JP2003169661A - Apparatus for analyzing nucleic acid and method for analyzing nucleic acid by using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inexpensive, simple and miniaturized apparatus for extracting and refining a nucleic acid from a sample containing the nucleic acid and analyzing the nucleic acid and to provide a method using the apparatus. <P>SOLUTION: This invention provides the apparatus for analyzing the nucleic acid in the sample, having the following means (a) to (d) and the analytic method using the apparatus. (a) A means for bringing the sample into contact with magnetism-responding particles having a ferromagnetic metal oxide and silica and carrying out treatment for absorbing the nucleic acid in the sample into the magnetism-responding particles. (b) A means for carrying out treatment for separating magnetism-responding particles, in which the nucleic acid is absorbed, from the sample by applying magnetic field thereto. (c) A means for carrying out treatment for separating the nucleic acid from the magnetism- responding particles by applying electric field thereto. (d) A means for carrying out treatment for amplifying the nucleic acid separated from the magnetism- responding particles. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an apparatus for analyzing nucleic acid in a sample containing a target nucleic acid and a method for analyzing nucleic acid using the apparatus.

According to the present invention, it becomes possible to efficiently analyze a nucleic acid on a small scale. That is, it is possible to downsize a nucleic acid extraction / purification device, which has been difficult to downsize by a conventional method, and analyze a nucleic acid with an inexpensive and simple structure. Then, it becomes possible to perform highly sensitive analysis even when the amount of sample used is extremely small.

[0003]

2. Description of the Related Art Extraction and purification of nucleic acid from biological materials such as cells containing nucleic acid is an important step in the field of genetic engineering and clinical diagnosis. For example, when trying to analyze a certain gene, it is first necessary to extract nucleic acids such as DNA and RNA from biological materials such as cells that retain the gene. Also, in DNA / RNA diagnosis for detecting infectious agents such as bacteria and viruses, after extracting nucleic acids of bacteria and viruses from biological materials such as blood,
It is necessary to detect.

Generally, DNA and RN contained in biological materials
Nucleic acid such as A does not exist in a free state but exists in the shell such as cell membrane and cell wall composed of protein, lipid and sugar, and in most cases, the nucleic acid itself forms a complex with the protein. is doing. Therefore, when extracting and purifying nucleic acids from biological materials, first, physical disruption by ultrasonic waves or heat or enzyme treatment by protease,
Nucleic acids are liberated by treatment with a surfactant or denaturant, and then crushed products by extraction operations with organic solvents such as phenol, ultracentrifugation, column chromatography using carriers such as ion exchangers, etc. It is necessary to purify the nucleic acid from the inside. These methods are combined and optimized according to the intended use of nucleic acids, starting materials, and extracted nucleic acids [Molecular
cloning: A Laboratory manual, 2nded. (Cold Spring
Harbor Laboratory Press, 1989)].

However, these methods are laborious because they involve complicated steps such as centrifugation. Furthermore, the nucleic acid sample extracted by these methods contains a large amount of contaminants such as proteins, which are harmful to the subsequent analysis. Therefore, in order to obtain nucleic acids with a high degree of purity, after performing these extraction operations, it is difficult and time-consuming to perform ultracentrifugation using a density gradient such as cesium chloride or phenol / chloroform extraction. It is necessary to perform the required protein removal operation.

On the other hand, as a simple nucleic acid extraction method, there is a method of using silica as a nucleic acid-binding solid-phase carrier (Japanese Patent Laid-Open No. HEI-2)
-289596). In this method, nucleic acids can be extracted from biological materials such as bacteria in one step, and since a low-concentration buffer such as water or TE buffer is used as an eluent, no special desalting and concentration operation is required. ,
There is an advantage that the extracted nucleic acid can be immediately used for the subsequent analysis. However, the nucleic acid obtained by elution from silica by this method had a low concentration and was not satisfactory in yield. Therefore, it can be applied on a normal scale for polymerase chain reaction (PCR), etc., which allows analysis with a small amount of nucleic acid, but for analysis such as Southern hybridization and Northern blot, a large scale and subsequent concentration step It requires a complicated operation. There is a nucleic acid isolation method using a magnetic carrier for nucleic acid binding as a nucleic acid extraction method with further improved convenience, for example, a magnetically responsive particle having a superparamagnetic iron oxide nucleus covered with a polymerizable silane coating capable of covalently binding a nucleic acid. Is known to be used (Japanese Patent Laid-Open No. 60-1564). However, this method also cannot eliminate dissatisfaction in the concentration and yield of the nucleic acid obtained by elution.

