNA by electrophoresis, said device comprising a first light source which emits light to a sample that has been introduced into a DNA amplification chamber, a second light source which emits light to the sample that has been sent from the DNA amplification chamber to an electrophoresis flow path, and a detector (111) to which the DNA amplification chamber and the electrophoresis flow path are connected in common, wherein: the detector (111) detects a first fluorescence intensity from the DNA amplification chamber; the same detector (111) detects a second fluorescence intensity from the electrophoresis flow path; and a control device (121) estimates, on the basis of the first fluorescence intensity, the amplification product amount resulting from the amplification, and analyzes the DNA on the basis of the second fluorescence intensity.

Description

DNA analysis apparatus and DNA analysis method

[0001] The present invention relates to a DNA analysis device and a DNA analysis method.

[0002] In electrophoresis analysis of deoxyribonucleic acid (DNA), separation is achieved based on the difference in the mobility of DNA molecules within the separation medium. In this analysis, a sample containing DNA molecules is injected into one end of a channel, such as a capillary filled with the separation medium, and a voltage is applied to both ends to move the DNA molecules in the sample by electrical force. For detection of the separated DNA, for example, fluorescence detection is used. Fluorescence detection involves binding a fluorescent dye to the DNA molecules and detecting them through a detection window provided in the middle of the channel or at the end of the channel opposite to the side where the sample was injected.

[0003] When analyzing DNA using electrophoresis, gene amplification is generally performed by polymerase chain reaction (PCR) or similar methods before injecting the sample into the separation medium. For example, by performing PCR using primers labeled with a fluorescent dye, the DNA of the target region labeled with the fluorescent dye is amplified.

[0004] In DNA amplification, the amplification must be performed in such a way that the amount of amplified product reaches an appropriate value. For example, if amplification by PCR is insufficient, the concentration of the fluorescently labeled target DNA will fall below the detection sensitivity. Conversely, if amplification by PCR is excessive, adverse effects such as an increase in nonspecific products or saturation of the detector due to exceeding the detection limit of the fluorescence detector will occur.

[0005] To ensure proper DNA amplification, amplification is sometimes performed while monitoring the amount of amplification product. For example, by using reagents such as an intercalator to create a situation where fluorescence of an intensity corresponding to the amount of amplification product is generated, the amount of amplification product can be monitored in real time by measuring the amount of fluorescence (real-time PCR). By stopping the amplification when the fluorescence level reaches the target range, the amount of amplification product can be set to the desired value.

[0006] Generally, DNA amplification is performed in disposable containers. However, even if the concentration of the fluorescent dye in the sample is the same, the amount of fluorescence detected may vary due to factors such as solution mixing, bubble generation during heating for PCR amplification, container contamination, and individual differences in container shape and light transmittance of the material. Since the amount of amplified product is estimated from the amount of fluorescence, variations in fluorescence that are not caused by changes in the amount of amplified product will result in an inaccurate estimate of the amount of amplified product.

[0007] To correct for fluorescence variability unrelated to the amount of amplified product, a reference fluorescent dye may be added to the sample solution. The reference fluorescent dye is not involved in amplification and its concentration remains constant. By taking the ratio of the fluorescence amount due to the reference fluorescent dye to the fluorescence amount due to the reagent used to measure the amount of amplified product, variability in fluorescence amount unrelated to changes in the amount of amplified product can be corrected.

[0008] A concrete example of DNA analysis using electrophoresis is personal identification using short tandem repeat (STR) polymorphisms. STRs are short repeats of 2 to 6 base pairs found in genes. STRs exist in multiple regions of human genes, and the pattern of how many repetitions each region has differs from person to person. By amplifying the STR region to be analyzed and analyzing the number of repetitions using electrophoresis, pattern information of an individual's STRs can be obtained. This pattern information is then used to identify the individual. Because it is necessary to accurately measure the number of repeats in the base sequence, capillary electrophoresis, which can separate DNA by size down to the single base unit, is often used for electrophoresis.

[0009] Personal identification using STR polymorphisms is used in criminal investigations, for example. However, it is often unclear how much of the DNA to be analyzed is present in a sample recovered from traces left at a crime scene. In such cases, monitoring the amount of amplified product while amplifying the DNA is particularly useful to ensure proper amplification before electrophoresis.

[0010] In personal identification using STR polymorphisms, automated devices (Rapid DNA devices) have been developed that perform everything from sample processing to electrophoresis and result analysis. In such cases, the entire sequence of operations described above is integrated into a single device.

[0011] For example, Patent Document 1 discloses a bioanalysis system including a sample preparation device that receives a crude sample to be processed into a sample of a suitable form for later analysis, and a sample analysis device that receives the sample from the sample preparation device to be subjected to analysis. Patent Document 1 discloses that the bioanalysis system is operable within an integrated automated system in which the sample preparation device and the sample analysis device are coupled. Patent Document 1 states that in this bioanalysis system, the sample output from the sample analysis device does not require further user intervention. Patent Document 1 also discloses that the sample preparation device is constructed and configured so that DNA amplification occurs by PCR in a biomolecular reaction. Furthermore, Patent Document 1 discloses that the analytical sample device includes a capillary electrophoresis system.

[0012] Furthermore, for example, Patent Document 2 discloses a pre-treatment integrated capillary electrophoresis apparatus. The pre-treatment integrated capillary electrophoresis apparatus in Patent Document 2 consists of a pre-treatment section and an analysis section. The DNA processed in the pre-treatment section is moved to the analysis section and analyzed. The pre-treatment section measures the concentration of DNA by the amount of laser light absorbed or by changes in fluorescence, and the analysis section separates and detects the DNA using a capillary.

[0013] Japanese Patent Publication No. 2007-510407, International Publication No. 2015 / 111443

[0014] In conventional techniques, as described in Patent Document 2, when the amount of amplified DNA is measured using fluorescence during DNA amplification in a sample before analysis, and then the DNA is detected by fluorescence during subsequent electrophoresis, two fluorescence detection devices are required. However, incorporating two fluorescence detection devices increases the size and cost of the device.

[0015] Furthermore, in conventional techniques, when correcting the amplification product amount using the fluorescence of a reference fluorescent dye, two types of fluorescent dyes are required: one for measuring the amplification product amount and another for the reference. To distinguish between the fluorescence from each fluorescent dye, the two dyes must have different fluorescence wavelengths. In addition, the detection device used to measure the amplification product amount must be capable of distinguishing between fluorescence wavelengths.

[0016] Furthermore, conventional techniques sometimes involve the use of multiple fluorescent dyes in DNA analysis by electrophoresis. In this case, the detector used for DNA detection in electrophoresis must also be capable of distinguishing fluorescence wavelengths. Generally, detectors capable of distinguishing wavelengths (detectors capable of acquiring the light spectrum) are more expensive than detectors that only detect the power of light. Equipping the system with two detectors capable of distinguishing wavelengths for measuring the amount of amplified product and detecting electrophoresis leads to increased costs.

[0017] In the aforementioned DNA analysis, three types of fluorescent dyes are used: a fluorescent dye for measuring the amount of amplified product, a reference fluorescent dye, and a fluorescent dye used for detection by electrophoresis. Furthermore, each of these can be a set of multiple fluorescent dyes. Increasing the number of types of fluorescent dyes used increases the cost of reagents required for the analysis, and the detector also needs to be able to distinguish and detect fluorescence across a wide range of wavelengths, which can increase the cost of the detector as well. In addition, exciting multiple fluorescent dyes requires light sources with different emission wavelengths that can excite each fluorescent dye, which also increases the cost of the light sources. In short, conventional techniques have the problem of increasing costs for various reasons.

[0018] This invention has been made in view of the above circumstances. The aim of this invention is to provide a DNA analysis device and a DNA analysis method with reduced costs.

[0019] The DNA analyzer according to the present invention that solves the above problems is a DNA analyzer that amplifies DNA in a sample to be measured and analyzes it by electrophoresis. It includes a first light source that irradiates light on the sample introduced into the chamber for amplifying DNA, a second light source that irradiates light on the sample sent from the chamber for amplifying DNA to the flow path for electrophoresis, and a detector commonly connected to the chamber for amplifying DNA and the flow path for electrophoresis. The detector detects the first fluorescence intensity from the chamber for amplifying DNA, and the same detector detects the second fluorescence intensity from the flow path for electrophoresis. A control device estimates the amount of amplification products by the amplification based on the first fluorescence intensity and analyzes DNA based on the second fluorescence intensity.

[0020] According to the present invention, a DNA analyzer and a DNA analysis method with reduced costs can be provided. Problems, configurations, and effects other than those described above will be clarified by the description of the following embodiments. Further features related to the present invention will become apparent from the description of this specification and the attached drawings.

[0021] This is a diagram showing the configuration of the DNA analyzer 100 according to the first embodiment. This is a top view of the spectrometer. This is a side view of the spectrometer. This is an explanatory diagram showing an example of a spectral image observed by the image sensor 204 in Figure 2. This is an explanatory diagram showing the state of DNA amplification in the DNA amplification unit 104 of the sample preprocessing mechanism 101. This is an explanatory diagram showing the fluorescence spectrum obtained by fluorescence detection in the sample preprocessing mechanism 101. This is an explanatory diagram showing the state of detection in the detection window 118 of the electrophoresis mechanism 102. This is an explanatory diagram showing the fluorescence spectrum obtained by fluorescence detection in the detection window 118 of the electrophoresis mechanism 102. This is an explanatory diagram showing a typical example of the change in intensity from fluorescent dye B with respect to the number of PCR cycles. This is an explanatory diagram showing the state of DNA amplification in the DNA amplification unit 104 of the sample preprocessing mechanism 101. This is an explanatory diagram showing the fluorescence spectrum obtained by fluorescence detection in the sample preprocessing mechanism 101. This is an explanatory diagram showing the state of detection in the detection window 118 of the electrophoresis mechanism 102. This is an explanatory diagram showing the fluorescence spectrum obtained by fluorescence detection in the detection window 118 of the electrophoresis mechanism 102. This is an explanatory diagram showing the DNA amplification process in the DNA amplification section 104 of the sample pretreatment mechanism 101. This is an explanatory diagram showing the fluorescence spectrum obtained by fluorescence detection in the sample pretreatment mechanism 101. This is an explanatory diagram showing the detection process in the detection window 118 of the electrophoresis mechanism 102. This is an explanatory diagram showing the fluorescence spectrum obtained by fluorescence detection in the detection window 118 of the electrophoresis mechanism 102. This is a flowchart showing an example of the operation steps of the DNA analyzer 100 according to the first embodiment. This is a structural diagram of the detector 111 used in the DNA analyzer 100 according to the second embodiment. This is a structural diagram showing a modified example of the detector 111 used in the DNA analyzer 100 according to the second embodiment. This is a top view of the spectrometer. This is a side view of the spectrometer. This is a side view showing a modified example of the spectrometer. This is a configuration diagram of the DNA analyzer 100 according to the fourth embodiment. This is a configuration diagram of the DNA analyzer 100 according to the fifth embodiment. This is a flowchart explaining the contents of the DNA analysis method according to the sixth embodiment.

