JP3817389B2 - Polynucleotide sorter - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method and apparatus for selectively separating and recovering only a polynucleotide having a specific base sequence from a mixed sample of cells having a plurality of different sequences or cells.
[0002]
[Prior art]
One of the devices used for detecting a specific base sequence of DNA at high speed and simply is a DNA chip. This utilizes micropatterning technology and DNA complementarity used in semiconductor microfabrication technology. A single-stranded oligonucleotide as a probe having a base length of about 8-9 having various sequences is immobilized on each region divided into two dimensions on the substrate, and DNA is contained on this substrate. A sample solution is dropped to bind to the probe in each region on the substrate, and at the same time, a fluorescent dye is bound to optically observe the degree of binding between the probe and the DNA in the sample solution. By doing so, the base sequence of DNA in the sample solution is analyzed.
[0003]
A method for capturing a specific oligonucleotide (DNA or RNA) as a probe on a solid phase is disclosed, for example, in US Pat. No. 4,446,237. In this method, a solution containing a specific oligonucleotide is heated to denature the oligonucleotide into a single strand, which is then fixed to the nitrocellulose membrane surface. Another method for capturing a specific oligonucleotide as a probe on a solid phase is described in SR Rasmussen et al, Analytical Biochemistry 198, 128-142 (1991). In this method, a polystyrene microplate in which a phosphate group at the 5 ′ end of an oligonucleotide is activated using 1-methylimidazole and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and a secondary amine is introduced. Fix to the surface of the. In this method, a secondary amine reacts with the activated 5′-terminal phosphate group, so that the 5′-terminal side of the oligonucleotide is covalently fixed to the microplate surface.
[0004]
In this way, if a specific oligonucleotide as a probe is immobilized on the membrane surface, a complementary polynucleotide (DNA or RNA) can be selectively bound thereto, so that The DNA chip is based on this concept.
[0005]
On the other hand, a method in which polynucleotides to be selectively separated and recovered are captured by a probe fixed on the chip, and then the entire chip is heated to separate the polynucleotide on the probe captured by thermal denaturation from the chip. Has already been reported in US Pat. No. 5,607,646.
[0006]
In addition, it is widely practiced to selectively separate and recover a specific polynucleotide by using a difference in electrophoresis speed of the polynucleotide in the gel.
[0007]
[Problems to be solved by the invention]
The gene analysis technique using a DNA chip described in the above prior art is based on a probe (specific single-stranded oligonucleotide) having a length of about 8-9 bases formed on a substrate and a polynucleotide (DNA or RNA) in a sample solution. ) This is a technique for capturing and observing by complementary binding to single-stranded polynucleotides, and further selectively separating DNA or RNA single-stranded polynucleotides captured by probes on the substrate from the substrate. However, no consideration was given to recovery.
[0008]
Conventionally, it has not been considered to directly extract polynucleotides to be separated and recovered from cells.
[0009]
Furthermore, in conventional separation techniques using electrophoresis, the mobility of polynucleotides is relative to each other and fluctuates due to changes in electrophoresis conditions, and it is necessary to identify the sample solution components that have been separated and recovered. . Further, there is a possibility that the polynucleotides undergoing migration may be mixed with each other due to diffusion, and it has been difficult to accurately separate and purify a very small amount of polynucleotides.
[0010]
An object of the polynucleotide separation method and apparatus of the present invention is to provide a method and means for selectively separating and recovering a minute amount of polynucleotide (DNA or RNA) having a specific base sequence with high accuracy and high speed.
[0011]
[Means for Solving the Problems]
In order to achieve the above object, the present invention modifies each region of the divided surface on the substrate with a probe (specific oligonucleotide) having a different base sequence, and the polynucleotide (DNA or DNA) in the sample solution is modified on the probe. RNA), and then selectively heating only a specific region of the substrate to release only the polynucleotides that are complementary to the probe from the probe, so that it is desired from the sample solution. It is proposed to selectively separate and recover only the polynucleotide.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
FIG. 1 is a flow chart of gene processing for explaining the positions occupied by the polynucleotide separating method and apparatus of the present invention in gene analysis and selective recovery processing. Reference numerals 801 and 802 denote preparation processes, which are a process for introducing a sample cell and a sample liquid, respectively. The present invention can be applied to both cases where the cell itself is used as a specimen and when body fluid is used as a specimen. Reference numeral 803 denotes a process of extracting the contents of the cell from the inside of the cell. Reference numeral 804 denotes a PCR amplification process which is executed when the concentration of the polynucleotide to be separated is low. Reference numeral 805 denotes a characteristic process of the present invention, which is a process of fixing sample components in a plurality of independent regions. Reference numeral 806 denotes a process of observing and analyzing the state of the fixed sample component. Reference numeral 807 denotes a process in which sample components are separated and recovered from independent regions.
[0013]
Hereinafter, as will be described in detail, the present invention has an efficient process by fixing a sample component to a plurality of independent regions and separating and recovering each component from this region. Sorting becomes possible.
[0014]
Example I
FIG. 2 is a schematic diagram showing the basic configuration of the first embodiment of the present invention. Reference numeral 1 denotes a substrate on which a target polynucleotide to be separated and recovered is captured and fixed. Reference numeral 13 denotes a substrate base. Reference numeral 14 denotes a target polynucleotide capturing region, which is divided into about 1 micron square on the surface of the substrate base 13 by a light exposure technique and developed in two dimensions. On the surface of the target polynucleotide capture region 14, a plurality of probes (single-stranded oligonucleotides) having a base length of about 8-9 bases having different sequences for each region are bound. This probe is an oligonucleotide that is complementary to the target polynucleotide in the sample solution. Therefore, when the sample solution comes to the surface of the target polynucleotide capture region 14, the probe and the target polynucleotide are bound. That is, the target polynucleotide is captured by the probe. When the probe and the target polynucleotide are bound, the fluorescent dye is bound at the same time. A heater 31 is provided on the back surface of the substrate 1 and can heat the entire surface of the substrate 1 from about room temperature to about 95 ° C. Reference numeral 15 denotes an objective lens. The substrate 1 is disposed perpendicular to the optical axis of the objective lens 15. Reference numeral 16 denotes a fluorescence observation light source, and reference numeral 17 denotes a focused light irradiation infrared laser light source. Each of the light sources is arranged perpendicular to the optical axis of the objective lens 15. Reference numerals 181 and 182 denote dichroic mirrors, which are arranged at positions where the optical axis of the objective lens 15 and the optical axes of the light sources 16 and 17 intersect, and guide the light emitted from the light sources 16 and 17 to the objective lens 15. To do. Further, it is desirable to use light sources 16 having different wavelengths from each other for the light of the light source 16, the fluorescence emitted by the fluorescent dye, and the light of the light source 17. Reference numeral 151 denotes a band-pass filter, and the light from the light source 16 passes through this to be narrowed down to an appropriate wavelength for exciting the fluorescent dye. The excitation light guided to the objective lens 15 is collected, and excites the fluorescent dye that is captured on the substrate 1 and bound together with the fixed target polynucleotide. The excited fluorescent dye emits fluorescence with an intensity proportional to the amount of target polynucleotide captured by the probe in each region on the substrate. Reference numeral 183 denotes a mirror, and the fluorescence generated with the intensity proportional to the amount of the target polynucleotide reflects the light collected through the objective lens 15 again. Reference numeral 152 denotes an emission filter that selectively transmits only light in the wavelength region of fluorescence desired to be detected. Reference numeral 191 denotes a detector that detects the intensity of fluorescence transmitted through the emission filter 152.
