JP2001056337A - Dna analyzer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a DNA analyzer capable of determining DNA sequences not through the use of gel electrophoresis but a small reaction cell chip. SOLUTION: This analyzer comprises a reaction cell 102 to house template DNA combined with primers, DNA polymerase, luciferase, apyrase, and sulfurylase, first to fourth cells 103-1 to 104-3 to house four types of dNTP separately, grooves 109-1 to 109-4 or capillaries to supply the four types of dNTP for the reaction cell 102 from any of the first to fourth cells in a predetermined order, means 104, 105-1 to 105-5, and 110-1 to 110-5 to impress an electric field to eccentrically move dNTP or a liquid containing dNTP to the reaction cell 102 through the grooves or capillaries, and a detector 106 to transform pyrophosphoric acids formed at the time of the formation of the elongation reaction of primers complementarily combined with template DNA into ATP and to detect phosphorescence generated by the light emission of luciferin due to the action of luciferase. By this constitution, it is possible to produce a portable inexpensive system of a size tens of times smaller than a DNA analyzing device frequently used at present.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to DNA diagnosis, DN
The present invention relates to a DNA analysis method for performing A base sequence determination and a DNA analyzer, and particularly to a small-sized handy type DNA analyzer.

[0002]

2. Description of the Related Art A fluorescent DNA sequencer or the like based on gel electrophoresis is used for DNA base sequence determination. In a conventional apparatus, a DNA fragment group produced by a Sanger method or the like and having a specific base type at each end is subjected to size separation by gel electrophoresis, and a base sequence is determined from the type and size information of the terminal base. To prepare a DNA fragment group, the DNA strand to be sequenced is used as a template.
NA complementary strand synthesis is used. An optical measurement technique using laser-induced fluorescence is used to measure a synthetic product.
Devices using these techniques are expensive and large in size.

On the other hand, a new DNA sequencing method has been reported (Anal. Biochem., 244, 344).
67-373 (1997), Science 281,
363-365 (1998)). In these reported techniques, pyrophosphate (i.
and the luciferase (L) is replaced with adenosine triphosphate (ATP).
luciferin (Luciferase) by the action of luciferase
ferin) to detect the light generated and illuminate the DNA
The progress of the complementary strand synthesis reaction is monitored to determine the sequence. Template DNA, primer (primr), DNA polymerase (DNA polymerase), sulfurylase, apyrase (A
Pyrose, and a reaction solution containing luciferase, luciferin, and the like are added to deoxynucleotide triphosphate (dN).
TP) (deoxyadenosine triphosphate (dATP), deoxycytosine triphosphate (dCTP), deoxyguanidine triphosphate (dGTP), and deoxythymine triphosphate (dTTP).
When TP)) is added, when a DNA complementary strand reaction is generated, pyrophosphate PPi is generated according to the following reaction formula.

[0004] Pyrophosphate PPi is converted to adenosine triphosphate (ATP) by sulfurylase according to the following reaction formula. n
Is the number of residues. The concentration of the generated ATP is detected by luciferase according to the following reaction formula.

[0005] On the other hand, ATP is produced by a nuclease such as apyrase.
It is decomposed into AMP (adenosine monophosphate) and inorganic monophosphate according to the following reaction formula. In addition, unreacted dNTP generates deoxynucleotide monophosphate and inorganic monophosphate by a nuclease such as apyrase according to the following reaction formula. Inorganic monophosphate does not make luciferin glow. Since the reaction for emitting luciferin and the reaction for decomposing ATP and dNTP simultaneously and competitively proceed, the luminescence stops after a certain period of time. Since the type of dNTP injected into the reaction solution can be known in advance, the type of dNTP incorporated into the template DNA by complementary strand synthesis can be determined, and the nucleotide sequence of the template DNA can be determined by the type of dNTP injected into the reaction solution and the presence or absence of light emission You can read from. An inkjet nozzle and an XY moving stage are used for dNTP injection.

[0006]

DISCLOSURE OF THE INVENTION Conventionally reported D
Each of the NA analyzers is large-scale, and is inconvenient to carry. However, with the growing need for DNA analysis, hospital bedside, outdoor,
In addition, there is a demand for the development of a notebook-sized or palm-sized, handy, small-sized DNA analyzer that can easily determine a DNA base sequence at various places such as an educational site. Further, in the prior art, four kinds of dNT which are reaction reagents are used.
For supplying P, spray or the like is used, and there is a problem that the apparatus becomes large.

SUMMARY OF THE INVENTION An object of the present invention is to solve the problems of the prior art and to provide an apparatus capable of determining a DNA sequence using a small chip-shaped reaction cell without using gel electrophoresis. It is an object of the present invention to provide a very small, portable, and inexpensive DNA analyzer which comprises a device comprising a chip provided with a photodetector and a photodetector.

[0008]

SUMMARY OF THE INVENTION The present invention relates to a DNA sequencer or a base sequence monitor, and more particularly to a DNA sequencer instead of gel electrophoresis conventionally used.
The sequential reaction of DNA complementary chain synthesis by converting pyrophosphoric acid (PPi) generated during complementary chain synthesis into ATP and illuminating luciferin by the action of luciferase and detecting the luminescence generated. The present invention relates to a DNA test chip for monitoring the progress of DNA and a test system using the same. In order to solve the problems of the prior art, in the present invention, a reaction cell and a reservoir for holding dNTP to be injected into the reaction cell are prepared on one plate, and each dNTP is determined in a predetermined order using an electric field. The base sequence is determined by successively introducing the light into the reaction cell repeatedly and detecting light emitted from the reaction cell. That is, in the present invention, an electric field for dNTP injection is supplied between the reservoir and the reaction cell. However, the arrangement can be determined from the timing of the application of the electric field and the correlation between the light emission and the arrangement without a large-scale device configuration. Can decide.
In the DNA test chip of the present invention and a test system using the same, light generated by the luciferase / luciferin reagent and proportional to the amount of ATP is detected. Less than,
The configuration of a typical DNA analyzer of the present invention will be described.

[0009] The first configuration of the apparatus comprises a template DNA to which a primer is bound, a DNA polymerase, a luciferase,
A reaction cell containing apyrase and sulfurylase, and four types of dNTPs (dATP, dCTP, dGT
P, and dTTP), and first, second, third, and fourth cells separately storing the four types of dNTPs in a predetermined order. A groove or a thin tube to be supplied to the reaction cell from any of the fourth cells;
a means for applying an electric field for electrically moving dNTP or a liquid containing the same through a groove or a capillary to a reaction cell, and converting pyrophosphate generated when an extension reaction of a primer complementary to a template DNA is generated to ATP; And a detector for detecting phosphorescence generated by causing luciferin or the like to emit light from the reaction cell.

[0010] The second configuration of the apparatus is as follows: template DNA to which primers are bound, DNA polymerase, luciferase,
A reaction cell containing luciferin, apyrase, and sulfurylase, and four types of dNTPs (dATP, dCT
P, dGTP, and dTTP) separately,
The second, third, and fourth cells and each of the four types of dNTPs are transferred from any one of the first, second, third, and fourth cells to the reaction cell in a predetermined order. Means for applying an electric field to supply and converting pyrophosphate generated when an elongation reaction of the primer complementary to the template DNA is generated to adenosine triphosphate, and adenosine triphosphate is generated by luciferase acting on luciferin Means for detecting chemiluminescence.

[0011] A third configuration of the apparatus includes a template DNA to which a primer is bound, a DNA polymerase, a luciferase,
A reaction cell containing luciferin, apyrase, and sulfurylase, and four types of dNTPs (dATP, dCT
P, dGTP, and dTTP) separately,
The second, third, and fourth cells and each of the four types of dNTPs are transferred from any one of the first, second, third, and fourth cells to the reaction cell in a predetermined order. Means for applying an electric field to supply, and pyrophosphoric acid generated when an elongation reaction of the primer complementary to the template DNA is generated,
A photodetector for converting to adenosine triphosphate and detecting chemiluminescence generated by the action of adenosine triphosphate on luciferin by luciferase, wherein each of the first, second, third, and fourth cells has The base sequence is determined based on the order of the applied electric field and the timing of chemiluminescence detection.

In the first to third configurations of the apparatus, the first, second, third, fourth cells and the reaction cell are formed on the same substrate, and the first, second, third, Another feature is that the substrate on which the fourth cell is formed and the substrate on which the reaction cell is formed are overlapped, and dNTP is supplied to the reaction cell by electrophoresis or electroosmotic flow. In the second and third configurations of the device, the first, second, third and fourth
It is also characterized in that each of the cells is connected to the reaction cell by a capillary or a groove.