[0022] Hereinafter, a DNA analyzer and a DNA analysis method according to an embodiment of the present invention will be described while appropriately referring to the drawings. In the following description and drawings, the same reference numerals may be given to common configurations, and duplicate descriptions may be omitted. Further, the present invention is not limited to the following embodiments. Furthermore, the description in this specification is merely a typical example and does not limit the claims or application examples in any sense.

[0023] In the following embodiments, when necessary for convenience, they will be described separately in a plurality of sections or embodiments. Unless otherwise specified, they are not unrelated to each other, and one is related to a modification, detail, supplementary explanation, etc. of part or all of the other. Also, in the following embodiments, when referring to the number of elements, etc. (including the number, numerical value, quantity, range, etc.), unless otherwise specified or clearly limited to a specific number in principle, it is not limited to that specific number, and it may be more than or less than the specific number.

[0024] Furthermore, in the following embodiments, it goes without saying that the constituent elements (including element steps, etc.) are not necessarily essential unless otherwise specified or clearly considered essential in principle. Similarly, in the following embodiments, when referring to the shape, positional relationship, etc. of the constituent elements, unless otherwise specified or clearly considered not to be so in principle, it includes those that are substantially approximate or similar to the shape, etc. This also applies to the above numerical values and ranges.

[0025] The configuration of the present invention is an apparatus that amplifies DNA in a sample and analyzes the amplified DNA by electrophoresis, and can be applied when both the measurement of the amount of the amplification product of DNA and the detection of DNA in electrophoresis are performed by fluorescence detection. Although the content of the analysis can be various, in the following, an apparatus for performing personal identification using STR polymorphism will be described with the assumption that it automatically performs from the pretreatment of the sample to capillary electrophoresis.

[0026] <First Embodiment> Figure 1 is a diagram of the DNA analyzer 100 according to the first embodiment. The DNA analyzer 100 amplifies DNA in a sample to be measured and analyzes it by electrophoresis. As shown in Figure 1, the DNA analyzer 100 has a first light source (excitation light source 108), a second light source (excitation light source 119), and a detector 111. The first light source irradiates light onto the sample introduced into the DNA amplification chamber (specifically, the DNA amplification unit 104). The DNA amplification unit 104 is provided in the pre-treatment cartridge 101A together with a sample dissolution chamber (sample dissolution unit 103) and a chamber for preparing for electrophoresis (electrophoresis preparation unit 105). The second light source irradiates light onto the sample sent from the pre-treatment cartridge 101A, which performs DNA amplification, to the electrophoresis channel (specifically, the capillary 114). In this embodiment, the detector 111 is connected in common to both the pre-treatment cartridge 101A, which performs DNA amplification, and the electrophoresis channel. The DNA analyzer 100 uses a detector 111 to detect a first fluorescence intensity from the pre-treatment cartridge 101A for DNA amplification, and the same detector 111 also detects a second fluorescence intensity from the electrophoresis channel. The control device 121 of the DNA analyzer 100 estimates the amount of amplification product based on the first fluorescence intensity and analyzes the DNA based on the second fluorescence intensity. The DNA analyzer 100 will be described in detail below.

[0027] The DNA analyzer 100 includes a sample pretreatment mechanism 101 and an electrophoresis mechanism 102. The sample pretreatment mechanism 101 extracts and amplifies DNA from a sample such as cells, making it ready for analysis by electrophoresis. The electrophoresis mechanism 102 separates the processed sample by size using electrophoresis and detects it by fluorescence measurement.

[0028] The sample pretreatment mechanism 101 has a pretreatment cartridge 101A for amplifying DNA. For example, the pretreatment cartridge 101A has a sample lysis unit 103, a DNA amplification unit 104, and an electrophoresis preparation unit 105, as described above. The sample pretreatment mechanism 101 also has mechanical mechanisms (not shown) that drive mechanisms such as a pump for transporting liquid within the pretreatment cartridge 101A and valves within the pretreatment cartridge 101A.

[0029] As an example, the pre-treatment cartridge 101A is a resin chip 101B, and the sample lysis section 103, DNA amplification section 104, and electrophoresis preparation section 105 are spaces provided on the resin chip. The sample lysis section 103, DNA amplification section 104, and electrophoresis preparation section 105 are formed by creating recessed portions on a plate-shaped resin chip 101B by cutting or injection molding, and then attaching another resin plate 101C on top of them. The resin plate 101C may be a thin film. The resin plate 101C has a sample inlet 106 and a sample outlet 101D. The sample is introduced through the sample inlet 106 and processed sequentially in the sample lysis section 103, DNA amplification section 104, and electrophoresis preparation section 105. The processed sample moves sequentially to the next processing section through a channel 107 provided on the chip. The sample that has undergone all processing moves to the electrophoresis mechanism 102 through the sample outlet 101D.

[0030] As another example, the sample lysis unit 103, the DNA amplification unit 104, and the electrophoresis preparation unit 105 may be in separate containers, and the sample solution may be moved between each container by an automated pipette or the like.

[0031] In the sample lysis section 103, cells in the sample are lysed and DNA is extracted from the cells. A generally known method can be used for DNA extraction. For example, sodium dodecyl sulfate is used to degrade the cell membrane and other structures, exposing the DNA molecules.

[0032] In the DNA amplification unit 104, the DNA in the sample is amplified to a concentration detectable by electrophoresis using methods such as PCR. At the same time, a fluorescent dye for detection is attached to the DNA molecules. For example, the fluorescent dye is bound to the primers used in PCR. The DNA strands created by extending these primers become labeled with the fluorescent dye.

[0033] As mentioned earlier, the DNA amplification unit 104 needs to amplify the DNA to an appropriate amount of amplification product. For this reason, the DNA analyzer 100 (sample pretreatment mechanism 101) detects the amount of amplification product and stops the amplification when the appropriate amount of amplification product is reached. As an example, a reagent for measuring the amount of amplification product is added to the solution used in the PCR reaction. The measuring reagents include intercalators, TaqMan® probes, molecular beacons, etc. With any of these measuring reagents, the amount of fluorescence signal increases in proportion to the amount of amplification product. The increase in the amount of fluorescence signal during amplification is monitored, and the amplification is stopped when the target amount of fluorescence signal is reached.

[0034] The DNA analyzer 100 is equipped with a mechanism for detecting fluorescence in the DNA amplification unit 104. For example, the DNA analyzer 100 has an excitation light source 108 (first light source), a dichroic mirror 109, an optical fiber 110, and a detector 111. Excitation light 112 from the excitation light source 108 passes through the dichroic mirror 109 and irradiates the solution in the DNA amplification unit 104. The fluorescent dye irradiated with excitation light 112 is excited and generates fluorescence 113. The generated fluorescence 113 is reflected by the dichroic mirror 109 and introduced into the optical fiber 110. The fluorescence 113 is guided by the optical fiber 110 to the detector 111 and detected.

[0035] In the electrophoresis preparation unit 105, the amplified sample is prepared for electrophoresis in the electrophoresis mechanism 102. Specifically, the salt concentration is reduced by mixing it with water or acrylamide, and then it is mixed with a size standard for measuring the DNA strand length in the sample by electrophoresis. The size standard is a DNA strand of known length and is labeled with a fluorescent dye for detection. The size standard is detected simultaneously with the sample in the electrophoresis mechanism 102, and the DNA strand length in the sample is estimated by comparing the time of the fluorescence signal generation caused by the size standard with the time of the signal generation caused by the sample.

[0036] The electrophoresis mechanism 102, as an example, includes a capillary 114, a cathode block 115, and an anode block 116. The cathode block 115 and the anode block 116 each have internal channels 115a and 116a, respectively. Electrodes 117 are installed in channels 115a and 116a, respectively, and the electrodes 117 are connected to a high-voltage power supply (not shown). Both ends of the capillary 114 are connected to the channel 115a of the cathode block 115 and the channel 116a of the anode block 116, respectively.

[0037] During electrophoresis, a polymer for DNA separation is filled into the capillary 114 through the channel 116a of the anode block 116 by a polymer injection mechanism (not shown). Next, the sample processed by the sample pretreatment mechanism 101 is injected into the channel 115a of the cathode block 115. After injection, a voltage is applied to both ends of the capillary 114 for a short time to inject the DNA in the sample, i.e., the PCR amplification product, into the capillary end. After that, the sample is discharged from the channel 115a of the cathode block 115, the capillary end is washed, and then channels 115a and 116a are filled with buffer. A voltage is applied between the electrodes 117 installed in the cathode block 115 and anode block 116 to perform electrophoresis of the DNA molecules.

[0038] Electrophoresed DNA molecules reach the detection window 118 on the capillary 114 at different times depending on their chain length. The coating of the capillary 114 is removed at the detection window 118. Shorter DNA reaches the detection window 118 earlier, while longer DNA reaches it later. By measuring the fluorescence signal from the DNA reaching the detection window 118 over time, a signal waveform reflecting the chain length of the DNA in the sample can be obtained.

[0039] The DNA analyzer 100 has a control device 121. The control device 121 controls the entire analysis process, including sample delivery and processing, electrophoresis, fluorescence detection, estimation of amplification product amount, and DNA analysis. The control device 121 may be a general-purpose personal computer equipped with a central processing unit (CPU), random access memory (RAM), read-only memory (ROM), storage media such as a solid-state drive (SSD), and input / output ports.

[0040] In the detection window 118, fluorescence excitation light from the excitation light source 119 (second light source) is irradiated onto the capillary 114. The fluorescent dye irradiated with fluorescence excitation light emits fluorescence. The generated fluorescence is taken up by the optical fiber 120 and guided to the detector 111 for detection. In the DNA analyzer 100 according to this embodiment, fluorescence detection in the sample pretreatment mechanism 101 and fluorescence detection in the electrophoresis mechanism 102 are performed by the same detector 111. As a result, the device can be made smaller and costs can be reduced compared to providing separate detectors for each type of fluorescence.

[0041] Since the sample is processed in the sample pretreatment mechanism 101 and then analyzed in the electrophoresis mechanism 102, the fluorescence detected by the amplification product measurement and the fluorescence detected by DNA detection in electrophoresis are generally not detected simultaneously by the detector 111. However, it is possible that the sample pretreatment mechanism 101 processes the next sample while the sample processed in the sample pretreatment mechanism 101 is being analyzed in the electrophoresis mechanism 102. In this case, the fluorescence detected by the amplification product measurement and the fluorescence detected by DNA detection in electrophoresis may be detected simultaneously by the detector 111.

