JP4094289B2 - Base sequence determination apparatus and base sequence determination method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a base sequence determination apparatus for reading a base sequence of a nucleic acid and a method for determining the base sequence of a nucleic acid. More specifically, in the present invention, the presence or absence of a fluorescent substance that labels nucleic acid bases by performing a reaction of cleaving nucleic acid bases one by one from the ends in a state where a carrier carrying nucleic acids is captured by laser light The present invention also relates to an apparatus and method for determining a base sequence according to type. Further, the base sequence determination apparatus of the present invention performs a predetermined reaction in a state where a carrier carrying an arbitrary substance to be subjected to the reaction is captured by a laser beam, and the result obtained by the reaction is determined based on the presence / absence of a fluorescent substance and / or Alternatively, it can be used as a device for determining by type.
[0002]
[Prior art]
Conventionally, gel electrophoresis apparatuses, capillary electrophoresis apparatuses, and the like are known as apparatuses for determining the base sequences of DNA and RNA nucleic acids. In a gel electrophoresis apparatus, a nucleic acid base sequence that is as long as possible can be read in principle by increasing the gel migration length. However, if the migration length is made longer, the apparatus becomes much larger, and it takes time and effort to prepare the sample. In addition, the capillary electrophoresis apparatus is excellent in that it can read a large amount of sample at a time, and in that it does not take much time to prepare the sample, but it can be read from 600 to 600 in one measurement. It is about 700 bases, and it is difficult to read a long nucleic acid base sequence.
[0003]
In addition, the base sequence determination of nucleic acids is generally performed by conventional radioisotopes (RI). ³² This was done using P. Although the RI technique is very sensitive, it is troublesome to handle radioactive waste and requires consideration for the environment.
[0004]
On the other hand, there is microspectrophotometry as a method for observing the internal structure of individual DNA or cells. In this method, a fluorescent dye is added to cells, and fluorescence is observed on a microscope preparation to measure the internal state and structure of the cell tissue. This method is not intended for reading a base sequence, and cannot be read.
[0005]
Further, flow cytometry is often used as a method for measuring living cells and fine particles in a fluid. Flow cytometry makes light such as laser light incident on each of these objects while transferring fine particles such as biological cells and latex particles in a fluid. Next, scattered light and fluorescence from the target cells and fine particles are observed by light receivers placed in a plurality of directions, and the shape and internal structure of the target are measured. In this method, scattered light and fluorescence from cells and fine particles are measured one after another in a fluid flowing at high speed, but a minute single molecule cannot be detected, and the base sequence cannot be read.
[0006]
In the 90's, research on detection and imaging of single molecules using fluorescence has been rapidly increasing. For example, as a method for detecting a single molecule, a method described in PMGoodwin et al., ACC. Chem. Res. 1996, 29, 607-613, fluorescence correlation spectroscopy (FCS), and the like can be mentioned. In fluorescence correlation spectroscopy, fluorescently labeled proteins and carrier particles are suspended in a solution in the field of view of a confocal laser microscope, and fluctuations in fluorescence intensity based on Brownian motion of these fine particles are analyzed. As a result, the number and size of the target fine particles can be measured. This technique is discussed in detail in, for example, Masataka Kaneshiro “Protein Nucleic Acid Enzyme” (1999) Vol. 44, No. 9, 1431-1437. With this method, only information about the size and amount of the fine particles can be obtained.
[0007]
Attempts have also been made to capture latex particles attached with DNA by a light trap in the channel, and to measure the behavior and force of the attached DNA by extending the attached DNA (Steven B. Smith et al, Science (1996). ) Vol.271, 795-). Furthermore, in a system that transports fine particles into the flow path, individual particles are captured (trapped) by laser light, and the movement of the trapped fine particles is controlled by turning on and off the light irradiation. Has been proposed (Japanese Patent Laid-Open No. 5-296914). As described above, in the method of optically trapping fine particles, a method of capturing fine particles by laser light or the like is disclosed, but a method of discriminating a fluorescent substance attached to the fine particles or measuring an amount of the fluorescent substance Is not disclosed.
[0008]
Attempts have also been made to determine the base sequence using a new technique. That is, mononucleotides (bases) are cut one by one from the end of one strand of DNA, fixed on the substrate while maintaining the order, and converted into a fluorescent derivative on the substrate. It is a technique that excites with a laser beam and shoots it with a video camera. This technique is described in detail in Mitsuru Ishikawa “Protein Nucleic Acid Enzyme” (1999) Vol.44, No13, 2019-2023. However, this approach must convert each base to a fluorescent derivative on the substrate. In addition, the bases separated on the substrate without the solvent must be arranged on the substrate while keeping the order in order, and handling of the substrate is very difficult.
[0009]
In general, when individual desired molecules are labeled using a fluorescent substance and detected / identified in a solution, a method of flowing a washing solution or a sample through the container using a reaction container is used. However, the size of the fluorescently labeled molecules that flow through the flow channel in the reaction vessel is extremely small compared to the size of the flow channel (inner diameter or width of the tube), so that weak fluorescence resulting from this can be reliably identified and It has been extremely difficult to detect.
[0010]
On the other hand, Keller et al. Have proposed the principle of a technique for detecting the fluorescence of a fluorescently labeled molecule flowing in a microchannel for each individual and thereby determining the DNA base sequence (Single-molecule fluoresence analysis in solution). Appl. Spectroscopy, Vol. 50, No. 7, 1996, PP12A-32A). This proposal uses a sample in which DNA labeled with a fluorescent dye is attached to a glass microparticle as a sample. This sample is captured by infrared laser light while flowing through a microchannel and decomposed into this channel. In this method, an enzyme is flowed to separate DNA bases one by one, and fluorescence from fluorescent molecules attached to each base is detected. However, Keller et al. Are not limited to the idea, and do not mention the configuration necessary to actually implement this method.
[0011]
Since then, Dorre et al. Have tried to make the proposal of Keller et al. Concrete, but as described in the report, the final goal of reading the base sequence has not been achieved ( Dorre K et al, Bioimaging 5, 139-152, 1997, Techniques for single molecule sequencing). As described above, there has been no report on how to specifically capture fine particles, move the captured fine particles to a predetermined position, and excite and measure fluorescence. In addition, there is no specific description regarding the mechanism by which the sample solution, decomposing enzyme solution, cleaning solution, and the like are carried in and out. Furthermore, it has never been clearly described how to process a fluorescence signal and read a base sequence.
[0012]
[Problems to be solved by the invention]
The present invention has been made in view of the above circumstances, and a means for concretely realizing a new base sequencing method that has improved the problems of the conventional base sequencing method that requires a separation operation by electrophoresis. The purpose is to provide. That is, an object of the present invention is to provide a base sequence determination device and a base sequence determination method capable of sequencing a nucleic acid of unlimited length continuously and simply.
[0013]
[Means for Solving the Problems]
In order to achieve the above object, the present invention provides means for reading the base sequence of a nucleic acid as follows. First, an analysis sample is prepared by identifying and labeling each base of a nucleic acid molecule to be sequenced with a fluorescent substance and attaching the nucleic acid to a carrier. This analysis sample is introduced into the flow channel of the measurement cell, and the carrier is captured by a laser. Next, in this state, a degradation enzyme solution is allowed to flow through the flow path, and the mononucleotides constituting the nucleic acid are cleaved sequentially from the end. Under the condition that the cleaved mononucleotide flows in the flow path in the cleaved order, the fluorescence emitted from the fluorescent substance labeled with the mononucleotide base is sequentially detected and the nucleic acid base sequence is read. .
[0014]
In particular, it is a means that can quickly transport sample solution, enzyme solution, washing solution, etc. to the flow channel of the measurement cell, and keeps the base of the nucleic acid cleaved by the degrading enzyme in the flow channel in the order of its cleavage. The present invention suggests means that can be transported.
[0015]
That is, the present invention has been achieved by the means described below.
[0016]
(1) An apparatus for judging the result obtained by a predetermined reaction based on the presence and / or type of a fluorescent substance, in which the carrier carrying the substance to be used for the reaction is captured in a light trap. In an apparatus for performing a predetermined reaction on a substance to be provided,
A measurement cell having a flow path therein and a transparent surface for transmitting a light beam into the flow path;
Means for transporting the suspension of the carrier to the flow path of the measurement cell;
A first laser light source for capturing the carrier;
A first optical system that collects the light beam from the first laser light source in the flow path of the measurement cell and captures the carrier;
Means for transporting a reaction solution for causing a substance carried by the carrier to perform a predetermined reaction while maintaining a constant flow rate in the flow path of the measurement cell;
A second laser light source for exciting the fluorescent material reflecting the result to determine the result obtained by the reaction;
A second optical system for condensing the light beam from the second laser light source in the flow path of the measurement cell and exciting the fluorescent material;
A third optical system for collecting fluorescence from the fluorescent material;
A photodetector for detecting the condensed fluorescence;
A determination apparatus comprising: analysis means for analyzing a result of the reaction from an identification signal generated by the photodetector.
[0017]
(2) The apparatus according to (1), wherein the means for conveying the suspension of the carrier to the flow path of the measurement cell and the substance carried by the carrier are caused to perform a predetermined reaction. None of the means for transporting the reaction liquid to the flow path of the measurement cell causes piping to transport each solution to the flow path of the measurement cell, and causes no pulsating flow in the transported liquid. An apparatus comprising a member for controlling a flow of liquid.
[0018]
(3) The apparatus according to (1) or (2), in which a means for transporting the suspension of the carrier to the flow path of the measurement cell and a predetermined reaction with the substance carried by the carrier Each of the means for transporting the reaction liquid for performing the measurement to the flow path of the measurement cell includes a pipe for transporting each solution to the flow path of the measurement cell, and the pipe is connected to the measurement cell. An apparatus is characterized in that, in the vicinity of the flow channel inlet, a branch is made in a direction toward the flow channel of the measurement cell and in a direction toward the waste liquid.
[0019]
(4) A base sequence determination apparatus using a carrier to which a nucleic acid labeled and associated with a type of base and a type of fluorescent substance is attached as an analysis sample,
A measurement cell having a flow path therein and a transparent surface for transmitting a light beam into the flow path;
Means for transporting a suspension of a carrier to which the nucleic acid as an analysis sample is attached to the flow path of the measurement cell;
A first laser light source for capturing the carrier to which the nucleic acid is attached;
A first optical system that collects the light beam from the first laser light source in the flow path of the measurement cell and captures the carrier to which the nucleic acid is attached;
Means for transporting the enzyme solution that cleaves mononucleotides constituting the nucleic acid in order from the end, while maintaining a constant flow rate in the flow path of the measurement cell;
A second laser light source for exciting the fluorescent substance that is identifying and labeling the base of the mononucleotide cleaved by the enzyme solution, in the order of cleavage;
A second optical system for condensing the light beam from the second laser light source in the flow path of the measurement cell and exciting the fluorescent materials in the order of cutting;
A third optical system for sequentially collecting the fluorescence from the fluorescent substance that identifies and labels the base of the nucleic acid;
A photodetector that sequentially detects the collected fluorescence and generates an identification signal corresponding to the detected fluorescence;
Analysis means for reading a base sequence of the nucleic acid from an identification signal generated by the photodetector;
A base sequence determination apparatus comprising:
[0020]
(5) The apparatus according to (4), wherein a means for transporting a suspension of a carrier to which the nucleic acid, which is an analysis sample, is attached to the flow path of the measurement cell, and the nucleic acid are configured The means for transporting the enzyme solution for cleaving the mononucleotide in order from the end to the flow path of the measurement cell are all pipes for transporting each solution to the flow path of the measurement cell, and the liquid to be transported An apparatus comprising a member for controlling a flow of liquid without causing a pulsating flow.
[0021]
(6) The apparatus according to (4) or (5), the means for transporting the suspension of the carrier to which the nucleic acid as the analysis sample is attached to the flow path of the measurement cell, and Each means for transporting an enzyme solution for sequentially cleaving mononucleotides constituting a nucleic acid from the end to the flow path of the measurement cell comprises a pipe for transporting each solution to the flow path of the measurement cell. The apparatus is characterized in that the pipe branches in the direction toward the flow path of the measurement cell and the direction toward the waste liquid near the flow path inlet of the measurement cell.