A fourth configuration of the apparatus comprises a template DNA to which a primer is bound, a DNA polymerase, a luciferase,
A reaction cell containing luciferin, apyrase, and sulfurylase, and four types of dNTPs (dATP, dCT
P, dGTP, and dTTP) separately,
First, second, and third cells connecting the second, third, and fourth cells, and the first, second, third, and fourth cells, and the reaction cell, respectively.
A third and a fourth groove or capillary, a first cell and a first
A first wiring connecting the second cell and the second electrode terminal, a second wiring connecting the second cell and the second electrode terminal, a third wiring connecting the third cell and the third electrode terminal, A substrate on which a fourth wiring connecting the fourth cell and the fourth electrode terminal, a fifth wiring connecting the reaction cell and the fifth electrode terminal are formed, and first, second, and third substrates. , A control device for selectively controlling any one of the first, second, third, and fourth electrode terminals to apply an electric field between any one of the fourth cell and the reaction cell, The pyrophosphoric acid generated when an elongation reaction of the primer complementary to the template DNA is generated by dNTP sequentially supplied to the reaction cell by the application of is converted into adenosine triphosphate by sulfurylase, and adenosine triphosphate is converted to luciferin by luciferase. A first reaction to generate phosphorescence generated by the action; A second reaction for decomposing nosin triphosphate by apyrase is performed in a reaction cell, and a photodetector for detecting phosphorescence generated by the first reaction; and a reaction cell when phosphorescence is detected by the photodetector. Supplied d
A display device for displaying the type of NTP.

In a fourth configuration of the device, the dNTPs are supplied to the reaction cell by electrophoresis or electroosmotic flow;
The base sequence of the template DNA is determined based on the order of the first, second, third, and fourth cells selectively controlled by the control device and applying an electric field to the reaction cell, and the timing of phosphorescence detection by the photodetector. Determining that the bottom of the reaction cell of the substrate is made of a transparent member, the photodetector detects phosphorescence from the bottom of the reaction cell, and the photodetector detects phosphorescence from the opening of the reaction cell. There is also a feature.

A fifth configuration of the device is composed of four types of dNTPs.
(DATP, dCTP, dGTP, and dTTP), each of which contains a first electrode.
A first substrate on which second, third, and fourth cells are formed; a template DNA, a DNA polymerase, a luciferase, a luciferin, an apyrase, and a sulfurylase to which primers are bound; A plurality of reaction cells are formed, and a second substrate disposed below the first substrate, and first, second, third, and fourth cells respectively connecting the first and second cells to each of the reaction cells. The second, third, and fourth capillaries and the first electrode of any of the first, second, third, and fourth cells are selectively controlled to provide first, second, third, and fourth cells. 4
A control device for applying an electric field between the first electrode of any one of the cells and the second electrode of the plurality of reaction cells; A first reaction in which pyrophosphate generated when an elongation reaction of a primer complementary to DNA is generated is converted to adenosine triphosphate by sulfurylase, and adenosine triphosphate acts on luciferin by luciferase to generate phosphorescence, A second reaction for decomposing adenosine triphosphate by apyrase is performed in a reaction cell, and a photodetector for detecting phosphorescence generated by the first reaction and a reaction cell when phosphorescence is detected by the photodetector. And a display device for displaying the type of the supplied dNTP.

In a fifth configuration of the apparatus, dNTPs are supplied to a plurality of reaction cells almost simultaneously by electrophoresis or electroosmotic flow, and an electric field is applied between the reaction cells under selective control by a control device. Determining the base sequence of the template DNA from the order of the first, second, third, and fourth cells and the timing of detecting phosphorescence by the photodetector; The bottom of the plurality of reaction cells of the second substrate is formed of a transparent member; the photodetector detects phosphorescence from the bottom of the reaction cell; and the plurality of reaction cells of the second substrate. The bottom of the reaction cell is composed of a transparent member, the photodetector detects phosphorescence from the bottom of the reaction cell, and a lens array is placed between the bottom of multiple reaction cells and the photodetector. There is.

A sixth configuration of the device is that a template DNA to which a primer is bound, a DNA polymerase, a luciferase,
A first substrate on which luciferin, apyrase, and sulfurylase are housed and in which a plurality of reaction cells in which first electrodes are arranged are formed, and four types of dNTPs (dATP, dCT
P, dGTP, and dTTP) are respectively formed, and first, second, third, and fourth cells in which the second electrodes are respectively formed are formed, and the first, second, and third cells are formed below the first substrate. A second substrate, first, second, third, and fourth capillaries connecting the first, second, third, and fourth cells to each reaction cell; The first electrode of any one of the third and fourth cells is selectively controlled, and the first electrode of any of the first, second, third, and fourth cells and the second electrode of the plurality of reaction cells are controlled. And a control device for applying an electric field between the electrodes, and pyrophosphoric acid generated when an extension reaction of the primer complementary to the template DNA is generated by dNTP sequentially supplied to the reaction cell by application of the electric field, Sulfurylase converts it to adenosine triphosphate, and adenosine triphosphate is converted to luciferase by luciferase. First a reaction for generating phosphorescence caused to act on phosphorus, and a second reaction decomposed by apyrase adenosine triphosphate was carried out in the reaction cell, the first
And a display device for displaying the type of dNTP supplied to the reaction cell when the phosphorescence is detected by the photodetector.

In a sixth configuration of the apparatus, dNTP is supplied to a plurality of reaction cells almost simultaneously by electrophoresis or electroosmotic flow, and an electric field is applied between the reaction cells under selective control by a control device. Determining the base sequence of the template DNA from the order of the first, second, third, and fourth cells and the timing of phosphorescence detection by the photodetector, wherein the photodetector is disposed on the first substrate. In addition, the photodetector is disposed on the first substrate, and a lens array is disposed between the photodetector and the first substrate.

A seventh configuration of the device comprises a transparent capillary in which luciferase is immobilized, and a plurality of microparticles to which different template DNAs to which primers are bound are immobilized, in which a plurality of fine particles are arranged in a predetermined order. dNTP
(DATP, dCTP, dGTP, and dTTP), and first, second, third, and fourth containers for storing DNA polymerase, apyrase, sulfurylase, and luciferin, and a buffer for storing buffer solution. 5, the first, second, third, fourth, and fifth containers, and the first, second, third, fourth, and fifth containers that respectively connect the liquid-feeding switching means. A liquid supply pipe, a sixth liquid supply pipe connecting the liquid supply switching means and the liquid supply port of the capillary, and a fifth liquid supply pipe is selected by the liquid supply switching means, and the buffer solution is supplied to the capillary. After flowing into the capillary, pyrophosphoric acid generated when an elongation reaction of the primer complementary to the template DNA is generated by dNTP sequentially supplied into the capillary by the liquid feeding switching means is added by sulfurylase. A first reaction in which adenosine triphosphate is converted into syn triphosphate and phosphorescence generated by the action of adenosine triphosphate on luciferin by luciferase, and a second reaction in which adenosine triphosphate is decomposed by apyrase are performed inside the capillary. A photodetector for detecting phosphorescence generated by the first reaction, and a display device for displaying the type of dNTP supplied into the capillary when phosphorescence is detected by the photodetector. .
The seventh configuration of the apparatus is also characterized by having dummy fine particles alternately arranged with a plurality of fine particles.

An eighth configuration of the device comprises a plurality of substantially parallel sections in which a plurality of microparticles to which luciferase is immobilized and different template DNAs to which primers are bound are immobilized are arranged in a predetermined sequence. Are transparent optical cells and four types of dNTPs (dATP, dCTP, dGT
P, and dTTP), and first, second, third, and fourth containers for storing DNA polymerase, apyrase, sulfurylase, and luciferin, and a fifth container for storing a buffer solution. First, second, third, fourth, and fifth liquid feed pipes respectively connecting the first, second, third, fourth, and fifth containers with liquid feed switching means; A sixth liquid supply pipe connecting the liquid supply switching means and the liquid supply port of the optical cell;
After the buffer solution is sent to the plurality of sections of the optical cell by selecting the liquid sending tube of (1), the primer complementarily bound to the template DNA by dNTP sequentially supplied to the plurality of sections of the optical cell by the solution sending switching means A first reaction in which pyrophosphate generated when an elongation reaction is generated is converted into adenosine triphosphate by sulfurylase, and adenosine triphosphate acts on luciferin by luciferase to generate phosphorescence, and adenosine triphosphate is converted by apyrase. Causing the second reaction to be decomposed to occur in a plurality of sections of the optical cell, detecting a phosphorescence generated by the first reaction, and a plurality of sections of the cell when the photodetector detects the phosphorescence. And a display device for displaying the type of dNTP supplied to the device. The eighth configuration of the apparatus is also characterized by having dummy fine particles alternately arranged with a plurality of fine particles.