Further, in the application to the diagnostic field, it is required that the analysis scale be miniaturized so that nucleic acid analysis can be carried out with a small amount of sample. However, in the conventional method, it is difficult to miniaturize the device because it is necessary to increase the scale in order to obtain a sufficient amount of nucleic acid. Furthermore, until now, no system has been developed that can carry all nucleic acid analysis steps including a nucleic acid extraction and purification step on a stage having an area of 40 cm ² or less, that is, on a chip.

[0008]

For these reasons,
There has been a demand for a device / method capable of extracting and purifying and analyzing nucleic acids simply and efficiently. In order to improve convenience, a series of operations for nucleic acid extraction and purification and analysis are integrated,
It is necessary to develop a system that can be linked. Further, by miniaturizing the scale, it becomes possible to analyze a small amount of nucleic acid, and it can be used in the diagnostic field. That is, an object of the present invention is to provide an apparatus and method for easily extracting and purifying nucleic acid from a sample containing nucleic acid and analyzing the nucleic acid. Furthermore, it is to provide a so-called miniaturized TAS (total analysis system) capable of miniaturizing from extraction and purification of nucleic acids to analysis.

[0009]

The inventors of the present invention can very efficiently purify a nucleic acid by using a magnetically responsive particle having a ferromagnetic metal oxide and silica and appropriately applying a magnetic field and an electric field. I found that I could do it. Based on this knowledge, the device including the stage of nucleic acid analysis can be downsized,
The present invention has been completed, which enables simple analysis of nucleic acids and can provide a miniaturized TAS for nucleic acid analysis.

The conventional method of using non-magnetically responsive silica as a nucleic acid-binding solid-phase carrier requires a sufficient amount of carrier for extraction and purification of nucleic acids. Therefore, it is difficult to miniaturize the system by the conventional method. Further, when automating the system by the conventional method, pipetting is required for various operations of stirring, separating and moving the carrier to which the nucleic acid is bound. Therefore, since a large-scale device having a robot arm for pipetting is required,
Miniaturization of the system was even more difficult. The method using magnetically responsive particles having a ferromagnetic metal oxide and silica, which the present inventors have found, allows various operations such as stirring, separation, and movement to be carried out easily and inexpensively by utilizing a magnetic field, thereby reducing the size. Became easier. Therefore, according to the method of the present invention, the nucleic acid extraction / purification process, which has been difficult to miniaturize by the conventional method, can be miniaturized, and the nucleic acid can be analyzed with an inexpensive and simple structure. Then, even if the amount of sample used is extremely small, highly sensitive analysis becomes possible. Further, even when the pipetting means is provided for the automation of the system, the means for isolating the nucleic acid and the like are miniaturized, so that it is expected that the whole system can be relatively miniaturized.

Further, it has been considered that almost all the nucleic acids bound to or adsorbed on the magnetically responsive particles having silica are eluted by a predetermined buffer or water. Therefore, the reason for the low yield of nucleic acids is that the nucleic acids of the magnetically responsive particles are used. It was thought to be due to its low binding capacity. Therefore, various improvements have been made to the material, particle shape, surface area, etc. of the magnetically responsive particles (Japanese Patent Laid-Open No. 2-289596, Japanese Patent Publication No. 7-1).
No. 3077, JP-A-9-12929). However, as a result of various studies, the present inventors have found that when a magnetically responsive particle having a ferromagnetic metal oxide and silica is used as a nucleic acid binding carrier, the nucleic acid bound to or adsorbed to the magnetically responsive particle is used. It was found that elution was difficult even with a predetermined buffer or water depending on the condition. Therefore, by utilizing the fact that nucleic acid has a negative charge because it has a phosphate-bonded skeleton, it is possible to release nucleic acid from magnetically responsive particles in high yield by placing the system containing magnetically responsive particles in an electric field. It was found that it becomes possible to construct a miniaturized TAS for nucleic acid analysis.