[0022]
(7) The apparatus according to any one of (1) to (6),
A light source for irradiating illumination light into the flow path of the measurement cell;
An optical system for irradiating the light from the light source into the flow path of the measurement cell and irradiating the carrier with illumination light;
An optical system for the carrier to capture an image captured by the first laser light source under the illumination light;
Imaging means for capturing an image of the carrier captured;
The apparatus further comprising:
[0023]
(8) As an analysis sample, a carrier to which a nucleic acid that is identified and labeled in association with a base type and a fluorescent substance type is used is attached. A method for determining a base sequence using a cell having a transparent surface for permeation into
Transporting a suspension of a carrier to which the nucleic acid, which is an analysis sample, is attached, to the flow path of the measurement cell;
Capturing the carrier to which the nucleic acid is attached with a laser trap in a flow path;
A flow of an enzyme solution for sequentially cleaving mononucleotides constituting the nucleic acid from the end in the flow path while maintaining a constant flow rate;
A step of sequentially exciting fluorescent substances that identify and label mononucleotide bases cleaved by the enzyme solution;
Sequentially detecting the fluorescence emitted by the fluorescent material excited by the above-mentioned steps, and reading the base sequence corresponding to the detected fluorescence; and
A nucleotide sequence determination method comprising:
[0024]
(9) The base sequence determination method according to (8), further comprising a step of capturing an image of the carrier to which the nucleic acid is attached captured by a laser trap.
[0025]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
[0026]
<Analytical sample>
First, an analysis sample used for reading a base sequence with the base sequence determination apparatus of the present invention will be described. As shown in FIG. 1, the analysis sample is “a nucleic acid that is identified and labeled in association with the type of base and the type of fluorescent substance attached to a carrier”.
[0027]
The “carrier” used in the present invention is not particularly limited as long as it is captured by laser light. As the carrier, any water-insoluble material such as glass fine particles, latex fine particles, metal fine particles such as gold colloid particles, magnetic fine particles, or living cells can be used. Further, the carrier particles to be laser trapped generally have a diameter of 0.01 μm to 10 μm, preferably a diameter of 0.05 μm to 2 μm, more preferably a diameter of 0.1 μm to 1 μm. As an example of the carrier, glass fine particles having a diameter of 1 μm can be mentioned. More specifically, streptavidin-coated silica beads manufactured by Bangs Laboratories Inc. (USA) can be used as the glass fine particles having a diameter of 1 μm.
[0028]
In the present invention, the “fluorescent substance” is not particularly limited as long as it can be detected that the fluorescent substance is labeled with the fluorescent substance. For example, rhodamine green (hereinafter also referred to as RG), tetramethylrhodamine-5-isothiocyanate (hereinafter also referred to as TMR), Cy5 (Sciive (registered trademark), Amersham Pharmacia Biotech), fluorescein isothiocyanate (FITC) ), Acridine Yellow, Texas Red. However, when each base is discriminated and labeled using two or more kinds of fluorescent substances, the respective fluorescent substances need to be distinguished and detected depending on the difference in the wavelength emitted.
[0029]
In the present invention, the “nucleic acid discriminated and labeled by associating the type of base and the type of fluorescent substance” is, as a specific example, an example shown in FIG. Examples include, but are not limited to, DNA. That is, in the present invention, “nucleic acid” refers to both DNA and RNA. Here, the “identification label” does not necessarily intend to label all four types of bases, and may include base types that are not fluorescently labeled. Further, two or three different base species may be labeled with the same fluorescent substance.
[0030]
Such variations of the identification mark are exemplified in specific samples (samples shown in FIGS. 16, 17, and 19) described later. For example, only one type (for example, adenine) of four types of nucleic acids may be identified and labeled using one type of fluorescent substance (see FIG. 16). Alternatively, two types of bases may be identified and labeled using two types of fluorescent substances having different emission wavelengths (see FIG. 17). Alternatively, two types of fluorescent substances having different emission wavelengths may be used, and one type of base may be labeled with one fluorescent substance and the other three types of bases may be identified and labeled with the other fluorescent substance (see FIG. 19). .
[0031]
“Nucleic acids that are identified and labeled by associating the types of bases with the types of fluorescent substances” can be prepared as follows, for example. Here, an example of preparing a sample in which the base of a nucleic acid is identified and labeled using two types of fluorescent substances will be described.
[0032]
First, as a substance to be attached to the carrier, a DNA single strand (or RNA single strand) in which the 5 ′ end is labeled with biotin and each base is discriminated and labeled is prepared. That is, the following reaction solution
{50 mM Tris-HCl (pH 8.5), 10 mM KCl, 15 mM (NH _Four ) ₂ SO _Four 7 mM MgSO _Four , 0.005% Triton X-100, 10 mM 2-Mercaptoethanol, 1 pmol of biotin-labeled primer per reaction, 20 μM of each fluorescence-labeled substrate (for example, RG-dTTP (RG-dUTP may be used instead), RG -DATP, TMR-dCTP, TMR-dGTP)}
A DNA single strand (template strand) and an extension enzyme are added to the sample, and the mixture is reacted at 37 ° C. for 30 minutes to prepare a sample. In this reaction, four kinds of fluorescently labeled mononucleotides are used as substrates, and a DNA strand (or complementary RNA strand) complementary to a single DNA strand (a strand serving as a template) occurs. The complementary DNA strand (or complementary RNA strand) synthesized here is used for actual sequence analysis. Hereinafter, this complementary DNA strand (or complementary RNA strand) is referred to as sample DNA (or sample RNA) and used as a sample. In this way, sample DNA (or sample RNA) having a predetermined sequence in which the 5 ′ end is labeled with biotin and each base is labeled with a fluorescent substance is prepared. However, conditions such as the composition of the buffer solution and the reaction temperature mentioned in the preparation examples are not limited to this description.
[0033]
Next, a reaction is performed in which the prepared sample DNA (or sample RNA) is attached to a carrier. As a means for attaching the sample DNA (or sample RNA) to the carrier, there may be mentioned a method using an avidin / biotin reaction shown in FIG. However, it is not limited to this as long as it fulfills such an adhesion function. Adhesion by avidin-biotin reaction uses the above-mentioned sample by using the above-mentioned Bangs Laboratories Inc. (USA) streptavidin-coated silica beads (with avidin immobilized on the surface of glass microparticles). This is performed by binding DNA (or sample RNA). More specifically, the silica beads and the sample DNA (or sample RNA) are added to a solution of 100 mM Tris-HCl (pH 8.0), 1.0 M LiCl, 0.1% (v / v) Tween 20, and at room temperature. It can be made to react. However, conditions such as the composition of the buffer solution and the reaction temperature are not limited to this description. Thus, if an analysis sample is prepared using a one-to-one reaction between avidin and biotin, one DNA strand (or RNA strand) can be attached to one carrier.
[0034]
The analytical sample used for the base sequence determination apparatus of the present invention, in which the nucleic acid base is discriminated and labeled, can be prepared by a known technique as described above. See also the description of Zeno Foldes-Papp et al, Nucleosides & Nucleosides, 16 (5 & 6), 781-787, 1997, Exonuclease degradation of DNA studied by fluorescence correlation spectroscopy for the preparation of analytical samples.
[0035]
<Base sequence determination device>
[First Embodiment]
Hereinafter, the base sequence determination apparatus according to the first embodiment of the present invention will be described with reference to FIG. In the apparatus of the present embodiment, an analysis sample in which a nucleic acid base is identified and labeled using one type of fluorescent substance is used.
[0036]
As shown in FIG. 2, the base sequence determination apparatus according to the present embodiment has the following configuration.
A measuring cell 1 having a flow path therein and having a transparent surface for transmitting a light beam into the flow path;
Means 2 for transporting a suspension of a carrier to which a nucleic acid as an analysis sample is attached to the flow path of the measurement cell;
A first laser light source 3 for capturing the carrier to which the nucleic acid is attached;
A first optical system 4 to 10 that collects a light beam from the first laser light source in a flow path of the measurement cell and captures the carrier to which the nucleic acid is attached;
Means 2 for transporting an enzyme solution for sequentially cleaving mononucleotides constituting the nucleic acid from the end to the flow path of the measurement cell;
A second laser light source 11 for exciting a fluorescent substance that has been identified and labeled with a base of a mononucleotide cleaved by the enzyme solution in the order of cleavage;
Second optical systems 12 to 14 and 10 for condensing the light beam from the second laser light source in the flow path of the measurement cell and exciting the fluorescent material in the order of cutting;
A third optical system 10, 15-19 for sequentially collecting the fluorescence from the fluorescent substance that is identifying and labeling the base of the nucleic acid;
A photodetector 20 that sequentially detects the collected fluorescence and generates an identification signal corresponding to the detected fluorescence;
Analysis means 21 to 23 for reading the base sequence of the nucleic acid from the identification signal generated by the photodetector;
A light source 24 for irradiating illumination light into the flow channel of the measurement cell;
Optical systems 25, 26, 15, 10 for irradiating light from the light source into the flow path of the measurement cell and irradiating illumination light to the carrier to which the nucleic acid is attached;
An optical system 10, 15, 27-29 for capturing an image captured by the carrier on which the nucleic acid, which is an analysis sample, is attached under the illumination light, by the first laser light source;
Imaging means 30 for capturing an image in which the carrier to which the nucleic acid is attached is captured.
[0037]
The base sequence determination apparatus of the present invention utilizes a confocal laser microscope. Thus, by making the optical system for detecting fluorescence into a confocal optical system, the bases of one molecule of the cleaved mononucleotide can be sequentially decoded.
[0038]
The base sequence determination apparatus of the present invention can be used to read a base sequence of a nucleic acid by performing a reaction of cleaving the base of the nucleic acid from the end while capturing the carrier carrying the nucleic acid with a laser beam. . The base sequence determination apparatus of the present invention is not limited to such use, but is used to capture a carrier carrying a substance to be subjected to a reaction with a laser trap, perform a predetermined reaction, and obtain the reaction result. You can also. The predetermined reaction refers to any reaction that can determine the result obtained by the reaction based on the presence and / or type of the fluorescent substance. For example, an antigen-antibody reaction may be carried out by reacting a fluorescently labeled antibody with a carrier carrying an antigen, or a single fluorescently labeled carrier carrying a single-stranded nucleic acid. A hybridization reaction may be carried out by reacting the strand nucleic acid.
[0039]
Hereafter, each structure and operation | movement which concern on 1st embodiment of a base sequence determination apparatus are demonstrated in order of the structure quoted previously.
[0040]
≪Measurement cell≫
First, the “measurement cell 1 having a flow path inside and a transparent surface for transmitting a light beam into the flow path” will be described. An example of the measurement cell is shown in detail in FIG. 3A is a perspective view showing the measurement cell, FIG. 3B is a plan view showing the measurement cell of FIG. 3A, and FIG. 3C is a view showing a part of a cross section taken along the line AA.
[0041]
First, the measurement cell 1 needs to have a flow path 1c therein and a transparent surface (surface 1b) for transmitting a light beam into the flow path. The objective lens is disposed on the side of the transparent surface (surface 1b), and laser light is incident from the side 1b.
[0042]
Moreover, when the measurement cell 1 is arranged in the base sequence determination apparatus of the present invention, it is necessary to provide the flow path at a position where the focal point of the objective lens can be adjusted in the flow path. In other words, it is necessary to appropriately set the thickness of the surface portion 1b that transmits the light beam so that the focus of the objective lens can be adjusted in the flow path. Furthermore, in the measurement cell 1, all surfaces through which light is transmitted when the light beam is guided into the flow path need to be flat so as not to cause light refraction.
[0043]
Furthermore, in the present invention, it is essential to transport mononucleotides cleaved in order from the end of the nucleic acid sample to be transported in the flow channel while maintaining the order. It needs to be a size.