A ninth configuration of the apparatus is such that a first electrode and a second electrode are arranged, a DNA polymerase and a luciferase are fixed in the vicinity of the first electrode, and a primer-bonded template DNA, apyrase, Reaction cell containing luciferin and sulfurylase, and 4 types of dNT
P (dATP, dCTP, dGTP, and dTTP), and first, second, third, and fourth cells in which third electrodes are respectively disposed, and first, second, third, and third cells. And the first connecting the fourth cell and the reaction cell, respectively.
By selectively controlling the substrate on which the second, third, and fourth pores are formed and the third electrode of any of the first, second, third, and fourth cells, the third electrode is formed. A control device for controlling an electric field applied between the electrode and the first electrode, and controlling an electric field applied between the first electrode and the second electrode;
An electric field applied between the third electrode and the first electrode of any one of the third and fourth cells causes the first, second, third, and fourth cells to change.
Is sequentially supplied to the reaction cell through any of the pores of
Pyrophosphate generated when an extension reaction of a primer complementary to a template DNA is generated by NTP is converted into adenosine triphosphate by sulfurylase, and adenosine triphosphate acts on luciferin by luciferase to generate phosphorescence. And a second reaction of decomposing adenosine triphosphate with apyrase in a reaction cell, and detecting a phosphorescence generated by the first reaction, and detecting the phosphorescence by the photodetector. A display device for displaying the type of dNTP supplied to the reaction cell at any time, between the third electrode and the first electrode of any of the first, second, third, and fourth cells. After applying the electric field, an electric field is applied between the first electrode and the second electrode to move the template DNA to the vicinity of the first electrode. Next, the configuration of a typical DNA analysis method of the present invention will be described.

The first configuration of the method includes a template DNA to which a primer is bound, a DNA polymerase, a luciferase,
Luciferin, apyrase, and sulfurylase are stored in a reaction cell, and four types of dNTPs (dATP, dCT
P, dGTP, and dTTP) are the first, second,
The process of storing in the third and fourth cells, and four types of dN
Each of the TPs, in a predetermined order, is first, second,
Supplying an electric field from one of the third and fourth cells to the reaction cell by using an electric field; and converting pyrophosphate generated when an extension reaction of the primer complementary to the template DNA is generated into adenosine triphosphate. And a step of detecting chemiluminescence generated by adenosine triphosphate acting on luciferin by luciferase.

In a first configuration of the method, after the step of detecting chemiluminescence, a step of supplying a buffer solution containing apyrase to the reaction cell is provided, wherein the first, second, third and fourth steps are performed.
4 through a capillary tube connecting each of the cells to the reaction cell.
Each kind of dNTP is supplied; a plurality of reaction cells are provided; each of the four kinds of dNTPs is supplied almost simultaneously to each reaction cell in a predetermined order; first, second, and third , And the order of the electric field applied to each of the fourth cells and the timing of the detection of chemiluminescence are also characterized in that the base sequence is determined.

The second configuration of the method comprises a template DNA to which a primer is bound, a DNA polymerase, a luciferase,
Luciferin, apyrase, and sulfurylase are stored in a reaction cell, and four types of dNTPs (dATP, dCT
P, dGTP, and dTTP) are the first, second,
A first step of accommodating in third and fourth cells, dNT
P is sequentially supplied to the reaction cell, and pyrophosphoric acid generated when an elongation reaction of the primer complementary to the template DNA is generated by dNTP sequentially supplied to the reaction cell is converted to adenosine triphosphate by sulfurylase A first reaction in which adenosine triphosphate acts on luciferin by luciferase to generate phosphorescence, and a second reaction in which adenosine triphosphate is decomposed by apyrase.
In the reaction cell to detect phosphorescence generated by the first reaction, and a fourth step of displaying the type of dNTP supplied to the reaction cell when the phosphorescence is detected. It is characterized by having

In a second configuration of the method, the base sequence of the template DNA is determined from the order in which dNTPs are supplied to the reaction cell and the timing of phosphorescence detection, and the bottom of the reaction cell is formed of a transparent member. The phosphorescence is detected from the bottom of the reaction cell, the phosphorescence is detected from the opening of the reaction cell, the first step includes a step of accommodating different template DNAs in a plurality of reaction cells, respectively. Step 2 is
Supplying dNTPs to the plurality of reaction cells at substantially the same time; a third step including a step of detecting phosphorescence generated in the plurality of reaction cells by the first reaction; and a fourth step; DN supplied to each reaction cell when detected
There is also a feature in displaying the type of TP. The first and second aspects of the method are also characterized in that the dNTP is supplied to the reaction cell by electrophoresis or electroosmosis.

A third configuration of the method is that a first electrode is disposed on each of the first, second, third, and fourth cells formed on the first substrate, and four types of dNTPs are provided on each of the first, second, third, and fourth cells.
(DATP, dCTP, dGTP, and dTTP), and a primer is bound to a plurality of reaction cells formed on a second substrate disposed below the first substrate and having second electrodes respectively disposed therein. Containing the prepared template DNA, DNA polymerase, luciferase, luciferin, apyrase, and sulfurylase, sequentially supplying dNTPs to the reaction cell, and sequentially supplying dNTPs to the reaction cell.
Pyrophosphate generated when an extension reaction of a primer complementary to a template DNA is generated by NTP is converted to adenosine triphosphate by sulfurylase, and adenosine triphosphate acts on luciferin by luciferase to generate phosphorescence. And a second reaction of decomposing adenosine triphosphate with apyrase in the reaction cell to detect phosphorescence generated by the first reaction, and supplying the phosphorescence to the reaction cell when the phosphorescence is detected. D
Displaying the type of NTP.

In a third configuration of the method, the dNTPs are supplied to the plurality of reaction cells almost simultaneously by electrophoresis or electroosmotic flow, the order in which the dNTPs are supplied to the plurality of reaction cells, and the detection of phosphorescence. From the timing of the template DNA
Is characterized in that the phosphorescence is detected through the transparent member at the bottom of the plurality of reaction cells on the second substrate.

A fourth configuration of the method is that a template DNA, a DNA polymerase, a luciferase, a luciferin, an apyrase, a primer-bonded template DNA, are formed on a plurality of reaction cells formed on a first substrate and each provided with a first electrode. And a sulfur free race, formed on a second substrate disposed below the first substrate, and provided on each of first, second, third, and fourth cells on which a second electrode is disposed, respectively. , 4 types of dNTPs (dATP, dCTP, dGT
P and dTTP), a step of sequentially supplying dNTPs to the reaction cell, and a step of sequentially supplying dNTPs to the reaction cell. Is converted to adenosine triphosphate by sulfurylase, and a first reaction in which adenosine triphosphate acts on luciferin by luciferase to generate phosphorescence and a second reaction in which adenosine triphosphate is decomposed by apyrase in a reaction cell. The method is characterized by comprising a step of detecting phosphorescence generated by the first reaction and a step of displaying the type of dNTP supplied to the reaction cell when the phosphorescence is detected.

In a fourth configuration of the method, the dNTPs are supplied to the plurality of reaction cells substantially simultaneously by electrophoresis or electroosmotic flow, the order in which the dNTPs are supplied to the plurality of reaction cells, and the photodetector. It is also characterized in that the base sequence of the template DNA is determined from the timing of the detection of phosphorescence.

A fifth configuration of the method is that a plurality of microparticles to which luciferase is immobilized inside a transparent capillary and different template DNAs to which primers are bonded are immobilized are arranged in a predetermined order. In the first, second, third, and fourth containers, four types of dNTPs (dATP, dATP,
dCTP, dGTP, and dTTP), containing DNA polymerase, apyrase, sulfurylase, and luciferin, and containing them in a fifth container that contains a buffer; and first, second, third, and fourth. , And the first, second, third, fourth, and fifth liquid supply pipes respectively connecting the fifth container and the liquid supply switching means, and the liquid supply switching means and the liquid supply port of the capillary. After the step of selecting the fifth liquid sending pipe by the liquid sending switching means from the sixth liquid sending pipe to be connected and sending the buffer solution into the capillary, the liquid sending switching means enters the inside of the capillary.
a step of sequentially supplying dNTPs, and a step of sequentially supplying dNTPs into the capillary to convert pyrophosphate generated when an extension reaction of a primer complementary to a template DNA is generated into adenosine triphosphate by sulfurylase; A first reaction in which triphosphate acts on luciferin by luciferase to generate phosphorescence,
Performing a second reaction of decomposing adenosine triphosphate by apyrase inside the capillary to detect phosphorescence generated by the first reaction; and detecting the phosphorescence generated by the photodetector when the phosphorescence is detected by the photodetector. Supplied d
Displaying the type of NTP.

