JP2004271384A - Dna analytical device and analytical method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To efficiently perform the hybridization of a sample DNA fragment even during electrophoresis. <P>SOLUTION: A first electrode 74 is provided on the left wall of a bathtub 71 into which an electrophoretic medium 82 is injected, and a second electrode 75 is provided on the right wall thereof. A solid imaging device 2 comprising pixels of nine rows and nine columns is provided on the bottom of the bathtub 71. Nine probe electrodes 35 are formed on the surface of a solid imaging device 25. Nine spots 60 are arrayed per probe electrode 35. A temperature adjustment element 72 abuts on the probe electrode 35, and the heat of the temperature adjustment element 72 is conducted through the probe electrodes 35, to thereby heat or cool the electrophoretic medium 82. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a DNA analyzer and an analysis method used for analyzing a sample DNA fragment.
[0002]
[Prior art]
2. Description of the Related Art In recent years, genetic information of living organisms has been used in a wide range of fields such as the medical field and the agricultural field. When using genetic information, elucidation of the structure of DNA is indispensable. DNA has two helically twisted polynucleotide chains, and each polynucleotide chain is composed of four types of bases (adenine: A, guanine: G, cytosine: C, thymine: T) in one dimension. It has an aligned base sequence (nucleotide sequence), and bases of one polynucleotide chain are linked to bases of the other polynucleotide chain based on complementarity of adenine and thymine and guanine and cytosine.
[0003]
The elucidation of the structure of DNA is to specify the base sequence. In order to specify the base sequence of DNA, a DNA microarray and a reading device therefor have been developed. Conventionally, the base sequence of a sample DNA is specified using a DNA microarray and its reader as follows.
[0004]
First, a plurality of single-stranded probe DNA fragments having a known base sequence are prepared, and the plurality of types of probe DNA fragments are spotted and aligned on a solid support such as a slide glass. On the obtained DNA microarray, a plurality of spots are arranged on the array. One spot is a group of one type of probe DNA fragment, and the base sequence of the probe DNA fragment differs for each spot.
[0005]
Next, the sample DNA collected from the specimen is denatured into a single-stranded sample DNA fragment, and a fluorescent substance or the like is bound to the denatured sample DNA fragment. Generally, there are a plurality of types of sample DNA fragments.
[0006]
Next, when a plurality of types of sample DNA fragments are added to the DNA microarray, the plurality of types of sample DNA fragments hybridize with their complementary probe DNA fragments. That is, a certain type of sample DNA fragment hybridizes with a complementary probe DNA fragment among a plurality of types of probe DNA fragments, but does not hybridize with a non-complementary probe DNA fragment. Since the sample DNA fragment is marked with a fluorescent substance, the probe DNA fragment bonded to the sample DNA fragment emits fluorescence.
For example, a sample DNA fragment having a nucleotide sequence of TCGGGAA binds to a probe DNA fragment having a nucleotide sequence of AGCCCTT, and fluorescence is emitted from a spot composed of the probe DNA fragment.
[0007]
Next, the DNA microarray is set in a reader and analyzed by the reader. The reader measures the fluorescence intensity distribution on the DNA microarray. The fluorescence intensity distribution on the DNA microarray is output as a two-dimensional image. A spot having a high fluorescence intensity in the output image indicates that a probe DNA fragment having a base sequence complementary to the base sequence of the sample DNA fragment is included. Therefore, the base sequence of the sample DNA fragment can be determined based on which spot in the two-dimensional image has the higher fluorescence intensity.
[0008]
However, in the above-mentioned DNA microarray, the distribution of the sample DNA fragments is not uniform because the sample DNA fragments are simply added onto the DNA microarray. That is, the sample DNA fragment may have a high concentration on some spots on the DNA microarray, but may have a low concentration on another spot. Therefore, when the probe DNA fragment of the spot has complementarity with any one of a plurality of types of sample DNA fragments, there is a possibility that the emission luminance of the fluorescence varies depending on the concentration of the sample DNA fragment on the spot.
[0009]
In order to solve the above problems, in Patent Document 1, electrodes are arranged in a matrix on a substrate, spots made of probe DNA fragments are fixed on each electrode, and a dispersion medium covers all of these electrodes and spots. It describes a DNA microarray that is coated on one surface of a substrate.