[0044]
Summarizing the above requirements, the measurement cell 1 has (1) the light incident on the flow path, the surface through which the light emitted from the flow path is transparent, and (2) the light incident on the flow path, the flow The surface through which light emitted from the path is transmitted is a flat plate having a uniform thickness and without unevenness. (3) The flat plate has an appropriate thickness so that the focal point of the objective lens can be adjusted in the flow path. (4) The width and depth of the flow path must be very small. In the present invention, the term “transparent” refers to incident light and does not necessarily match the transparency seen by the human eye.
[0045]
In FIG. 3, the measurement cell 1 is composed of a main body portion 1a and a transparent surface portion 1b through which laser light can pass, and a flow path 1c is formed therebetween. It goes without saying that the measurement cell is not limited to such a two-part configuration. The size of the measurement cell 1 is not particularly limited, but is approximately 1 to 25 mm in the length direction of the flow path, 5 to 15 mm in the width direction of the flow path, and 0.3 to 1 mm in the depth direction (thickness) of the flow path. be able to. For example, the size of the measurement cell 1 can be about 10 mm in the length direction of the channel, about 5 mm in the width direction of the channel, and about 1 mm in the depth direction (thickness) of the channel.
[0046]
In FIG. 3, a main body portion 1a is made of a silicon wafer, and a flow path 1c (groove) is formed in the main body portion by etching or the like. The thickness of the main body portion 1a is preferably 0.2 to 1 mm. The channel width is preferably 5 to 100 μm, more preferably 10 to 50 μm, and the channel depth is preferably 5 to 100 μm, more preferably 10 to 50 μm. For example, the channel width is about 50 μm, The channel depth can be about 50 μm. The length of the flow path is preferably 1 to 25 mm, and can be about 10 mm, for example. In FIG. 3, through holes are provided in the silicon wafer at both ends of the flow path, and tubes for solution loading / unloading are connected to the holes through the connection terminals 1d.
[0047]
In FIG. 3, the surface portion 1b is a transparent flat plate (glass cover) made of glass. As described above, the surface portion 1b is transparent and needs to be a flat plate having the same thickness without unevenness because light incident on the channel and light emitted from the channel pass therethrough. Further, as described above, since the thickness of the surface portion 1b affects the focal position of the objective lens, the thickness is preferably 0.13 to 0.21 mm, and more preferably 0.169 to 0.171 mm. The surface portion 1b is bonded to the main body portion (silicon portion) 1a by a method such as anodic bonding.
[0048]
Moreover, the measurement cell 1 is not limited to the cell provided with the above-mentioned flow path, and a minute hollow cylindrical measurement cell such as a glass capillary, or a thin sheet measurement cell can also be used.
[0049]
In FIG. 3, the flow path 1 c is a single linear groove. In this case, when a solution different from the previously flowed solution is flowed, it is necessary to once wash the flow path with a buffer solution or the like. Such a washing operation can also be efficiently performed by appropriately branching and increasing the flow paths. For example, the flow path may be branched to produce a Y-shaped flow path.
[0050]
Further, the measurement cell is a flow channel having a shape as shown in FIG. 4 (plan view of the measurement cell), that is, a linear shape, but the width and depth of the flow channel are both narrowed at the center of the flow channel. You may have a flow path of the shape. The narrow portion of the flow path is irradiated with a carrier capturing laser and an excitation laser. Width of the narrowed part (W in the figure ₂ And the depth is preferably 5 to 50 μm. The length of the narrow channel portion (indicated by L in the figure) is preferably 1 to 10 mm. The width of the non-narrow portion of the flow path (W in the figure) ₁ And the depth is 5 to 200 μm. Thus, by narrowing the width and depth of the flow path only at the central portion of the flow path, a narrow range in which the solution is difficult to transport can be minimized.
[0051]
≪Solution loading / unloading mechanism≫
Next, “Means 2 for transporting a suspension of a carrier with a nucleic acid as an analysis sample attached to the flow path of the measurement cell” will be described. This means is also called a solution carry-in / carry-out mechanism, and its outline is shown in FIG.
[0052]
The reason why such a solution carrying-in / out mechanism is provided in the present invention is that the following problems have been found in conveying a solution by electro-osmotic flow (EOF) proposed by Dorre et al. . That is, when conveying a solution by electroosmotic flow, it is necessary to apply a high voltage. Therefore, if the electroosmotic flow is used for a long time or the flow rate is increased, heat is generated in the flow path, and the activity of proteins such as enzymes may be lowered, or bubbles may be generated. Therefore, it is necessary to take measures such as cooling the periphery of the flow path. In particular, the narrower the flow path in the reaction section, the more easily heat and bubbles are generated.
[0053]
In addition, when a solution loading / unloading mechanism is provided as in the present invention and the solution is conveyed by gravity or pump driving, the flow of liquid in the flow path becomes “laminar flow” and the flow is fastest near the center of the flow path. The flow velocity distribution in which the flow becomes slower as it goes to the periphery of the flow path is shown. However, in the electroosmotic flow, the liquid in the flow path becomes a flow called “plug flow”, and the flow of molecules (mononucleotides) to be conveyed does not concentrate on the central portion of the flow path, and the liquid in the flow path There is also a possibility that the horizontal direction of the flow will be dispersed. Thus, when the mononucleotide is dispersed in the peripheral portion of the flow path, it becomes difficult to identify the base of the mononucleotide flowing through the flow path in the confocal region. Therefore, in consideration of such problems, in the present invention, a solution loading / unloading mechanism as described below is provided.
[0054]
The sample solution (suspension of the carrier) used in the present invention is preferably one in which the carrier is suspended at a predetermined concentration. That is, the sample solution is set to an appropriate concentration at which one carrier is easily captured by the laser trap. If the carrier concentration is too high, there is a high possibility that another carrier will be caught behind the trapped carrier or that the carrier once trapped will be repelled by other carriers that have flown later. On the other hand, if the carrier concentration is too low, the possibility of trapping becomes low. Therefore, suspensions containing 0.01 to 0.3% by weight of carrier are generally used. For example, a glass fine particle suspended in a phosphate buffer at a concentration of 0.1% by weight can be used.
[0055]
In the solution carry-in / carry-out mechanism shown in FIG. 5, the measurement cell 1 is accommodated in a holder 1e. Since the measurement cell 1 is small in size as described above, it is easier to accommodate the measurement cell 1 in a holder. A carry-in tank 2a and a carry-out tank 2c are connected to each of the junction terminals 1d attached to both ends of the holder 1e via a carry-in tube 2b and a carry-out tube 2d, respectively. The loading tank 2a is filled with a solution for transporting to the flow path. As the solution, a sample solution (carrier suspension), a channel cleaning solution (buffer solution), a buffer solution containing a degrading enzyme solution, and the like are used. As the tubes 2b and 2d, for example, a Teflon material can be used.
[0056]
Carrying in the sample liquid and the like from the carry-in tank 2a to the flow path and carrying out into the carry-out tank 2c can be performed by moving the upper and lower height positions of both tanks. By controlling the height positions of both tanks, the flow rate and flow rate of the solution into the flow path can be controlled. Alternatively, the solution can be carried in and out by directly driving a syringe or the like using gas pressure to perform suction and discharge. In this case, the flow rate and flow rate of the solution into the flow path can be controlled by controlling the gas pressure. Further, the solution can be carried in and out by a motor-driven pump.
[0057]
The flow rate and flow rate of the solution into the flow path must be controlled with attention to the following points. That is, in the present invention, when the flow rate is too high, the carrier trapped by the laser trap escapes, so the upper limit is a flow rate that does not escape the carrier from the trapped state. Thus, the upper limit of the flow velocity is set in relation to the force (laser light output) for laser trapping the carrier. If the flow rate is too slow, the cleaved mononucleotides may move in any direction due to Brownian motion, and the mononucleotides may not be aligned in the cleaved order. A flow rate of about In general, the flow rate is set to 0.5 mm / s to 10 mm / s. For example, the flow rate is set to 1 mm / s.
[0058]
In the present invention, in embodying the configuration of the base sequence determination apparatus, attention was paid to the solution loading / unloading mechanism. This is because the following problems were newly found when the configuration of the base sequence determination apparatus of the present invention was embodied. That is, the carrier trapped by the laser (trap state) is released from the trap state due to a rapid change (pulsating flow) of the liquid flow rate, and the mononucleotide cleaved by the degrading enzyme However, due to a rapid change in the flow rate of the liquid, a problem has been found that the cutting order is not maintained and the fluid is not conveyed in the flow path. In order to accurately read the base sequence targeted by the present invention, it is most important to transport mononucleotides cut in order from the end of the nucleic acid in the flow path of the measurement cell in the order. It becomes. Therefore, in order to solve these problems, in addition to making the width and depth of the flow channel of the measurement cell minute, the solution transport in the flow channel must be maintained at a constant flow rate. The present inventors paid attention to. However, generally, when the width and depth (inner diameter) of the flow path are 1 mm or less, it becomes difficult to transport the solution. Therefore, the present inventors have thought that it is necessary to construct a solution loading / unloading mechanism that can smoothly transport a solution into such a minute flow path and can maintain a constant flow rate. It came.
[0059]
A specific configuration is shown in FIGS. 6 and 7 as an example of a solution carrying-in / out mechanism that satisfies such requirements. That is, in the present invention, the solution carry-in / carry-out mechanism preferably has the following two characteristics.
[0060]
First, this mechanism includes piping for transporting each solution to the flow channel of the measurement cell in order to smoothly transport the solution to the minute flow channel, and the piping is connected to the flow channel inlet of the measurement cell. In the vicinity, it is branched into a direction toward the flow path of the measurement cell and a direction toward the waste liquid. In this specification, “piping” means all pipes that form a flow path for guiding a solution to the flow path of the measurement cell and a flow path for discharging the solution from the flow path of the measurement cell as waste liquid.
[0061]
Here, near the flow channel inlet of the measurement cell, the pipe branched in the direction toward the flow channel of the measurement cell and the direction toward the waste liquid is difficult to transport the solution to the flow channel of the minute measurement cell. It is provided in order to solve a certain problem, and has the meaning of assisting the conveyance of the solution to the flow path of the measurement cell. The piping toward the waste liquid functions to temporarily release the solution transported to the vicinity of the flow channel inlet of the measurement cell in the direction of the waste liquid. Once the solution is flowed in the direction of the waste liquid, the solution can be quickly transported to the vicinity of the flow channel inlet of the measurement cell by switching the flow channel through which the solution flows and guiding the solution to the flow channel of the measurement cell. it can. Note that the piping toward the waste liquid direction can also function to release air in the piping.
[0062]
Here, “close to the flow channel inlet” is preferably as close as possible to the flow channel inlet of the measurement cell in the sense of assisting the conveyance of the solution to the flow channel of the measurement cell. Specifically, it is preferable that the branch point of the pipe is located within about 10 mm from the connection portion (for example, the connection terminal) that connects the flow path of the measurement cell and the “pipe”.
[0063]
Second, the solution carry-in / carry-out mechanism is a member that controls the flow of the liquid flowing in the pipe without causing a pulsating flow in the conveyed liquid in order to maintain a constant flow rate of the solution flowing through the flow path. It is characterized by comprising. “A member that controls the flow of liquid” is an arbitrary element that can control the flow of liquid by flowing the liquid, stopping the flow, or switching the flow path of the liquid flowing in the branched pipe. It is a member. In general, a valve can be used as the member.
[0064]
In the present invention, in order to maintain the flow of the solution at a constant flow rate in the flow path (that is, without causing a pulsating flow in the solution), the “member for controlling the flow of the liquid (valve)” is examined as follows. did. For example, a commonly used solenoid valve is excellent in that it is easily available and its operation can be easily controlled by a computer. However, when this solenoid valve is used in the solution loading / unloading mechanism according to the present invention, a sudden volume change occurs when an operation for controlling the flow of the liquid is performed. It cannot be transported. Therefore, in the present invention, it is appropriate to use a valve that does not cause a sudden volume change when an operation for controlling the flow of the liquid, such as switching the flow direction of the liquid, is performed in the branched pipe. I found out. That is, a valve that gradually changes the flow rate of the liquid when performing an operation for controlling the flow of the liquid, such as a valve that controls the flow of the solution by rotation (specifically, a rubbing valve is preferable) is used. Found that is appropriate. However, the valve used in the present invention is not particularly limited as long as it can maintain the flow of the solution at a constant flow rate without causing a pulsating flow in the liquid when the operation of controlling the flow of the liquid is performed. It is not limited. That is, as long as the mononucleotide cleaved in order from the end of the sample DNA (or sample RNA) can be transported in the flow path in the order by maintaining the flow of the solution at a constant flow rate, the limitation is imposed. Not.