That is, the present invention has the following configuration.
(1) An apparatus for analyzing nucleic acid in a sample, which comprises the following means (a) to (d). (A) a means for contacting the nucleic acid with magnetically responsive particles having a ferromagnetic metal oxide and silica to adsorb the nucleic acid in the sample and the magnetically responsive particles; (b) applying a magnetic field Means for separating the adsorbed magnetically responsive particles from the sample, (c) means for applying an electric field to separate the nucleic acid from the magnetically responsive particles, and (d) amplifying the nucleic acid separated from the magnetically responsive particles. A means of performing processing. (2) The device according to (1), further including the following means (e). (E) A means for performing a process of electrophoresing the amplified nucleic acid. (3) the (a) all of the processing by means of ~ (e) is configured to be performed in one stage, according to the area of the stage is 1cm ² ~40cm ^{2 (2)} . (4) At least (b), (c), and
A groove for performing the processing by each means of (e) is formed, and the width of the groove is 5 μm to 1 mm (3)
The device according to. (5) The device according to any one of (1) to (4), further including control means capable of automatically controlling at least one of the respective means. (6) Further comprising means for pipetting, (5)
The device according to. (7) A means for performing a process for separating nucleic acid is further provided,
The apparatus according to any one of (1) to (6). (8) The device according to (7), further including means for performing a hybridization treatment of the separated nucleic acid. (9) The device according to (7), further including a unit that performs at least one measurement of absorption spectroscopy, fluorescence spectroscopy, or emission spectroscopy of the separated nucleic acid. (10) A method for analyzing nucleic acid in a sample, which comprises using the device according to any one of (1) to (9).

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION In the present specification, to analyze a nucleic acid means to purify and amplify the nucleic acid, to subject the nucleic acid to an electrophoretic treatment, to collect a specific nucleic acid, to perform hybridization, various spectroscopic methods. This is a concept that includes performing measurement by the method.

The present invention is characterized by including the above-mentioned means (a) to (d). Hereinafter, each means (a) to (d) will be described in order. In the following description, more preferable aspects of the present invention (“stage”, “groove”, “automating means”, etc.) will also be described, but they are essential for the present invention (the invention according to claim 1). It is not a configuration requirement, but (a)
By including the means of (d), it becomes possible to easily isolate the nucleic acid from the sample, and it is possible to contribute to miniaturization of the device.

The means (a) of the apparatus according to the present invention, that is, the means for bringing the nucleic acid into contact with the magnetically responsive particles and adsorbing both of them does not require any special equipment and can simply mix both. It is sufficient if there is a region (hereinafter, also referred to as "reaction chamber"). The conditions for bringing them into contact with each other are not particularly limited, but it is preferable that they are mild conditions that do not impair the properties of the nucleic acid and the magnetically responsive particles, and both may be allowed to coexist. By the treatment of the means (a), the nucleic acid in the sample is adsorbed on the magnetically responsive particles.

The means (b) is a means for separating the nucleic acid from the sample by applying a magnetic field to the magnetically responsive particles to which the nucleic acid has been adsorbed by the means (a) and moving them. A method using a permanent magnet or a method using an electromagnet can be considered as a means for applying a magnetic field in order to separate and move the magnetically responsive particles to which nucleic acid is bound, but it is necessary that the magnetically responsive particles have a sufficient magnetic force. is there. For example, a magnet with a magnetic flux density of about 300 Gauss can be used.
Thus, by using the magnetically responsive particles as a carrier for nucleic acids,
By controlling the magnetic field, nucleic acids can be agitated, separated, and moved, and the number of steps using a pipette, which causes an increase in size of the device, can be reduced, which can contribute to miniaturization of the device.