[0042] The fluorescence detected in the amplification product measurement and the fluorescence detected in DNA detection in electrophoresis may be detected simultaneously by detector 111. However, considering the dynamic range of detector 111, the appropriate detection conditions for detector 111 may differ for detecting both. For example, the photodetector element of detector 111 is a camera element, and with an exposure time optimized for DNA detection in electrophoresis, the fluorescence signal in the amplification product measurement may exceed the dynamic range, potentially causing the element to saturate.

[0043] When simultaneously detecting fluorescence detected by amplification product measurement and fluorescence detected by DNA detection in electrophoresis, the above problem can be resolved by switching the emission timing of excitation light sources 108 and 119. As an example, excitation light sources 108 and 119 are turned on alternately to detect fluorescence. The detection conditions of the detector 111, for example, the exposure time of the camera element in the above example, are also changed alternately.

[0044] Figure 1 illustrates an electrophoresis mechanism 102 having one sample pretreatment mechanism 101 and one capillary tube 114, each processing and analyzing one sample simultaneously. However, the number of samples processed simultaneously by the sample pretreatment mechanism 101 and the electrophoresis mechanism 102 may be multiple. For example, the sample pretreatment mechanism 101 may have multiple sets of sample processing structures to process multiple samples simultaneously. Alternatively, the DNA analyzer 100 may have multiple sample pretreatment mechanisms 101.

[0045] Furthermore, the DNA analyzer 100 may have multiple electrophoresis mechanisms 102. Alternatively, the electrophoresis mechanism 102 may have multiple capillaries 114, allowing for the simultaneous analysis of multiple samples. When the electrophoresis mechanism 102 has multiple capillaries 114, the cathode block 115 has multiple independent channel structures, each injecting a different sample into a separate capillary 114. In addition, all capillaries 114 may be connected to a common channel in the anode block 116.

[0046] In the fluorescence detection in the sample pretreatment mechanism 101 and the fluorescence detection in the electrophoresis mechanism 102, fluorescence from two or more types of fluorescent dyes may be detected. Multiple types of measurement reagents may be added in the DNA amplification unit 104, and DNA amplification may be measured at two or more sites. In addition, DNA fragments with different fluorescent dyes attached may be detected in the detection window 118.

[0047] The detection of multiple fluorescent dyes will be explained using STR polymorphism for personal identification as an example. In STR polymorphism for personal identification, individuals are identified based on the number of repetitions of repetitive sequences in the genome, but using only one repetitive sequence does not provide sufficient personal identification ability. By determining the number of repetitions of multiple repetitive sequences and identifying individuals using that set, the probability of different people being identified as the same person is reduced. In some cases, the number of repetitions of 20 or more repetitive sequences is measured to enhance personal identification ability.

[0048] When measuring the number of repetitions of a repeat sequence at multiple locations, DNA amplification in the DNA amplification unit 104 is performed on the repeat sequences at multiple locations. In this case, the amplification does not necessarily proceed at the same rate at all locations. To monitor the amplification state at each site, multiple types of reagents for measuring amplification at each site may be added. To distinguish the signals from each reagent, fluorescent dyes emitting fluorescence at different wavelengths are used. As an example, multiple TaqMan probes designed to hybridize downstream of the repeat sequence are used. Different fluorescent dyes are used for each TaqMan probe.

[0049] Furthermore, when measuring the number of repetitions using the electrophoresis mechanism 102, amplification products from different locations may be labeled with different fluorescent dyes. Since it is desirable to determine the number of repetitions for as many repeating sites as possible in a single capillary electrophoresis, the amplification products for each repeating site are generally identified by the chain length of the amplification product and the fluorescent dye. That is, when measuring the number of repetitions for the first, second, third, and fourth repeating sites, as an example, the amplification product for the first repeating site is labeled with the first fluorescent dye, and the amplification reaction is designed to produce a product with a length of 100 bp to 200 bp. Similarly, the amplification product for the second repeating site is labeled with the first fluorescent dye, and the amplification reaction is designed to produce a product with a length of 200 bp to 300 bp. The amplification product for the third repeating site is labeled with the second fluorescent dye, and the amplification reaction is designed to produce a product with a length of 100 bp to 200 bp. The amplification product of the fourth repeat region is labeled with a second fluorescent dye, and the amplification reaction is designed to produce a product of length 200 bp to 300 bp. This can be achieved by selecting the hybridization position of the primers for amplifying each repeat region and the fluorescent dye to be labeled on the primers. This method makes it possible to determine the number of repeats for more than 20 repeat regions in a single electrophoresis.

[0050] When using multiple types of fluorescent dyes, the detector 111 needs to have the function of separating and detecting light by wavelength. As an example, the detector 111 is a spectrometer composed of lens 201, lens 202, grating 203, and image sensor 204 (Figures 2A and 2B). Figure 2A is a top view of the spectrometer. Figure 2B is a side view of the spectrometer. As shown in Figures 2A and 2B, the light introduced by the optical fiber 205 is collimated by lens 201 and spectrally separated by grating 203. Then, it is focused by lens 202 and the spectrum is projected onto image sensor 204.

[0051] The detector 111 has multiple detection channels and detects multiple different spectra. As an example, multiple optical fibers 205 are arranged in a vertical line at the light incidence position of the spectrometer. Figure 3 is an explanatory diagram showing an example of a spectral image observed by the image sensor 204 in Figure 2. Figure 3 shows the case where the detector 111 has four input fibers (optical fibers 1 to 4) and they are arranged in the vertical direction of the paper. In this example, the wavelength dispersion direction is parallel to the paper, and the spectrum of each input fiber is projected horizontally on the image sensor 204.

[0052] For example, two of the four channels are used for detecting fluorescence in the DNA amplification unit 104, and two are used for detecting fluorescence in the electrophoresis mechanism 102. The detector 111 needs to cover the wavelength range of fluorescence detected by both. For example, the 700 nm to 800 nm wavelength range is used for detecting the amount of DNA amplification product in the DNA amplification unit 104 (optical fibers 1 and 2), and the 500 nm to 700 nm wavelength range is used for detecting DNA in the electrophoresis mechanism 102 (optical fibers 3 and 4).

[0053] In the DNA analyzer 100, the same sample undergoes two fluorescence detections for different purposes in the sample pretreatment mechanism 101 and the electrophoresis mechanism 102. Since each fluorescence detection has a different purpose, different types of fluorescent dyes need to be used for each. On the other hand, by detecting some fluorescent dyes in both, the number of types of fluorescent dyes required can be reduced. Specifically, the fluorescent dye used as a reference in measuring the amount of amplification product in the DNA amplification unit 104 and the fluorescent dye used for detection in the electrophoresis mechanism 102 can be the same.

[0054] Figure 4 shows a case where the above method is not used, and different fluorescent dyes are used for detection. Figure 4A is an explanatory diagram showing the DNA amplification process in the DNA amplification section 104 of the sample pretreatment mechanism 101. Figure 4B is an explanatory diagram showing the fluorescence spectrum obtained by fluorescence detection in the sample pretreatment mechanism 101. Figure 4C is an explanatory diagram showing the detection process in the detection window 118 of the electrophoresis mechanism 102. Figure 4D is an explanatory diagram showing the fluorescence spectrum obtained by fluorescence detection in the detection window 118 of the electrophoresis mechanism 102. In the example shown in Figure 4, template DNA 401 is amplified with primer 402, and the amount of amplification product is detected with TaqMan probe 403. During capillary electrophoresis, the amplification product 404 produced by the extension of primer 402 is detected.

[0055] Primer 402 is coated with fluorescent dye A for detection during electrophoresis. TaqMan probe 403 contains fluorescent dye B and quencher C. TaqMan probe 403 is designed to hybridize downstream of primer 402. TaqMan probe 403 is degraded by DNA polymerase 405 as primer 402 extends. Before degradation, the fluorescence of fluorescent dye B is suppressed by quencher C. After degradation, fluorescent dye B and quencher C are physically separated, and fluorescent dye B begins to emit fluorescence. The intensity of fluorescence due to fluorescent dye B correlates with the degradation of TaqMan probe 403, i.e., the extension of primer 402, and the amount of amplification product can be measured by measuring the fluorescence intensity of fluorescent dye B. As shown in Figures 4B and 4D, the fluorescence wavelength of fluorescent dye B is obtained by irradiation with excitation wavelength Ex. B. The fluorescence wavelength of reference fluorescent dye D is obtained by irradiation with excitation wavelength Ex. D. By irradiating with excitation wavelength Ex. A, the fluorescence wavelength of fluorescent dye A can be obtained.

[0056] In measuring the amount of amplified product from the fluorescence intensity of fluorescent dye B, a reference fluorescent dye D may be used to improve measurement accuracy. Reference fluorescent dye D is a fluorescent dye that is not related to DNA amplification and always emits fluorescence of a constant intensity. For example, if air bubbles or debris get inside the DNA amplification unit 104, that part will not emit fluorescence, and the fluorescence intensity of fluorescent dye B will be lower than its original value. In addition, the fluorescence intensity of fluorescent dye B may also be lower than its original value if dirt accumulates on the part that detects fluorescence from the DNA amplification unit 104, or if the position of the resin chip forming the DNA amplification unit 104 shifts.

[0057] Therefore, the decrease in fluorescence signal is corrected by acquiring the signal from both the fluorescent dye B and the reference fluorescent dye D. The decrease in fluorescence intensity due to the above-mentioned causes occurs in the same ratio for fluorescence from fluorescent dye B and fluorescence from the reference fluorescent dye D. Therefore, the effect can be corrected by taking the ratio of the fluorescence intensity from fluorescent dye B to the fluorescence intensity from the reference fluorescent dye D, which emits a constant intensity.

[0058] Figure 5 shows the correction of fluorescence intensity. Figure 5 is an explanatory diagram illustrating a typical example of the change in fluorescence intensity from fluorescent dye B with respect to the number of PCR cycles. When amplification is performed normally, the fluorescence intensity from fluorescent dye B changes as shown by the solid line 51 in Figure 5. A certain threshold for fluorescence intensity is set for this curve (PCR termination threshold), and when this threshold value is exceeded, sufficient amplification is considered to have been achieved, and the PCR amplification is stopped.