[0065]
As shown in FIG. 6 and FIG. 7, an example of the solution carrying-in / out mechanism having such characteristics is provided with branched pipes, valves, and T-type joints. It can be brought into and out of the road. Furthermore, this mechanism can maintain a constant flow rate by using a specific valve that does not cause a sudden volume change (ie, does not cause a pulsating flow of liquid) during transport of the solution.
[0066]
Note that the solution carry-in / carry-out mechanism described in this specification can be used when any liquid is carried into a cell provided with a microchannel.
[0067]
Hereinafter, a specific example of the solution loading / unloading mechanism will be described in detail with reference to FIG. However, it goes without saying that the solution carrying-in / out mechanism in the present invention is not limited to the example shown in FIG. 6 as long as the solution can be carried into a minute flow path while maintaining a constant flow rate.
[0068]
The solution loading / unloading mechanism shown in FIG. 6 includes a degradation enzyme introduction syringe 44, a sample introduction syringe 45, buffer tanks 46 and 47, valves 1 to 9, T-type joints 48 and 49, a negative pressure pump 52, A waste tank 53 and a pipe connecting them are configured.
[0069]
In FIG. 6, the piping of the solution carry-in / carry-out mechanism is connected to the flow path of the measurement cell 1 via connection terminals 50 and 51. In this way, the measurement cell is installed in a state where it can be exchanged via the connection terminals 50 and 51 in consideration of the fact that the flow path has a very narrow width and thus it takes time to wash after flowing the sample liquid. It is desirable to keep it. In the following description, the flow channel inside the measurement cell and the flow channel outside the measurement cell are referred to as “measurement cell flow channel” and “pipe flow channel”, respectively, with the connection terminal as a boundary. The piping is composed of, for example, Teflon.
[0070]
Each component part of the solution carrying in / out mechanism shown in FIG. 6 will be described.
[0071]
{Syringe and buffer tank}
From the decomposing enzyme introduction syringe 44 and the sample introduction syringe 45, the decomposing enzyme solution and the sample solution are injected into the “piping flow path”, respectively. The decomposing enzyme introduction syringe 44 and the sample introduction syringe 45 are respectively provided with buffer tanks 46 and 47 for diluting the decomposing enzyme solution and the sample solution and washing the flow path through which the solution flows.
[0072]
In FIG. 6, the degrading enzyme solution is conveyed from left to right (in the direction from valve 4 to valve 2) in the flow channel of the measurement cell, and the sample solution is transferred from right to left (valve in the flow channel of the measurement cell. 2 to the direction of the valve 4). It is not limited to the example shown in FIG. 6, You may install each syringe and a buffer solution tank so that a degradation enzyme solution and a sample solution may flow in the flow path of a measurement cell toward the same direction. However, when the degrading enzyme solution flows in the flow channel of the measurement cell, the carrier capture laser (Trap laser) is irradiated upstream, and the excitation laser (in the figure, Excitation laser) needs to be irradiated. That is, first, upstream of the flow path, the decomposing enzyme solution acts on the carrier (which carries the nucleic acid) captured by the carrier capture laser, and then the labeled nucleotide cleaved by this action is moved downstream of the flow path. It needs to be excited with an excitation laser.
[0073]
{Valve and T-type fitting}
Valves are installed to control the flow of the solution in the piping of the solution loading / unloading mechanism, such as closing or opening the flow path of the liquid, or selecting the flow direction of the liquid in the branched piping. Yes. In FIG. 6, valves 1 to 9 are two-way valves or three-way valves. In the figure, NO displayed on the valves is Normal Open (normally open), NC is Normal Close (normally shut off), and COM is Common ( Common). As described above, preferably, a valve that does not cause pulsation is used.
[0074]
On the other hand, the T-type joint is generally used to form a branched pipe. In FIG. 6, T-shaped joints 48 and 49 function to form a pipe branched in a direction toward the flow path of the measurement cell and a direction toward the waste liquid near the flow path inlet of the measurement cell. That is, by installing the T-shaped joints 48 and 49 very close to the flow path of the measurement cell and branching the pipe, the solution conveyed to the vicinity of the flow path entrance of the measurement cell is temporarily released to the waste liquid tank, The solution to be transported into the flow channel of the measurement cell can be quickly guided to near the entrance of the flow channel. Thereby, conveyance of the solution to the flow path of the measurement cell which is difficult to convey the solution can be assisted. For example, the T-shaped joint 48 is preferably located as close as possible to the connection terminal 50, and can be located, for example, at a position about 20 mm away from the connection terminal 50. Similarly, the T-shaped joint 49 is preferably located as close as possible to the connection terminal 51, and can be located, for example, at a position about 20 mm away from the connection terminal 51.
[0075]
Here, it is preferable that all the inner diameters of the “piping flow paths” have a width that is somewhat larger than the flow path width (for example, 50 μm) of the measurement cell. Thus, in order to carry the solution smoothly, it is preferable to make the flow path width of the pipe wider than the flow path width of the measurement cell, for example, the flow path width is 50 to 800 μm, preferably about 250 μm. Can do. On the other hand, the width and depth of the flow path of the measurement cell need to be as small as 50 μm, for example, as described above so as to meet the purpose of the base sequence determination of the present invention.
[0076]
Further, in the solution carry-in / carry-out mechanism shown in FIG. 6, the piping is branched into a direction toward the flow path of the measurement cell and a direction toward the waste liquid at each of the locations where the valve 4 and the valve 2 are installed. Also at the locations where the valve 4 and the valve 2 are installed, the solution to be transferred into the flow path of the measurement cell can be quickly guided to the valve by branching the piping and allowing the solution to temporarily escape to the waste liquid tank. Thereby, conveyance of the solution to the flow path of a measurement cell can be assisted.
[0077]
For example, the valve 4 can be positioned at a position away from the connection terminal 50 by about 200 mm, and the valve 2 can be positioned at a position away from the connection terminal 51 by about 200 mm. In addition, as shown in FIG. 7 described later, a single pipe may be used as the pipe for conveying the solution from the valve 48 to the waste liquid tank and the pipe for conveying the solution from the valve 4 to the waste liquid tank. Similarly, a pipe for conveying the solution from the valve 49 to the waste liquid tank and a pipe for conveying the solution from the valve 2 to the waste liquid tank can be combined with one pipe.
[0078]
{Negative pressure pump}
The negative pressure pump 52 functions to guide the solution to the waste liquid tank 53 by suction. However, the solution can be guided to the waste tank by gravity without using a negative pressure pump. The use of a negative pressure pump can transport the solution more efficiently in the minute flow path, but when the influence of the vibration of the pump is to be avoided, the solution is transported by gravity without using the pump. In the present invention, once the carrier is captured by the laser in the flow channel of the measurement cell, it is preferable not to use a pump in order to avoid vibrations in all subsequent operations.
[0079]
Next, the operation of loading each solution (sample solution, decomposing enzyme solution, buffer solution) into the flow path of the measurement cell by the solution loading / unloading mechanism will be described.
[0080]
Each solution is preferably deaerated before being carried into the flow path.
[0081]
First, the sample liquid is conveyed into the flow path of the measurement cell, and then the carrier contained in the sample liquid is captured in the flow path of the measurement cell by a carrier capturing laser (Trap laser). Carriers other than the trapped carrier are washed away from the flow path of the measurement cell. Next, the degradation enzyme solution is conveyed into the flow channel of the measurement cell, and the mononucleotides of the nucleic acid carried by the carrier are cleaved in order from the end. The fluorescent substance labeled with the mononucleotide is excited by an excitation laser, and the kind of fluorescent substance (that is, the kind of base) is read. The sample liquid or the like that has been measured is carried out to the waste liquid tank 53 by flowing a buffer solution.
[0082]
Details will be described below.
[0083]
A) Transport of buffer solution
(A-1) The valve 8 is opened, the valve 1 is set to the NC side (COM and NC are connected), and the valve 2 is set to the NO side (COM and NO are connected). The buffer solution tank 47 is pushed upward, and the buffer solution is guided to the waste solution tank by gravity.
[0084]
(A-2) The valve 2 is set to the NC side, the valve 5 is set to the NO side, and the buffer solution tank 47 is pushed upward to guide the buffer solution to the waste solution tank via the T-shaped joint 49. At this time, the valve 5 may be set to the NC side, the valve 7 may be set to the NC side, and suction may be performed by a negative pressure pump, or the valve 5 may be set to the NO side and guided to the waste liquid tank by gravity. By the operations (A-1) and (A-2), the solution can be quickly guided to the T-type joint 49.
[0085]
(A-3) The valve 5 is closed, the valve 6 is opened, and the buffer tank 47 is pushed upward to transfer the buffer into the flow path of the measurement cell. The buffer solution flowing through the flow path of the measurement cell is guided to the waste liquid tank through the opened valve 6. At this time, the valve 6 may be set to the NC side, the valve 7 may be set to the NO side, and suction may be performed by a negative pressure pump, or the valve 6 may be set to the NO side and guided to the waste liquid tank by gravity.
[0086]
(A-4) Open the valve 9, set the valve 3 to the NC side, and set the valve 4 to the NC side. The valve 6 is set to the NO side, the buffer solution tank 46 is pushed upward, and the buffer solution is guided to the waste solution tank by gravity. At this time, the valve 6 may be set to the NC side, the valve 7 may be set to the NO side, and suction may be performed by a negative pressure pump, or the valve 6 may be set to the NO side and guided to the waste liquid tank by gravity.
[0087]
(A-5) The valve 4 is set to the NO side, the buffer solution tank 46 is pushed upward, and the buffer solution is guided to the waste solution tank by gravity. By the operations (A-1) to (A-5), all the pipes are filled with the buffer solution.
[0088]
B) Sample input
Valve 1 is set to the NO side, valve 2 is set to the NC side, and valve 5 is closed. By opening the valve 6 and pushing the piston of the syringe 45, a sample liquid (for example, a glass bead suspension, a particle diameter of 1 μm, a concentration of 0.1% by weight) is guided to the flow path of the measurement cell. The sample liquid that has passed through the flow path of the measurement cell is guided to the waste liquid tank by suctioning with a negative pressure pump with the valve 6 on the NC side and the valve 7 on the NO side. Note that the sample liquid may be guided to the waste liquid tank by gravity.
[0089]
C) Cleaning of carriers other than the trapped carrier (B / F separation)
After capturing the carrier with the laser for capturing the carrier, the valve 1 is set to the NC side, the buffer solution tank 47 is pushed upward, the buffer solution is guided to the flow channel of the measurement cell, and the uncaptured carrier in the flow channel is washed away. At this time, the valve 6 may be set to the NC side, the valve 7 may be set to the NO side, and suction may be performed by a negative pressure pump, or the valve 6 may be set to the NO side and guided to the waste liquid tank by gravity. However, the use of a pump is not desirable because the trapped carrier may be washed away by the vibration. By this operation, carriers other than the carrier captured by the laser beam are washed away, and the flow path of the measurement cell is washed.
[0090]
D) Transport of degradation enzyme solution
The valve 9 is closed, the valve 3 is set to the NO side, the valve 4 is set to the NC side, and the valve 6 is closed. By opening the valve 5 and pushing in the piston of the syringe 44, the degradation enzyme solution is guided to the flow path of the measurement cell. The degradation enzyme solution that has passed through the flow path of the measurement cell may be led to the waste liquid tank by suctioning with a negative pressure pump with the valve 5 on the NC side and the valve 7 on the NC side, or led to the waste liquid tank by gravity. May be. However, even in this operation, it is not desirable to use the pump because the trapped carrier may be washed away by the vibration of the pump.