The means (c) applies an electric field to
It is a means for releasing and separating nucleic acids from magnetically responsive particles. The conditions for applying an electric field are preferably mild conditions that do not impair the properties of nucleic acids and magnetically responsive particles. For example, 10 to 200 V in the flow path immersed in the electrolyte solution.
By energizing at a voltage of about a certain level, only the nucleic acid is moved in the positive electrode direction, so that the magnetically responsive particles and the nucleic acid are separated, and a purified nucleic acid can be obtained. Alternatively, a magnetically responsive particle having a nucleic acid bound is injected into a groove in a gel matrix immersed in an electrolyte solution, and the gel matrix is filled with 10 to 20
By energizing at a voltage of about 0 V, the solid-phase carrier does not move in the gel matrix but only the nucleic acid moves toward the positive electrode, so that the magnetically responsive particles and the nucleic acid are separated and a purified nucleic acid can be obtained. . Also, a membrane having appropriate holes can be used. That is, the nucleic acid is released from the magnetically responsive particles by covering the liquid containing the magnetically responsive particles to which the nucleic acid is bound with a membrane and energizing at a suitable voltage, so that the purified nucleic acid can be recovered.
As described above, by using an electric field, nucleic acids can be released from magnetically responsive particles in a higher yield than when they are simply eluted in water or a buffer, which can contribute to the realization of minute amount analysis. .

No special equipment is required as a device for applying an electric field to the system containing magnetically responsive particles.
For example, a commercially available electrophoresis apparatus or power supply for separating and analyzing nucleic acids and proteins can be used as it is. However, in order to functionally perform the extraction and purification of nucleic acids according to the present invention, it is considered that an automated device equipped with a dispensing mechanism, electrodes, and solid-liquid separation means will be useful. The production of an automated device equipped with these means is sufficiently possible by the conventional technique, and the automated device is also an embodiment of the present invention.

The nucleic acid in the sample can be purified by subjecting it to the treatment by the above means. The purified nucleic acid is stored in an area for storing (hereinafter, referred to as “collection chamber”) if necessary, and then subjected to treatment by the following means. As described above, the region for performing the processing of the means (a) to the means (c) and the recovery chamber (when present) are collectively referred to as “nucleic acid purification section”.
The processes of the means (a) to the means (c) may be performed in different areas, but from the viewpoint of saving space in the device,
It is preferable to share as little area as possible. Eg 3
Approximately 10 flow paths can be used as regions for performing each processing of the means (a) to (c), and the regions can be shared by applying a magnetic field or an electric field according to the need of each means.

The means for carrying out the treatment for amplifying nucleic acid (means (d), hereinafter also referred to as "nucleic acid amplification section") may have at least a space in which the purified nucleic acid is present.
It is preferable to add a polymerase, a substrate, a buffer and the like as reagents to provide a suitable reaction temperature. Temperature control by a Peltier element is more preferable because it dramatically improves the reaction.

The nucleic acid in the sample can be purified and amplified by each of the above means. As a more preferred embodiment of the present invention, a device further having a means (means (e)) for electrophoresing the nucleic acid amplified as described above can be mentioned. The area in which the means (e) is performed is hereinafter referred to as "analyzer".
The groove (flow channel) of the analysis unit is exemplified by a flow channel having a width necessary for general capillary electrophoresis, preferably 5 μm to 1 mm, and more preferably 5 to 200 μm. The means (e) may be combined with a light source such as a light emitting diode that generates excitation light or a CCD camera as a device for detecting nucleic acid. A device for applying an electric field to the flow channel during electrophoresis may be used in combination with a device for applying an electric field to a system containing magnetically responsive particles, or a commercially available electric device for separate analysis of nucleic acids and proteins. An electrophoretic device or a power supply may be used. In any case, no special equipment is needed.