[0059] Here, let's assume that the intensity of fluorescent dye B decreases due to the factors mentioned above, and the curve changes from the solid line 51 in Figure 5 to the dotted line 52. In this example, because the fluorescence intensity has decreased, the number of PCR cycles required to exceed the PCR termination criterion increases, resulting in excessive amplification. For example, if there are too many PCR cycles in the amplification of the STR region for personal identification, nonspecific amplification may occur, and signals that should not appear may be observed in the electrophoretic waveform. Also, during PCR amplification, adenine can be added to the end of the amplified product, but with an excessive number of cycles, the amplified product may become a mixture of adenine-added and non-adenine-added products, making it impossible to accurately measure the number of STR repeats. If the decrease in fluorescence intensity is even greater, the fluorescence intensity may not reach the PCR termination criterion, and the termination cannot be determined.

[0060] By taking the ratio of the fluorescence intensity from fluorescent dye B to the fluorescence intensity from the reference fluorescent dye D, the curve represented by the dotted line 52 is corrected to a curve like the dashed line 53 shown in Figure 5. This eliminates the problem associated with the decrease in fluorescence intensity, and allows the number of PCR cycles to be set to the optimal number even if there is attenuation of the fluorescence signal due to air bubbles, etc.

[0061] As described above, when fluorescent dye A is used for detection during capillary electrophoresis, fluorescent dye B is used for measuring the amount of amplified product in DNA amplification, and fluorescent dye D is used as a reference for fluorescent dye B, the DNA amplification unit 104 requires a light source to excite fluorescent dyes B and D and a detector that can distinguish and detect the fluorescence of both. Furthermore, the electrophoresis mechanism 102 requires a light source to excite fluorescent dye A and a detector that detects the fluorescence of fluorescent dye A. However, as shown in Figures 4 and 5, even when fluorescent dyes A, B, and D are used, the DNA analyzer 100 according to the first embodiment detects them all with the same detector 111, so the device can be made smaller and costs can be reduced compared to providing separate detectors for each fluorescence.

[0062] Figure 6 shows a more preferred example of the method for measuring the amount of amplified product in this embodiment. Figure 6A is an explanatory diagram showing the DNA amplification process in the DNA amplification section 104 of the sample pretreatment mechanism 101. Figure 6B is an explanatory diagram showing the fluorescence spectrum obtained by fluorescence detection in the sample pretreatment mechanism 101. Figure 6C is an explanatory diagram showing the detection process in the detection window 118 of the electrophoresis mechanism 102. Figure 6D is an explanatory diagram showing the fluorescence spectrum obtained by fluorescence detection in the detection window 118 of the electrophoresis mechanism 102.

[0063] As shown in Figure 6, in a more preferred example of the amplification product amount measurement method in this embodiment, fluorescence detection in the DNA amplification unit 104 and fluorescence detection in the electrophoresis mechanism 102 both involve excitation of fluorescent dye A by irradiation with excitation wavelength Ex. A (Figures 6B and 6D) and detection of fluorescence from fluorescent dye A. As can be seen by comparing Figure 6A and Figure 4A, in this example, the reference fluorescent dye D is not used. In DNA amplification, the primer 402 is extended by DNA polymerase 405. At this time, DNA extension does not affect the fluorescence of fluorescent dye A. Taking advantage of this, as shown in Figure 6B, in fluorescence detection in the DNA amplification unit 104, the fluorescence of fluorescent dye A is used as a reference, and the amount of amplification product is detected by the ratio of the intensity with the fluorescence of fluorescent dye B. Also, as shown in Figure 6D, the fluorescence of fluorescent dye A is again used for detection in the electrophoresis mechanism 102.

[0064] In other words, the sample contains a fluorescent dye that emits fluorescence, and fluorescence detection is performed from the same fluorescent dye (fluorescent dye A in this example) in both the DNA amplification chamber (specifically, the DNA amplification unit 104) and the electrophoresis channel (electrophoresis mechanism 102, more specifically, the capillary 114).

[0065] More specifically, for example, in the detection of fluorescence in the DNA amplification chamber (specifically, the DNA amplification unit 104), fluorescence from fluorescent dye B, which detects the amount of amplification product, and fluorescence from fluorescent dye A, which serves as a reference for fluorescent dye B, are detected. Similarly, in the detection of fluorescence in the electrophoresis channel, fluorescence from fluorescent dye A, which detects the DNA separated by electrophoresis, is detected. In other words, the fluorescent dye A that serves as the reference and the fluorescent dye A that detects the DNA separated by electrophoresis are the same fluorescent dye.

[0066] As shown in these embodiments, there are several advantages to using fluorescent dye A as a reference fluorescent dye. First, by not using fluorescent dye D, the cost of fluorescent dye D can be reduced. Also, in the preferred method shown in Figure 6, the DNA amplification unit 104 requires a light source to excite fluorescent dyes A and B and a detector to distinguish and detect the fluorescence of both, while the electrophoresis mechanism 102 requires a light source to excite fluorescent dye A and a detector to detect the fluorescence of fluorescent dye A. In this embodiment, as mentioned above, the same detector 111 is used. Furthermore, in this preferred example, fluorescent dye A is excited in common in both the DNA amplification unit 104 and the electrophoresis mechanism 102. Therefore, it is possible to share the excitation light source for exciting fluorescent dye A (not shown in Figure 1, see, for example, Figure 12), and costs are further reduced.

[0067] In Figure 6, as shown in Figure 6B, fluorescent dye B is excited at a different excitation wavelength, Ex. B, than fluorescent dye A. However, fluorescent dye B may also be excited at the same wavelength as fluorescent dye A. In this case, all fluorescent dyes are excited by the same light source (not shown in Figure 1, see, for example, Figure 12). That is, the first light source and the second light source may be the same light source. In this case, fluorescent dyes A and B are selected so that their absorption spectra overlap, but their peak wavelengths are different. A method is also known for producing a set of fluorescent dyes with different peak wavelengths at the same excitation wavelength by using fluorescence resonance energy transfer and combining the same donor fluorescent dye with different acceptor fluorescent dyes.

[0068] Furthermore, in the method of this embodiment, as shown in Figure 1, the detector 111 detects both the fluorescence from the DNA amplification unit 104 and the fluorescence from the detection window 118. In the method of Figure 4, the detector 111 needs to be able to detect all wavelength bands of fluorescent dyes A, B, and D. On the other hand, in the method of Figure 6, it is sufficient to cover the wavelength bands of fluorescent dyes A and B. Since the detection wavelength band to be covered is smaller, a further reduction in the material cost of the detector 111 can be expected. For example, if the detector 111 has the structure shown in Figure 2, a smaller detection wavelength band to be covered means that the required size of the image sensor 204 becomes smaller, and a lower-cost element can be used.

[0069] Note that the fluorescent dye detected in both the DNA amplification unit 104 and the electrophoresis mechanism 102 does not have to be the fluorescent dye attached to the primer. Figure 7 shows an example in which fluorescence from the size standard 701 is used as a reference. Figure 7A is an explanatory diagram showing the DNA amplification process in the DNA amplification unit 104 of the sample pretreatment mechanism 101. Figure 7B is an explanatory diagram showing the fluorescence spectrum obtained by fluorescence detection in the sample pretreatment mechanism 101. Figure 7C is an explanatory diagram showing the detection process in the detection window 118 of the electrophoresis mechanism 102. Figure 7D is an explanatory diagram showing the fluorescence spectrum obtained by fluorescence detection in the detection window 118 of the electrophoresis mechanism 102. As mentioned above, the size standard 701 is a DNA molecule with a known chain length and is used to correlate the detection time of the fluorescence signal originating from each DNA fragment with the chain length of the DNA fragment during electrophoresis.

[0070] As shown in Figure 7A, a size standard 701 is added during DNA amplification. The size standard 701 has a sequence that is not amplified by the added primer. The size standard 701 has a fluorescent dye D used as a reference. Similar to the example described above, as shown in Figures 7B and 7D, in both fluorescence detection in the DNA amplification unit 104 and fluorescence detection in the electrophoresis mechanism 102, excitation of the fluorescent dye D by irradiation with excitation wavelength Ex. D and detection of fluorescence from the fluorescent dye D are performed. As shown in Figure 7B, in fluorescence detection in the DNA amplification unit 104, the fluorescence of the fluorescent dye D is used as a reference, and the amount of amplified product is detected by the ratio of its intensity to the fluorescence of the fluorescent dye B. Also, as shown in Figure 7D, since it functions as a size standard 701 in the electrophoresis mechanism 102, the fluorescence of the fluorescent dye D is used again for detection. Needless to say, even in the case shown in Figure 7, in the DNA analyzer 100 according to the first embodiment, these are detected by the same detector 111, so the device can be made smaller and costs can be reduced compared to providing separate detectors for each fluorescence.

[0071] In the DNA analysis process, the control device 121 can change the parameters at each stage based on the results from the detector 111 (the amount of amplified product estimated based on the first fluorescence intensity). Specifically, it can adjust one or more parameters selected from the group of conditions that can be used to detect fluorescence from the electrophoresis channel (specifically, the DNA amplification chamber 104), the injection conditions when injecting the sample into the electrophoresis channel (specifically, the capillary 114), and the detection conditions. Based on the amount of amplified product estimated based on the first fluorescence intensity, DNA amplification and analysis can be performed under suitable conditions.

[0072] In the DNA amplification unit 104, the amount of amplification product is measured (estimated) from the fluorescence intensity detected by the detector 111 (i.e., the first fluorescence intensity), and the parameters (amplification conditions) for amplification are adjusted based on the estimated amount of amplification product. For example, in DNA amplification by PCR, DNA denaturation, annealing, and extension are performed by changing the temperature of the sample to multiple temperatures. The amount of amplification product increases logarithmically by repeating the cycle of denaturation, annealing, and extension, but the temperature change cycle is stopped and amplification is stopped when the desired amount of amplification product is reached.

[0073] In the electrophoresis mechanism 102, the amount of sample injected into the electrophoresis channel (capillary 114) can be adjusted based on the estimated amount of amplified product. The amplified DNA molecules in the sample are, for example, electrically injected into the capillary 114. Electrical sample injection is performed by bringing the sample solution into contact with the end of the capillary 114 and applying a voltage for a short time. The injection conditions, such as the voltage and injection time, can be adjusted according to the estimated amount of amplified product.

[0074] Furthermore, in fluorescence detection by detector 111, the exposure time of the image sensor 204 can be adjusted based on the estimated amount of amplification product. The intensity of the detected fluorescence spectrum is estimated from the estimated amount of amplification product, and the detection conditions, such as the exposure time, are adjusted so that the estimated fluorescence spectrum intensity falls within the dynamic range of detector 111.