[0010]
The identification method using the DNA microarray described in Patent Document 1 is as follows. First, a sample DNA fragment is injected into a dispersion medium. Then, point scanning is performed by sequentially applying a positive voltage to each electrode. Since the sample DNA fragment in the dispersion medium migrates toward the selected electrode when a positive voltage is applied among the plurality of electrodes, the sample DNA fragment has a high concentration at the spot of the selected electrode. . By performing such a point scan, the spots are sequentially brought into a high concentration state of the sample DNA fragment one by one. Therefore, all spots having complementarity with the sample DNA fragment have substantially the same fluorescence intensity. As described above, the technique described in Patent Document 1 utilizes the fact that a sample DNA fragment electrophoreses in a dispersion medium.
[0011]
[Patent Document 1]
Japanese Patent Publication No. 11-512605
[0012]
[Problems to be solved by the invention]
However, in the conventional DNA microarray, when a plurality of types of sample DNA fragments have a partially complementary base sequence, the sample DNA fragments partially hybridize during electrophoresis. Will combine. Therefore, even if a partially hybridized sample DNA fragment migrates to a complementary spot, it does not hybridize at that spot, so that the base sequences of a plurality of types of sample DNA fragments cannot be specified. Therefore, the entire electrophoretic medium is heated in order to denature the sample DNA fragment to a single-stranded state, but then the entire electrophoretic medium is cooled to hybridize, resulting in poor efficiency. In addition, it was not possible to hybridize only spots at arbitrary positions.
[0013]
Thus, an object of the present invention is to efficiently hybridize a sample DNA fragment even during electrophoresis.
[0014]
[Means for Solving the Problems]
In order to solve the above problems, the DNA analyzer according to the first aspect of the present invention is, for example, as shown in FIGS.
A tub containing the electrophoretic medium (eg, tub 71);
A plurality of probe electrodes (for example, probe electrodes 35, 35, ...) arranged in the bathtub;
A plurality of spots (for example, spots 60, 60,...) Consisting of a probe DNA fragment having a known base sequence and arranged on the plurality of probe electrodes;
A temperature adjusting element (eg, a temperature adjusting element 72) for adjusting the temperature of the plurality of spots via the probe electrode;
It is characterized by having.
[0015]
According to the first aspect of the present invention, the sample DNA fragment in the electrophoresis medium in the bath can be attracted by the probe electrode by electrophoresis. At this time, in order to maintain the denatured state of the sample DNA fragment before it hybridizes with the probe DNA fragment, the spot, that is, the probe DNA fragment and the electrophoresis medium around it are in a relatively high temperature state. If the temperature continues, the sample DNA fragment and the probe DNA fragment cannot hybridize, but the temperature control element controls the temperature of a plurality of spots via the probe electrode, so that the vicinity of the spot where hybridization is performed is locally cooled. By doing so, the sample can be cooled efficiently, and thereby the sample DNA fragment and the probe DNA fragment complementary to each other can be easily hybridized.
[0016]
According to a second aspect of the present invention, in the DNA analyzer according to the first aspect, the temperature control element is in contact with each of the plurality of probe electrodes, and the plurality of the plurality of probe electrodes are partially connected to the plurality of probe electrodes. The temperature of a part of the spot is selectively adjusted.
[0017]
According to the second aspect of the invention, the temperature control element selectively conducts heat to an arbitrary probe electrode, so that it is possible to hybridize only an arbitrary spot. That is, it is preferable that the probe DNA fragments at the predetermined probe electrode spots are hybridized, and that the probe DNA fragments at the probe electrode spots other than the predetermined probe electrode are not hybridized (for example, step S19). In the above, the probe DNA fragments 61 on the seventh to ninth column probe electrodes 35 are preferably hybridized, and the probe DNA fragments 61 on the fourth to sixth column probe electrodes 35 are hybridized. Is not hybridized), the temperature is lowered to such an extent that the probe DNA fragments at the predetermined probe electrode spot can hybridize, and the probe DNA fragments at the probe electrode spots other than the predetermined probe electrode are hybridized. Raise the temperature to an extent that Accordingly, when the stirring, it is possible to selectively form a dispersed easily spot and dispersed hard spot.