With the device having the above means, the nucleic acid in the sample can be efficiently isolated by a simple operation.
Of the nucleic acids thus isolated, a means for fractionating the nucleic acid (hereinafter referred to as “preparation unit”) for the purpose of performing measurement / detection by hybridization, absorption, fluorescence or luminescence of a specific nucleic acid That is, the device including the above) is also an embodiment of the present invention. There is no particular limitation on the shape of the preparative section, and for example, the same shape as the analysis section does not pose any problem. For example, only the nucleic acid having the target size can be collected by intersecting the collecting section with the flow path of the analyzing section. Further, the flow channel of the analysis unit and the flow channel of the sorting unit may share the same flow channel.

In the present invention, all the processing by the above means (a) to (e) can be configured to be performed in one stage, and an apparatus configured in such a manner can be used in the apparatus of the present invention. This is one of the preferred embodiments. Further, by the area of the stage is a 1cm ² ~40cm ^2,
It is more preferable because a miniaturized TAS from extraction and purification of nucleic acid to analysis can be provided.

Further, the stage (b),
Grooves (flow paths) for forming the treatments by the means (c) and (e) are formed, and the width of the grooves is 5 μm to 1 m.
From the viewpoint of miniaturization of the device, m is preferable. Further, as the material of the stage, a general plastic such as glass, polypropylene, or polycarbonate, or a known or novel polymer material such as heat resistant resin can be used. The processing of the pattern of each part of the stage is
Although known methods may be used, since the surface processing accuracy has an important influence on stirring, separation, and movement of magnetically responsive particles, for example, injection molding method, glass molding method, dry etching method, laser cutting method, A method such as the 2P method is preferable.

Further, by providing a control means capable of automatically controlling at least one of each means of the above apparatus, it is possible to automate a part or all of the steps from extraction and purification of nucleic acid to analysis and fractionation. It will be possible. Further, by providing a means for further pipetting in the above apparatus and transporting the sample between the respective means, the analysis / preparation can be continuously performed. Although it is inevitable that the device will be slightly enlarged in size by providing the pipetting means, since the device of the present invention is relatively minute as described above, even if the pipetting means is provided, it will be better than the conventional device. Thus, it is possible to reduce the size as a whole. In the practice of the present invention, the pipetting means and the control means are not particularly limited, and commonly used ones can be used.

The apparatus may further have means for measuring the specific nucleic acid collected by the collecting section by various spectroscopic methods and for carrying out a hybridization treatment. By having these means, it becomes possible to perform the steps from purification of nucleic acid to measurement and hybridization with one device. As the spectroscopic method, any spectroscopic method useful for the analysis of nucleic acids can be adopted, and examples thereof include absorption spectroscopy, fluorescence spectroscopy, and emission spectroscopy. As the means for carrying out the hybridization treatment, the means usually used in the art can be used.

More specific examples of the magnetic responsive particles having a ferromagnetic metal oxide and silica used in the present invention include coating a ferromagnetic metal oxide such as magnetite, γ-iron oxide, manganese zinc ferrite with silica. And particles obtained by dispersing these ferromagnetic oxides in silica.

The sample containing nucleic acid used in the present invention is a biological material containing protein, biomembrane, DNA or RNA, low molecular weight nucleic acid and the like. Examples of such biological materials include blood, animal cells, plant cells, insect cells, yeast, animal tissues, plant tissues, bacteriophages, viruses, bacteria, and combinations thereof containing them. Also, for purification purposes, the biological material may be a nucleic acid in a plasmid or amplification product.

The nucleic acid extraction and purification solution used in each of the means (a) to (c) of the present invention includes a buffer containing a chaotropic substance, EDTA (ethylenediaminetetraacetic acid), Tris-hydrochloric acid and the like. In the presence of chaotropic ions, nucleic acids are specifically adsorbed on carriers such as silica particles and separated from other cell components. Examples of chaotropic substances include guanidine salts, sodium iodide, potassium iodide, sodium (iso) thiocyanate, urea and the like. They may be used in combination. The concentration is preferably about 1 to 10 mol / l. Preferred nucleic acid extraction and purification solution in the present invention,
For example, guanidine thiocyanate, Triton X
-100, Tris / HCl buffer. Further, the magnetically responsive particles to which the nucleic acid is bound are washed several times with, for example, about 70% ethanol, the carrier is dried, and then sterile water or T is added.
The addition of a low ionic concentration solution such as E buffer gives favorable results for improving the purity of the purified nucleic acid and releasing the nucleic acid from the magnetically responsive particles.