[0015]
Therefore, the objective lens 15 and the substrate 1 are relatively moved, and the surface of the substrate 1 is sequentially scanned by the excitation light guided to the objective lens 15 to obtain a spatial distribution of the fluorescence intensity emitted by the fluorescent dye as a fluorescence image. By knowing more, the spatial distribution of the amount of the target polynucleotide in the sample solution captured by the probe immobilized on the substrate can be obtained as a fluorescence image. Here, as the excitation light used when observing the fluorescence of the target polynucleotide, it is desirable to use a fluorescent dye that emits fluorescence with light having a wavelength in the visible region when exciting the fluorescent dye bound together with the target polynucleotide. Instead of light emission by the fluorescent dye bound together with the target polynucleotide, autofluorescence of the target polynucleotide can be observed. In this case, it is desirable to use a near ultraviolet wavelength region of 280 nm to 400 nm as the wavelength of the excitation light.
[0016]
The fluorescence intensity of the fluorescent dye decreases according to the rate of temperature increase due to the thermal quenching effect. By utilizing this, in this example, it is possible to measure a local temperature rise in the target polynucleotide capture region on the substrate. In the apparatus shown in FIG. 2, the change in the fluorescence intensity of the fluorescent dye bound together with the target polynucleotide detected by the detector 191 is analyzed based on the temperature dependence of the fluorescence intensity change, and the target polynucleotide is analyzed. The temperature of the trapping region can be estimated.
[0017]
Infrared light generated by the infrared laser light source 17 is reflected downward by the dichroic mirror 182, collected by the objective lens 15, and applied to the target polynucleotide capture region 14 formed on the substrate 1. In the target polynucleotide capturing region 14, the thin film layer or fine particle layer formed on the substrate 1 absorbs infrared light and generates heat, so that the local temperature in the sample solution can be raised. The focusing position of the focused light from the laser light source focused on the surface of the substrate 1 by the objective lens 15 can be arbitrarily determined by moving the objective lens 15 or moving the substrate 1. In order to avoid damage to the captured target polynucleotide, it is desirable not to use light in the wavelength region where the target polynucleotide has absorption. For example, near infrared light having a wavelength of 800 nm or more and visible light having a wavelength of 400 nm or more are preferably used.
[0018]
In general, when a single-stranded polynucleotide such as DNA or RNA has a complementary relationship with another single-stranded polynucleotide such as a probe, a hydrogen-bonded base pair is formed to form a double-stranded polynucleotide. It is known that when the temperature of the sample solution is raised to about 95 ° C. or more, the double-stranded polynucleotide of the nucleic acid base pair derived from this hydrogen bond is dissociated into a single-stranded polynucleotide. Therefore, by irradiating the specific target polynucleotide capture region 14 on the substrate with infrared focused light and raising the temperature to 95 ° C., the target polynucleotide bound to the probe in the specific target polynucleotide capture region is selected. Can be separated.
[0019]
Further, when a surface electrode is arranged on the substrate 1 and an alternating electric field is applied between the surface electrode and the substrate 1, the probe on the substrate 1 and the polynucleotide in the sample solution on the substrate 1 can be extended. it can. As a result, nonspecific binding between the probe and the polynucleotide in the sample solution can be reduced, and dissociation of the probe and target polynucleotide pair by focused light can be effectively achieved.
[0020]
In addition, the dissociation temperature of the target polynucleotide hybridized with the probe increases in proportion to the amount of bound hydrogen bonds. Therefore, by adjusting the dissociation temperature, the degree of matching of the target polynucleotide and the probe can be matched. Depending on the selection, it is possible to sort the polynucleotides selectively. That is, the target polynucleotide bound to the probe begins to dissociate from the target polynucleotide with few hydrogen bonds as the temperature is gradually raised. Therefore, the output of the laser light source 17 is feedback-controlled based on the temperature information obtained by the analyzer unit 192, and the temperature of the target polynucleotide capture region 14 is adjusted to take into account the degree of specific binding. However, the target polynucleotide can be selectively collected.
[0021]
Further, by adding a quaternary ammonium salt such as tetraethylammonium chloride to the sample solution, it is possible to make the variation in the dissociation temperature of the target polynucleotide bound to the probe almost uniform.
[0022]
FIG. 3 shows a first means for heating a specific region of the target polynucleotide capture region 14 on the substrate. In this embodiment, the substrate base 13 is composed of a conductive film 131 and a heat conductive insulating substrate 132 disposed on the surface. On the conductive film 131, a plurality of target polynucleotide capture regions 141, 142, 143, 144, 145, and 146 are arranged. Each target polynucleotide capturing region has a two-layer structure of a light absorbing layer 21 such as an aluminum oxide thin layer and a heat insulating layer 22. In addition, probes (oligonucleotides) 41, 42, 43, 44, 45, 46 having a base length of about 8-9 bases are bound to the surface of each target polynucleotide capture region. In the figure, a state in which target polynucleotides 41 ′, 42 ′, 43 ′, and 46 ′ are bound to only probes 41, 42, 43, and 46 among these probes is schematically shown. Therefore, by identifying the target polynucleotide capture region by fluorescence observation, it is possible to estimate the correspondence of the target polynucleotide bound to the probe among the polynucleotides in the sample solution. In the example shown in the figure, the probes and the target polynucleotide have complementary binding in the regions 141, 142, 143, and 146, but the regions 144 and 145 have no complementary binding. This shows that there is no polynucleotide in the sample solution having a complementary relationship with the probes arranged in the regions 144 and 145.
[0023]
Next, the target polynucleotide capture region 142 in which the target polynucleotide 42 ′ is bound to the probe 42 is irradiated with the focused light 51 from the objective lens 15. The light absorbing layer 21 in the region 142 absorbs the focused light 51 and generates heat. When the vicinity of the region 142 reaches a high temperature of about 95 ° C. due to heat generation of the light absorption layer 21 in the region 142, the hydrogen bond between the probe 42 and the target polynucleotide 42 ′ is released, and the target polynucleotide captured in the region 142 Only 42 ′ is selectively removed from the substrate 1. In this case, if the size of the focused light focused region is smaller than the size of one target polynucleotide capture region, the optical axis may be adjusted so that the focused region is within the target polynucleotide capture region. . In addition, when the target polynucleotide capture region is smaller than the focus region of the focused light, the target polynucleotide capture region is heated so that only one region is heated by the focused light, with a sufficient gap between each target polynucleotide capture region. It is desirable to arrange the areas. In this figure, only one probe is shown in the target polynucleotide capture region in order to make the drawing easier to see, but actually, a plurality of probes having the same base sequence in each region are fixed. Is normal.
[0024]
In this embodiment, since the conductive film 131 is disposed on the surface of the substrate base 13, as described above, an electrode plate (not shown) facing the upper surface of the substrate 1 is provided, and an AC electric field is interposed between the two. The probe or polynucleotide can be stretched by applying. Accordingly, erroneous binding at the time of binding, and steric entanglement when the target polynucleotide is dissociated from the probe by heating the target polynucleotide capture region can be prevented, and processing can be performed efficiently.
[0025]
Furthermore, by using the thermally conductive insulating substrate 132 for the substrate base 13, the cooling effect when the focused light 51 is not irradiated can be improved.
[0026]
The light absorption layer 21 in the present embodiment may be produced by vapor deposition, but may be produced by coating and spreading. The shape of the target polynucleotide capture region may be a square, a rectangle, or a circle or an ellipse that matches the shape of the focused light by using an optical lithography mask.
[0027]
FIG. 4 shows a second means for heating a specific area on the substrate 1. In the example of FIG. 3, the thin layer 21 having the light absorption property is formed in the target polynucleotide capture region. However, in this embodiment, the thin layer 21 has the light absorption property and is larger than the size of the target polynucleotide capture region. In addition, fine particles 23 having sufficiently small light absorption characteristics are arranged dispersed in the target polynucleotide capture region. It is assumed that one or more fine particles are placed in each region. Also in this embodiment, the heat insulating layer 22 is provided in each region, and the fine particles 23 are arranged on the upper surface thereof. The substrate 1 is the same as the example of FIG. 3 in that the substrate 1 is composed of a substrate base 13 composed of a conductive film 131 and a thermally conductive insulating substrate 132.