[0018]
The invention according to claim 3 provides the DNA analyzer according to claim 1 or 2, for example, as shown in FIGS.
A first electrode (for example, a first electrode 74) and a second electrode (for example, a second electrode 75) are disposed in the bathtub and are opposed to each other in the width direction of the bathtub.
[0019]
According to the third aspect of the present invention, the sample DNA fragment can be efficiently electrophoresed by applying a voltage to the first electrode and the second electrode.
[0020]
The invention according to claim 4 is the DNA analyzer according to claim 3,
The plurality of probe electrodes are divided into a plurality of electrode sets including one probe electrode or several adjacent probe electrodes,
The spots arranged on a predetermined electrode set among the plurality of electrode sets have different base sequences of the probe DNA fragments, and the number of bases of one probe DNA fragment is the same as each other or the predetermined number of bases are different. More closely approximate to each other than the number of bases of one probe DNA fragment of the spot arranged on the probe electrode other than the electrode set,
The one probe DNA fragment located on the second electrode side has a smaller number of bases than the one probe DNA fragment located on the first electrode side.
[0021]
The electrophoretic mobility of a DNA fragment increases as the number of bases decreases. Therefore, when a plurality of types of sample DNA fragments having different numbers of bases are injected into the first electrode side of the electrophoretic medium, the sample DNA fragments having a small number of bases are easily migrated to the second electrode side, and the sample DNA fragments having a large number of bases are Since the fluid resistance is high, it is difficult to migrate to the second electrode side. Here, in the invention according to claim 4, since the number of bases of the probe DNA fragment in the plurality of spots decreases as the electrode set approaches the second electrode, the sample DNA fragment has a voltage between the first electrode and the second electrode. Thus migrates onto a set of electrodes having a probe DNA fragment having substantially the same number of bases. Since the plurality of probe electrodes and the plurality of spots are arranged at positions deeper than the first electrode and the second electrode, when the sample DNA fragment migrates from the first electrode to the second electrode, the sample DNA fragment Does not reach the spot, but when a voltage higher than the voltage of the first electrode that attracts the sample DNA fragment is applied to the probe electrode, the sample DNA fragment also migrates in the depth direction.
Therefore, the sample DNA fragment migrates to the spot of the probe DNA fragment having substantially the same number of bases. Therefore, since the sample DNA fragment can be hybridized only with the complementary probe DNA fragment having the same base number, the base sequence of each of a plurality of types of sample DNA fragments having different base numbers and different electrophoresis migration speeds can be obtained. It can be analyzed efficiently.
[0022]
The invention according to claim 5 provides the DNA analyzer according to claim 3 or 4, as shown in FIGS.
A voltage control circuit (Step SS1, Step S6, Step S11) for applying a voltage for a predetermined time so that the potential of the second electrode is higher than the potential of the first electrode for the number of sets of the electrode sets ( For example, a voltage control circuit 73) is provided.
[0023]
In the invention according to claim 5, the voltage application step of applying a voltage for a predetermined time so that the potential of the second electrode is higher than the potential of the first electrode is repeated, so that the sample DNA fragment is moved from the first electrode to the second electrode. Electrophoreses towards electrodes. Thereby, the movable sample DNA fragment can be appropriately moved.
[0024]
According to a sixth aspect of the present invention, in the DNA analyzer according to the fifth aspect, when the voltage application step by the voltage control circuit is repeated, the temperature control element performs a heating operation, and the voltage control circuit After the repetition of the voltage application step is completed, a temperature control circuit for ending the heat generation operation of the temperature adjustment element is provided.
[0025]
In the invention according to claim 6, since the temperature control circuit causes the temperature control element to perform the heat generation operation, the sample DNA fragment in the electrophoresis medium is easily migrated because hybridization does not occur. After that, the temperature control circuit terminates the heating operation of the heating element, so that the temperature of the electrophoretic medium decreases, and the sample DNA fragments are likely to hybridize.
[0026]
The invention according to claim 7 is the DNA analyzer according to claim 5,
The voltage control circuit, after each of the voltage applying step, selects one of the plurality of electrode sets from the second electrode in order from the second electrode, one or more probe electrodes in the selected electrode set Is applied so that the potential of the first electrode and the second electrode is higher than the potential of at least one of the first electrode and the second electrode.