[0091]
E) Cleaning the flow path
After cleaving each base of nucleic acid with a degrading enzyme, valve 9 is opened, valve 3 is set to the NC side, valve 4 is set to the NC side, valve 6 is closed, buffer tank 46 is pushed upward, and the buffer solution is measured. To the flow path. This operation cleans the flow path. This washing operation can be efficiently performed by suctioning with a negative pressure pump with the valve 5 on the NC side and the valve 7 on the NC side.
[0092]
Next, another example of the solution carry-in / carry-out mechanism will be described with reference to FIG. This specific example is basically the same as the example shown in FIG. 6, and the same reference numerals are given to the same components. 7 includes a degradation enzyme introduction syringe 44, a sample introduction syringe 45, buffer tanks 46 and 47, valves 21 to 25, T-type joints 48, 49, 54 and 55, and a negative electrode. It is composed of a pressure pump 52, a waste tank 53, and piping connecting them.
[0093]
The solution loading / unloading mechanism shown in FIG. 7 is provided with branched pipes, valves, and T-shaped joints as in the example shown in FIG. 6, so that each solution can be efficiently and smoothly carried into and out of the microchannel. It is. Similarly, this mechanism can maintain a constant flow rate by using a specific valve that does not cause a sudden volume change (ie, does not cause a pulsating flow of liquid) during transport of the solution. is there.
[0094]
Similarly to the example shown in FIG. 6, the T-type joints 48 and 49 are provided in the vicinity of the flow channel inlet of the measurement cell to form a pipe branched into the direction toward the flow channel of the measurement cell and the direction toward the waste liquid. It is functioning. By using this branched pipe to temporarily release the solution to the waste liquid tank, the solution to be transported into the flow channel of the measurement cell can be quickly guided to the vicinity of the flow channel inlet of the measurement cell. Thereby, conveyance of the solution to the flow path of the measurement cell which is difficult to convey the solution can be assisted. For example, the T-type joint 48 can be positioned at a position about 20 mm away from the connection terminal 50, and the T-type joint 49 can be located at a position about 20 mm away from the connection terminal 51.
[0095]
In addition, as in the example shown in FIG. 6, it is preferable to make the flow path width of the pipe outside the measurement cell wider than the flow path width (for example, 50 μm) inside the measurement cell in order to smoothly transport the solution.
[0096]
Below, operation | movement of the solution carrying in / out mechanism shown in FIG. 7 is demonstrated.
[0097]
A) Transport of buffer solution
Prior to sample introduction, all the flow paths are filled with a buffer solution by the following operation.
[0098]
(A-1) Open the valve 21 and set the valve 23 to the NO side. The buffer solution tank 47 is pushed upward, and the buffer solution is guided to the waste solution tank by gravity. At this time, the valve 23 may be on the NC side, the valve 25 may be on the NC side, and suction may be performed by a negative pressure pump, or the valve 23 may be on the NO side and guided to the waste liquid tank by gravity. By this operation, the solution can be quickly guided to the T-type joint 49.
[0099]
(A-2) The valve 23 is closed, the valve 24 is set to the NC side or the NO side, and the buffer solution tank 47 is pushed upward to convey the buffer solution into the flow path of the measurement cell. The buffer solution flowing through the flow path of the measurement cell is led to the waste liquid tank through the valve 24. At this time, the valve 24 may be set to the NC side, the valve 25 may be set to the NO side, and suction may be performed by a negative pressure pump, or the valve 25 may be set to the NO side and guided to the waste liquid tank by gravity.
[0100]
(A-3) The valve 22 is opened, the valve 24 is set to the NC side or NO side, the buffer solution tank 46 is pushed upward, and the buffer solution is guided toward the waste solution tank by gravity. At this time, the valve 24 may be set to the NC side, the valve 25 may be set to the NO side, and suction may be performed by a negative pressure pump, or the valve 24 may be set to the NO side and guided to the waste liquid tank by gravity. Through the operations (A-1) to (A-3), all the flow paths are filled with the buffer solution.
[0101]
B) Sample input
The valve 21 is closed, the valve 23 is closed, the valve 24 is opened, and the piston of the syringe 45 is pushed in, so that the sample liquid (for example, glass bead suspension, particle diameter 1 μm, concentration 0.1% by weight) flows through the measurement cell. Lead to. The sample liquid that has passed through the flow path of the measurement cell is guided to the waste liquid tank by suctioning with a negative pressure pump with the valve 24 on the NC side and the valve 25 on the NO side. Note that the sample liquid may be guided to the waste liquid tank by gravity.
[0102]
C) Cleaning of carriers other than the trapped carrier (B / F separation)
After capturing the carrier with laser light, the valve 21 is opened, the buffer tank 47 is pushed upward, the buffer solution is guided to the flow path of the measurement cell, and the uncaptured carrier in the flow path is washed away. At this time, the valve 24 may be set to the NC side, the valve 25 may be set to the NO side, and suction may be performed by a negative pressure pump, or the valve 24 may be set to the NO side and guided to the waste liquid tank by gravity. However, the use of a pump is not desirable because the trapped carrier may be washed away by the vibration. By this operation, carriers other than the carrier captured by the laser beam are washed away, and the flow path of the measurement cell is washed.
[0103]
D) Transport of degradation enzyme solution
The valve 22 is closed, the valve 24 is closed, the valve 23 is opened, and the piston of the syringe 44 is pushed in, whereby the degrading enzyme solution is guided to the flow path of the measurement cell. The degradation enzyme solution that has passed through the flow path of the measurement cell may be led to the waste liquid tank by suctioning with a negative pressure pump with the valve 23 on the NC side and the valve 25 on the NC side, or led to the waste liquid tank by gravity. May be. However, even in this operation, it is not desirable to use the pump because the trapped carrier may be washed away by the vibration of the pump.
[0104]
E) Cleaning the flow path
After the cleavage of each base of the nucleic acid by the degrading enzyme, the valve 22 is opened, the valve 24 is closed, the buffer solution tank 46 is pushed upward, the buffer solution is guided to the flow channel of the measurement cell, and the flow channel is washed. At this time, the flow path can be efficiently cleaned by suction with a negative pressure pump with the valve 23 on the NC side and the valve 25 on the NC side.
[0105]
≪Laser light source for carrier capture≫
Next, the “first laser light source 3 for capturing the carrier to which the nucleic acid is attached” will be described. The type and irradiation wavelength of the laser emitted from the first laser light source (hereinafter also referred to as carrier capturing laser) can be appropriately selected according to the size of the carrier to be captured. Alternatively, after selecting a laser for capturing a carrier, the size of the carrier captured by the laser may be selected.
[0106]
The laser used in the present invention is not limited as long as it is conventionally used in a laser trap, and generally a laser of 488 nm (argon laser) to 1064 nm (Nd: YAG laser) can be used. For example, Cr. A LiSAF laser can be used. Alternatively, an infrared solid laser such as an Nd: YAG laser or a dye laser may be used. Preferably, an infrared laser having a wavelength of 700 nm or more is used. For example, in order to capture glass fine particles having a diameter of 1 μm with a laser trap, Cr. A LiSAF laser can be used.
[0107]
For details on the relationship between laser wavelength and trapped particle size, see WHWright, GJSonek, and MW Berns, Appl.Phys.Lett., Vol.63 (6), 1993, Radiation trapping forces on microspheres with See the description of optical tweezers.
[0108]
The higher the output of the carrier capture laser, the stronger the ability to capture the carrier, which is desirable. However, the device (objective lens), the analytical sample, and the degrading enzyme that cleaves the bases constituting the nucleic acid from the end are damaged. There is. Therefore, the output is set high (preferably 2 W or less) so as not to cause such a harmful effect. Generally, a laser power of 100 to 1000 mW is used to capture the carrier.
[0109]
≪First optical system≫
Next, “first optical systems 4 to 10 that collect the light beam from the first laser light source in the flow path of the measurement cell and capture the carrier to which the nucleic acid is attached” will be described. The optical system includes a collimating lens 4, a beam expander 5, a lens 6, a mirror 7, a lens 8, a dichroic half mirror 9, and an objective lens 10. These optical systems 4 to 10 are arranged in this order on the optical path of the light beam emitted from the carrier capturing laser.
[0110]
In the apparatus of the present invention, the condensing optical system using the objective lens is a confocal optical system, and the laser beam is focused on a very narrow area determined by the numerical aperture (NA) of the objective lens. Yes. For example, the numerical aperture (NA) of the objective lens used in the apparatus of the present invention is 0.9.
[0111]
The light beam of the carrier capturing laser is collimated by the collimating lens 4 and the beam diameter is increased by the beam expander 5. Thereafter, the light is condensed on the surface of the mirror 7 by the lens 6 and converted into parallel light by the lens 8, and then the optical path is bent by the dichroic half mirror 9 and guided to the objective lens 10. The light beam through the objective lens 10 is condensed at a predetermined position in the flow path of the measurement cell 1.
[0112]
The capturing position of the carrier in the flow path can be adjusted in the length direction of the flow path by changing the angle of the mirror 7. Further, the capture position in the optical axis direction can be controlled by the movement of the objective lens 10 in the optical axis direction. By adjusting and controlling in this way, it becomes possible to irradiate a predetermined position in the channel with the carrier capturing laser. Here, the predetermined position is determined by the positional relationship with the excitation laser at the time of base sequence analysis, which will be described later.
[0113]
Note that when capturing the carrier by irradiating the carrier capturing laser, the sample liquid may or may not flow in the flow path.
[0114]
The light beam condensed in the flow path as described above can capture one carrier moving along the flow in the flow path. This is shown in FIG. As shown in FIG. 8, the light beam of the carrier capturing laser is focused by the objective lens 10, passes through the surface portion 1 b of the measurement cell 1, and is focused on the central portion of the solution in the flow path 1 c, Irradiate a suspension of the carrier. Thereby, one carrier is captured in the flow path.
[0115]
≪Enzyme solution transport means≫
Next, “means for transporting an enzyme solution that cleaves mononucleotides constituting the nucleic acid in order from the end to the flow path of the measurement cell” will be described. In the present embodiment, the means for flowing the enzyme solution can be shared with the means 2 for transporting the carrier suspension. Therefore, see also the above description regarding the solution carry-in / carry-out mechanism.
[0116]
Here, “cleave in order from the end” means that, when the sample nucleic acid is attached to the carrier on the 5 ′ end side, the mononucleotide constituting the nucleic acid is cleaved in order from the 3 ′ end. . On the contrary, when the nucleic acid used as a sample is attached to the carrier on the 3 ′ end side, it means that the mononucleotides constituting the nucleic acid are sequentially cleaved from the 5 ′ end.
[0117]
The “enzyme solution” can be appropriately selected from exonuclease that cleaves mononucleotides one by one only from the 3 ′ side, or exonuclease that cleaves mononucleotides one by one from the 5 ′ side. For example, exonuclease I, T7 polymerase can be used. The enzyme activity possessed by the enzyme solution is appropriately set within a range in which a desired decomposition reaction is performed, but preferably an enzyme solution having an activity of 10 to 200 nM is used.
[0118]
In the solution carry-in / carry-out mechanism shown in FIG. 5, the enzyme solution is caused to flow from the carry-in tank 2a to the flow path at a predetermined flow rate and is discharged from the flow path to the carry-out tank 2c. As described above, the enzyme solution can be caused to flow through the flow path at a predetermined flow rate by controlling the vertical positions of both tanks or controlling the gas pressure of the syringe. Further, the solution can be carried in and out by a motor-driven pump.
[0119]
As described above, in the transportation of the enzyme solution, to maintain a predetermined flow rate constantly, it is required in the present invention that mononucleotides cut in order from the end are transported in the flow path in the order. Has an important meaning. In the present invention, when the flow velocity is too high, particles captured by the laser trap escape, so the upper limit is a flow velocity at which particles do not escape from the trapped state. If the flow rate is too slow, the cleaved mononucleotides may move in any direction due to Brownian motion, and the mononucleotides may not be aligned in the cleaved order. A flow rate of about In order to increase the analysis accuracy of the base sequence of the nucleic acid, it is preferable that the flow rate be high in a range where the captured particles do not escape. That is, the flow velocity is set in relation to the force (laser output) that is laser trapping particles. Generally, the flow rate is generally set to 0.5 mm / s to 10 mm / s. For example, the flow rate is set to 1 mm / s.