[0028]
When the specific target polynucleotide capture region 142 is irradiated with the focused light 51, the microparticles 23 in the region absorb the light and generate heat, and only the vicinity of the region 142 can be heated. Only the target polynucleotide bound to can be specifically dissociated from the probe. In this embodiment, by using the fine particles 23, it is possible to specifically heat a region smaller than the focusing range of the focused light in the example of FIG. 3, but the target polynucleotide capture region and the focused light focusing region The size relationship is the same as in the example of FIG. The light absorption layer composed of the fine particles 23 in this embodiment can be produced by applying and dispersing fine particles. Also, the shape can be any shape as in the example of FIG.
[0029]
FIG. 5 shows a third means for heating a specific area on the substrate 1. In the present embodiment, similarly to FIG. 4, the temperature rise by the fine particles 24 having absorption in the wavelength region of the focused light is used. However, the fine particles 24 are captured by the focused light 51 in the manner of optical tweezers. The difference is that the microparticles 24 are arranged in a target polynucleotide capture region where it is desired to dissociate the probe and the target polynucleotide. At this time, the numerical aperture of the objective lens 15 is desirably 12 or more based on the characteristics of the optical tweezers. Also in this embodiment, the heat insulating layer 22 is provided in each region. The substrate 1 is the same as the example of FIG. 3 in that the substrate 1 is composed of a substrate base 13 composed of a conductive film 131 and a thermally conductive insulating substrate 132. Since it is the same as that of FIG. 4 except that the fine particles 24 are captured in the manner of optical tweezers, the other explanation regarding the drawing is omitted.
[0030]
In this embodiment, since the temperature rise by the fine particles 24 is only in the vicinity of the fine particles 24 in the region where the fine particles 24 are arranged, the size of the target polynucleotide capturing region and the size of the focused region of the focused light are essentially related. Absent. That is, even when the focused region of the focused light covers a plurality of target polynucleotide capture regions, the temperature rise by the microparticles 24 is only in the vicinity of the microparticles 24 in the region where the microparticles 24 are disposed. Is limited to that of the target polynucleotide capture region in which the microparticles 24 are arranged. Therefore, in this example, there is an advantage that the density of the target polynucleotide capture region can be increased.
[0031]
FIG. 6 shows an example of a more detailed structure centering on the relationship with the substrate 1 of the polynucleotide separation cell 7 using the basic configuration of this embodiment shown in FIG.
[0032]
In this figure, a part of the polynucleotide separation cell 7 is cut away so that the inside can be seen. FIG. 7 shows an AA cross section of this polynucleotide separation cell, and FIG. 8 shows a BB cross section.
[0033]
The polynucleotide separation cell 7 has three sample solution chambers 731, 732, and 733 that are partitioned by a cell upper plate 721, a cell lower plate 722, and a plurality of spacers 723. The cell upper plate 721 is made of a light transmissive material. Each of the three sample solution chambers can take in and out the sample solution from the sample solution inlet / outlet 711, 712, and 713. Further, communication holes 714 and 715 are provided between the sample solution chambers, and the sample solution is moved through these holes. The substrate 1 on which the probe shown in FIG. 2 is fixed is fixed on the cell lower plate 722 of the sample solution chamber 732, and an electrode plate 131 for applying an electric field is incorporated on the substrate 1. Further, electrodes 751, 752, 754, and 755 are added to the side wall surfaces of the sample solution chambers 731, 732, and 733, respectively, and an electric field is applied to the inner surface of the cell upper plate above the sample solution chamber 732. Electrodes 753 are arranged in a mesh shape. The temperature of the substrate 1 can be adjusted from 0 ° C. to 95 ° C. by the Peltier elements 32, 34, and 35 with the heat sink 33. The temperature of each of the sample solution chambers 731, 732, and 733 can be adjusted from 0 ° C. to 95 ° C. by operating each of the Peltier elements 32, 34, and 35 independently. A PCR reaction or a denaturation reaction can be performed without changing. Further, in order to move the position of the substrate 1 to be observed with the objective lens 15, the cell 7 is fixed on rails 742, 743, and 744 by a jig 741, and freely on a two-dimensional surface by a stepping motor (not shown). Can be moved.
[0034]
9A to 9C show time tables for explaining the nucleic acid separation process using the polynucleotide separation cell shown in FIGS. 6-8. First, the sample solution containing the polynucleotide introduced into the sample solution chamber 731 is subjected to PCR amplification as a pretreatment by giving a temperature change with time as shown in FIG. 9A for about 20 to 30 cycles. That is, a double-stranded polynucleotide is converted into a single-stranded polynucleotide during the first denaturation process. Subsequently, annealing with the probe in the sample solution and subsequent polymerase extension reaction are performed, and the process of doubling the polynucleotide is repeated. The temperature control at this time is performed by the Peltier element 32 and is performed on the entire sample solution in the sample solution chamber 731. Next, in the sample solution containing the amplified polynucleotide, only the polynucleotide having a specific base sequence is separated in the process as shown in FIG. 9B. That is, in the sample solution introduction process, the electrodes 751 and 755 are used as the cathode, the electrode 754 and the electrode on the substrate 1 are used as the anode, and the polynucleotide in the sample solution in the sample solution chamber 731 is guided to the sample solution chamber 732. Next, the sample solution in the sample solution chamber 732 is heated to 95 ° C. by the Peltier element 34 to remove the hydrogen bond of the polynucleotide in the sample solution to form a single strand. Next, the temperature is lowered to 37 ° C., and binding with the probe in the target polynucleotide capture region on the substrate 1 is performed. Then, the polynucleotide in the sample solution that has not bound to the probe in the capture region is returned to the sample solution chamber 731 again by using the electrode 751 as an anode. Next, to which region on the substrate 1 the target oligonucleotide is bound is confirmed by a fluorescence observation technique. Then, based on the information of the region where the target polynucleotide is bonded to the probe on the substrate 1 (output of the fluorescence intensity detector 191 in FIG. 2), one target polynucleotide capture region on the substrate 1 is irradiated with focused light. Thus, the target polynucleotide bound to the probe in that region can be removed from the probe. At this time, an alternating electric field is generated between the electrode 753 and the electrode 131 on the surface of the substrate 1 to extend the target polynucleotide. Furthermore, the focused light 51 is applied from the lens 15 and the target target is placed in the sample solution chamber 733 by using the electrode 755 as an anode while dissociating only the target polynucleotide in the specific target polynucleotide capture region on the substrate 1 from the probe. Only the polynucleotide is introduced. At this time, by keeping the electrode 751 at the anode and the electrode 752 as the cathode, the polynucleotide not bound to the probe in the sample solution in the sample solution chamber 731 is held in the sample solution chamber 731, and the sample solution chamber It does not move to 732 or 733. Furthermore, components necessary for PCR are introduced into the sample solution chamber 733 from the solution inlet / outlet 713 while applying an electric field to the electrode. Thereafter, the desired polynucleotide is selectively separated from the sample solution introduced into the sample solution chamber 733 with high accuracy and high speed by performing the post-processing PCR shown in FIG. 9C for about 20 to 30 cycles by the Peltier element 35. It can be recovered and amplified. Also, once the amplified sample solution in the sample solution chamber 733 was recovered, the substrate 1 was moved, and the other target polynucleotide capture region on the substrate 1 was again shown in FIG. By repeating the separation process and the post-processing PCR process shown in FIG. 9C, the polynucleotide captured in each target polynucleotide capture region of the substrate 1 can be selectively recovered and amplified sequentially.
[0035]
In the time schedule described with reference to FIGS. 9A to 9C, no special operation has been described for the temperature decrease. For example, the Peltier elements 32, 34 and 35 in FIG. 6 are actively used for heat dissipation. Accordingly, the entire room can be cooled in a short time. In addition, it is possible to provide a Peltier element for each target polynucleotide capture region for cooling after local denaturation to dissociate the polynucleotide bound to the probe of the target polynucleotide capture region. Needless to say.
[0036]
Example II
In this example, in order to dissociate the target polynucleotide bound to the probe in Example I, the temperature of each target polynucleotide capture region was controlled by convergent light irradiation, and the heat generated embedded in the substrate 1 was used. It differs only in the point of using a structure that utilizes the body, but is otherwise essentially the same. This will be described below with reference to FIGS.