[0027]
In the invention according to claim 7, the sample DNA fragment migrates from the first electrode to the second electrode in the voltage application step, and the migration distance increases as the number of bases of the sample DNA fragment decreases. For example, a sample DNA fragment having a small number of bases migrates closest to the electrode set closest to the second electrode in the first voltage application step. Since the potential is higher than the potential of at least one of the first electrode and the second electrode, the sample DNA fragment having a small number of bases migrates in the depth direction to the selected electrode set.
In this manner, the voltage application step is repeated, and when one of the plurality of electrode sets is selected from the plurality of electrode sets in order of being closer to the second electrode during each of the voltage application steps, the sample DNA fragment is applied to the second electrode. It is distributed to each electrode set so that the number of bases decreases as the electrode set becomes closer.
Thereafter, since the temperature control circuit terminates the heat generation operation of the temperature control element, the temperature of the electrophoretic medium decreases, and the sample DNA fragment easily hybridizes with a complementary probe DNA fragment having the same base number.
[0028]
The invention according to claim 8 is the DNA analyzer according to claim 7,
The voltage control circuit is characterized in that a voltage is applied such that the potential of at least one of the first electrode and the second electrode is higher than the potentials of the plurality of probe electrodes after selecting all the electrode sets. I do.
[0029]
In the invention according to claim 8, by applying a voltage so that the potential of at least one of the first electrode and the second electrode is higher than the potential of a plurality of probe electrodes after selecting all the electrode sets. The unhybridized sample DNA fragment moves away from the spot to at least one of the first electrode and the second electrode. However, the sample DNA fragment hybridized with the complementary probe DNA fragment does not leave the spot.
Therefore, since the sample DNA fragments are distributed only in the spots of the complementary probe DNA fragments, only the hybridized spots can be detected accurately.
[0030]
According to a ninth aspect of the present invention, there is provided the DNA analyzer according to any one of the first to eighth aspects, wherein a plurality of photosensors are provided corresponding to the plurality of spots arranged on the plurality of probe electrodes. It has an element.
[0031]
According to the ninth aspect of the present invention, since each photosensor element can detect the presence or absence of hybridization for each spot, a plurality of spots can be simultaneously detected.
[0032]
The analysis method of the invention according to claim 10 is
An electrophoresis step of applying a voltage to a plurality of probe electrodes provided in a bath containing an electrophoresis medium to attract sample DNA fragments in the electrophoresis medium to the probe DNA fragments provided in the probe electrodes;
A temperature adjusting step of adjusting the temperature of the probe DNA fragment and the surrounding electrophoretic medium through the probe electrode,
It is characterized by including.
[0033]
In the invention according to claim 10, the temperature of the probe DNA fragment and the temperature of the electrophoresis medium around the probe DNA fragment can be adjusted via the probe electrode if necessary, so that any probe DNA fragment can be selectively hybridized. In addition, by maintaining the denatured state of the sample DNA fragment near the probe DNA fragment other than the arbitrary probe DNA fragment, the electrophoresis can be quickly performed.
[0034]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, specific embodiments of the present invention will be described with reference to the drawings. However, the scope of the invention is not limited to the illustrated example.
[0035]
FIG. 1 is a cutaway front view of a DNA identification device 100 for analyzing DNA, and FIG. 2 is a top view of the DNA identification device 100.
[0036]
The DNA identifying apparatus 100 includes a bathtub 71, an ultraviolet irradiator 77 disposed above the bathtub 71, a first electrode 74 disposed in an upper portion inside the bathtub 71 and horizontally opposed to the width direction of the bathtub 71, and a second electrode 74. It has two electrodes 75 and a DNA sensor 1 that is detachable from the bottom of the bathtub 71.
[0037]
The DNA sensor 1 will be described with reference to FIGS. FIG. 3 is a plan view of the DNA sensor 1, FIG. 4 (a) is a plan view showing one pixel of the DNA sensor 1, and FIG. 4 (b) is a broken line II in FIG. 4 (a). It is sectional drawing fractured | ruptured and shown by -II.