[0120]
<< Laser light source for excitation >>
Next, “excitation laser light source 11 for exciting a fluorescent substance that has been identified and labeled with a base of a mononucleotide cleaved by the enzyme solution in the order of cleavage” will be described. The excitation laser generally has a wavelength in the visible range, and a laser having a wavelength for exciting the fluorescent substance used is used. For example, a solid blue laser (473 nm) is used when rhodamine green is used as the fluorescent material, and an argon laser (514.5 nm) is used when TMR is used.
[0121]
In this embodiment, there is one excitation laser light source, and therefore, only one type of fluorescent substance excited by the laser light source is used for labeling the nucleobase.
[0122]
≪Second optical system≫
Subsequently, “second optical systems 12 to 14 and 10 for condensing the light beam from the excitation laser light source in the flow path of the measurement cell and exciting the fluorescent materials in the order of cutting” will be described. The optical system includes a collimating lens 12, a beam expander 13, a dichroic half mirror 14, and an objective lens 10. These optical systems 12 to 14 and 10 are arranged in this order on the optical path of the light beam emitted from the laser light source 11.
[0123]
The light beam from the excitation laser light source 11 is converted into a parallel beam by the collimator lens 12, the beam diameter is increased by the beam expander 13, the optical path is bent by the dichroic half mirror 14, and the light beam is guided to the objective lens 10. The light beam that has passed through the objective lens 10 is condensed at a predetermined position in the flow path of the measurement cell 1.
[0124]
The irradiation position of the excitation laser in the flow path can be adjusted in the length direction of the flow path by changing the angle of the dichroic half mirror 14. Further, the irradiation position in the optical axis direction can be controlled by moving the objective lens 10 in the optical axis direction. By adjusting and controlling in this way, it becomes possible to irradiate a predetermined position in the flow path with the excitation laser.
[0125]
Therefore, in the present embodiment, both the light beam of the carrier capturing laser and the light beam of the excitation laser described above are collected by one objective lens 10 and focused in the flow path of the measurement cell 1.
[0126]
In the present invention, at the time of base sequence analysis, the light beam of the carrier capturing laser and the light beam of the excitation laser need to be irradiated in a predetermined positional relationship within the flow path of the measurement cell. Specifically, at the time of base sequence analysis, both beams need to be irradiated into the flow path in a state as shown in FIG. That is, it is necessary that the carrier is captured by the carrier capturing laser upstream of the channel through which the solution flows, and the excitation laser is irradiated downstream thereof. With such a predetermined positional relationship, the fluorescent substance in which the mononucleotides constituting the nucleic acid supported on the carrier are sequentially allowed to flow into the irradiation region of the excitation laser in the order of cleavage and then the bases of the mononucleotides are labeled Can be sequentially excited (see FIG. 9).
[0127]
Therefore, the optical path of the light beam is set in advance so that both laser beams are irradiated in the predetermined positional relationship at the time of base sequence analysis, or the carrier capturing laser beam or the excitation laser beam is analyzed before the analysis. It is necessary to move at least one beam to an appropriate position. In the present embodiment shown in FIG. 2, such control of the optical path of the light beam is performed by an optical system for irradiating the light beam (specifically, the dichroic half mirror 9 or the dichroic half mirror 14).
[0128]
The predetermined position of both laser beams at the time of base sequence analysis is not particularly limited as long as both lasers are located in the objective lens field of view, but preferably both lasers are located farthest in the objective lens field of view. (Approximately 25 μm apart).
[0129]
As described above, in this embodiment, it is possible to focus two laser beams on a predetermined distance with one objective lens. However, the base sequence determination apparatus of the present invention is not limited to a mode in which two light beams are condensed at a position separated by one objective lens, and the apparatus may be assembled by two objective lenses (described later). (Refer to the fourth embodiment and FIG. 13).
[0130]
≪Third optical system≫
Next, the “third optical systems 10 and 15 to 19 that sequentially collect fluorescence from a fluorescent substance that identifies and labels the bases of the nucleic acids” will be described. The optical system includes an objective lens 10, a lens 15, an aperture 16, a condenser lens 17, an Ir cut filter 18, and a band pass filter 19. These optical systems 15 to 19 are arranged in this order on the optical path of the fluorescence emitted from the fluorescent material. In FIG. 2, an optical system that connects the objective lens 10 and the light receiver 20 that detects the condensed fluorescence is referred to as a “measurement optical system”.
[0131]
Fluorescence from the fluorescent material passes through the objective lens 10, forms an image on the aperture 16 by the lens 15, and then focuses on the light receiving surface of the light receiver 20 by the condenser lens 17. An Ir cut filter 18 and a band pass filter 19 are arranged in front of the light receiving surface of the light receiver 20 so that only the fluorescence from the fluorescent material reaches the light receiver 20. The Ir cut filter 18 cuts noise light such as light reflected by the optical system in the middle of the optical path such as the wall surface of the measurement cell 1 and the back surface of the mirror. The band pass filter 19 efficiently allows only the light having the fluorescence wavelength to pass therethrough.
[0132]
≪Photodetector≫
Next, “the photodetector 20 that sequentially detects collected fluorescence and generates an identification signal corresponding to the detected fluorescence” will be described. The photodetector 20 can be a weak photodetector such as an avalanche photodiode or a photomultiplier tube. The photodetector 20 detects fluorescence corresponding to the type of base of the mononucleotide in the order of the cleaved mononucleotide. Then, the detected fluorescence is converted into an identification signal corresponding to the detected fluorescence and transmitted to an analysis unit described later. Specifically, it is converted into photoelectrons by the photodetector 20.
[0133]
≪Analysis means≫
Finally, “analyzing means 21 to 23 for reading the base sequence of the nucleic acid from the identification signal generated by the photodetector” will be described. The analysis means processes the signal obtained from the photodetector 20 by the signal processing unit 21 and displays the result on the monitor screen of the computer 23. Specifically, the optical signal converted into photoelectrons by the photodetector 20 is subjected to processing such as waveform shaping by the signal processing unit 21, and then guided to the computer 23 as a photon pulse, and the number of pulses is counted. Displayed on the screen. The signal control unit 22 can release the trap of the carrier by feeding back the final information sent to the computer to the laser light source for capturing the carrier.
[0134]
≪Configuration for observing the capture state of the carrier≫
In the base sequence determination apparatus of the present embodiment shown in FIG. 2, a light source 24 for irradiating illumination light into the flow path of the measurement cell in order to photograph the captured state of the carrier, an illumination optical system 25, 26, 15, and 10 are installed. In FIG. 2, the light source 24 is a halogen lamp, and the illumination optical systems 25, 26, 15, and 10 include a lens 25, a movable mirror 26, a lens 15, and the objective lens 10. Illumination light from the light source 24 passes through the lens 25, bends in the direction of the measurement cell by the movable mirror 26, passes through the lens 15, and is then condensed on the analysis sample by the objective lens 10.
[0135]
The light source and the illumination optical system are not necessarily required in the apparatus of the present invention. Therefore, when not in use, the movable mirror 26 may be removed from the measuring optical system as necessary by moving it.
[0136]
In addition, the base sequence determination apparatus according to the present embodiment shown in FIG. 2 includes imaging optical systems 10, 15, 27 to 29 for imaging a state in which a carrier is captured under the illumination light, and imaging means. 30 is installed. In FIG. 2, the photographing optical systems 10, 15, and 27 to 29 are composed of an objective lens 10, a lens 15, a movable prism 27, a band pass filter 28, and a lens 29, and the imaging means 30 is a CCD camera. The reflected light from the analysis sample irradiated with the illumination light passes through the objective lens 10 and the lens 15, and then is bent toward the CCD camera 30 by the movable prism 27, and only the light having the necessary wavelength is obtained by the band pass filter 28. The light is selectively transmitted and sent to the image pickup means (CCD camera) 30 through the lens 29. In this way, the photographing optical system selectively transmits light having a wavelength that is efficiently received by the CCD camera 30 and forms an image on the television monitor.
[0137]
In FIG. 2, the imaging optical system and the illumination optical system irradiate the captured carrier in the flow channel of the measurement cell and image it, so that it is necessary to match the measurement optical system with the movable prism 27. is there. Further, the photographing optical system and the imaging means are not necessarily required in the apparatus of the present invention. Therefore, it may be used only when capturing the carrier, and may be removed from the measuring optical system as necessary by moving the movable prism 27 at other times.
[0138]
[Second Embodiment]
Next, a base sequence determination apparatus according to the second embodiment of the present invention will be described with reference to FIG.
[0139]
The basic configuration of the second embodiment is the same as that of the first embodiment. In the second embodiment of FIG. 10, the same reference numerals are given to the same portions (reference numerals 1 to 30) as those of the first embodiment shown in FIG. In the apparatus of the present embodiment, an analysis sample in which a nucleic acid base is identified and labeled using two types of fluorescent substances is used.
[0140]
Therefore, in this embodiment, two types of lasers having different emission wavelengths, for example, an argon ion laser (wavelength 514.5 nm) and a solid blue laser (wavelength 473 nm) are used as the excitation laser light source. Therefore, in the apparatus of the present embodiment in FIG. 10, a second excitation laser light source 31 is installed in addition to the excitation laser light source 11.
[0141]
Similarly to the light beam from the excitation laser light source 11, the light beam from the second excitation laser light source 31 is converted into a parallel beam by the collimator lens 32 and the beam diameter is increased by the beam expander 33. Thereafter, the optical path is bent by the mirror 34 and the two dichroic half mirrors 35 and 14 and guided to the objective lens 10. The light beams from the two excitation laser light sources 11 and 31 are combined into one optical path by the dichroic half mirror 35 and guided to the objective lens 10.
[0142]
Thus, two types of fluorescent materials can be excited by using two types of lasers having different wavelengths. As the two kinds of fluorescent substances, for example, rhodamine green (RG) and tetramethylrhodamine-5-isothiocyanate (TMR) can be used. Rhodamine green is excited by a solid blue laser (473 nm) and emits fluorescence at a wavelength of 550 nm, while tetramethylrhodamine-5-isothiocyanate is excited by an argon laser (514.5 nm) and emits fluorescence at a wavelength of 570 nm. Radiate. By emitting fluorescence of different wavelengths using two types of fluorescent substances, it becomes possible to distinguish and detect two types of bases.
[0143]
In the present embodiment, in order to detect emitted fluorescence having different wavelengths, it is necessary to provide two measurement optical systems. That is, the fluorescence from two types of fluorescent materials is adjusted by the dichroic half mirror 36 so that the transmitted light intensity is maximized in accordance with the emission wavelength of each fluorescent material, and the optical path is divided into two. One of the two divided lights passes through the band pass filter 19 and is received by the light receiver 20. The optical signal converted into photoelectrons by the light receiver is processed by the signal processing unit 21 and sent to the computer 23 as a photon pulse, and the number of pulses is counted. Similarly, the other light passes through the band pass filter 37 and is received by the light receiver 38. A signal from the light receiver 20 is processed by the signal processing unit 39 and sent to the computer 23. The signal control unit 22 can release the trap of the carrier by feeding back the received information to the laser light source for capturing the carrier.
[0144]
The configuration of such two measurement optical systems is basically the same as that of the one measurement optical system in the first embodiment. The areas measured by the two light receivers are the same area. In this way, fluorescence can be detected independently for each of the two optical paths.
[0145]
The apparatus of the second embodiment has the following advantages. That is, the position of two types of fluorescence can be read simultaneously with one measurement, and the corresponding base sequence can be read. Therefore, the number of measurements can be reduced and the measurement accuracy can be improved as compared with the apparatus of the first embodiment.
[0146]
In addition, the base sequence determination apparatus of this invention can also determine the base sequence of the nucleic acid which identified and labeled the base using three types of fluorescent substances which can install and detect three excitation laser light sources. Furthermore, a base sequence determination apparatus provided with more than three excitation laser light sources is possible, and such an apparatus is also included in the scope of the present invention.