In the means (c) of the present invention, a releasing buffer such as TE buffer or sterilized water may be used to finally release and recover the nucleic acid.

One aspect of the present invention is a method capable of extracting, purifying and analyzing a nucleic acid simply and efficiently. Further, one embodiment of the present invention is a system capable of integrating and linking a series of operations of nucleic acid extraction purification and analysis. Furthermore, another embodiment of the present invention is a miniaturizable total system from extraction and purification of nucleic acids to analysis.

The present invention may enable a miniaturized system for extracting, purifying and analyzing nucleic acids, which has been difficult with conventional techniques. Further, it can be used in the field of gene diagnosis.

In the above description, the main focus was on the analysis of DNA, but the nucleic acid analysis method of the present invention is not limited to RN.
The same can be applied to A. In this case, RNA may be subjected to a reverse transcription reaction in the nucleic acid amplification section (means (d)) to form target DNA or probe DNA.

Embodiments of the present invention will be described below in more detail with reference to the drawings, but the present invention is not limited thereto. FIG. 1 shows one embodiment of the present invention.

A sample of biological material or the like is purified in the nucleic acid purification section A, and nucleic acids are recovered. The nucleic acid purifying section A has a plurality of diameters of about 6 mm which can serve as reaction chambers and / or recovery chambers.
Of hemispheres (hereinafter, simply referred to as “hemispheres”) 1 and channels 2 to 4 having a width of about 900 μm that connect the hemispheres and can apply a magnetic field and / or an electric field. There is no particular limitation on the route for connecting the hemispheres 1, but it is preferable that the hemispheres 1 are linearly connected to each other.

First, in the hemisphere 1, the nucleic acid is adsorbed on the magnetically responsive particles having a ferromagnetic metal oxide and silica (use as a reaction chamber). That is, the processing of the means (a) is performed in the hemisphere 1. Next, the magnetically responsive particles to which the nucleic acid is adsorbed are moved from the hemisphere 1 to the adjacent hemisphere by the magnetic field via the flow path 3, and the particles are separated from the sample. That is, this means that the processing by means (b) has been performed. Next, in the hemisphere, a reagent for eluting the above-mentioned nucleic acid is added to the particles by using pipetting, valve dispensing or the like. Furthermore, the nucleic acid is released by applying an electric field. Next, from there, via channel 4,
The nucleic acid is moved to the adjacent hemisphere by an electric field, and only the nucleic acid is separated and stored (use as a recovery chamber). As a result, the processing by means (c) is performed.

In the nucleic acid purifying section A, there is no particular limitation as to which hemisphere or flow channel the treatment by each means is performed.
If necessary, a step of washing with a buffer solution, ethanol or other organic solvent may be added. Further, if necessary, a plurality of steps may be simultaneously carried out, such as simultaneously treating two or more samples or treating the same sample by dividing it into two or more. Then, at this time, in order to separate the hemisphere / flow channel used for each sample from those of other samples, a cut may be provided in the flow channel. Further, when moving the sample, it is not necessary to use a magnetic field / electric field for all movements, and a known means such as pipetting may be used for some of the movements.

The nucleic acid purified and recovered in the nucleic acid purifying section A is transferred to the nucleic acid amplifying section B and amplified therein. That is, the processing by means (d) is performed. The nucleic acid amplification section B is a hemisphere 5 having a diameter of about 6 mm. The hemisphere 5 is connected to the hemisphere of the nucleic acid purifying section A via a channel 6 having a width of about 900 μm capable of applying a magnetic field and / or an electric field.
The transfer from the nucleic acid purifying section A to the nucleic acid amplifying section B can be performed by using an electric field, and if necessary, other known methods such as pipetting may be used. In the hemisphere 5 of the nucleic acid amplification unit B, the purified nucleic acid is amplified by a known method such as the PCR method.