[0075] As an example, consider a scenario where, despite performing a sufficient number of PCR cycles, a sufficient amount of amplification product is not obtained. This situation might occur, for example, if the sample introduced into the sample pretreatment mechanism 101 is contaminated with a PCR inhibitor, preventing sufficient DNA amplification. In this case, the fluorescence signal obtained by fluorescence detection in the electrophoresis mechanism 102 becomes small, and a good electrophoretic waveform (size distribution of DNA in the sample) cannot be obtained. Even in such cases, in order to obtain the best possible analytical results, the control device 121 sets the sample injection time and the exposure time during fluorescence detection to be longer than when a sufficient amount of amplification product is obtained, based on the detection result of the detector 111 (estimated amount of amplification product), thereby increasing the amount of fluorescence signal obtained.

[0076] Figure 8 is a flowchart showing an example of the operation steps of the DNA analyzer 100 according to the first embodiment. As shown in Figure 8, first, the sample is introduced through the sample inlet 106 (S801). Next, a sample processing reagent is added to dissolve the sample and extract the DNA (S802). After dissolution, the sample is transported to the DNA amplification unit 104 (S803). Subsequently, the PCR reagent is introduced into the DNA amplification unit 104 (S804). A reagent for measuring the amount of amplification product is also added to the PCR reagent.

[0077] In the DNA amplification unit 104, DNA in the sample is amplified by PCR. First, one cycle of DNA denaturation, annealing, and extension is performed (S805). Next, the amount of PCR product (amount of amplified product) of the amplified DNA is measured (S806). Based on the measurement result of the amount of amplified product, it is determined whether or not to terminate the DNA amplification (S807). If the amplification is not terminated (No in S807), DNA denaturation, annealing, and extension are performed again (S805). If the amplification is terminated (Yes in S807), the amplified sample is introduced into the electrophoresis preparation unit 105, mixed with water and a size standard, and prepared for separation by electrophoresis (S808).

[0078] Next, the chain length of the amplified DNA fragments is measured by capillary electrophoresis. First, polymer is injected into capillary 114 (S809). Then, the sample is moved to the end of the capillary, and a voltage is applied for a short time to inject the DNA in the sample, i.e., the PCR amplification product, into the end of the capillary (S810). The sample (sample solution) is discarded from the channel 115a of the cathode block 115, buffer is introduced, and a voltage is applied to both ends of the capillary to perform electrophoresis (S811). The data obtained from electrophoresis is processed to obtain the chain length of each DNA fragment and to determine the number of repeats of each STR site (S812).

[0079] The detector 111 in this embodiment has multiple detection channels. Some of the multiple detection channels are used to detect fluorescence from the DNA amplification chamber (specifically, the DNA amplification unit 104). The remaining channels of the multiple detection channels are used to detect fluorescence from the electrophoresis channel (electrophoresis mechanism 102, more specifically, the capillary 114). As mentioned above, multiple fluorescent dyes can be used to measure the amount of amplification product in the DNA amplification unit 104 and to detect the separated DNA in the electrophoresis mechanism 102. Generally, the emission spectra of fluorescent dyes have a width of several tens of nanometers, so when multiple fluorescent dyes are used, overlaps may occur in the fluorescence spectra of each dye.

[0080] In particular, DNA detection in capillary electrophoresis utilizes fluorescence resonance energy transfer, and a set of fluorescent dyes designed to emit different fluorescence at the same excitation wavelength can be used. For example, the excitation wavelength is 505 nm, and 4 to 8 different fluorescent dyes emit fluorescence in the wavelength range of 520 to 700 nm. The fluorescence spectra of these fluorescent dyes have different peak wavelengths, but there is some overlap in the spectra. Therefore, a process is needed to estimate the mixing ratio of each fluorescent dye from the mixed spectrum of multiple fluorescent dyes observed by the detector 111.

[0081] Furthermore, when using a spectrometer like the one shown in Figure 2, crosstalk between detection channels can occur. For example, when fluorescence is introduced into an optical fiber 205 and detected at the position on the image sensor 204 corresponding to the optical fiber 205 (channel 1), fluorescence from the optical fiber 205 may also be observed at other positions on the image sensor 204 (other channels) due to optical system aberrations, multiple reflections between surfaces such as lenses and gratings, etc. The occurrence of crosstalk is undesirable as it hinders accurate measurement.

[0082] A method is known for numerically resolving the overlap of fluorescent dye spectra and crosstalk between detection channels using matrix operations (see, for example, Japanese Patent Publication No. 7282880). Based on this, for example, suppose that the detector 111 has L detection channels (optical fibers), and each channel detects the wavelength range of the target to be measured as data at M points. Suppose there are N fluorescent dyes to be detected (N ≤ M). Here, the spectral output from the detector 111 is represented as a vector with L × M elements (denoted as S). The output from the detector 111 can be expressed as S = RC using a matrix (denoted as R, size L × M rows L × N columns) representing the response of each channel when the fluorescence spectrum of a specific fluorescent dye in a specific optical fiber is input, and a vector with L × N elements (denoted as C) representing the concentration ratio of the fluorescent dyes present in the target detected by each fiber.

[0083] If the fluorescence spectra from each fluorescent dye are previously injected into each optical fiber and the matrix R is obtained, then R － = (A ｔ A) －１ A ｔ The generalized inverse matrix calculated by gives C = R － The fluorescence dye concentration can be obtained from the spectrum acquired by detector 111 as S.

[0084] In this embodiment, the detector 111 of the DNA analyzer 100 uses different detection channels for measuring the amount of amplified product in the DNA amplification unit 104 and for detecting separated DNA in the electrophoresis mechanism 102, detecting the fluorescence spectra of different sets of fluorescent dyes. To obtain the concentration of the fluorescent dye from the fluorescence spectra detected in each, multiple matrices R for different sets of fluorescent dyes and sets of detection channels are used. － You may have different options and use them depending on the detection situation.

[0085] As an example, assume that the sample pretreatment mechanism 101 processes four samples simultaneously, and the processed samples are sent to the electrophoresis mechanism 102 and analyzed simultaneously by four capillaries 114. The detector 111 has eight detection channels. It is assumed that channels 1 to 4 are used for detecting the amount of amplification products in the sample pretreatment mechanism 101, and channels 5 to 8 are used for DNA detection in the electrophoresis mechanism 102.

[0086] At this time, the control device 121 obtains a matrix R of the concentrations of each fluorescent dye from the fluorescence spectra obtained in channels 1 to 4 １ － and a matrix R of the concentrations of each fluorescent dye from the fluorescence spectra obtained in channels 5 to 8 ２ － and uses them separately for each detection.

[0087] Note that it is arbitrary which detection channel is used in detecting fluorescence from the DNA amplification unit 104, detecting fluorescence from the electrophoresis mechanism 102, etc. For example, the detector 111 is a spectroscope as shown in FIG. 2, and in the above example, it has a total of eight input optical fibers (detection channels). The detection channels are numbered 1 to 8 in order, and channels 1, 3, 5, and 7 may be used for detecting fluorescence from the DNA amplification unit 104, and channels 2, 4, 6, and 8 may be used for detecting fluorescence from the electrophoresis mechanism 102.

[0088] When the detector 111 has the structure shown in FIG. 2, crosstalk between detection channels is basically the largest between adjacent channels. In such a case, crosstalk can be reduced by alternately providing the channels used for detecting fluorescence from the DNA amplification unit 104 and detecting fluorescence from the electrophoresis mechanism 102 as described above. Usually, since the analysis by the electrophoresis mechanism 102 is carried out after the processing in the sample pretreatment mechanism 101 is completed, the detection of fluorescence from the DNA amplification unit 104 and the detection of fluorescence from the electrophoresis mechanism 102 are carried out at different timings. Therefore, in the previous example, only four channels are used for measurement at a time. Since adjacent channels are not used simultaneously, crosstalk is further reduced.

[0089] As mentioned above, crosstalk between channels can be eliminated by signal processing of the fluorescence spectrum. However, since it is not always possible to eliminate all crosstalk by correcting it through signal processing, it is desirable to design the detector 111 in a way that minimizes crosstalk as much as possible.

[0090] Furthermore, when the sample pretreatment mechanism 101 processes multiple samples and the electrophoresis mechanism 102 analyzes multiple samples, the set of fluorescent dyes used for each sample may be different. Also, the number of samples processed by the sample pretreatment mechanism 101 and the number of samples analyzed by the electrophoresis mechanism 102 may be different.

[0091] The calculations described above are generalized below. The sample preprocessing mechanism 101 (DNA amplification chamber (DNA amplification unit 104)) processes a maximum of M samples, and the electrophoresis mechanism 102 (electrophoresis channel) analyzes a maximum of N samples. The detector 111 has M + N detection channels.

[0092] Here, the sample preprocessing mechanism 101 processes m samples, where m is a number such that m ≤ M. Each of the m samples is A ｉ Assume that the amount of amplified product is detected using (i = 1 to m) fluorescent dyes. The electrophoresis mechanism 102 analyzes n samples, where n is a number such that n ≤ N. Note that if m ≥ N, the analysis is performed using multiple electrophoresis cycles. Each of the n samples is B ｊ Assume that DNA fragments labeled with (j=1 to n) fluorescent dyes are detected.

[0093] The sample preprocessing mechanism 101 internally holds information on the response to all possible input patterns through a prior calibration operation. That is, the sample preprocessing mechanism 101 holds information on the signal patterns detected in a particular channel among the M+N channels when the spectrum of one of the fluorescent dyes that may be used is detected in that channel and the other channels, for all combinations of fluorescent dyes and channels.

[0094] In estimating the amplification product concentration (amount of amplification product) from the fluorescence spectrum in the sample pretreatment mechanism 101, first, matrix R １ This generates L １ ×m row ΣA ｉ It is a matrix of columns. L １ This represents the number of spectral data points used to estimate the amplification product concentration. Matrix R １ This is generated by extracting some of the aforementioned information. Based on this matrix, the generalized inverse matrix R is generated. １ － The number of elements L is composed of the output from the channel used by the detector 111. １ Vector S representing the spectrum of ×m １ From R, the concentration estimate is obtained １ － S １ This is obtained as follows: In other words, the control device 121 estimates the amount of amplified product based on the first fluorescence intensity from the spectrum detected by m detection channels. １ +A ２ +...+A ｍ The concentration of each fluorescent dye is estimated.

[0095] In measuring the concentration ratio of each DNA fragment from the fluorescence spectrum in the electrophoresis mechanism 102, first, matrix R ２ This generates L ２ ×n rows ΣB ｊ It is a matrix of columns. L ２ This represents the number of spectral data points used for detection in electrophoresis. Matrix R ２ This is generated by extracting some of the aforementioned information. Based on this matrix, the generalized inverse matrix R is generated. ２ － The number of elements L is composed of the output from the channel used by the detector 111. ２ Vector S representing the spectrum of ×n ２ From R, the concentration estimate is obtained ２ － S ２ This is obtained as follows: In other words, the control device 121 obtains B from the spectrum detected by n detection channels in the DNA analysis based on the second fluorescence intensity. １ +B ２ +...+B ｎThe concentration of each fluorescent dye is estimated.