[0147]
[Third Embodiment]
Next, a base sequence determination device according to a third embodiment of the present invention will be described with reference to FIG.
[0148]
The basic configuration of the third embodiment is the same as that of the first embodiment. In the third embodiment shown in FIG. 11, the same reference numerals are given to the same portions (reference numerals 1 to 30) as in the first embodiment shown in FIG. 2. As in the first embodiment, the apparatus of this embodiment uses an analysis sample in which a nucleic acid base is identified and labeled using one type of fluorescent substance.
[0149]
In the present embodiment, a completely transparent measurement cell 1 ′ as shown in FIG. 12 is used as a measurement cell having a flow path. FIG. 12 is a view corresponding to FIG. 3C and a cross-sectional view taken along the flow path.
[0150]
The structure of the measurement cell 1 ′ is basically the same as that of the measurement cell 1 shown in FIG. The measurement cell 1 is different in that only the surface through which the laser beam is transmitted is transparent, whereas the measurement cell 1 ′ used here is a transparent cell. The measurement cell 1 ′ can be composed of, for example, a glass plate, Pyrex, or a transparent plastic plate such as acrylic or vinyl chloride resin.
[0151]
By using such a measurement cell 1 ′, the imaging optical system for observing the state where the carrier particles are captured can be separated from the measurement optical system. In other words, in FIG. 11, the photographing optical systems 43 and 28 to 29 are separated from the optical system from the objective lens 10 to the light receiver 20 which is a measurement optical system.
[0152]
In the present embodiment, the illumination light from the halogen lamp 24 and the light beam from the carrier capturing laser 3 are expanded through the objective lens 43, and then only the light having the required wavelength is selectively transmitted by the bandpass filter 28. The image is picked up by the image pickup means (CCD camera) 30 through the lens 29.
[0153]
Moreover, the base sequence determination apparatus of this invention may be equipped with the shutter opened and closed corresponding to operation | movement of each optical system, as FIG. 11 shows in figure. Specifically, in FIG. 11, a first optical system that guides the carrier capturing laser from the light source 3 to the measurement cell, a second optical system that guides the excitation laser from the light source 11 to the measurement cell, and the objective lens 10 Shutters 40, 41, and 42 are sequentially installed in each of the measurement optical systems from to the light receiver 20.
[0154]
Thus, by separating the imaging optical system from the measurement optical system, it is possible to remove the interference with the measurement light of the optical system for observation and observe the transmitted light. Becomes brighter, and it is possible to clearly detect the state of carrier particle transport and particle trapping.
[0155]
[Fourth Embodiment]
Next, a base sequence determining apparatus according to the fourth embodiment of the present invention will be described with reference to FIG.
[0156]
The basic configuration of the fourth embodiment is the same as that of the first embodiment. In the fourth embodiment shown in FIG. 13, the same parts (reference numerals 1 to 30) as those in the first embodiment shown in FIG. As in the first embodiment, the apparatus of this embodiment uses an analysis sample in which a nucleic acid base is identified and labeled using one type of fluorescent substance.
[0157]
In the present embodiment, as in the apparatus according to the third embodiment, a completely transparent measurement cell 1 ′ as shown in FIG. 12 is used as a measurement cell having a flow path.
[0158]
By using such a measurement cell 1 ′, the imaging optical system for observing the state where the carrier particles are captured can be separated from the measurement optical system. That is, in FIG. 13, the photographing optical systems 43 and 28 to 29 are separated from the optical system from the objective lens 10 to the light receiver 20 which is a measurement optical system.
[0159]
Thus, by separating the imaging optical system from the measurement optical system, it is possible to remove the interference with the measurement light of the optical system for observation and observe the transmitted light. Becomes brighter, and it is possible to clearly detect the state of carrier particle transport and particle trapping.
[0160]
Further, in the present embodiment, by using the entire transparent measurement cell 1 ′, the first optical system for guiding the carrier capturing laser to the measurement cell is used, and the second optical system for guiding the excitation laser to the measurement cell is used. It becomes possible to separate from the optical system and the measuring optical system. That is, in FIG. 13, the first optical systems 4 to 9 and 43 are separated from the second optical systems 11 to 14 and 10 and the measurement optical systems 10 and 14 to 20. In FIG. 13, the carrier capturing laser is incident from the upper surface of the measurement cell and condensed in the flow channel, and the excitation laser is incident from the lower surface of the measurement cell and condensed in the flow channel.
[0161]
As described above, the laser capturing laser (infrared light) and the excitation laser (visible light) are shared by the same optical system and are not condensed in the flow path, so that the focal positions of the respective laser beams are adjusted. It becomes easy. In general, a correction optical system is required to adjust the focus position of infrared light and visible light to the same position through the same optical system. However, as shown in FIG. 13, it is possible to independently adjust the focal position by passing the carrier capturing laser and the excitation laser through separate optical systems.
[0162]
For details of the operations of the base sequence determination apparatuses according to all the embodiments described above, refer to the following description of the base sequence determination method.
[0163]
<Base sequence determination method>
Next, the base sequence determination method of the present invention will be described. In addition, the base sequence determination method of this invention can be performed using the above-mentioned apparatus. Please refer to the configuration of the above-mentioned apparatus as appropriate.
[0164]
The base sequence determination method of the present invention comprises:
As an analysis sample, a carrier to which a nucleic acid labeled and associated with a base type and a fluorescent substance type is attached is used. As a measurement cell, a flow channel is provided inside, and a light beam is transmitted into the flow channel. A base sequencing method using a cell with a transparent surface for,
1) a step of transporting a suspension of a carrier to which the nucleic acid, which is an analysis sample, is attached, to the flow path of the measurement cell;
2) capturing the carrier to which the nucleic acid is attached with a laser trap in the flow path;
3) flowing an enzyme solution for cleaving mononucleotides constituting the nucleic acid in order from the end into the flow path;
4) a step of sequentially exciting a fluorescent substance that discriminates and labels the base of the mononucleotide cleaved by the enzyme solution;
5) a step of sequentially detecting the fluorescence emitted by the fluorescent material excited by the above step and reading a base sequence corresponding to the detected fluorescence;
It is the method which comprises.
[0165]
In a preferred embodiment, the base sequence determination method of the present invention comprises:
As an analysis sample, a carrier to which a nucleic acid labeled and associated with a base type and a fluorescent substance type is attached is used. As a measurement cell, a flow channel is provided inside, and a light beam is transmitted into the flow channel. A base sequencing method using a cell with a transparent surface for,
1) a step of transporting a suspension of a carrier to which the nucleic acid, which is an analysis sample, is attached, to the flow path of the measurement cell;
2-1) capturing the carrier to which the nucleic acid is attached with a laser trap in the flow path;
2-2) A step of confirming that the nucleic acid molecule identified and labeled with a fluorescent substance is attached to the carrier captured in the step by irradiating with excitation light;
2-3) A step of positioning a laser irradiation position for capturing the carrier in the flow channel upstream of the excitation light irradiation position;
3) flowing an enzyme solution for cleaving mononucleotides constituting the nucleic acid in order from the end into the flow path;
4) a step of sequentially exciting a fluorescent substance that discriminates and labels the base of the mononucleotide cleaved by the enzyme solution;
5) a step of sequentially detecting the fluorescence emitted by the fluorescent material excited by the above step and reading a base sequence corresponding to the detected fluorescence;
It is the method which comprises.
[0166]
The analysis sample and measurement cell used in the base sequence determination method of the present invention are as described above. Hereinafter, each process of 1) -5) is demonstrated in order. See also the flowchart shown in FIG. 14 for the overall flow of the base sequence determination method.
[0167]
1) Transport of analysis sample
An analysis sample (a carrier suspension to which a nucleic acid whose base is discriminated and labeled with a fluorescent substance) is conveyed to a measurement cell by a solution loading / unloading mechanism as shown in FIG.
[0168]
2-1) Carrier capture
The carrier capturing laser is turned on, and one carrier is captured by the laser trap. When one carrier is captured, other uncaptured carriers present in the channel may be washed away with a buffer solution. Here, the state where one carrier is captured can be confirmed by an imaging means (CCD camera).
[0169]
2-2) It can be confirmed as follows that the nucleic acid molecule identified and labeled with the fluorescent substance is attached to the captured carrier. The excitation laser is turned on while the carrier is captured by the laser trap. At this time, the light beam of the excitation laser irradiates the carrier captured through the same optical path as the carrier capturing laser. Thereby, the fluorescent substance labeling the nucleic acid carried by the carrier is excited.
[0170]
Next, the presence or absence of fluorescence emitted from the fluorescent material is measured. The emitted fluorescent signal is converted into an electric signal (photon pulse) and detected. An example of the result of fluorescence measurement is shown in FIG. A certain threshold value is determined in advance, and it is determined that the fluorescence is detected only when the number of photon pulses is measured more than this. This threshold value can be appropriately set in consideration of the background noise of the apparatus.
[0171]
When the number of photon pulses is lower than the threshold and fluorescence is not detected (in the case of “NO” in the flowchart), it is determined that no nucleic acid molecule is attached to the carrier. At this time, the signal control unit issues a command, stops the irradiation of the carrier capturing laser, and releases the trap of the carrier. The carrier suspension is again poured into the measurement cell, and the same operation is repeated until fluorescence is detected by fluorescence measurement.
[0172]
When the number of photon pulses is higher than the threshold and fluorescence is detected (in the case of “YES” in the flowchart), it is determined that nucleic acid molecules are attached to the carrier.
[0173]
2-3) When it is confirmed that nucleic acid molecules are attached to the captured carrier, the irradiation positions of the carrier capturing laser and the excitation laser that are irradiating the carrier through the same optical path are A predetermined state (predetermined state shown in FIG. 9) necessary for analysis is set. That is, when a decomposing enzyme solution for cleaving nucleobases one by one is conveyed into the flow path, a state is created in which the carrier is captured upstream and the excitation laser is irradiated downstream. Specifically, a predetermined state can be created by moving the carrier capturing laser beam that was on the same optical path as the excitation laser beam in the direction perpendicular to the optical axis (length direction of the flow path). Alternatively, the predetermined state shown in FIG. 9 may be created by shifting the optical path of the excitation laser beam from the optical path of the carrier capturing laser beam.
[0174]
Here, the distance for moving the carrier capturing laser and / or the excitation laser beam is not particularly limited as long as both lasers are located in the objective lens field, but preferably both lasers are in the objective lens field. And move to the most distant position (about 25 μm).
[0175]
3) Introduction of degrading enzymes
After both laser beams are set in a predetermined state for analyzing the base sequence, a buffer solution containing a degrading enzyme is caused to flow through the flow path of the measurement cell by the solution carry-in / carry-out mechanism. By introducing the degrading enzyme, the mononucleotide of the nucleic acid attached to the carrier is cleaved sequentially from the end.
[0176]
Buffers containing degrading enzymes include 100 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ 2.5 mM DTT can be used. As the degrading enzyme, exonuclease having an activity of 10 to 200 nM, T7 polymerase and the like can be used. The decomposition reaction is performed at room temperature in this buffer. The composition, temperature, etc. of the buffer solution are not limited to those described above.
[0177]
As described above, the flow rate is a speed (lower limit) at which the cleaved mononucleotide can flow in the flow path in accordance with the cleaving order, and the speed at which the carrier does not escape from the laser trapped state ( The upper limit is not limited. For example, it can be about 1 mm / s.
[0178]
The operation of transporting the solution into the flow path needs to be performed so that no pulsating flow occurs after the carrier is captured in the flow path. This is to prevent the trapped carrier from being washed away by the pulsating flow, and to ensure that the cut bases are transported in the flow path in the order.
[0179]
4) Fluorescence emission
The bases of the mononucleotide cleaved by the degrading enzyme flow into the excitation laser irradiation position (that is, the measurement region) in the flow path in the order of being cleaved under the control of the flow rate. As a result, the fluorescent substances that identify and label the bases are sequentially excited, and the fluorescence emitted at this time is sequentially guided to the measurement optical system via the objective lens.