A sixth configuration of the method is that, in each of a plurality of substantially parallel sections of the optical cell, luciferase and a plurality of microparticles to which different template DNAs to which primers are bound are fixedly arranged in a line in a predetermined order. And four types of dNTPs in the first, second, third, and fourth containers.
(DATP, dCTP, dGTP, and dTTP), and DNA polymerase, apyrase, sulfurylase, and luciferin, and a buffer in a fifth container, and first, second, and third containers. , Fourth, fifth, and fifth liquid supply pipes connecting liquid supply switching means with each of the first, fourth, and fifth containers, and liquid supply switching means and liquid supply of the optical cell. The sixth that connects the mouth
The fifth liquid feed pipe is selected by the liquid feed switching means from among the liquid feed pipes, and the buffer solution is sent to the plurality of sections of the optical cell. , A step of sequentially supplying dNTPs, and a step of sequentially supplying dNTPs to a plurality of compartments of the optical cell to convert pyrophosphate generated when an extension reaction of a primer complementary to a template DNA is generated to adenosine triphosphate by sulfurylase. Then, a first reaction for generating phosphorescence generated by adenosine triphosphate acting on luciferin by luciferase and a second reaction for decomposing adenosine triphosphate by apyrase are performed in a plurality of compartments of the optical cell. 1
And a step of displaying the type of dNTP supplied to the plurality of sections of the optical cell when the phosphorescence is detected by the photodetector. A sixth aspect of the method is also characterized in that the phosphorescence is detected from the transparent bottom of a plurality of sections of the optical cell.

[0032] A seventh configuration of the method includes forming a first electrode of a reaction cell formed on a substrate and having a first electrode and a second electrode disposed therein.
In the vicinity of the electrode, DNA polymerase and luciferase are immobilized, and the template DNA, apyrase, luciferin, and sulfurylase to which the primers are bound are stored,
Four types of dNTPs are provided in the first, second, third, and fourth cells formed on the substrate and in which the third electrodes are respectively disposed.
(DATP, dCTP, dGTP, and dTTP) separately, and selecting and controlling a third electrode of any of the first, second, third, and fourth cells to form a third electrode First
Controlling an electric field applied between the first and second electrodes;
An electric field applied between the third electrode and the first electrode of any of the second, third, and fourth cells causes the first, second, third, and fourth cells to react with the reaction cell. Through the first, second, third and fourth pores respectively connecting the
a step of sequentially supplying dNTP, a step of controlling an electric field applied between the first electrode and the second electrode, and a step of elongating a primer complementary to the template DNA by dNTP sequentially supplied to the reaction cell. Pyrophosphate generated when the reaction is generated is converted into adenosine triphosphate by sulfurylase, the first reaction in which adenosine triphosphate acts on luciferin by luciferase to generate phosphorescence, and adenosine triphosphate is decomposed by apyrase. Second
And a step of detecting phosphorescence generated by the first reaction in the reaction cell, and a step of displaying a type of dNTP supplied to the reaction cell when the phosphorescence is detected. After applying an electric field between the third electrode and the first electrode of any of the first, second, third, and fourth cells, an electric field is applied between the first electrode and the second electrode. Apply the template DNA
Is moved to the vicinity of the first electrode.

The second to seventh structures of the method are also characterized in that a step of supplying a buffer containing apyrase to the reaction cell is provided after the step of detecting phosphorescence.

As described above, in the present invention, a reaction cell and a sample reservoir holding dNTP are prepared on the same plate by processing a small solid plate and used as a DNA test chip. Performing DNA complementary strand synthesis on a small chip, supplying four dNTPs as reaction reagents to the reaction cells of the chip in a predetermined order in order, and monitoring PPi generated as a by-product during DNA complementary strand synthesis. Thus, a simple, inexpensive, and compact DNA analyzer can be provided without using gel electrophoresis. The configuration of the system of the present invention is a simple configuration comprising a reaction chip and a photodetector. The system can be made very small, portable anywhere, inexpensive, and can be used in places that were previously difficult to use, such as outdoors, bedside, educational sites, and the like. In addition, the components are inexpensive, reflecting their simplicity, and the operation is simple.

[0035]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below with reference to embodiments.

Embodiment 1 FIG. 1 is a perspective view showing a configuration example of a DNA sequence determination apparatus according to Embodiment 1 of the present invention. The DNA sequencing apparatus of Example 1 includes a small plate (DNA sequencing board) 101 of about 2 cm × 5 cm, a photomultiplier tube for detecting light generated in the reaction cell 102, and the like.
Power supply 10 for supplying NTP solution to reaction cell 102
4. a detection system 107 for controlling the operation of the photodetector 106 to process the detected signal, and a detection system 107
And a display device 108 for displaying the result obtained in the step (c). When the bottom of the reaction cell 102 is made of a transparent member, the light generated in the reaction cell 102 is emitted from the DNA sequencing substrate 1.
01 can be detected from below. Of course, light generated in the reaction cell 102 can also be detected from above the DNA sequencing substrate 101.

The DNA sequence determination substrate 101 has a reaction cell 102 and a dATP holding cell 103 for holding dATP.
-1, dCTP holding cell 103 holding dCTP
2. dGTP holding cell 103-3 holding dGTP,
dTTP holding cell 103-4 holding dTTP, dA
DATP supply narrow groove 109-1 for supplying TP to the reaction cell 102, dCTP supply narrow groove 109-2 for supplying dCTP to the reaction cell 102, dGTP supply for supplying dGTP to the reaction cell 102 Narrow groove 109
-3, dT for supplying dTTP to the reaction cell 102
TP supply narrow groove 109-4, reaction cell 102 and electrode 10
5-5, and a lead 110- connecting the dATP holding cell 103-1 and the electrode 105-1.
1. Lead wire 110-2 connecting dCTP holding cell 103-2 and electrode 105-2, dGTP holding cell 103-3
Lead 110-3 connecting the electrode 105-3 and the electrode 105-3, dTT
A lead 110-4 connecting the P holding cell 103-4 and the electrode 105-4 and a lead 110-5 connecting the reaction cell 102 and the electrode 105-5 are formed.

For example, the volume of the reaction cell 102 is 10 μL
(Microliter), the volume of the dNTP holding cell 103-i (i = 1, 2, 3, 4) holding dNTP is 20
μL.

The reaction cell 102 contains primers, template DNA, DNA polymerase, luciferase,
Luciferin, apyrase, sulfurylase and others are retained. dATP holding cell 103-1 and reaction cell 10
When an electric field is applied between the two, d
ATP is supplied to the reaction cell 102. The flow rate of the osmic flow depends on the electric field strength, but is about 1 mm / s when the flow rate is about 200 V / cm.
Here, the distance between (i = 1, 2, 3, 4) and the reaction section 102 was 3 mm. dNTP holding cell 103-i
The distance between (i = 1, 2, 3, 4) and the reaction section 102 is not a fixed value but may be changed, but may be 10 m.
m or less is good. However, this is not always the case when the speed is increased by pressing or the like.

The nucleic acid to be incorporated in the complementary strand synthesis is dA
In the case of TP, when dATP is added, the complementary strand is extended and pyrophosphate (PPi) is released as one of the reaction products. PPi is converted to ATP by sulfurylase. The concentration of ATP can be monitored by measuring the chemiluminescence (phosphorescence) generated from luciferin by the action of luciferase.

On the other hand, ATP is decomposed by apyrase and its number is reduced. The phosphorescence decreases accordingly. dAT
After P, dCTP, dGTP and dTTP are sequentially injected into the reaction cell 102. The dNTP reaction cell 10
2 is selected by the selection control of the detection system 107, the electrode 105-i (i = 1, 2, 3, 4) to which an electric field is applied is selected, and the electrode 105-5 and the electrode 105-i (i = 1) are selected. ,
2, 3, 4), and a dNTP holding cell 103-i (i = 1, 2,...) Holding dNTP is applied.
This is performed by sequentially supplying an electric field between (3, 4) and the reaction cell 102.