[0096] In the estimation of amplification product concentrations from fluorescence spectra in the sample pretreatment mechanism 101 and the measurement of concentration ratios of each DNA fragment from fluorescence spectra in the electrophoresis mechanism 102, calculations may be performed collectively on the outputs of all M+N channels without distinguishing between them. However, in this case, for example, when estimating amplification product concentrations from fluorescence spectra in the sample pretreatment mechanism 101, calculations are also performed on the outputs of channels that are detected in electrophoresis, even though detection is not performed in electrophoresis. As mentioned above, by performing calculations only on channels used by separate matrices, unnecessary calculations on channels that do not detect fluorescence can be eliminated, thereby reducing computational costs.

[0097] <First Embodiment: Summary> The DNA analyzer 100 according to the first embodiment comprises a sample pretreatment mechanism 101 and an electrophoresis mechanism 102. The sample pretreatment mechanism 101 includes a DNA amplification unit 104, in which the amount of amplified product is measured (estimated) using fluorescence detection. The electrophoresis mechanism 102 detects the target DNA by fluorescence. The DNA analyzer 100 includes a detector 111. The detector 111 is used for both fluorescence detection in the sample pretreatment mechanism 101 and fluorescence detection in the DNA amplification unit 104. Since the DNA analyzer 100 uses the same detector 111 for both fluorescence detection in the sample pretreatment mechanism 101 and fluorescence detection in the DNA amplification unit 104, the device is smaller and simpler and costs can be reduced compared to providing separate detectors for each type of fluorescence. Furthermore, the fluorescent dye used as a reference in fluorescence detection in the DNA amplification unit 104 and the fluorescent dye used in detection in the electrophoresis mechanism 102 may be the same fluorescent dye. In this case, the cost of reagents is reduced. Furthermore, since fluorescent dyes are shared, the number of required dye types decreases, and the number of excitation light sources can be reduced. Therefore, costs are reduced. Moreover, the light source used for fluorescence excitation in the DNA amplification unit 104 and the light source used for fluorescence excitation in the electrophoresis mechanism 102 may be the same light source. The apparatus can be made smaller and simpler than if separate light sources were provided, and costs can be reduced.

[0098] <Second Embodiment> Next, a second embodiment will be described. Figure 9A is a structural diagram of the detector 111 used in the DNA analyzer 100 according to the second embodiment. The DNA analyzer 100 according to the second embodiment has the same structure as the DNA analyzer 100 according to the first embodiment, but the form of the detector 111 used is different.

[0099] The detector 111 used in the DNA analyzer 100 according to the second embodiment uses multiple types of dichroic mirror arrays 111A and 111B for fluorescence spectroscopy, thereby performing fluorescence spectroscopy. Figure 9 shows, as an example, the structure of the detector 111 when four types of fluorescent dyes are used for DNA detection in the electrophoresis mechanism 102 and two types of fluorescent dyes are used for fluorescence detection in the DNA amplification unit 104.

[0100] Dichroic mirrors reflect light with a specific wavelength as the boundary, while transmitting light with a shorter wavelength. Conversely, they transmit light with a wavelength longer than a certain wavelength, while reflecting light with a shorter wavelength. In a dichroic mirror array, fluorescence is spectrally analyzed using multiple dichroic mirrors, each with different wavelengths at which reflection and transmission switch.

[0101] As shown in Figure 9A, light introduced by the optical fiber 120 that guides the fluorescence from the electrophoresis mechanism 102 is collimated by the lens 902 and irradiated onto the dichroic mirrors 903 that constitute the dichroic mirror array 111A. For example, the wavelength range of the fluorescence detected by this is 500 to 700 nm. For example, the dichroic mirror 903 transmits light with shorter wavelengths than 550 nm and reflects light with longer wavelengths. Dichroic mirrors 904 and 905 reflect light with shorter wavelengths than 600 nm and 650 nm, respectively, and transmit light with longer wavelengths. Mirror 906 reflects all light from 500 to 700 nm.

[0102] With the above configuration, the light beams 908, 909, 910, and 911 detected by the detection element 907 positioned after the dichroic mirror array 111A have wavelengths of 500-550 nm, 550-600 nm, 600-650 nm, and 650-700 nm, respectively. The wavelength bands of each beam divided by the dichroic mirror array 111A do not need to be equally spaced and may be adjusted according to the fluorescence spectrum of the fluorescent dye used for detection. For example, the transmission and reflection wavelengths of the dichroic mirrors may be adjusted so that the central wavelengths of the light beams 908, 909, 910, and 911 are the peak wavelengths of the four types of fluorescent dyes used for DNA detection in the electrophoresis mechanism 102.

[0103] Meanwhile, light from the optical fiber 110 that guides fluorescence from the DNA amplification unit 104 is collimated by the lens 913 and incident on the dichroic mirror 914 that constitutes the dichroic mirror array 111B. For example, the wavelength range of fluorescence from the DNA amplification unit 104 is 700 to 800 nm. The dichroic mirror 914 transmits light with wavelengths shorter than 750 nm and reflects light with longer wavelengths. Mirror 915 reflects all light with wavelengths of 700 to 800 nm. The wavelengths of the light beams 916 and 917 separated by the dichroic mirror array 111B are 700 to 750 nm and 750 to 800 nm, respectively.

[0104] <Modifications in the Second Embodiment> (Modification 1) In Figure 9A, one each of dichroic mirror arrays 111A and 111B with different structures of four and two mirrors is provided. In Modification 1, the detector 111 may be provided with multiple dichroic mirror arrays 111A and 111B with different structures of four and two mirrors (the embodiment in which multiple dichroic mirror arrays 111A and 111B with different structures according to Modification 1 is provided is not shown). As an example, as mentioned above, analysis may be performed with multiple capillaries 114. In that case, for example, multiple sets of dichroic mirror arrays 111A and 111B with different structures of four and two mirrors as shown in Figure 9A may be provided for each of the multiple capillaries 114 (this embodiment is not shown). In this way, by providing multiple sets of dichroic mirror arrays 111A and 111B, fluorescence detection from the DNA amplification unit 104 in multiple sample pretreatment mechanisms 101 and fluorescence detection in the electrophoresis mechanism 102, which can perform electrophoresis of multiple samples simultaneously, can be performed at the same time.

[0105] (Modification 2) The detector 111 may also have multiple dichroic mirror arrays 111C with the same structure, rather than having dichroic mirror arrays 111A and 111B with different structures, such as a dichroic mirror array 111A composed of four dichroic mirrors and a dichroic mirror array 111B composed of two dichroic mirrors, as shown in Figure 9A. Figure 9B shows this embodiment as Modification 2. Figure 9B is a structural diagram showing a modified version of the detector 111 used in the DNA analyzer 100 according to the second embodiment.

[0106] That is, as shown in Figure 9B, the dichroic mirror array 111C in the detector 111 according to Modification 2 is composed of six dichroic mirrors. In this Modification 2, light from 500 to 800 nm may be detected in 50 nm increments by detecting six light beams. In this case, the mirror 906 described above is replaced with a dichroic mirror 906B. The dichroic mirror 906B transmits light with wavelengths longer than 700 nm and reflects light with shorter wavelengths. Therefore, it becomes possible to detect the six light beams.

[0107] In this modified example 2, multiple sets of optical fibers and lenses (optical fiber 120 and lens 902, and optical fiber 110 and lens 913) are provided for the same dichroic mirror array 111C. The multiple sets of optical fibers and lenses are arranged in the depth direction of the paper in Figure 9B. Light introduced by the optical fiber 120 that guides fluorescence from the electrophoresis mechanism 102 is separated by the dichroic mirrors 903 to 906B and detected by the detection element 907. Light from the optical fiber 110 that guides fluorescence from the DNA amplification unit 104 passes through the dichroic mirrors 903 to 906B, is separated by the dichroic mirror 914, is completely reflected by the mirror 915, and is detected by the detection element 907.

[0108] In this case, it is simple in that there is no need to fabricate dichroic mirror arrays with different structures. However, the detection region of 500-700 nm is not used in fluorescence detection from the DNA amplification unit 104, and the detection region of 700-800 nm is not used in fluorescence detection in the electrophoresis mechanism 102. Unused regions are created on the detection element 907, which increases the required size of the detection element 907 and may lead to increased costs. The DNA analyzer 100 according to the second embodiment can employ dichroic mirror arrays having structures suitable for each detection shown in Figures 9A and 9B, thereby improving the problem of increased costs and enabling a simpler configuration.

[0109] <Second Embodiment: Summary> The DNA analyzer 100 according to the second embodiment has a detector 111 that performs spectroscopy using dichroic mirror arrays 111A to 111C. The detector 111 may have one or more dichroic mirror arrays 111A and 111B with different structures, or it may have multiple dichroic mirror arrays 111C with the same structure. This increases the design flexibility of the detector 111, and also improves the problem of increased costs and allows for a simpler configuration.

[0110] <Third Embodiment> Figure 10 is a structural diagram of the spectrometer of the detector 111 used in the DNA analyzer 100 according to the third embodiment. Figure 10A is a top view of the spectrometer. Figure 10B is a side view of the spectrometer. The DNA analyzer 100 according to the third embodiment has the same structure as the DNA analyzer 100 according to the first and second embodiments, but the form of the detector 111 used is different.

[0111] The detector 111 shown in Figures 10A and 10B spectrally separates light using lenses 201, 202, and a grating 203, similar to the spectrometer shown in Figures 2A and 2B of the first embodiment. On the other hand, the image sensor 204 used in the detector 111 shown in Figures 2A and 2B is replaced by a line sensor 1001 in the detector 111 shown in Figures 10A and 10B, with optical fibers 110 and 120 connected to a single line sensor 1001. The optical fibers 110 and 120 are arranged to be detected by the line sensor 1001.

[0112] In other words, in this embodiment, the detector 111 has two input fibers (i.e., two detection channels) for a single line sensor 1001. One of the two input fibers introduces fluorescence from the DNA amplification chamber (specifically, the DNA amplification unit 104) (i.e., it is used to detect fluorescence). In this embodiment, the remaining input fiber introduces fluorescence from the electrophoresis channel (specifically, the capillary 114) (i.e., it is used to detect fluorescence). The fluorescence from the DNA amplification chamber (DNA amplification unit 104) and the fluorescence from the electrophoresis channel (capillary 114) are imaged (detected) in the same area on the image sensor of the detector 111 (line sensor 1001 in this description). With this configuration in this embodiment, the detector 111 (spectrometer) detects fluorescence from both the DNA amplification chamber and the electrophoresis channel.