[0037]
FIG. 10 is a diagram showing the relationship between the substrate 1 having the target polynucleotide capture region and the optical system for detecting the capture state in the target polynucleotide capture region according to the second embodiment of the present invention. As is clear in comparison with FIGS. 10 and 2 and 3, in this embodiment, the substrate 1 is composed of the conductive film 131 having the target polynucleotide capture region on the surface, and then the temperature controller 133 and the thermal conductivity. The substrate base 13 is composed of an insulating substrate 132. In addition, a plurality of target polynucleotide capture regions 141, 142, 143, 144, 145, and 146 are disposed on the surface of the conductive film 131, and each target polynucleotide capture region is placed inside the temperature control unit 133. An electrode for independently controlling the temperature and a heating element that generates heat are embedded. Each target polynucleotide capture region has a two-layer structure of a probe binding layer 221 such as a thin aluminum oxide layer and an electrical insulator layer 222. Furthermore, probes (oligonucleotides) 41, 42, 43, 44, 45, 46 having a base length of about 8-9 bases are bound to the surface of each target polynucleotide capture region. In the figure, the target polynucleotides 41 ′, 42 ′, 43 ′, and 46 ′ having a complementary relationship with only the probes 41, 42, 43, and 46 among these probes are schematically illustrated. Is shown.
[0038]
Also in this example, when the target polynucleotides 41 ′, 42 ′, 43 ′, and 46 ′ are combined with the probes 41, 42, 43, and 46, the fluorescent dyes are excited simultaneously. Then, the fluorescence emitted from this can be detected, and the degree of binding between the probe and the target polynucleotide in each region can be estimated from the fluorescence intensity. Furthermore, the local temperature rise of the target polynucleotide capture region can be measured by utilizing the fact that the fluorescence intensity of the fluorescent dye decreases according to the rate of temperature rise due to the thermal quenching effect due to the temperature rise. Further, the change in the fluorescence intensity of the fluorescent dye can be analyzed based on the temperature dependence of the fluorescence intensity change, and the temperature of the target polynucleotide capture region can be estimated. In this example, the same reference numerals are assigned to the same or equivalent parts as in Example I.
[0039]
FIG. 11 is a cross-sectional view taken along the line AA of the substrate 1 shown in FIG. The substrate 1 has a structure in which a heat conductive insulating substrate 132, a temperature control unit 133, and a conductive film 131 are laminated. On the surface of the uppermost conductive film 131, a two-layer structure of a probe binding layer 221 serving as a target polynucleotide capturing region and an electrical insulator layer 222 is formed. The temperature control unit 133 includes a surface electrode 226 layer, a heating element 225 layer, and a surface electrode 224 layer. The electrodes of the surface electrode 226 layer and the surface electrode 224 layer intersect each other in a matrix shape, and the intersecting position corresponds to the position of each heating element 225 and corresponds to the position of each target polynucleotide capture region. It is formed to do. A layer of a thermistor 231 for temperature measurement corresponding to each target polynucleotide capture region is embedded in the heat conductive insulating substrate 132, and the temperature of each target polynucleotide capture region can be measured.
[0040]
12A to 12C are cross-sectional views of a BB cross section, a CC cross section, and a DD cross section of the temperature control unit 133 of the substrate 1 of the second embodiment. The surface electrode 226 and the surface electrode 224 are orthogonal to each other, and the heating element layer 225 is sandwiched between the intersections. Therefore, when a current is applied by applying a potential difference between the surface electrode 226 and the surface electrode 224, only the heating element 225 at this intersection generates heat. That is, when the surface electrode 226 and the surface electrode 224 are appropriately selected and a potential difference is applied, the temperature of the target polynucleotide capture region at the position corresponding to this intersection can be increased.
[0041]
Although the substrate 1 shown in FIG. 10 can be replaced as the substrate 1 of the polynucleotide separation cell 7 shown in FIGS. 6-8, a simpler example will be described here.
[0042]
FIG. 13 shows a block diagram of the polynucleotide separation cell 251 and related devices of the second embodiment. FIG. 14 shows a GG sectional view of the polynucleotide separation cell 251 of the second embodiment. The polynucleotide separation cell 251 is made of a light transmissive container so that the substrate 1 in the cell can be optically observed. The cell 251 is joined with a sample solution injection tube 252 for injecting a sample containing a polynucleotide and an extraction tube 253 for taking out a target polynucleotide captured in a specific target polynucleotide capture region of the substrate 1. The solution can flow in the direction of. Further, a transparent electrode (not shown) is disposed on the upper surface of the cell 251, and a potential difference can be given to the conductive film 131 of the substrate 1. In order to generate a heating element corresponding to a specific target polynucleotide capture region or to generate a DC electric field or an AC electric field in the target polynucleotide capture region, this embodiment uses a DC power supply 254, an AC power supply 255, and a control circuit 256. The state of the substrate 1 can be controlled based on the temperature information from the thermistor 231 according to the procedure shown later.
[0043]
FIG. 15 shows a timetable for explaining the separation process of the polynucleotide separation cell of the second embodiment.
[0044]
From the sample introduced into the cell 251, only a polynucleotide having a specific base sequence is separated in the process shown in FIG.
[0045]
(1) In the process of introducing the sample solution, the conductive film 131 of the substrate 1 is used as an anode and the transparent electrode on the upper surface of the cell is used as a cathode, so that the polynucleotide in the solution is guided to the surface of the substrate 1. Alternatively, the polynucleotide may be elongated by generating an alternating electric field between the conductive film 131 of the substrate 1 and the transparent electrode on the upper surface of the cell. In this state, all the electrodes 224 and 226 in the temperature control unit 133 are made conductive, all the heating elements 225 generate heat, and the temperature of the surface layer of the substrate 1 is raised to 95 ° C.
[0046]
(2) Next, the temperature is lowered to 37 ° C. As a result, the probe immobilized on the target polynucleotide capture region and the target polynucleotide in the sample solution bind to each other.
[0047]
(3) Next, the surface layer of the substrate 1 is raised to about 72 ° C., and a polynucleotide non-specifically bound to the probe with the conductive film 131 of the substrate 1 as a cathode and the transparent electrode on the upper surface of the cell as an anode is sampled. Remove with solution.
[0048]
(4) After exchanging the solution in the cell 251, each one of the electrodes 224 and 226 in the temperature controller 133 is turned on, one of the heating elements 225 is heated, and one of the target polynucleotide capture regions is The temperature is raised to 95 ° C., and the target polynucleotide bound to the probe in this region is dissociated and collected together with the solution.
[0049]
(5) By performing the same procedure for each target polynucleotide capture region, the target polynucleotide bound to the probe in each region can be sequentially taken out.
[0050]
In this case, in the same manner as described in Example I, the binding state of the target polynucleotide for each target polynucleotide capture region is detected in advance based on the temperature information from the detector 191 and the analysis device 192 and recovered. If the above-described temperature control is performed only for the target polynucleotide capture region to which the desired polynucleotide is bound, the processing efficiency can be increased.
[0051]
Example III
The third embodiment relates to the configuration of the substrate 1 having the target polynucleotide capture region devised to stably fix the probe. Specifically, the present invention proposes a substrate 1 having a structure in which the surface of the capture region is a metal film having an oxide film layer and the probe can be stably fixed by a silane coupling reaction.
[0052]
A procedure for manufacturing the substrate 1 according to this embodiment will be described. A glass substrate having a thickness of 04 mm and a square of 24 mm is prepared, immersed in a NaOH (1M) solution, and subjected to ultrasonic cleaning for 30 minutes. After washing with running ultrapure water, bake at 110 ° C. for 15 minutes. Chromium (Cr) is deposited using a vacuum deposition apparatus. The thickness is 3 nm. Wash with ethanol. Immerse in a stock solution of 3-glycidoxypropyltrimethoxysilane for 5 minutes, then immerse in a solution of 4% 3-glycidoxypropyltrimethoxysilane dissolved in 50% ethanol solvent for 30 minutes and occasionally stir. The substrate 1 is obtained by removing from the solution, baking at 110 ° C. for 30 minutes, and introducing a glycidoxy group onto the metal surface with a silane coupling reagent.