[0180]
5) Reading the base sequence
The base sequence is sequentially read by the fluorescent substance that identifies and labels each base of the nucleic acid.
[0181]
Hereinafter, specific examples of identifying and labeling the types of bases of nucleic acids and the types of fluorescent substances in association with each other will be given.
[0182]
For example, FIG. 16 shows the result of reading the base sequence of an analysis sample in which one type of DNA base is identified and labeled using one type of fluorescent substance. The analysis sample can be read in sequence using the apparatus of the first embodiment of the present invention.
[0183]
In the figure, “◯” indicates that the base is labeled with a fluorescent substance, and “X” indicates that the base is not labeled with a fluorescent substance. Of the four types of bases in the sample DNA, the sample labeled with all adenine is labeled sample 1-1, the sample labeled with all guanine is labeled with sample 1-2, and the sample labeled with all thymine is labeled with sample 1-3. .
[0184]
In this way, when only a single base is labeled, at least three measurement times are required to read the entire base sequence, but the DNA base sequence can be read as indicated by the reading result. .
[0185]
FIG. 17 shows the result of reading the base sequence of an analysis sample in which two types of bases are discriminated and labeled using two types of fluorescent substances. The analysis sample can be read in sequence using the apparatus of the second embodiment of the present invention.
[0186]
In the figure, “◯” indicates that the base is labeled with fluorescent substance A (eg, TMR), “●” indicates that the base is labeled with fluorescent substance B (eg, RG), and “×” indicates that the base is labeled with Indicates that the base is not labeled with a fluorescent substance. Of the four types of bases in the sample DNA, adenine and guanine are identified and labeled with two types of fluorescent substances A and B, respectively. Sample 2-1, and thymine and cytosine are identified and labeled with two types of fluorescent substances A and B, respectively. This was designated as Sample 2-2. The reading results are collectively shown in FIG.
[0187]
When two types of fluorescent substances are used in this way, the positions of two types of bases can be detected simultaneously in one measurement, and the number of measurements for reading the entire base sequence can be made at least twice. Compared with the case where one kind of fluorescent substance is used (example shown in FIG. 16), it becomes possible to perform sequencing in a short time.
[0188]
FIG. 19 shows the result of reading the base sequence of an analysis sample in which one type of base and the other three types of bases are discriminated using two types of fluorescent substances. The analysis sample can be read in sequence using the apparatus of the second embodiment of the present invention.
[0189]
In the figure, “◯” indicates that the base is labeled with a fluorescent substance A (eg, TMR), and “●” indicates that the base is labeled with a fluorescent substance B (eg, RG). Of the four bases of sample DNA, adenine is labeled with fluorescent substance A, the other bases (G, T, C) are labeled with fluorescent substance B, sample 3-1 and guanine is labeled with fluorescent substance A. Other bases (A, T, C) labeled with fluorescent substance B are sample 3-2, thymine is labeled with fluorescent substance A, and other bases (A, G, C) are labeled with fluorescent substance B. The labeled product is designated as Sample 3-3.
[0190]
Thus, if all four types of bases are discriminated and labeled using two types of fluorescent substances, fluorescence will be observed for all four types of bases, so that the measurement accuracy can be improved. That is, it is not necessary to determine the number of bases for which fluorescence was not observed as in the examples shown in FIGS. 16 and 17 based on the length of time during which fluorescence was not detected.
[0191]
In the method of the present invention, it is desirable to obtain the sequencing data for a plurality of times by changing the DNA molecule for one analysis sample, thereby improving the reliability of the data. The pattern for identifying and labeling the base of the nucleic acid is not limited to the examples shown in FIGS. 16, 17, and 19 and can be set as appropriate.
[0192]
【The invention's effect】
As described above, the present invention provides a novel base sequence determination apparatus and base sequence determination method. According to the present invention, the base sequence of a nucleic acid can be read easily and in a short time without requiring skill. In addition, the present invention has the advantage that the length of the nucleic acid serving as a sample is not limited in principle.
[Brief description of the drawings]
FIG. 1 is a view showing an analysis sample used for base sequence determination of the present invention.
FIG. 2 is a diagram showing a configuration of a base sequence determination device according to the first embodiment of the present invention.
FIG. 3 is a diagram showing a configuration of a measurement cell.
FIG. 4 is a diagram showing another example of a measurement cell.
FIG. 5 is a diagram showing a configuration of a solution carry-in / carry-out mechanism.
FIG. 6 is a diagram showing an example of automatic operation of a solution carry-in / carry-out mechanism.
FIG. 7 is a diagram showing an example of automatic operation of a solution carry-in / carry-out mechanism.
FIG. 8 is a diagram showing a state where one carrier is captured in the flow path of the measurement cell.
FIG. 9 is a view showing a state in a flow channel of a measurement cell at the time of base sequence analysis.
FIG. 10 is a diagram showing a configuration of a base sequence determination device according to a second embodiment of the present invention.
FIG. 11 is a diagram showing a configuration of a base sequence determination device according to a third embodiment of the present invention.
FIG. 12 is a diagram showing the configuration of a completely transparent measurement cell.
FIG. 13 is a diagram showing a configuration of a base sequence determination device according to a fourth embodiment of the present invention.
FIG. 14 is a flowchart showing the flow of the base sequence determination method of the present invention.
FIG. 15 is a diagram showing an example of the result of measuring the presence or absence of fluorescence in an analysis sample.
FIG. 16 is a diagram showing an example of the result of analyzing the base sequence of DNA.
FIG. 17 is a diagram showing an example of the result of analyzing a DNA base sequence.
18 is a diagram showing the measurement result of FIG.
FIG. 19 is a diagram showing an example of a result of analyzing a DNA base sequence.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1, 1 '... measurement cell, 1a, 1a' ... main-body part, 1b ... surface part, 1c, 1c '... flow path, 1d, 1d' ... connection terminal, 1e ... holder of measurement cell provided with flow path, 2, ... Solution loading / unloading mechanism, 2a ... Tank for loading, 2b ... Tube for loading, 2c ... Tank for unloading, 2d ... Tube for unloading, 3 ... Laser light source for capturing carrier, 4 ... Collimator lens, 5 ... Beam expander, 6 ... Lens, 7 ... Mirror, 8 ... Lens, 9 ... Dichroic half mirror, 10 ... Objective lens, 11 ... Excitation laser light source, 12 ... Collimating lens, 13 ... Beam expander, 14 ... Dichroic half mirror, 15 ... Lens, 16 ... Aperture, 17 ... Condensing lens, 18 ... Ir cut filter, 19 ... Band pass filter, 20 ... Light receiver, 21 ... Signal processing unit, 22 ... Signal 23, computer, 24 ... halogen lamp, 25 ... lens, 26 ... movable mirror, 27 ... movable prism, 28 ... bandpass filter, 29 ... lens, 30 ... CCD camera, 31 ... second excitation laser Light source, 32 ... collimating lens, 33 ... beam expander, 34 ... mirror, 35 ... dichroic half mirror, 36 ... dichroic half mirror, 37 ... band pass filter, 38 ... light receiver, 39 ... signal processing unit, 40 ... shutter (1 , 41 ... Shutter (2), 42 ... Shutter (3), 43 ... Objective lens, 44 ... Degrading enzyme introduction syringe, 45 ... Sample introduction syringe, 46 ... Buffer tank, 47 ... Buffer tank, 48 ... T type Fitting, 49 ... T-type fitting, 50 ... connection terminal, 51 ... connection terminal, 52 ... negative pressure pump, 53 ... Liquid tank, 54 ... T-type joints, 55 ... T-type joints

Claims (5)

An apparatus for determining the result obtained by a predetermined reaction based on the presence or absence and / or type of a fluorescent substance, in a state where a carrier carrying the substance for the reaction is captured by a light trap, In an apparatus for performing a predetermined reaction,
A measurement cell having a flow path therein and a transparent surface for transmitting a light beam into the flow path;
Means for transporting the suspension of the carrier to the flow path of the measurement cell;
A first laser light source for capturing the carrier;
A first optical system that collects the light beam from the first laser light source in the flow path of the measurement cell and captures the carrier;
Means for transporting a reaction solution for causing a substance carried by the carrier to perform a predetermined reaction while maintaining a constant flow rate in the flow path of the measurement cell;
A second laser light source for exciting the fluorescent material reflecting the result to determine the result obtained by the reaction;
A second optical system for condensing the light beam from the second laser light source in the flow path of the measurement cell and exciting the fluorescent material;
A third optical system for collecting fluorescence from the fluorescent material;
A photodetector for detecting the condensed fluorescence;
Analyzing means for analyzing the result of the reaction from the identification signal generated by the photodetector,
Means for transporting the suspension of the carrier to the flow path of the measurement cell, and a reaction solution for causing the substance carried by the carrier to perform a predetermined reaction are transported to the flow path of the measurement cell. All of the means for providing a pipe for transporting each solution to the flow path of the measurement cell, and a member for controlling the flow of the liquid without causing a pulsating flow in the transported liquid, Judgment device to do.   The apparatus according to claim 1, wherein means for transporting the suspension of the carrier to the flow path of the measurement cell and a reaction solution for causing a substance carried by the carrier to perform a predetermined reaction Each of the means for transporting the solution to the flow path of the measurement cell comprises a pipe for transporting each solution to the flow path of the measurement cell, and the pipe is near the flow path inlet of the measurement cell. An apparatus that branches into a direction toward the flow path of the measurement cell and a direction toward the waste liquid. A base sequence determination device using a carrier to which a nucleic acid labeled and associated with a base type and a fluorescent material is attached as an analysis sample,
A measurement cell having a flow path therein and a transparent surface for transmitting a light beam into the flow path;
Means for transporting a suspension of a carrier to which the nucleic acid as an analysis sample is attached to the flow path of the measurement cell;
A first laser light source for capturing the carrier to which the nucleic acid is attached;
A first optical system that collects the light beam from the first laser light source in the flow path of the measurement cell and captures the carrier to which the nucleic acid is attached;
Means for transporting the enzyme solution that cleaves mononucleotides constituting the nucleic acid in order from the end, while maintaining a constant flow rate in the flow path of the measurement cell;
A second laser light source for exciting the fluorescent substance that is identifying and labeling the base of the mononucleotide cleaved by the enzyme solution, in the order of cleavage;
A second optical system for condensing the light beam from the second laser light source in the flow path of the measurement cell and exciting the fluorescent materials in the order of cutting;
A third optical system for sequentially collecting the fluorescence from the fluorescent substance that identifies and labels the base of the nucleic acid;
A photodetector that sequentially detects the collected fluorescence and generates an identification signal corresponding to the detected fluorescence;
From the identification signal generated by the photodetector, comprising an analysis means for reading the base sequence of the nucleic acid,
The means for transporting the suspension of the carrier to which the nucleic acid, which is the analysis sample, is attached to the flow channel of the measurement cell, and the enzyme solution for sequentially cleaving the mononucleotide constituting the nucleic acid from the end are measured. Any means for transporting to the flow path of the cell includes a pipe for transporting each solution to the flow path of the measurement cell, and a member for controlling the flow of the liquid without causing a pulsating flow in the transported liquid A base sequence determination apparatus comprising: 4. The apparatus according to claim 3 , wherein means for transporting a suspension of the carrier to which the nucleic acid, which is an analysis sample, is attached to the flow path of the measurement cell, and a mononucleotide that constitutes the nucleic acid. Each of the means for transporting the enzyme solution that cleaves in order from the end to the flow path of the measurement cell includes a pipe for transporting each solution to the flow path of the measurement cell, and the pipe is connected to the measurement cell. The apparatus is characterized by branching in a direction toward the flow path of the measurement cell and a direction toward the waste liquid near the flow path inlet of the liquid crystal. The apparatus according to any one of claims 1 to 4 ,
A light source for irradiating illumination light into the flow path of the measurement cell;
An optical system for irradiating the light from the light source into the flow path of the measurement cell and irradiating the carrier with illumination light;
An optical system for the carrier to capture an image captured by the first laser light source under the illumination light;
The apparatus further comprising imaging means for imaging an image in which the carrier is captured.

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