[0113] As described above, in this embodiment, fluorescence from the sample pretreatment mechanism 101 is introduced into the optical fiber 110, and fluorescence from the electrophoresis mechanism 102 is introduced into the optical fiber 120. Therefore, in this embodiment, if fluorescence is introduced into the two optical fibers simultaneously, the two spectra will be mixed. However, since fluorescence measurements from both are basically performed at different timings, each spectrum can be obtained independently. Thus, in this embodiment, fluorescence from the DNA amplification unit 104 and fluorescence from the electrophoresis mechanism 102 can be measured separately and appropriately.

[0114] The spectrometer shown in Figures 2A and 2B of the first embodiment requires an image sensor 204 capable of capturing all measurable spectra. However, as mentioned above, in the example of Figures 10A and 10B, the image sensor 204 is replaced with a single line sensor 1001. This reduces costs. If multiple channels are required, multiple structures of Figures 10A and 10B may be used, or multiple line sensors 1001 may be used as shown in Figure 11. Figure 11 is a side view showing a modified example of the spectrometer.

[0115] Furthermore, in the example shown in Figures 10A and 10B, the structure for performing spectroscopy may be a dichroic mirror array 111A to 111C as shown in Figures 9A and 9B, instead of lenses 201 and 202 and grating 203. In this case, the detection element 907 shown in Figures 9A and 9B is a line sensor 1001.

[0116] <Third Embodiment: Summary> The DNA analyzer 100 according to the third embodiment has a detector 111 that detects light using a line sensor 1001. The detector 111 has two input fibers (optical fibers 110, 120) for one line sensor 1001, and fluorescence from the sample pretreatment mechanism 101 and fluorescence from the electrophoresis mechanism 102 are introduced into each input fiber. Since the emission timing of the fluorescence from each is different, each spectrum can be obtained independently. In addition, since the line sensor 1001 is used instead of the image sensor 204 and detection element 907, costs can be reduced.

[0117] <Fourth Embodiment> Figure 12 is a configuration diagram of the DNA analyzer 100 according to the fourth embodiment. In the DNA analyzer 100 according to the first embodiment, the detector 111 was shared between the sample pretreatment mechanism 101 and the electrophoresis mechanism 102. On the other hand, in the DNA analyzer 100 according to the fourth embodiment, the detector is not shared. In the fourth embodiment, as shown in Figure 12, the sample pretreatment mechanism 101 and the electrophoresis mechanism 102 each have detectors 1201 and 1202.

[0118] On the other hand, in the DNA analyzer 100 according to the fourth embodiment, the excitation light source 108 is shared by the sample pretreatment mechanism 101 and the electrophoresis mechanism 102. The excitation light 112 irradiated from the excitation light source 108 is split by the beam splitter 1203. One of the split excitation light 112 passes through the dichroic mirror 109 and is used for fluorescence excitation in the sample pretreatment mechanism 101. The fluorescence 113 generated by the irradiation of the excitation light 112 in the sample pretreatment mechanism 101 is reflected by the dichroic mirror 109 and detected by the detector 1201. The other split excitation light 112 is reflected by mirrors 1204, 1204 respectively and then used for fluorescence excitation in the electrophoresis mechanism 102. The fluorescence 113 generated by the irradiation of the excitation light 112 in the detection window 118 is detected by the detector 1202. The excitation light source 108 may output light that is a combination of multiple wavelengths of light, so as to excite multiple fluorescent dyes. In addition, a separate light source may be provided for use only by the sample pretreatment mechanism 101 or the electrophoresis mechanism 102, in addition to the excitation light source 108.

[0119] In the DNA analyzer 100 according to the fourth embodiment, as in the DNA analyzer 100 according to the first embodiment, the fluorescent dye used for detection in the sample pretreatment mechanism 101 and the electrophoresis mechanism 102 may be shared. Specifically, the method of sharing fluorescent dyes shown in Figures 6A to D and 7A to D in the description of the first embodiment can be used.

[0120] Specific aspects of the DNA analyzer 100 according to the fourth embodiment described above are as follows. The DNA analyzer 100 according to the fourth embodiment amplifies DNA in a sample to be measured and analyzes it by electrophoresis. The aforementioned sample contains a fluorescent dye that emits fluorescence. The DNA analyzer 100 has a light source (excitation light source 108), a first detector (detector 1201), and a second detector (detector 1202). The light source irradiates light onto the sample introduced into the DNA amplification chamber (specifically, the DNA amplification unit 104) and the sample sent from the DNA amplification chamber to the electrophoresis channel (specifically, the capillary 114). The first detector is connected to the DNA amplification chamber. The second detector is connected to the electrophoresis channel. Based on the first fluorescence intensity from the DNA amplification chamber detected by the first detector, the control device 121 estimates the amount of amplification product due to amplification. The DNA analyzer 100 analyzes DNA based on the second fluorescence intensity from the electrophoresis channel detected by the second detector, and the control device 121 analyzes the DNA.

[0121] <Fourth Embodiment: Summary> In the DNA analyzer 100 according to the fourth embodiment, the excitation light source 108 is shared between the sample pretreatment mechanism 101 and the electrophoresis mechanism 102. With this configuration, the device is smaller and simpler, and costs can be reduced, compared to providing separate light sources for each.

[0122] <Fifth Embodiment> Figure 13 is a configuration diagram of the DNA analyzer 100 according to the fifth embodiment. In the DNA analyzer 100 according to the fifth embodiment, the detector and excitation light source are not shared. On the other hand, in the DNA analyzer 100 according to the fifth embodiment, some of the fluorescent dyes used for detection are shared by the method of sharing fluorescent dyes shown in Figures 6A to D and 7A to D in the description of the first embodiment.

[0123] Specific aspects of the DNA analyzer 100 according to the fifth embodiment described above are as follows. The DNA analyzer 100 according to the fifth embodiment amplifies DNA in a sample to be measured and analyzes it by electrophoresis. The aforementioned sample contains a fluorescent dye that emits fluorescence. The DNA analyzer 100 has a first light source (excitation light source 108), a second light source (excitation light source 119), a first detector (detector 1201), and a second detector (detector 1202). The first light source irradiates light onto the sample introduced into the DNA amplification chamber (specifically, the DNA amplification unit 104). The second light source irradiates light onto the sample sent from the DNA amplification chamber to the electrophoresis channel (specifically, the capillary 114). The first detector is connected to the DNA amplification chamber. The second detector is connected to the electrophoresis channel. The DNA analyzer 100 uses a control device 121 to estimate the amount of amplified product based on a first fluorescence intensity from a chamber where DNA is amplified, as detected by the first detector. The DNA analyzer 100 uses a control device 121 to analyze DNA based on a second fluorescence intensity from an electrophoretic channel detected by the second detector. The DNA analyzer 100 uses the same fluorescent dye for fluorescence detection in both the first and second detectors (see Figures 6A-D and 7A-D).

[0124] The following are preferred embodiments of the fifth embodiment (see Figures 6A to D). Specifically, in the detection of fluorescence in the first detector, fluorescence from fluorescent dye B, which detects the amount of amplified product, and fluorescence from fluorescent dye A, which serves as a reference for fluorescent dye B, are detected. In this preferred embodiment, in the detection of fluorescence in the second detector, fluorescence from fluorescent dye A, which detects DNA separated by electrophoresis, is detected. Furthermore, in this preferred embodiment, the fluorescent dye A that serves as the reference and the fluorescent dye A that detects DNA separated by electrophoresis are the same fluorescent dye A. In this preferred embodiment, since fluorescent dye A is used as the reference fluorescent dye without using fluorescent dye D, the cost of fluorescent dye D can be reduced. Also, in this preferred embodiment, fluorescent dye A is excited in common by the DNA amplification unit 104 and the electrophoresis mechanism 102. Therefore, the excitation light source that excites fluorescent dye A can be shared by both, further reducing costs.

[0125] <Fifth Embodiment: Summary> In the DNA analyzer 100 according to the fifth embodiment, the sample pretreatment mechanism 101 and the electrophoresis mechanism 102 share some of the fluorescent dyes used for detection (the same fluorescent dye is used). In the fifth embodiment, the same fluorescent dye is used, which reduces the number of types of fluorescent dyes, and thus reduces the cost of fluorescent dyes.

[0126] <Sixth Embodiment> A DNA analysis method according to the sixth embodiment (hereinafter sometimes referred to as "this DNA analysis method") will now be described. Figure 14 is a flowchart illustrating the contents of the DNA analysis method according to the sixth embodiment. This DNA analysis method amplifies the DNA in the sample to be measured and analyzes it by electrophoresis. This DNA analysis method uses a DNA analyzer 100 with the following configuration. The DNA analyzer 100 has a first light source (excitation light source 108), a second light source (excitation light source 119), and a detector 111. The first light source irradiates light onto the sample introduced into the DNA amplification chamber (specifically, the DNA amplification unit 104). The second light source irradiates light onto the sample sent from the DNA amplification chamber to the electrophoresis channel (specifically, the capillary 114). The detector 111 is connected in common to both the DNA amplification chamber and the electrophoresis channel. For details of the configuration of the DNA analyzer 100, refer to the first embodiment, for example.

[0127] Using a DNA analyzer 100 having the above configuration, this DNA analysis method performs the following steps in this order: first detection step S1, estimation step S2, second detection step S3, and analysis step S4. In the first detection step S1, the detector 111 detects a first fluorescence intensity from the chamber where DNA amplification takes place. In the estimation step S2, the control device 121 estimates the amount of amplification product based on the first fluorescence intensity. In the second detection step S3, the same detector 111 as described above detects a second fluorescence intensity from the electrophoresis channel. In the analysis step S4, the control device 121 analyzes the DNA based on the second fluorescence intensity.

[0128] (Preferred Embodiment in the Sixth Embodiment) In the DNA analysis method, a preferred embodiment is that, based on the amount of amplification product estimated based on the first fluorescence intensity, one or more conditions selected from the group of amplification conditions in the chamber for DNA amplification, injection conditions when injecting the sample into the electrophoresis channel, and detection conditions for detecting fluorescence from the electrophoresis channel can be adjusted.