The DNA sensor 1 includes a light detection device 2 that is an imaging device, an electromagnetic shield layer 32 formed on the entire surface of the light detection device 2, an overcoat layer 33 formed on the electromagnetic shield layer 32, An ultraviolet shielding layer 34 formed on the layer 33, a plurality of probe electrodes 35, 35,... Arranged on the surface of the ultraviolet shielding layer 34, each of which is a group of single-stranded probe DNA fragments 61 and a probe. , A plurality of spots 60, 60,... Arranged and fixed on the electrodes 35, 35,.
[0038]
The light detection device 2 includes a substantially flat transparent substrate 17 and a plurality of double gates arranged on the surface of the transparent substrate 17 in a matrix of n rows and m columns (m and n are integers of 2 or more). , Each of which is constituted by a field-effect transistor. Note that a row refers to an arrangement of m photosensor elements 20, 20,... Aligned in a horizontal direction along a source line 42 and a drain line 43 described later. The column refers to an arrangement of n photosensor elements 20, 20,... Aligned in a vertical direction along a top gate line 44 and a bottom gate line 41, which will be described later.
[0039]
The transparent substrate 17 has a light transmitting property (hereinafter, simply referred to as a light transmitting property) and an insulating property, and is a glass substrate such as quartz glass or a plastic substrate such as polycarbonate. The back surface of the transparent substrate 17 forms the back surface of the light detection device 2. Note that a substrate having a light-shielding property may be used instead of the transparent substrate 17 having a light-transmitting property.
[0040]
These photosensor elements 20, 20,... Are photoelectric conversion elements that become pixels. The photo sensor element 20 includes a bottom gate electrode 21 formed on the transparent substrate 17, a bottom gate insulating film 22 formed on the bottom gate electrode 21, and a bottom gate insulating film 22 between the bottom gate electrode 21. A semiconductor film 23 sandwiched and opposed to the bottom gate electrode 21; a channel protective film 24 formed on a central portion of the semiconductor film 23; and an impurity semiconductor film 25 formed on both ends of the semiconductor film 23 so as to be separated from each other. , 26, a source electrode 27 formed on one impurity semiconductor film 25, a drain electrode 28 formed on the other impurity semiconductor film 26, and a top gate formed on the source electrode 27 and the drain electrode 28. The top gate insulating film 29 and the channel protection film 24 are interposed between the insulating film 29 and the semiconductor film 23 and are opposed to the semiconductor film 23. And Pugeto electrode 30 comprises a.
[0041]
On the transparent substrate 17, a bottom gate electrode 21 is formed for each photosensor element 20. Also, on the transparent substrate 17, m bottom gate lines 41, 41,... Extending in the vertical direction are formed, one for each row, and each photo sensor element in the same row arranged in the vertical direction. The 20 bottom gate electrodes 21 are formed integrally with a common bottom gate line 41. The bottom gate electrode 21 and the bottom gate line 41 have conductivity and light shielding properties, and are made of, for example, chromium, a chromium alloy, aluminum, an aluminum alloy, or an alloy thereof.
[0042]
On the bottom gate electrode 21 and the bottom gate line 41, a bottom gate insulating film 22 common to all the photosensor elements 20, 20,... Is formed. The bottom gate insulating film 22 has an insulating property and a light transmitting property, for example, silicon nitride (SiN) or silicon oxide (SiO 2). ₂ ).
[0043]
On the bottom gate insulating film 22, a semiconductor film 23 is formed for each photosensor element 20. The semiconductor film 23 has a substantially rectangular shape in plan view, and is a layer formed of amorphous silicon or polysilicon. On the semiconductor film 23, a channel protection film 24 is formed. The channel protective film 24 has a function of protecting the interface of the semiconductor film 23 from an etchant used for patterning, has insulating properties and translucency, and is made of, for example, silicon nitride or silicon oxide. The semiconductor film 23 exhibits sensitivity to light, and when light enters the semiconductor film 23, an amount of electron-hole pairs corresponding to the amount of incident light is centered around the interface between the channel protective film 24 and the semiconductor film 23. Is to occur. In this case, holes are generated as carriers on the semiconductor film 23 side, and electrons are generated on the channel protection film 24 side.