If the nucleic acid base to be taken in is dCTP, phosphorescence occurs when an electric field is supplied between the dCTP holding cell 103-2 holding it and the reaction cell 102. If it is not a base species to be incorporated, there is no phosphorescence. The duration of the phosphorescence is adjusted by the concentration of Apirase, but the duration of the phosphorescence is appropriately several seconds. The base species to be incorporated changes depending on the elongation status of the complementary strand, and the incorporated base species corresponds to the sequence of the DNA. To increase the accuracy of sequencing, it is necessary to ensure that the dNTP supply is switched. Despite switching the dNTP to be supplied, it is inconvenient if the old dNTP supplied earlier is continuously supplied.

In this case, the buffer solution is dNTP
Supply narrow groove 109-i (i = 1, 2,
The dNTP solution near the reaction tank (reaction cell 102) may be pushed out toward the reaction tank (reaction cell 102) in addition to the dNTP solution from the middle of the steps 3 and 4). At the end of the first dNTP supply, the buffer solution is supplied by the osmotic flow and is removed from the flow path. The elimination is more perfect if you mix the apylace in the buffer solution.

Next, the next dNTP is supplied. If the supply order is determined in advance, the arrangement can be sequentially determined from the timing of applying a voltage and the presence or absence of phosphorescence emission. When the same base species is continuously taken in, the emission intensity is almost doubled, so that it can be recognized.

In Example 1, the reaction cell 10 was mounted on a small chip (a DNA sequencing substrate) of about 2 cm × 5 cm.
2, dNTP holding cell 103-i (i = 1, 2, 3,
4), dNTP supply narrow groove 109-i (i = 1, 2,
3, 4), lead wire 110-i (i = 1, 2, 3, 4)
Etc. can all be configured.

In the present invention, unlike the case of fluorescence detection, no light source is required, and a large-scale device such as a spray nozzle is not required for injecting a reagent as in the prior art.
In the present invention, this chip and an optical detector (photomultiplier tube,
A simple DNA sequence reading device that only requires a photodiode or the like can be provided.

(Embodiment 2) In the configuration example of Embodiment 1, one reaction cell is used. However, Embodiment 2 is an example in which reagents are supplied to a plurality of reaction cells and multiple kinds of DNA are simultaneously sequenced.

FIG. 2 is a perspective view showing a DNA sequencing apparatus (multi-reaction cell type DNA sequencing chip) according to a second embodiment of the present invention, and FIG. 3 is a DNA sequencing apparatus according to the second embodiment of the present invention.
It is a figure showing the example of composition of a sequence determination device.

As shown in FIG. 2, the multi-reaction cell type DNA sequencing chip of Example 2
01 and a second substrate 202. The first substrate 201 has a dATP holding cell 20 for holding dATP.
3-1. DCTP holding cell 203 holding dCTP
2. dGTP holding cell 203-3 for holding dGTP,
A dTTP holding cell 203-4 holding dTTP is formed.

As shown in FIG. 3, a plurality of reaction cells 206 are formed on the second substrate 202, and the plurality of reaction cells 206 are added to the dNTP holding cells 203-i (i = 1, 2, 3, 4) of the first substrate 201. Has electrodes 204-i (i = 1, 2, 3, 4). Each reaction cell 206 is provided with an electrode 207. As in Example 1, each reaction cell 206 holds primers, template DNA, DNA polymerase, luciferase, luciferin, apyrase, sulfurylase, and the like.

Each reaction cell 206 of the second substrate 202,
The dNTP holding cell 203-i (i =
1, 2, 3, 4) means a dNTP supply capillary tube 205-i (i = 1, 2) penetrating the bottom of the dNTP holding cell 203-i (i = 1, 2, 3, 4, 4) of the first substrate 201. 2, 3,
4). That is, each reaction cell 206
Is coupled to the dATP holding cell 203-1 by the dATP supply capillary 205-1, and the dCTP supply capillary 20
5-2 to dCTP holding cell 203-2, and d
It is connected to the dGTP holding cell 203-3 by the GTP supply capillary 205-3, and the dTTP supply capillary 205-4.
To the dTTP holding cell 203-4. By applying a voltage between the electrodes 204-i (i = 1, 2, 3, 4) and the electrodes 207, dNTP is injected into each reaction cell 206.

DNTP is dATP, dCTP, dGT
Each of the four substrates, P and dTTP, is held in a different dNTP holding cell 201-i (i = 1, 2, 3, 4), and is independently injected into each reaction cell 206. In this example, the order of injection of dNTPs is dATP, dCTP, dGT.
P, dTTP, and the dNTP supply capillary 20
5-i (i = 1, 2, 3, 4) is supplied to each reaction cell 206 using an osmotic flow. Of course, the liquid containing the substrate may be supplied by pressurization or the like. In order to clarify the starting point and the ending point of the reaction when the substrate is changed, a solution containing apyrase is caused to flow through the substrate flowing channel each time the reaction is carried out, and the excess substrate is washed away as in Example 1. .

At least the bottom surface of each reaction cell 206 of the first substrate is made of an optically transparent member and serves as a window for detecting chemiluminescence. Lens array 20
8, and using a cooled CCD or the like as the two-dimensional detector 209, the light emission from each reaction cell 206 can be distinguished and detected.

In the second embodiment, since a cooled array sensor is used, the size of the detector is slightly increased. However, the size of the sensor system can be considered only. Can be provided.

In the example shown in FIG. 2, the dNTP holding cell 20
The first substrate on which 3-i (i = 1, 2, 3, 4) is formed is disposed on the upper side, and the second substrate on which each reaction cell 206 is formed is disposed on the lower side. It is good also as composition.

FIG. 4 is a diagram showing a configuration example of a DNA sequencing apparatus (multi-reaction cell type DNA sequencing chip) which is a modification of the second embodiment of the present invention.

As shown in FIG. 4, a plurality of reaction cells 206 'are formed in the second substrate 202', and the first substrate 20 '
1 ′ dNTP holding cell 203′-i (i = 1, 2,
3 and 4) have electrodes 204-i (i = 1, 2, 3, 4)
Is arranged. Each reaction cell 206 ′ has an electrode 207.
Is arranged. Each reaction cell 2 of the second substrate 202 '
06 ′ and the dNTP holding cell 20 of the first substrate 201 ′
3′-i (i = 1, 2, 3, 4) means that the second substrate 20
2 'are connected by dNTP supply capillaries 205'-i (i = 1, 2, 3, 4) penetrating the bottom of each reaction cell 206.

The second substrate 202 ′ is arranged on the upper part and the first substrate 201 ′ is arranged on the lower part.
Using the lens array 208 and the two-dimensional detector 209, light emitted from each reaction cell 206 'can be distinguished and detected from above 2' in the same manner as in the configuration example of FIG.

In the configuration example described above, for example, the volume of each reaction cell 206 is 10 μL (microliter), and the dNTP holding cell 203-i holding dNTP is used.
The volume of (i = 1, 2, 3, 4) is 20 μL.

(Embodiment 3) In Embodiment 3, the target DN
This is an example in which A and luciferase are immobilized on a solid surface.
Various target DNAs are respectively immobilized on different beads or fine particles. Luciferase is also immobilized on the beads or microparticles.

FIG. 5 shows a DNA sequencing system according to Example 3 of the present invention, in which beads or fine particles having a DNA sample immobilized thereon are arranged inside a transparent capillary having a stopper 303 at the end. It is a figure showing the example of composition of an arrangement system. As shown in FIG.
DNA sample (primer-bound template D
The beads or fine particles 302 to which NA) is fixed are packed in the capillary 301. That is, beads or fine particles 302 having different target DNAs are arranged inside the capillary 301 in a predetermined order.

By switching the switching valve 305,
From the dNTP holding vessel 306-i (i = 1, 2, 3, 4), a reaction solution (including luciferin, apyrase, sulfurylase, etc.) containing DNA polymerase and dNTP is added in a predetermined order to a purge solution (buffer solution). In turn,
The liquid flows into the reaction liquid channel 304 and is injected from the inlet of the reaction liquid in the capillary. When changing the type of dNTP, if the previously injected dNTP species remains, it may cause a sequencing error. Therefore, by switching the switching valve 305, the buffer solution for purging is caused to flow from the buffer solution holding container 306-5. Check that the light emission has turned off.