[0129] Furthermore, in this DNA analysis method, a preferred embodiment is that a commonly connected detector 111 has M + N detection channels that detect the M DNA amplification chambers and the N electrophoresis channels. In this preferred embodiment, m samples (m ≤ M) introduced into the M DNA amplification chambers are A ｉ It contains (i = 1 to m) fluorescent dyes. Also, n samples (n ≤ N) detected in N electrophoretic channels are B ｊ It contains (j = 1 to n) fluorescent dyes. And in this preferred embodiment, in estimation step S2, the control device 121 estimates the amount of amplified product based on the first fluorescence intensity from the spectrum detected by m detection channels A １ +A ２ +...+A ｍ The concentration of the fluorescent dye is estimated. In this preferred embodiment, in analysis step S4, the control device 121 determines the B from the spectrum detected in the n detection channels in the DNA analysis based on the second fluorescence intensity. １ +B ２ +...+B ｎ The concentration of each fluorescent dye is estimated.

[0130] In this DNA analysis method, a preferred embodiment is that the sample contains a fluorescent dye that emits fluorescence, and fluorescence detection is performed from the same fluorescent dye in both the DNA amplification chamber and the electrophoresis channel.

[0131] Furthermore, the following are preferred embodiments of this DNA analysis method (see Figures 6A to D). Specifically, in the detection of fluorescence in the DNA amplification chamber, fluorescence from fluorescent dye B, which detects the amount of amplification product, and fluorescence from fluorescent dye A, which serves as a reference for fluorescent dye B, are detected. In this preferred embodiment, in the detection of fluorescence in the electrophoresis channel, fluorescence from fluorescent dye A, which detects the DNA separated by electrophoresis, is detected. Furthermore, in this preferred embodiment, the fluorescent dye A that serves as the reference and the fluorescent dye A that detects the DNA separated by electrophoresis are the same fluorescent dye A.

[0132] Furthermore, the preferred embodiments listed in the sixth embodiment are matters already described in the first embodiment, so their explanation will be omitted.

[0133] <Modifications> The DNA analysis apparatus 100 and DNA analysis method according to the present invention have been described in detail above with reference to embodiments. However, the present invention is not limited to the embodiments described above, and various modifications are included. For example, the embodiments described above are described in detail in order to explain the present invention in an easy-to-understand manner, and are not necessarily limited to those having all the configurations described. Furthermore, it is possible to replace a part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. In addition, it is possible to add, delete, or replace parts of the configuration of each embodiment with other configurations.

[0134] 100 DNA analyzer 101 Sample pretreatment mechanism 101A Pretreatment cartridge 102 Electrophoresis mechanism 103 Sample lysis section 104 DNA amplification section (chamber for DNA amplification) 105 Electrophoresis preparation section 108 Excitation light source (first light source, light source) 111 Detector 111A, 111B, 111C Dichroic mirror array 114 Capillary (channel for electrophoresis) 119 Excitation light source (second light source) 121 Control device 204 Image sensor 1201 Detector (first detector) 1202 Detector (second detector) A Fluorescent dye B Fluorescent dye C Quencher D Reference fluorescent dye

Claims

1. A DNA analyzer that amplifies DNA in a sample to be measured and analyzes it by electrophoresis, comprising: a first light source that irradiates light onto a sample introduced into a DNA amplification chamber; a second light source that irradiates light onto the sample sent from the DNA amplification chamber to an electrophoresis channel; and a detector connected in common to the DNA amplification chamber and the electrophoresis channel, wherein the detector detects a first fluorescence intensity from the DNA amplification chamber and the same detector detects a second fluorescence intensity from the electrophoresis channel, and a control device estimates the amount of amplified product based on the first fluorescence intensity and analyzes the DNA based on the second fluorescence intensity.

2. The DNA analyzer according to claim 1, characterized in that, based on the amount of amplification product estimated based on the first fluorescence intensity, one or more conditions selected from the group of amplification conditions in the chamber for amplifying the DNA, injection conditions when injecting the sample into the electrophoresis channel, and detection conditions for detecting fluorescence from the electrophoresis channel are adjusted.

3. The commonly connected detector has M + N detection channels that detect M DNA amplification chambers and N electrophoresis channels, and m samples (m ≤ M) introduced into the M DNA amplification chambers are A ｉ The sample contains (i = 1 to m) fluorescent dyes, and n samples (n ≤ N) detected in N electrophoretic channels are B ｊ The device contains (j = 1 to n) fluorescent dyes, and in estimating the amount of the amplification product based on the first fluorescence intensity, the control device uses the spectrum detected by m detection channels to determine the amount of the amplification product. １ +A ２ +...+A ｍ The concentration of each fluorescent dye is estimated, and the control device analyzes the DNA based on the second fluorescence intensity, using the spectrum detected by n detection channels to determine B １ +B ２ +...+B ｎ The DNA analyzer according to claim 1, characterized by estimating the concentration of individual fluorescent dyes.

4. The DNA analyzer according to claim 1, characterized in that the sample contains a fluorescent dye that emits fluorescence, and fluorescence detection from the same fluorescent dye is performed in the detection of fluorescence in the chamber for amplifying the DNA and in the detection of fluorescence in the channel for electrophoresis.

5. The DNA analyzer according to claim 4, characterized in that, in the detection of fluorescence in the chamber for DNA amplification, fluorescence from a fluorescent dye for detecting the amount of amplification product and fluorescence from a fluorescent dye that serves as a reference for the fluorescent dye for detecting the amount of amplification product are detected, in the detection of fluorescence in the channel for electrophoresis, fluorescence from a fluorescent dye for detecting DNA separated by electrophoresis is detected, and the fluorescent dye that serves as the reference and the fluorescent dye for detecting DNA separated by electrophoresis are the same fluorescent dye.

6. The DNA analyzer according to claim 1, characterized in that the detector performs fluorescence spectroscopy using multiple types of dichroic mirror arrays.

7. The DNA analyzer according to claim 1, wherein the detector has two detection channels for a single image sensor, one of the two detection channels is used to detect fluorescence from the chamber for amplifying the DNA, and the remaining channel of the two detection channels is used to detect fluorescence from the electrophoretic channel, and the fluorescence from the chamber for amplifying the DNA and the fluorescence from the electrophoretic channel are imaged in the same area on the image sensor of the detector.

8. The DNA analyzer according to claim 1, characterized in that the first light source and the second light source are the same light source.

9. A DNA analyzer that amplifies DNA in a sample to be measured and analyzes it by electrophoresis, wherein the sample contains a fluorescent dye that emits fluorescence, and comprises: a first light source that irradiates light onto the sample introduced into a DNA amplification chamber; a second light source that irradiates light onto the sample sent from the DNA amplification chamber to a channel for electrophoresis; a first detector connected to the DNA amplification chamber; and a second detector connected to the channel for electrophoresis, wherein a control device estimates the amount of amplification product based on a first fluorescence intensity detected by the first detector from the DNA amplification chamber, and the control device analyzes the DNA based on a second fluorescence intensity detected by the second detector from the channel for electrophoresis, and the same fluorescent dye is used for fluorescence detection in the first detector and fluorescence detection in the second detector.

10. The DNA analyzer according to claim 9, characterized in that, in the detection of fluorescence in the first detector, fluorescence from a fluorescent dye that detects the amount of amplification product and fluorescence from a fluorescent dye that serves as a reference for the fluorescent dye that detects the amount of amplification product are detected, and in the detection of fluorescence in the second detector, fluorescence from a fluorescent dye that detects the DNA separated by electrophoresis is detected, and the fluorescent dye that serves as the reference and the fluorescent dye that detects the DNA separated by electrophoresis are the same fluorescent dye.

11. A DNA analyzer for amplifying DNA in a sample to be measured and analyzing it by electrophoresis, comprising: a light source for irradiating light onto a sample introduced into a DNA amplification chamber and onto the sample sent from the DNA amplification chamber to a channel for electrophoresis; a first detector connected to the DNA amplification chamber; and a second detector connected to the channel for electrophoresis, wherein a control device estimates the amount of amplification product based on a first fluorescence intensity detected by the first detector from the DNA amplification chamber, and the control device analyzes the DNA based on a second fluorescence intensity detected by the second detector from the channel for electrophoresis.

12. A DNA analysis method for amplifying DNA in a sample to be measured and analyzing it by electrophoresis, comprising: a DNA analyzer having a first light source that irradiates light onto a sample introduced into a DNA amplification chamber; a second light source that irradiates light onto the sample sent from the DNA amplification chamber to a channel for electrophoresis; and a detector commonly connected to the DNA amplification chamber and the channel for electrophoresis, wherein the DNA analysis method comprises: a first detection step in which the detector detects a first fluorescence intensity from the DNA amplification chamber; an estimation step in which a control device estimates the amount of amplification product based on the first fluorescence intensity; a second detection step in which the same detector as the first detection device detects a second fluorescence intensity from the channel for electrophoresis; and an analysis step in which the control device analyzes DNA based on the second fluorescence intensity.

13. The DNA analysis method according to 12, characterized in that, based on the amount of amplification product estimated based on the first fluorescence intensity, one or more conditions selected from the group of amplification conditions in the chamber for amplifying the DNA, injection conditions when injecting the sample into the electrophoresis channel, and detection conditions for detecting fluorescence from the electrophoresis channel are adjusted.

14. The commonly connected detectors have M + N detection channels for detecting a chamber for amplifying M pieces of the DNA and N flow paths for electrophoresis. m samples (m ≤ M) introduced into the chamber for amplifying M pieces of the DNA contain A ｉ fluorescent dyes of (i = 1 to m) pieces, and n samples (n ≤ N) detected in the N flow paths for electrophoresis contain B ｊ fluorescent dyes of (j = 1 to n) pieces. In the estimation step, the control device estimates the concentrations of A １ + A ２ + ··· + A ｍ fluorescent dyes from the spectra detected by m detection channels in the estimation of the amount of the amplification product based on the first fluorescence intensity. In the analysis step, the control device estimates the concentrations of B １ + B ２ + ··· + B ｎ fluorescent dyes from the spectra detected by n detection channels in the analysis of the DNA based on the second fluorescence intensity. The DNA analysis method according to claim 12, characterized in that the above is performed.

15. The DNA analysis method according to claim 12, characterized in that the sample contains a fluorescent dye that emits fluorescence, and fluorescence detection from the same fluorescent dye is performed in the detection of fluorescence in the chamber for amplifying the DNA and in the detection of fluorescence in the channel for electrophoresis.

16. The DNA analysis method according to 15, characterized in that, in the detection of fluorescence in the chamber for DNA amplification, fluorescence from a fluorescent dye for detecting the amount of amplification product and fluorescence from a fluorescent dye that serves as a reference for the fluorescent dye for detecting the amount of amplification product are detected, in the detection of fluorescence in the channel for electrophoresis, fluorescence from a fluorescent dye for detecting DNA separated by electrophoresis is detected, and the fluorescent dye that serves as the reference and the fluorescent dye for detecting DNA separated by electrophoresis are the same fluorescent dye.

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