[0053]
FIG. 16 is a diagram showing a state in which the probe can be stably fixed on the substrate 1 according to the third embodiment. In the substrate 1 of this embodiment, a Cr layer 303 is deposited on the surface of a glass substrate 302, and the surface thereof is an oxide film 304. The probe is fixed by the silane coupling layer 305. In order to confirm that the probe can be stably immobilized, in this example, the ends of the probes 311 and 312 immobilized on the target polynucleotide capture regions 306 and 307 via the silane coupling layer 305 are terminated with a fluorescent dye. Polynucleotides 521 bp and 625 bp long labeled with followodamine 101 were conjugated, respectively.
[0054]
The result of a comparative experiment conducted to confirm the effect of the substrate 1 according to this example will be described.
[0055]
First, a substrate having the structure of this embodiment, a substrate in which aluminum (Al) is deposited on the surface of the glass 302 instead of Cr, and a substrate in which the surface of the glass 302 is directly subjected to silane coupling are compared. Two substrates were prepared by depositing Cr over the entire surface of the glass 302 with a thickness of 54 nm as substrates having the structure of this example. The light transmittance at 600 nm to 800 nm was 53 to 56%. The thickness of the Al film of the substrate on which Al was deposited over the entire surface of the glass 302 instead of Cr was 15 nm. The manufacturing procedure is the same as in this embodiment. The light transmittance of 600 nm to 800 nm of this substrate was 19 to 25%. The light transmittance of 600 nm to 800 nm of the glass itself on which no metal film is deposited is approximately 84%. With such a transmittance, a target polynucleotide labeled with a fluorescent dye such as sulforhodamine 101 (absorption maximum 594 nm, fluorescence 615 nm) is excited with argon (Ar, 514 nm) or helium-neon (He-Ne, 545 nm). ) To detect the target polynucleotide captured by the probe by fluorescence.
[0056]
FIG. 17A to FIG. 17C are diagrams for explaining experimental results for confirming the effect of the substrate of the third embodiment.
[0057]
17A is a substrate of the third embodiment in which Cr is deposited to 54 nm, FIG. 17B is a substrate in which Al is deposited to a thickness of 15 nm instead of Cr, and FIG. 17C has a metal deposited film. Each of the non-substrates was subjected to a treatment of binding 521 bp and 625 bp long polynucleotides labeled with the fluorescent dye sulfododamine 101 to the probes 311 and 312 immobilized via the silane coupling layer 305, respectively. It was. The result of having measured with the laser confocal microscope in this state about the relative fluorescence intensity and the transmittance | permeability is shown. The horizontal axis represents the scan position on the substrate, and the vertical axis represents the solid line 330 showing the fluorescence (580 nm or more) distribution and transmitted light intensity distribution detected by the laser microscope. A wavy line 331 shows a transmittance distribution from 380 nm to 450 nm.
[0058]
As can be seen from FIG. 17 (A), in the substrate of this example, a constant fluorescence intensity 330 was obtained over the entire area CA where the polynucleotide labeled with the fluorescent dye sulfododamine 101 was bound. The fluorescence intensity 330 from other parts is sufficiently low. Further, the transmittance from 380 nm to 450 nm is almost constant at any part. This indicates that the Cr layer is stably held in various reaction operations from the probe fixation to the binding of the target polynucleotide, and there is no peeling from the surface of the glass 302.
[0059]
As can be seen from FIG. 17 (B), in the substrate on which Al is deposited instead of Cr, the transmittance of the ridge region CA to which the polynucleotide labeled with the fluorescent dye sulfodamine 101 is bonded is remarkably increased. ing. This means that the Al vapor deposition surface is dissolved in the probe fixing reaction. As for the Al vapor deposition surface, the result which dissolves with the 10mM Tris hydrochloric acid-1mM EDTA buffer (pH75) which is the general solution which dissolves DNA and RNA is obtained, the substrate where Al vapor deposition is done in the reaction system which uses aqueous solution You can see that it is not available. As a result, the probe is not substantially immobilized and, therefore, there is no binding of the polynucleotide labeled with the fluorescent dye sulfodamine 101, and the fluorescence intensity 330 is sufficiently low as a whole.
[0060]
As can be seen from FIG. 17C, in the substrate on which the probe is fixed by direct silane coupling reaction on the glass surface, the region CA to which the polynucleotide labeled with the fluorescent dye sulfodamine 101 is bonded is another probe. Since the fluorescence intensity 330 is higher than the portion where the probe is not fixed, it can be seen that the function of fixing the probe can be achieved. However, the fluorescence intensity 330 varies from place to place as compared to the substrate of this example. This suggests that the probe is not fixed uniformly.
[0061]
As is clear from these results, it can be seen that the substrate according to the present example on which Cr was deposited is advantageous for stably holding the probe and capturing the target polynucleotide. In addition, it turns out that what was vapor-deposited on the glass substrate to the thickness of several nanometers to 10 nm with the metal stable not only in Cr but a weak alkaline solution is excellent. Specifically, it is sufficient if the surface is covered with an oxide film and difficult to dissolve in a weak alkaline solution at 95 ° C. However, since gold and platinum do not form an oxide film, a silane coupling reaction cannot be performed. So it is not available. Stainless steel is stable to weak alkali and has properties similar to Cr, such as forming an oxide film. However, it is excluded because the active residue cannot be introduced using a silane coupling reaction for unknown reasons. From the same examination, it is known that at least one of Ti, V, Cr, Fe, Co, Ni, Mo, and W is effective.
[0062]
Next, with reference to FIG. 18 (A) and FIG. 18 (B), it will be described that the target polynucleotide captured by the probe can be effectively dissociated and recovered by the substrate according to this example. 18A and 18B show the regions 306 and 307 shown in FIG. 16 in which the 5 ′ end captured by the probe immobilized through the silane coupling layer 305 is sulfodermine 101, respectively. It is an experimental result about the labeled polynucleotide of 521 bp and 625 bp. The substrate surface is covered with 20 microliters of 20 mM Tris-HCl buffer (pH 75).
[0063]
18A and 18B show the results of fluorescence detection (510 nm to 580 nm) by laser scanning (514 nm) across the respective regions 306 and 307. The horizontal axis represents the scan position on the substrate, and the vertical axis represents the relative fluorescence intensity distribution detected by the laser microscope. Reference numeral 335 denotes a line indicating the relative fluorescence intensity, and almost constant fluorescence is detected over the entire area of each of the regions 306 and 307. Next, only the region 306 is scanned with a 1053 W YAG laser (spot diameter 5 nm). As a result, as indicated by a broken line 336 in FIG. 18A, the relative fluorescence intensity distribution after scanning was reduced to about 1/10 in the portion 333 scanned by the YAG laser. In the region 307, there is no change in both the relative fluorescence intensities 335 and 336.
[0064]
FIG. 19 is a diagram schematically showing the results of electrophoresis for explaining the fractionation of polynucleotides according to this example.
[0065]
Reference numeral 340 denotes a marker lane. Reference numerals 341 and 342 denote lanes showing the results of preparing the same polynucleotides captured in the regions 306 and 307 of the substrate 1 and measuring them by electrophoresis. Respectively, 521 bp and 625 bp bands appear clearly. 343 and 344 collect the buffer solution on the surface of the substrate 1 after scanning the region 306 of the substrate 1 with 1053 nm YAG laser 10 mW (spot diameter 5 nm), amplify the PCR, and 2% agarose gel electrophoresis of the PCR amplification product It is a lane which shows the result measured by. A 521 bp band is clearly detected in the lane 343, but no 625 bp band appears in the lane 344. This indicates that only the polynucleotide dissociated from the region 306 scanned with YAG laser 10 mW (spot diameter 5 nm) was present in the solution obtained by PCR amplification of the recovered buffer. According to this example, the probe can be immobilized and the captured polynucleotide can be effectively separated.