When dNTPs are newly inserted and light is emitted, the reaction solution is not allowed to flow, but is kept near beads or fine particles where PPi generated by the DNA polymerase reaction is generated. Thus, it is possible to determine which beads or microparticles emit light due to the complementary strand synthesis reaction that occurs with the DNA immobilized on the template. To make this more certain,
Beads or microparticles with DNA and luciferase,
It is also effective to adopt a configuration in which dummy beads or fine particles are alternately arranged in a capillary.

The reaction of various dNTPs in a predetermined order to generate PPi, the generation of ATP from PPi, and the emission of luciferin by the action of luciferase to detect luminescence is the same as in the previous embodiment. 3 can be used.

As beads or fine particles usable in Example 3, beads or fine particles of several μm to 100 μm are commercially available. Of course, beads or fine particles of a special size can also be prepared and used.

FIG. 6 shows a modification of the third embodiment of the present invention, in which a plurality of microparticles to which different template DNAs to which the primers are bound are respectively fixed are arranged in a predetermined order in a row. It is a top view which shows the example of a structure of the DNA sequencing system of the type using the optical cell with the bottom which has a partition and is transparent. As shown in FIG. 6, the optical cell 310 includes a plurality of substantially parallel sections 312-1, 312-2, and 31-2.
12-3, 312-4, 312-5 are formed, and a stopper 303 'is arranged at the end of each section.

Each section shown in FIG. 6 corresponds to the capillary 301 shown in FIG. 5, and in the configuration example of the optical cell 310 shown in FIG. 6, the common reaction liquid flow path 304 is used to form the configuration shown in FIG. Is an example of a configuration that simultaneously realizes a plurality of. In the configuration example shown in FIG. 6, light emitted from the vicinity of the beads or fine particles 302 to which the DNA sample (primer-bound template DNA) in each compartment is fixed is emitted from the transparent bottom of the optical cell or from above the optical cell. , Using the detection system shown in FIG.

The DNA sequence determination system of the configuration example shown in FIG. 6 can simultaneously determine the base sequences of many more types of target DNA than the DNA sequence determination system of the configuration example shown in FIG.

Example 4 Example 4 is an example of a DNA sequencing chip in which target DNA and dNTP are collected and localized by an electric field to improve the reaction efficiency. By localizing the reaction, an efficient reaction becomes possible.

FIG. 7 is a perspective view showing a configuration example of a DNA sequencing chip of Example 4 of the present invention, and FIG. 8 is a partial cross section of the DNA sequencing chip of Example 4 (A shown in FIG. 7). −
(A 'cross section). The chip for DNA sequencing of Example 4 is a chip substrate 405 for DNA sequencing.
And a cover 406. A reaction cell 401, a dATP holding cell 402-1, a dCTP holding cell 402-2, a dGTP
The holding cell 402-3 and the dTTP holding cell 402-4 are formed.

The dNTP holding cell 402-i (i = 1,
2, 3 and 4) and the reaction cell 401
It is tied at 7. An electrode 408 is arranged at the bottom of each of the dNTP holding cells 402-i. Reaction cell 40
A focusing electrode 410 is arranged on the four inner walls near the pores 407 formed in the four walls, and a DNA focusing electrode 409 is arranged on the bottom of the reaction cell 401. The cover 406 is formed of the dNT of the DNA sequencing chip substrate 405.
Opening of each cell of P holding cell 402-i and reaction cell 4
01 opening.

The four dNTPs are different dNTPs.
It is held in the holding cell 402-i (i = 1, 2, 3, 4). dNTP holding cell 402-i (i = 1, 2,
A pore 407 is provided between the reaction cell 401 and the reaction cell 401 according to the electric field.
Can be moved to

One continuous focusing electrode 410 is provided near each of the pores 407 formed on the four walls of the thin-film reaction cell 401. DNA polymerase and DNA polymerase are located near the focusing electrode 410. Luciferase is immobilized. The reaction cell 401 holds primers, template DNA, luciferin, apyrase, sulfurylase, and the like.

DATP holding cell 4 holding dATP
As the DNA gathers near the pore 407 of 02-1,
When an electric field is applied between the electrode 408 of the dATP holding cell 402-1, the DNA focusing electrode 409 of the reaction cell 401, and the focusing electrode 410 of the reaction cell 401, the dATP in the dATP holding cell 402-1 becomes fine pore 407. The DNA target moves from the solution in the reaction cell 401 toward the focusing electrode 410, and the complementary strand synthesis proceeds by the polymerase near the focusing electrode 410.

Electrode 40 of dATP holding cell 402-1
8. The potential of the DNA focusing electrode 409 is
Negative potential is applied relative to the potential of
DNTP holding cell 402- other than P holding cell 402-1
The potential of the electrode 408 at i (i = 2, 3, 4) is applied with a positive potential relatively to the potential of the focusing electrode 410.

The resulting PPi in reaction cell 401
Is converted to ATP. The detection of luminescence from luciferin is the same as in the previous example, and the detection system shown in FIG. 3 can be used. ATP is gradually decomposed by apyrase and the reaction is terminated.

Subsequently, an electric field is applied so that DNA gathers near the pore 407 near the dCTP holding cell 402-2 holding dCTP. In this case, the dNTP holding cells 402-i (i =
The potential of the electrode 408 of (1, 3, 4) is
Positive potential is applied relative to the potential of
dATP, dGTP, dTTP are dNTP holding cells 40
2-i (i = 1, 3, 4).

The dNTP holding cells 402-i (i =
Focusing electrode 41 near pores 407 of 1, 2, 3, 4)
The reaction is carried out so that dNTPs and DNAs gather around 0, and in the same manner as in the above-described embodiment, the type of base incorporated by complementary strand synthesis can be determined from the generated luminescence, and the sequence can be determined.

In the configuration example described above, for example, the volume of the reaction cell 401 is 10 μL (microliter),
dNTP holding cell 402-i (i =
1, 2, 3, and 4) have a volume of 20 μL, and the diameter of the pore 407 is 20 μm.

[0080]

As described above, according to the present invention, the DN is achieved by using a small chip-shaped reaction cell without using gel electrophoresis.
A can be sequenced. This system can be composed of a chip having a reaction cell of 2 cm × 5 cm or smaller and a photodetector, and can provide a very small DNA analyzer. At present, a system that is only a few tenths of the size of a frequently used DNA analyzer can be manufactured. The number of constituent parts is small, it is inexpensive, and the device itself can be produced at low cost. Such a compact device is useful for carrying or conducting DNA tests quickly at the bedside.
is important. Furthermore, the simple and inexpensive device provided by the present invention is also suitable for use in educational settings in educational settings.

[Brief description of the drawings]

FIG. 1 is a perspective view showing a configuration example of a DNA sequencing apparatus according to a first embodiment of the present invention.

FIG. 2 is a perspective view showing a DNA sequencing apparatus according to a second embodiment of the present invention.

FIG. 3 is a diagram illustrating a configuration example of a DNA sequencing apparatus according to a second embodiment of the present invention.

FIG. 4 is a diagram showing a configuration example of a DNA sequence determination device that is a modification of the second embodiment of the present invention.

FIG. 5 is a diagram showing a configuration example of a DNA sequencing system of Example 3 of the present invention, in which beads or fine particles having a DNA sample immobilized thereon are arranged in a capillary.

FIG. 6 is a plan view showing a configuration example of a DNA sequencing system of a type using an optical cell having a plurality of substantially parallel sections, which is a modification of the third embodiment of the present invention.

FIG. 7 is a perspective view showing a configuration example of a DNA sequencing chip according to a fourth embodiment of the present invention.

FIG. 8 is a perspective view including a partial cross section of a DNA sequencing chip of Example 4 of the present invention.