The nucleic acid amplifying section B is further provided with an analyzing section C1 for electrophoresing nucleic acid and / or a separating section for collecting nucleic acid via a channel 8 having a width of about 100 μm which can be applied with an electric field. It is preferably connected to the take-up portion (not shown in FIG. 1, but reference numeral C2 in FIG. 2). This configuration allows the electric field to be utilized for analysis and / or transition to preparative. Alternatively, the nucleic acid amplification unit B (ie, hemisphere 5) is separately provided.
It is also possible to move the nucleic acid purified and recovered in the nucleic acid purifying section A to the analyzing section and / or the preparative section without amplifying the nucleic acid and directly amplifying it in the recovery chamber without moving. That is, the processing of the means (d) is performed by the means (a) to (c).
That is, it is also possible to perform the treatment in a hemisphere or a channel. Further, a plurality of steps may be performed at the same time, such as treating two or more samples at the same time or treating the same sample by dividing it into two or more.

As shown in FIG. 1, the amplified nucleic acid is electrophoresed in the analysis section C1 (processing by means (e)).
The analysis section C1 is composed of a channel 7 having a width of about 100 μm for electrophoresis. Channel 7 and nucleic acid amplification section B (ie hemisphere 5)
Is a width of about 10 for moving nucleic acids using an electric field.
They are connected via a 0 μm flow path 8. The amplified nucleic acid is transferred to the intersection with the flow path 7 through the flow path 8 so that the transfer to the electrophoresis can be performed smoothly.

FIG. 2 also shows an example of the device according to the present invention. Reference numeral C2 in this figure is a sorting section having a channel 8 for sorting nucleic acids. The amplified nucleic acid moves to the end through the channel 8 and is then separated. As in the present example, an apparatus for performing fractionation immediately after amplification (that is, treatment by means (d)) without performing electrophoresis is also an embodiment of the present invention. The width of the flow path 8 of the sorting section C2 is about 1
It is 00 μm.

FIG. 3 also shows an example of the device according to the present invention. In this device, the analysis unit C1 and the sorting unit C2 are combined. The nucleic acid extraction and purification method and amplification method in this embodiment are the same as the methods in the above-described embodiments,
Detailed description thereof will be omitted. The amplified nucleic acid is transferred to the intersection with the flow path 7 via the flow path 8. After that, when the nucleic acid separated by electrophoresis (treatment by means (e)) and then separated by electrophoresis moves to another channel 9 that intersects channel 7 at an appropriate position, the flow By applying an electric field in the direction of flowing in the path 9, nucleic acids of a specific size can be collected. The thus-collected nucleic acid can be measured by hybridization, absorption spectroscopy, fluorescence spectroscopy, or emission spectroscopy. Alternatively, the nucleic acid may be amplified (treated by the means (d)) and then separated without electrophoresis. Alternatively, the amplified nucleic acid may be subjected to electrophoresis, and then the nucleic acid having a specific size may be collected.

Nucleic acid purification section A, nucleic acid amplification section B, analysis section C1
And / or the sorting unit C2 has a length of 95 mm and a width of 38 mm.
Fits on the plate (stage). Further, a magnetic field generation source and its movable device 10, a power supply 11 for generating an electric field, and a CCD device 12 for detection in an analysis unit are provided at the bottom of the plate, and an excitation light for fluorescence detection of nucleic acid is provided at the top. It has a generation source (LED) 13. Further, reference numeral 14 is an image output / control device such as a computer capable of processing an output signal from the CCD device 12 and outputting an image, and can also control the magnetic field generation source and its movable device 10 and power supply 11. .

[0044]

Industrial Applicability According to the present invention, it is possible to provide an apparatus / method capable of easily and efficiently extracting and purifying and analyzing a nucleic acid, which was difficult with the conventional techniques. As a result, by miniaturizing the scale of the device, it becomes possible to analyze a small amount of nucleic acid, and it can be used in the diagnostic field. Furthermore, it is possible to provide a so-called miniaturized TAS (total analysis system) capable of miniaturizing from extraction and purification of nucleic acids to analysis by utilizing the superiority of the present method.