[0066]
Example IV
While the substrate 1 is flat in Example I-III described above, in the fourth example, the structure utilizing the inner surface of the capillary tube reduces the amount of sample solution required for polynucleotide separation. To do.
[0067]
20 and 21 are an outer surface of a thin tube that can be adopted in the fourth embodiment and an AA cross-sectional view.
[0068]
Reference numeral 401 denotes a thin tube, which is a light-transmitting thin tube such as a glass thin tube. Reference numeral 412 denotes a tube wall, and an inner tube coating 413 made of a metal such as chromium shown in Example III is applied to the inner surface thereof. The surface of the coated surface is a stable oxide generated by air oxidation of metal. Target polynucleotide capture regions 414 and 415 each having a probe immobilized in a cylindrical shape are disposed on the coated surface. In each target polynucleotide capture region 414, 415, a probe specific to each region is closely fixed, and only polynucleotides complementary to these among the polynucleotides in the sample solution are captured in the respective regions. Can do. Further, a marker 411 is marked on the outer surface of the thin tube 401 at the boundary position of each target polynucleotide capture region on the inner surface, and the position of each target polynucleotide capture region can be confirmed from the outer surface of the thin tube.
[0069]
FIG. 22 is a diagram for explaining an example of a structure for fixing a probe to a target polynucleotide capture region.
[0070]
In fact, as shown in FIG. 21, in order to form concentrically independent oligonucleotide probe layers on the inner surface of the capillary tube, for example, the following technique is used. First, the entire area of the thin tube 401 is reacted with a bivalent reagent having silanol groups and epoxy groups at both ends, so that the epoxy groups 416 are grown on the entire surface of the tube. Then, the inner wall of the tube has water repellency and repels the sample aqueous solution. Next, a nucleic acid sample solution to be bound to the surface of the specific region 415 is introduced into the tube 401 in the form of droplets. Then, since the inner wall of the tube has water repellency, the liquid becomes spherical and comes into concentric contact with the inner wall of the tube with a minimum contact area. This droplet is guided to the region 415 and left for a while to bind the free end of the epoxy group 416 and the nucleic acid probe whose amino group is modified at the 5 end only in a predetermined region. The sample droplet containing the reacted nucleic acid probe is taken out from the thin tube 401 immediately after the reaction. Similarly, a structure as shown in FIG. 21 can be formed by repeatedly introducing different sample droplets and allowing them to react in different regions.
[0071]
The time table for sorting the target polynucleotides in the sample solution according to the fourth embodiment is the same as that shown in FIG.
[0072]
FIG. 23 is a diagram showing the overall configuration of the polynucleotide separating apparatus of the fourth embodiment. Reference numeral 431 denotes a polynucleotide separation module, in which the thin tube 401 described in FIGS. 20 and 21 is incorporated. Both ends of the thin tube 401 are joined to the thin tube joint portions 432 and 433. The sample solution held in the container 434, the cleaning liquid held in the container 435, the cleaning liquid held in the container 436, and the air that has passed through the air filter 437 are selected by the selector 438 and introduced into the narrow tube joint 432 by the pump 439. . Reference numeral 400 denotes a controller, which is representative of the control and power supply means of the apparatus of this embodiment. The solution discharged from the polynucleotide separation module 431 passes through the capillary joint 433 and is sent to a post-processing process 441 such as PCR amplification. The controller 400 collects necessary data from each part of the device, and sends necessary operation signals to each part accordingly. Details on the specific configuration and connection between each part of the device will be described in detail. Omitted. Those skilled in the art can easily realize the above-described time table and the following description.
[0073]
FIG. 24 is a diagram showing a BB cross section of the polynucleotide separation module 431 of the fourth embodiment. In the module 431, a light source 442 that introduces light into the tube wall of the thin tube 401, a donut-shaped heat source 443 corresponding to each target polynucleotide capture region of the thin tube 401, and excitation light that is plasmon excited from the thin tube to the inner surface A light detection probe 444 for detecting the fluorescence of the excited target polynucleotide capture region or the marker 411 on the surface of the capillary tube and a heat detection probe 445 for detecting the temperature of the capillary tube are arranged in parallel. Electrodes 451 and 452 are disposed in the thin tube joints 432 and 433, respectively, and the sample solution components in the thin tube 401 can be collected by electrophoresis. In addition, an electric potential can be applied to the inner surface coating 413 of the thin tube 401 by the electrode connector 453, whereby the nucleic acid component can be moved away from or pulled away from the surface of the inner surface of the thin tube.
[0074]
FIG. 25 is a diagram showing a modification of the polynucleotide separation module 431 of the embodiment shown in FIG. In this example, the basic configuration is the same as that of the example shown in FIG. 24, except that the cleaning liquid introduced when recovering the separated polynucleotide is changed to droplets 471. By adjusting the selector 438 shown in FIG. 23, the droplet 471 of the cleaning liquid in the tank 436 is introduced into the narrow tube 401, and then the droplet 471 is pushed out by the air that has passed through the air filter 437 to capture the target polynucleotide to be collected. Advance to the area so that the droplet 471 covers the area. Next, the leak valves 461 and 462 are opened to release the air in the narrow tube 401 expanded by the local heat generation by the heat generation source 443 so that the position of the droplet 471 does not move. Thereafter, the temperature of the region is raised above the dissociation temperature to dissociate the target polynucleotide from the probe. Next, the leak valves 461 and 462 are closed, and the target polynucleotide is recovered by pushing out the droplet 471 to the capillary joint 433 by the air that has passed through the air filter 437. In this example, since the droplet contacts the specific target polynucleotide capture region, even if the temperature of the other region reaches the dissociation temperature, it does not get mixed in the droplet and is higher. Recovery separated with high accuracy can be performed. In this example, the heat generation is local heat generation by the heat generation source 443 in the polynucleotide separation module 431. However, when one heat generation source having a shape covering the entire target polynucleotide capture region is used, high-precision separation is similarly performed. Can be recovered.
[0075]
FIG. 26 is a diagram showing a modification of the polynucleotide separation module 431 of the embodiment shown in FIG. In this example, the light source 442, the heat generation source 443, the light detection probe 444 and the heat detection probe 445 provided inside the narrow tube 401 are omitted, and an optical system instead of this is provided outside the thin tube 401. In other words, the structure shown in FIGS. 2 and 6 which is an embodiment of the embodiment I is a form using the thin tube 401 instead of the substrate 1. Therefore, although the same thing as the optical system of FIG. 2 can be used, only the objective lens 15 is shown in the figure for simplification.
[0076]
The capillary 401 joined to the capillary joints 432 and 433 is moved on the rail 446, and the objective lens 15 observes the state of the predetermined polynucleotide capture region in the capillary and simultaneously irradiates the region of the capillary with the focused light. Thus, only the vicinity of the focusing point in the narrow tube can be locally heated. At this time, if the tubule has a structure capable of rotating in the circumferential direction, the capture state of the polynucleotide in the entire region in the circumferential direction in the tubule can be observed, heated, and recovered.
[0077]
FIG. 27 is a diagram showing a modification of the polynucleotide separation module 431 of the embodiment shown in FIG. This example is the same as FIG. 26 in that an optical system is provided outside the capillary tube 401, but only one set of the same optical system provided in each target polynucleotide capture region inside the capillary tube is provided outside the capillary tube. The module 431 is different in that it is structured to move on the rail 481 with respect to the thin tube 401. Thereby, the apparatus can be reduced in size as a whole.
[0078]
Example V
In Examples I-IV above, the target polynucleotide was in the sample solution, but this example is intended to fractionate the target polynucleotide directly from the cell itself. is there. Of course, even in this example, the sample solution is eventually collected in the form of a sample solution containing the polynucleotide, but after the cells are introduced into the target polynucleotide capture region, the sample solution is formed. As a result, the presence ratio of the target polynucleotide in the sample solution becomes high, and the number of cases where PCR or the like as pretreatment can be omitted increases. Moreover, according to the present Example, contents, such as a nucleic acid of a cell and a protein, can also be obtained for every cell.