[Explanation of symbols]

101: DNA sequencing substrate, 102: Reaction cell, 1
03-1 ... dATP holding cell, 103-2 ... dCTP holding cell, 103-3 ... dGTP holding cell, 103-4 ...
dTTP holding cell, 104 power supply, 105-1, 105
-2, 105-3, 105-4, 105-5 ... electrodes, 1
06: photodetector, 107: detection system, 108: display device, 109-1: dATP supply narrow groove, 109-2 ...
dCTP supply narrow groove, 109-3 ... dGTP supply narrow groove, 109-4 ... dTTP supply narrow groove, 110-1, 1
10-2, 110-3, 110-4, 110-5 ... lead wires, 201, 201 '... first substrate, 202, 20
2 ': second substrate, 203-1; 203'-1: dAT
P holding cell, 203-2, 203'-2 ... dCTP holding cell, 203-3, 203'-3 ... dGTP holding cell,
203-4, 203'-4... DTTP holding cell, 204
-1, 204-2, 204-3, 204-4 ... electrodes, 2
05-1, 205'-1... Thin tube for dATP supply, 205
-2,205'-2 ... dCTP supply capillary, 205-
3, 205'-3 ... dGTP supply thin tube, 205-4,
205'-4 ... a thin tube for supplying dTTP, 206, 206 '
... reaction cell, 207 ... electrode, 208 ... lens array, 2
09 ... two-dimensional detector, 301 ... capillary, 302 ...
Beads or microparticles immobilized with DNA, 303, 303 '
... Stopper, 304 ... Reaction liquid flow path, 305 ... Switching valve, 306-1 ... dATP holding container, 306-2 ...
dCTP holding container, 306-3 ... dGTP holding container, 3
06-4: dTTP holding container, 306-5: buffer liquid holding container, 310: optical cell having a plurality of substantially parallel sections, 312-1, 312-2, 312-3, 312-
4, 312-5 ... section, 401 ... reaction cell, 402-1
... dATP holding cell, 402-2 ... dCTP holding cell,
402-3 ... dGTP holding cell, 402-4 ... dTTP
Holding cell, 405: chip substrate for DNA sequencing, 40
6 ... cover, 407 ... pore, 408 ... electrode, 409 ... D
NA focusing electrode, 410: Focusing electrode.

Claims (35)