[Brief description of drawings]

FIG. 1 schematically shows the overall configuration of an apparatus for carrying out nucleic acid extraction and purification, nucleic acid amplification and nucleic acid electrophoresis analysis as a nucleic acid analysis method of the present invention.

FIG. 2 schematically shows the overall configuration of an apparatus for carrying out extraction and purification of nucleic acids, amplification of nucleic acids, and fractionation of nucleic acids as a method for analyzing nucleic acids of the present invention.

FIG. 3 schematically shows the overall configuration of an apparatus for carrying out extraction and purification of nucleic acids, amplification of nucleic acids, analysis of nucleic acids by electrophoresis, and fractionation for nucleic acid analysis as a method for analyzing nucleic acids of the present invention.

[Explanation of symbols]

A: Nucleic acid purification section B: Nucleic acid amplification section C1 ... Analysis part C2 ... Preparation section 1 ... Hemispherical depression ("hemisphere") 2 ... Channel 3 ... Flow path 4 ... Channel 5 ... hemispherical depression ("hemisphere") 6 ... Flow path 7 ... Flow path 8 ... Flow path 9 ... Flow path 10 ... Magnetic field generation source and its movable device 11 ... Power supply 12 ... CCD device 13 ... Excitation light source 14 ... Image output and control device

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/533 G01N 33/566 33/566 37/00 101 37/00 101 27/26 315K 325B (72) Inventor Katsunori Ikeda 10-24 Toyo-cho, Tsuruga-shi, Fukui Prefecture Toyobo Co., Ltd. Tsuruga Bio Research Laboratory (72) Inventor Mikio Kishimoto 1-88 Tora-cho, Ibaraki-shi, Osaka Hitachi Maxell Co., Ltd. (72) Inventor Nobuhiro Umebayashi 1-88 1-Tora, Ibaraki-shi, Osaka Prefecture F-term (reference) within Hitachi Maxell Co., Ltd. (reference) 2G045 BA11 BA13 BB03 BB50 CA25 CB01 CB21 DA12 DA13 DA14 FA19 FA36 FB02 FB05 FB12 FB13 GC10 JA01 JA07 4A23 ABB CC01 FA12 4B063 QA01 QA12 QA13 QQ42 QR32 QR55 QS34 QS36 QX02

Claims (10)

[Claims] 1. Having the following means (a) to (d):
A device for analyzing nucleic acids in a sample. (A) means for contacting the sample with magnetically responsive particles having a ferromagnetic metal oxide and silica to adsorb the nucleic acid in the sample and the magnetically responsive particles, (b) applying a magnetic field Means for separating the adsorbed magnetically responsive particles from the sample, (c) means for applying an electric field to separate the nucleic acid from the magnetically responsive particles, and (d) amplifying the nucleic acid separated from the magnetically responsive particles. A means of performing processing. 2. The apparatus according to claim 1, further comprising the following means (e). (E) A means for performing a process of electrophoresing the amplified nucleic acid. 3. will be configured so that all processing by means of the (a) ~ (e) is carried out in one stage, according to claim 2 area of the stage is 1 cm ² ～40Cm ² Equipment. 4. The stage has at least (b),
The apparatus according to claim 3, wherein a groove for performing the processing by each of the means (c) and (e) is formed, and the width of the groove is 5 μm to 1 mm. 5. The apparatus according to claim 1, further comprising control means capable of automatically controlling at least one of the respective means. 6. The apparatus of claim 5, further comprising means for pipetting. 7. The apparatus according to claim 1, further comprising a means for performing a process for separating nucleic acids. 8. The apparatus according to claim 7, further comprising means for performing a hybridization treatment of the separated nucleic acid. 9. The apparatus according to claim 7, further comprising means for performing at least one measurement of absorption spectroscopy, fluorescence spectroscopy or emission spectroscopy of the separated nucleic acid. 10. A method for analyzing nucleic acid in a sample, which comprises using the apparatus according to claim 1.