[0079]
FIG. 28 is a diagram showing basic elements of this embodiment.
[0080]
Reference numeral 1 denotes a substrate, on which a target polynucleotide capture region 511 similar to that described in the previous example is formed. Reference numeral 521 denotes a controller, and in order to capture and fix the cells 561 in the target polynucleotide capture region 511, a direct current or an alternating electric field is applied between the electrode of the capture region 511 and the ground electrode 514 provided to face the substrate 1. Apply. 526 is a power supply circuit for applying or grounding a DC or AC electric field, and 525 is a selection switch. 512 is a Peltier element, and 513 is a temperature sensor, which is embedded in the substrate 1 in the vicinity of each target polynucleotide capture region. A temperature measurement and control unit 523 inputs a signal from the temperature sensor 513, and controls the surface temperature of each target polynucleotide capture region independently by the Peltier element 512. Which region to control the temperature depends on an instruction from the controller 521.
[0081]
FIG. 29 is a diagram showing a basic configuration of this example in which a polynucleotide separation cell having the basic elements described in FIG. 28 and an optical system are combined. FIG. 30 is a view showing an AA cross section of the separation cell described in FIG.
[0082]
541 is a polynucleotide separation cell, 542 and 543 are a sample introduction tube and a sample discharge tube, through which a cell sample or a washing solution is introduced into the separation cell, and cell debris, protein, and purified nucleic acid from the separation cell Samples are collected. As shown in FIG. 30, a target polynucleotide capture region is formed on the substrate 1 inside the separation cell 541. In this embodiment, a cell sample introduced from the sample introduction tube 542 is used as a target polynucleotide capture region. Electrodes 551, 552, 553, ---- are arranged, and wirings are connected to them. In this figure, the upper electrode 514 does not appear. Probes that are single-stranded oligonucleotides complementary to the respective target polynucleotides are immobilized on each target polynucleotide capture region.
[0083]
The inside of the polynucleotide separation cell 541 can be monitored by the same optical system as described in FIG. That is, the substrate 1 is arranged perpendicular to the optical axis of the objective lens 15, and excitation light from the fluorescence observation light source 16 is guided to the objective lens 15 via the bandpass filter 151 and the dichroic mirror 181. The fluorescent dye bound to the target polynucleotide captured and immobilized on the substrate 1 is excited by the excitation light induced by the objective lens 15, and the excited fluorescent dye is captured by the probe in each region on the substrate. Emits fluorescence with an intensity proportional to the amount of target polynucleotide. Fluorescence generated with an intensity proportional to the amount of the target polynucleotide is collected again through the objective lens 15, and only light in the wavelength region of the fluorescence to be detected is selectively passed through the emission filter 152 to the detector 191. It can be introduced to know the inside of the separation cell 541.
[0084]
In general, when a cell is heated, part of the cell membrane is broken and the contents such as nucleic acid and protein are released. Therefore, after introducing a sample solution containing cells into the polynucleotide separation cell 541, the temperature of a specific target polynucleotide capture region on the substrate 1 is raised to 95 ° C., thereby destroying the cells in that region and As well as releasing the product, the double-stranded polynucleotide can be dissociated into a single-stranded polynucleotide. Thereafter, when the temperature of the capture region is lowered to 37 ° C., a single-stranded polynucleotide complementary to the probe in that region of these target polynucleotides can be captured by the probe. In order to control the temperature of a specific capture region, a Peltier element 512 corresponding to this region may be selected to pass a current.
[0085]
Hereinafter, with reference to FIG. 31 (A) -FIG. 31 (E) and FIG. 32 (A) -FIG. 32 (D), a method for fractionating cell contents and target polynucleotides directly from blood cells according to this example. Will be explained.
[0086]
First, the ionic strength of a blood sample is reduced, erythrocytes are destroyed, and the cells in the blood sample are prepared so that only white blood cells are present. FIG. 31A is a diagram showing a state where target polynucleotide capture regions 551, 552, and 553-- are formed on the surface of the substrate 1. FIG. FIG. 31B is a diagram showing a state in which a blood sample is introduced into the polynucleotide separation cell 541 and leukocytes 561 are straying on the surface of the substrate 1. In this state, an alternating electric field is applied between the electrodes of the target polynucleotide capture regions 551, 552, 553 --- and the electrode 514, and the leukocytes 561 are attracted to the target polynucleotide capture region. At this time, the gradient of the electric field density generated between the electrode 514 and the electrode of each target polynucleotide capture region becomes dense in each target polynucleotide capture region. Moreover, as an alternating electric field used at this time, the frequency of 1 kHz or more and 10 V / mm or more are desirable. FIG. 31 (C) is a diagram showing a state in which white blood cells are attracted to the target polynucleotide capture region by the alternating electric field, and the white blood cells are arranged in each region. In this state, the fluorescently labeled antigen substance is introduced into the cell 541. FIG. 31D is a diagram showing a state in which only white blood cells (white blood cells 562 and 563 hatched in the figure) that react with an antibody against an antigen substance emit labeled fluorescence. However, at this time, it is desirable that the temperature of the substrate surface is cooled to about 4 ° C. and controlled so that the amount of mRNA or the like in the cell does not change due to binding with the marker antigen. Even when the temperature is lowered, there is no significant difference in the binding ability of the antibody portion present on the surface of B cells or the like. Therefore, the position of the target polynucleotide capture region where the fluorescent white blood cells are present can be identified by the output of the detector 191 by the optical system described in FIG. Thereafter, the contents of the cells having reactivity with the antigenic substance can be released into the sample solution by heating and destroying them one by one. FIG. 31E is a diagram showing a state in which only the white blood cell 563 is destroyed among the white blood cells fixed in each region, and as a result, the white blood cell 563 disappears from the substrate 1. Therefore, in this state, if the sample solution in the polynucleotide separation cell 541 is collected while the AC electric field is applied to other cells, the contents in specific leukocytes can be taken out.
[0087]
FIG. 32A is a diagram showing a situation where the probe 671 is fixed to one of the target polynucleotide capture regions 616. FIG. 32 (B) is a diagram showing a state in which leukocytes 662 are attracted and captured in the capture region 616 as described above. After the leukocytes 662 are attracted to the capture region 616 in this way, the sample solution is exchanged and the pH is lowered to about 4. This is because the protein has a pK value of about 4 or more, and when the pH is set to 4 or less, the total charge of the protein becomes positive and the charge of the polynucleotide is negative, so it is easily separated in a DC electric field. It is because it is possible. Under such pH conditions, the temperature of the capture region 616 is raised by the Peltier element 512 to destroy white blood cells. Therefore, if the electrode of the target polynucleotide capture region 616 is the anode and the electrode 514 on the opposite surface is the cathode, the polynucleotide 664 gathers in the capture region 616 and the protein 663 is electrophoresed on the surface of the opposite electrode 514. FIG. 32C is a diagram schematically showing how the polynucleotide 664 and the protein 663 move by electrophoresis. By exchanging the solution in the container in this state, the protein and the polynucleotide can be easily separated. At this time, as a matter of course, a polynucleotide complementary to the probe 671 fixed to the target polynucleotide capture region binds thereto.
[0088]
Next, when the electrode of the target polynucleotide capture region is the cathode and the opposite electrode 514 is the anode, the target polynucleotide 666 bound to the probe 671 on the capture region 616 remains on the region 616 but not bound. Is liberated in the sample solution. Therefore, when the sample solution at this time is collected, among the polynucleotides in the leukocytes 662, those not bound to the probe 671 can be collected in the solution. FIG. 32D is a diagram schematically showing a state in which the polynucleotide 665 not bound to the probe 671 moves by electrophoresis.