[Claims] 1. A template DNA or DNA to which a primer is bound.
Reaction cells containing polymerase, luciferase, apyrase, and sulfurylase, and four types of dNTPs
(DATP, dCTP, dGTP, and dTTP), first, second, third, and fourth cells, which are stored separately, and the four types of dNTPs, respectively, in the first order in a predetermined order. A groove or a thin tube which is supplied from any one of the second, third and fourth cells to the reaction cell;
Means for applying an electric field for electrically transferring P or a liquid containing the same to the reaction cell through the groove or the capillary, and pyrophosphoric acid generated when an elongation reaction of the primer complementary to the template DNA is generated. A detector for detecting phosphorescence generated by emitting luciferin or the like by converting into ATP from the reaction cell.
A analyzer. 2. The DNA analyzer according to claim 1, wherein the first, second, third, and fourth cells and the reaction cell are formed on the same substrate. DNA
Analysis equipment. 3. The DNA analyzer according to claim 1, wherein the substrate on which the first, second, third, and fourth cells are formed and the substrate on which the reaction cell is formed. A DNA analyzer characterized by being superimposed. 4. The DNA analyzer according to claim 1, wherein the dNTP is supplied to the reaction cell by electrophoresis or electroosmotic flow. 5. A template DNA or DNA having a primer bound thereto.
Reaction cells containing polymerase, luciferase, luciferin, apyrase, and sulfurylase, and four types of dNTPs (dATP, dCTP, dGTP, and d
First, second, third and fourth housing TTPs separately
And an electric field for supplying each of the four types of dNTPs in a predetermined order from any one of the first, second, third, and fourth cells to the reaction cell. Means for applying, converting pyrophosphoric acid generated when an elongation reaction of a primer complementary to the template DNA is generated to adenosine triphosphate, and causing chemiluminescence generated by the adenosine triphosphate acting on the luciferin by the luciferase. DNA comprising: means for detecting
Analysis equipment. 6. The DNA analyzer according to claim 5, wherein each of the first, second, third, and fourth cells and the reaction cell are connected by a capillary or a groove. DNA analyzer. 7. The DNA analyzer according to claim 5, wherein the first, second, third, and fourth cells and the reaction cell are formed on the same substrate. DNA
Analysis equipment. 8. The DNA analyzer according to claim 5, wherein the substrate on which the first, second, third, and fourth cells are formed and the substrate on which the reaction cell is formed. A DNA analyzer characterized by being superimposed. 9. The DNA analyzer according to claim 5, wherein the dNTP is supplied to the reaction cell by electrophoresis or electroosmotic flow. 10. A primer-bonded template DNA, DN
A reaction cell containing A polymerase, luciferase, luciferin, apyrase, and sulfurylase;
First, second, third, and fourth cells that separately store different types of dNTPs (dATP, dCTP, dGTP, and dTTP), and each of the four types of dNTPs, in a predetermined order, A means for applying an electric field to supply the reaction cell from any one of the first, second, third, and fourth cells; and a means for generating an extension reaction of a primer complementary to the template DNA. And a photodetector for detecting chemiluminescence generated by the adenosine triphosphate acting on the luciferin by the luciferase, wherein the first, second and third pyrophosphates are converted to adenosine triphosphate. And a sequence of the electric field applied to each of the fourth cell and the fourth cell, and a base sequence is determined from a timing of detection of the chemiluminescence. 11. The DNA analyzer according to claim 10, wherein each of the first, second, third, and fourth cells and the reaction cell are connected by a capillary or a groove. DNA analyzer. 12. The DNA analyzer according to claim 10, wherein the first, second, third, and fourth cells and the reaction cell are formed on the same substrate. DN
A analyzer. 13. The DNA analyzer according to claim 10, wherein the substrate on which the first, second, third, and fourth cells are formed, and the substrate on which the reaction cell is formed. A DNA analyzer characterized by being superimposed. 14. The DNA analyzer according to claim 10, wherein the dNTP is supplied to the reaction cell by electrophoresis or electroosmotic flow. 15. A template DNA, DN having a primer bound thereto.
A reaction cell containing A polymerase, luciferase, luciferin, apyrase, and sulfurylase;
First, second, third, and fourth cells that separately store different types of dNTPs (dATP, dCTP, dGTP, and dTTP); and the first, second, third, and fourth cells. First, second, third, and fourth narrow grooves or capillaries respectively connecting the reaction cells; a first wiring connecting the first cell and a first electrode terminal; A second wiring connecting the cell and the second electrode terminal, a third wiring connecting the third cell and the third electrode terminal, and a fourth wiring connecting the fourth cell to the fourth electrode terminal;
A fourth wiring connecting the electrode terminals of
A substrate on which a fifth wiring connecting the electrode terminals is formed;
Any one of the first, second, third, and fourth electrode terminals for applying an electric field between the first, second, third, or fourth cell and the reaction cell; A control device for selecting and controlling, the pyrophosphoric acid generated when the extension reaction of the primer complementary to the template DNA is generated by the dNTP sequentially supplied to the reaction cell by applying the electric field, A first reaction in which phosphorylase is converted into adenosine triphosphate by sulfurylase and the adenosine triphosphate acts on the luciferin by the luciferase to generate phosphorescence, and a second reaction in which the adenosine triphosphate is decomposed by the apyrase. A photodetector for detecting the phosphorescence generated by the first reaction, which is performed in the reaction cell, and the photodetector detects the phosphorescence when the phosphorescence is detected. D, characterized in that it comprises a display unit for displaying the type of the dNTP supplied to response cells
NA analyzer. 16. The DNA analyzer according to claim 15, wherein the dNTP is supplied to the reaction cell by electrophoresis or electroosmotic flow. 17. The DNA analyzer according to claim 15, wherein the first, second, third, and fourth cells are selectively controlled by the control device and an electric field is applied to the reaction cell. A DNA analyzer, wherein a base sequence of the template DNA is determined from a cell order and a timing of detecting the phosphorescence by the photodetector. 18. The DNA analyzer according to claim 15, wherein the bottom of the reaction cell on the substrate is formed of a transparent member, and the photodetector detects the phosphorescence from the bottom of the reaction cell. A DNA analyzer, characterized in that: 19. The DNA analyzer according to claim 15, wherein the photodetector detects the phosphorescence from an opening of the reaction cell. 20. Four types of dNTPs (dATP, dCT
P, dGTP, and dTTP), a first substrate on which first, second, third, and fourth cells are respectively formed on which first electrodes are disposed, and a template to which primers are bonded. A second substrate that contains DNA, DNA polymerase, luciferase, luciferin, apyrase, and sulfurylase, in which a plurality of reaction cells in which a second electrode is arranged are formed, and a second substrate that is arranged below the first substrate; First, second, third, and fourth capillaries respectively connecting the first, second, third, and fourth cells to the respective reaction cells; and the first, second, third, and fourth capillaries. The first electrode of any one of the fourth cells is selectively controlled, and the first electrode of any of the first, second, third, and fourth cells and the first electrode of the plurality of reaction cells are controlled. A control device for applying an electric field between the first electrode and the second electrode, The dNTP sequentially supplied to the reaction cell by application of a field converts pyrophosphate generated when an elongation reaction of the primer complementary to the template DNA is generated to adenosine triphosphate by the sulfurylase, and converts the adenosine triphosphate to adenosine triphosphate. The first reaction in which phosphoric acid is generated by triphosphate acting on the luciferin by the luciferase and the second reaction in which the adenosine triphosphate is decomposed by the apyrase are performed in the reaction cell. A photodetector for detecting the phosphorescence generated by the reaction, and the dN supplied to the reaction cell when the phosphorescence is detected by the photodetector.
A display device for displaying the type of TP. 21. The DNA analyzer according to claim 20, wherein the dNTP is supplied to the plurality of reaction cells almost simultaneously by electrophoresis or electroosmotic flow. 22. The DNA analyzer according to claim 20, wherein the first, second, third, and fourth electric cells are selectively controlled by the control device and an electric field is applied to the reaction cell. A DNA analyzer, wherein a base sequence of the template DNA is determined from a cell order and a timing of detecting the phosphorescence by the photodetector. 23. The DNA analyzer according to claim 20, wherein the bottom of the plurality of reaction cells of the second substrate is formed of a transparent member. 24. The DNA analyzer according to claim 20, wherein the bottom of the plurality of reaction cells on the second substrate is formed of a transparent member, and the photodetector applies the phosphorescence to the reaction cell. A DNA analyzer characterized by detecting from the bottom of the DNA. 25. The DNA analyzer according to claim 20, wherein the bottom of the plurality of reaction cells on the second substrate is formed of a transparent member, and the photodetector applies the phosphorescence to the reaction cell. A DNA array, wherein a lens array is disposed between the bottoms of the plurality of reaction cells and the photodetector. 26. A template DNA, DN having a primer bound thereto.
A first substrate containing a plurality of reaction cells containing A polymerase, luciferase, luciferin, apyrase, and sulfurylase;
First, second, third, and fourth cells in which different types of dNTPs (dATP, dCTP, dGTP, and dTTP) are accommodated, and in which second electrodes are respectively arranged, are formed, and the first substrate is formed. A second substrate disposed below the
First, second, third, and fourth capillaries respectively connecting the first, second, third, and fourth cells to the respective reaction cells; and the first, second, third, and fourth capillaries. By selectively controlling the first electrode of any one of the fourth cells, the first, second, third,
A control device for applying an electric field between the first electrode of any of the fourth cells and the second electrodes of the plurality of reaction cells, and sequentially applying the electric field to the reaction cells. By the supplied dNTP, pyrophosphate generated when an elongation reaction of the primer complementary to the template DNA is generated is converted to adenosine triphosphate by the sulfurylase, and the adenosine triphosphate is converted to the luciferin by the luciferase. A first reaction for generating phosphorescence generated by the action and a second reaction for decomposing the adenosine triphosphate by the apyrase are performed in the reaction cell, and the phosphorescence generated by the first reaction is detected. And the dNTP supplied to the reaction cell when the phosphorescence is detected by the photodetector.
And a display device for displaying the type of DNA. 27. The DNA analyzer according to claim 26, wherein the dNTP is supplied to the plurality of reaction cells almost simultaneously by electrophoresis or electroosmotic flow. 28. The DNA analyzer according to claim 26, wherein the first, second, third, and fourth electric cells are selectively controlled by the control device, and an electric field is applied to the reaction cell. A DNA analyzer, wherein a base sequence of the template DNA is determined from a cell order and a timing of detecting the phosphorescence by the photodetector. 29. The DNA analyzer according to claim 26, wherein said photodetector is disposed on said first substrate. 30. The DNA analyzer according to claim 26, wherein the photodetector is disposed on the first substrate, and a lens is provided between the photodetector and the first substrate. A DNA analyzer, wherein an array is arranged. 31. A transparent capillary in which a plurality of microparticles to which luciferase is immobilized and different template DNAs to which primers are respectively immobilized are arranged in a predetermined order, and four types of dNTPs (dATP, dCT
P, dGTP, and dTTP), and first, second, third, and fourth containers each containing DNA polymerase, apyrase, sulfurylase, and luciferin, and a fifth container containing a buffer solution. And each of the first, second, third, fourth, and fifth containers;
First, second, third,
A fourth liquid supply pipe, a sixth liquid supply pipe connecting the liquid supply switching means and a liquid supply port of the capillary, and a fifth liquid supply pipe provided by the liquid supply switching means. Is selected, and after the buffer solution is sent into the capillary, the dNTP sequentially supplied into the capillary by the solution sending switching means causes the template DN to be supplied.
A pyrophosphoric acid generated when an elongation reaction of the primer complementary to A is generated is converted to adenosine triphosphate by the sulfurylase, and the adenosine triphosphate acts on the luciferin by the luciferase to generate phosphorescence. A photodetector for detecting the phosphorescence generated by the first reaction by allowing the reaction of No. 1 and a second reaction of decomposing the adenosine triphosphate by the apyrase to be performed inside the capillary; A display device for displaying a type of the dNTP supplied into the capillary when the phosphorescence is detected by a detector. 32. The DNA analyzer according to claim 31, further comprising dummy fine particles alternately arranged with the plurality of fine particles. 33. An optical cell having a plurality of substantially parallel sections in which a plurality of microparticles to which luciferase is immobilized and different template DNAs to which primers are respectively immobilized are arranged in a line in a predetermined order, and a bottom is transparent. 4 types of dN
TP (dATP, dCTP, dGTP, and dTTP)
, Each containing DNA polymerase, apyrase, sulfurylase, and luciferin,
A second, third, and fourth container and a fifth
, The second, third, fourth, and fifth containers, and the first, second, third, fourth, and fifth containers that respectively connect the liquid-feeding switching means. A liquid supply pipe, a sixth liquid supply pipe connecting the liquid supply switching means and the liquid supply port of the optical cell, and selecting a fifth liquid supply pipe by the liquid supply switching means; After the buffer solution is sent to the plurality of sections of the optical cell, the dN sequentially supplied to the plurality of sections of the optical cell by the solution sending switching means.
By TP, pyrophosphate generated when an extension reaction of the primer complementary to the template DNA is generated is converted to adenosine triphosphate by the sulfurylase, and the adenosine triphosphate is generated by the luciferase acting on the luciferin. A first reaction for generating phosphorescence and a second reaction for decomposing the adenosine triphosphate by the apyrase are performed in the plurality of compartments of the optical cell, and the phosphorescence generated by the first reaction is generated. DNA analysis comprising: a photodetector for detecting; and a display device for displaying a type of the dNTP supplied to the plurality of sections of the optical cell when the phosphorescence is detected by the photodetector. apparatus. 34. The DNA analyzer according to claim 33, further comprising dummy fine particles alternately arranged with the plurality of fine particles. 35. A first electrode and a second electrode, wherein a DNA polymerase and a luciferase are immobilized in the vicinity of the first electrode, and a primer-bound template DNA, apyrase, luciferin and sulfurylase are bound. Reaction cells to be stored and four types of dNTPs (dAT
P, dCTP, dGTP, and dTTP), and first, second, third, and fourth cells in which third electrodes are respectively arranged, and the first, second, third, and fourth cells. First, second, and fourth connecting the fourth cell and the reaction cell, respectively.
By selectively controlling a substrate on which third and fourth pores are formed and the third electrode of any of the first, second, third, and fourth cells, the third electrode is formed. A control device for controlling an electric field applied between an electrode and the first electrode, and controlling an electric field applied between the first electrode and the second electrode; The first, second, third, and fourth cells are formed by the electric field applied between the third electrode and the first electrode of any of the second, third, and fourth cells. The dN supplied sequentially to the reaction cell through any of the holes
By TP, pyrophosphate generated when an extension reaction of the primer complementary to the template DNA is generated is converted to adenosine triphosphate by the sulfurylase, and the adenosine triphosphate is generated by the luciferase acting on the luciferin. A photodetector for performing a first reaction for generating phosphorescence and a second reaction for decomposing the adenosine triphosphate with the apyrase in the reaction cell, and detecting the phosphorescence generated by the first reaction And a display device for displaying a type of the dNTP supplied to the reaction cell when the phosphorescence is detected by the photodetector, wherein the display device displays any one of the first, second, third, and fourth. After applying the electric field between the third electrode and the first electrode of the cell, the electric field is applied between the first electrode and the second electrode. It applied to, DNA analysis apparatus characterized by moving the template DNA in the vicinity of the first electrode.