[0089]
Finally, by raising the temperature of the specific target polynucleotide capture region to about 95 ° C., the specific target polynucleotide bound to the probe in that region is released. By collecting the sample solution at this time, The target polynucleotide such as the target mRNA can be recovered.
[0090]
After completing this procedure for one leukocyte, if the same procedure is followed for the next leukocyte, the target polynucleotide such as the target mRNA can be recovered one by one. Alternatively, cells that match a plurality of conditions may be selected and collected by staining with two or more different markers by staining with a marker label and observing with different filters.
[0091]
Further, the total amount of the target polynucleotide can be quantitatively measured by the signal of the optical system in a state where the target polynucleotide is bound to the probe in the target polynucleotide capture region. Therefore, this example can also be applied to the examination of the total amount of mRNA in leukocytes such as B cells, and by estimating the activity of leukocytes reactive to a specific antigen, the disease state can be obtained quickly and easily. You can also know.
[0092]
Of course, it goes without saying that this embodiment is not limited to leukocytes but can be applied to arbitrary cells.
[0093]
【The invention's effect】
According to the present invention, a target polynucleotide can be fractionated efficiently and with high accuracy.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a flow chart of gene processing for explaining positions occupied by a polynucleotide separation method and apparatus of the present invention in gene analysis and selective recovery processing.
FIG. 2 is a schematic diagram showing a basic configuration of a first exemplary embodiment of the present invention.
FIG. 3 is a schematic view showing a first means for heating a specific region on the substrate of the first embodiment.
FIG. 4 is a schematic view showing a second means for heating a specific region on the substrate of the first embodiment.
FIG. 5 is a schematic view showing a third means for heating a specific region on the substrate of the first embodiment.
FIG. 6 is a schematic diagram showing a specific structure of the polynucleotide separation cell of the first example.
7 is a cross-sectional view taken along line AA of the cell shown in FIG.
8 is a cross-sectional view of the cell shown in FIG. 6 taken along the line BB.
FIGS. 9A to 9C are timetables showing a nucleic acid separation process according to the first embodiment of the present invention. FIGS.
FIG. 10 is a diagram showing a relationship between a substrate 1 having a target polynucleotide capture region and an optical system for detecting a capture state in the target polynucleotide capture region according to the second embodiment of the present invention.
11 shows an AA cross-sectional view of the substrate 1 shown in FIG.
FIGS. 12A to 12C are cross-sectional views taken along a line BB, a line CC, and a line DD of the temperature control unit 133 of the substrate 1 according to the second embodiment. FIGS.
FIG. 13 shows a block diagram of a polynucleotide separation cell 251 and related devices of the second embodiment.
FIG. 14 shows a GG cross-sectional view of a polynucleotide separation cell 251 of the second example.
FIG. 15 shows a timetable for explaining the separation process of the polynucleotide separation cell of the second embodiment.
FIG. 16 is a view showing a state in which a probe can be stably fixed on a substrate according to a third embodiment of the present invention.
FIGS. 17A to 17C are diagrams for explaining experimental results for confirming the effect of the substrate of the third embodiment. FIGS.
FIGS. 18A and 18B are diagrams illustrating that the target oligonucleotide captured by the probe by the substrate according to the third embodiment can be effectively dissociated and recovered.
FIG. 19 is a diagram schematically showing the results of electrophoresis for explaining the sorting of double-stranded polynucleotides according to the present example.
FIG. 20 is an external view of a thin tube that can be employed in the fourth embodiment.
FIG. 21 is an AA cross-sectional view of a thin tube that can be employed in the fourth embodiment.
FIG. 22 is a diagram for explaining an example of a structure for immobilizing a probe to a target polynucleotide capture region.
FIG. 23 is a diagram showing an overall configuration of a polynucleotide separating apparatus according to a fourth embodiment.
FIG. 24 is a diagram showing a BB cross section of a polynucleotide separation module 431 of a fourth example.
FIG. 25 is a diagram showing a modification of the polynucleotide separation module 431 of the embodiment shown in FIG. 24.
FIG. 26 is a diagram showing a modification of the polynucleotide separation module 431 of the embodiment shown in FIG. 24.
27 is a diagram showing a modification of the polynucleotide separation module 431 of the embodiment shown in FIG. 26. FIG.
FIG. 28 is a diagram showing basic elements of a fifth embodiment.
FIG. 29 is a diagram showing a basic configuration of this example in which a polynucleotide separation cell having the basic elements described in FIG. 28 and an optical system are combined.
30 is a diagram showing an AA cross section of the separation cell described in FIG. 29;
FIG. 31A is a diagram showing a state where a target polynucleotide capture region is formed on the surface of the substrate described in FIG. 28, and FIG. 31B is a diagram in which a blood sample is introduced into a polynucleotide separation cell and leukocytes are formed on the substrate. The figure which shows the state which is stray | floating on the surface of (1), (C) is a figure which shows the state which leukocytes were attracted to the target polynucleotide capture area | region by the alternating current electric field, and the leukocyte was arrange | positioned in each area | region, (D) is an antigenic substance The figure which shows the state which came to emit the fluorescence which only the leukocyte which reacts with an antibody reacts with the label | marker, (E) is a result of having destroyed only one leukocyte fixed to each area | region of the target polynucleotide capture area | region, It is a figure which shows the state where this was lost from the board | substrate.
32A is a diagram showing a situation where a probe is fixed to one of target polynucleotide capture regions, and FIG. 32B is a diagram showing a state where leukocytes are attracted to and captured by the target polynucleotide capture region. , (C) is a diagram schematically showing how polynucleotides and proteins move in opposite directions by electrophoresis, and (D) shows that polynucleotides that did not bind to the probe in the target polynucleotide capture region move by electrophoresis. It is a figure which shows a mode typically.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Substrate, 13 ... Substrate base, 131 ... Conductive film, 132 ... Thermally conductive insulating substrate, 14, 141, 142, 143, 144, 145, 146 ... Nucleic acid binding region, 15 ... Objective lens, 151 ... Band pass Filter, 152 ... Emission filter, 16 ... Light source for fluorescence observation, 17 ... Laser light source, 181, 182 ... Dichroic mirror, 183 ... Mirror, 191 ... Detector, 192 ... Analyzer unit, 21 ... Light absorption layer, 22 ... Adiabatic Layers 23, 24 ... fine particles, 31 ... heaters, 32, 34, 35 ... Peltier elements, 33 ... cooling plates, 41, 43, 44, 47 ... nucleic acid samples bound to oligonucleotides fixed on the substrate, 42, 45 46 ... Oligonucleotide immobilized on the substrate, 51 ... Focused light, 7 ... Nucleic acid separation cell, 711, 712, 713 ... Sample entrance / exit, 14 ... Hole, 721 ... Cell upper plate, 722 ... Cell lower plate, 723 ... Spacer, 731, 732, 733 ... Sample chamber, 741 ... Nucleic acid separation cell holding jig, 742, 743, 744 ... Rail, 751, 752, 753, 754, 755 ... electrodes.

Claims (6)

Means for setting a substrate having a plurality of regions to which oligonucleotide probes having different base sequences are fixed on the surface;
Means for supplying a sample solution containing a polynucleotide on the substrate;
And a means for selectively heating the region by irradiating the region with light having a wavelength that is not absorbed by the polynucleotide.   2. The polynucleotide sorting apparatus according to claim 1, further comprising means for recovering the polynucleotide liberated by heating with the oligonucleotide probe fixed to the region and heating the heating means. .   The polynucleotide sorting apparatus according to claim 1, further comprising means for generating an alternating electric field in the region.   The means for collecting includes a first electrode placed on the substrate, at least one other electrode, and a means for controlling the polarity of the first electrode and the other electrode. Item 3. The polynucleotide sorting device according to Item 2.   The polynucleotide sorting apparatus according to claim 1, wherein the substrate has a light absorption layer in the region.   The polynucleotide sorting apparatus according to claim 1, wherein the substrate has a heat insulating layer and a light absorption layer disposed thereon in the region.

Images