[0044]
On one end of the semiconductor film 23, an impurity semiconductor film 25 is formed so as to partially overlap the channel protective film 24, and on the other end of the semiconductor film 23, an impurity semiconductor film 26 is partially formed on the channel protection film. It is formed so as to overlap with the protective film 24. The impurity semiconductor films 25 and 26 are patterned for each photosensor element 20. The impurity semiconductor films 25 and 26 are made of amorphous silicon (n) containing n-type impurity ions. ⁺ Silicon).
[0045]
On the impurity semiconductor film 25, a source electrode 27 patterned for each photosensor element 20 is formed. On the impurity semiconductor film 26, a drain electrode 28 patterned for each photosensor element 20 is formed. Also, on the bottom gate insulating film 22, m source lines 42, 42,... Extending in the horizontal direction are formed, one for each row, and m drain lines extending in the horizontal direction. .. Are formed one per line. The source electrodes 27 of the photosensor elements 20 in the same row arranged in the horizontal direction are formed integrally with the common source line 42, and the drain electrodes of the photosensor elements 20 in the same row arranged in the horizontal direction are formed. The electrode 28 is formed integrally with the common drain line 43. The source electrode 27, the drain electrode 28, the source line 42, and the drain line 43 have conductivity and light-shielding properties, and are made of, for example, chromium, a chromium alloy, aluminum, an aluminum alloy, or an alloy thereof.
[0046]
On the channel protective film 24, the source electrode 27 and the drain electrode 28, and the source lines 42, 42,... And the drain lines 43, 43,. Is formed. The top gate insulating film 29 has an insulating property and a light transmitting property, and is made of, for example, silicon nitride or silicon oxide.
[0047]
On the top gate insulating film 29, a top gate electrode 30 patterned for each photosensor element 20 is formed. Also, n top gate lines 44 extending in the vertical direction are formed on the top gate insulating film 29, one for each column, and the top of each photosensor element 20 in the same column arranged in the vertical direction is formed. The gate electrode 30 is formed integrally with a common top gate line 44. The top gate electrode 30 and the top gate line 44 have conductivity and translucency, for example, indium oxide, zinc oxide, tin oxide, or a mixture containing at least one of them (for example, tin-doped indium oxide ( (ITO) and zinc-doped indium oxide).
The photosensor element 20 configured as described above is a photoelectric conversion element using the semiconductor film 23 as a light receiving unit. The photosensor elements 20, 20,... Are collectively covered by a common protective insulating film 31, and the protective insulating film 31 is formed on the top gate electrode 30 and the top gate line 44. The protective insulating film 31 has an insulating property and a light transmitting property, and is made of, for example, silicon nitride or silicon oxide.
[0048]
On the protective insulating film 31, an electromagnetic shield layer 32 is formed on one surface in common to all the photosensor elements 20, 20,. The electromagnetic shield layer 32 has conductivity and translucency, and is formed of, for example, indium oxide, zinc oxide, tin oxide, or a mixture containing at least one of them.
[0049]
On the electromagnetic shield layer 32, an overcoat layer 33 is formed on one surface in common to all the photosensor elements 20, 20,. The overcoat layer 33 has an insulating property and a light transmitting property, and is made of, for example, silicon nitride or silicon oxide.
[0050]
On the overcoat layer 33, an ultraviolet shielding layer 34 is formed on one surface in common to all the photosensor elements 20, 20,. The ultraviolet shielding layer 34 has a property of shielding ultraviolet rays that excite a fluorescent substance to be described later and transmitting fluorescence (mainly visible light) emitted from the fluorescent substance excited by the ultraviolet rays. Examples of the ultraviolet shielding layer 34 include a layer made of anatase or rutile titanium oxide. In addition, a dielectric multilayer film in which a dielectric H layer having a high refractive index and a dielectric L layer having a lower refractive index than the dielectric H layer are alternately laminated with an optical film thickness of ４ of the ultraviolet wavelength. Can be
[0051]
On the ultraviolet shielding layer 34, m probe electrodes 35, 35,... Extending in the vertical direction are formed, one for each row. These probe electrodes 35 are arranged so that their longitudinal directions are parallel to each other. Further, each probe electrode 35 is formed so as to collectively cover n photosensor elements 20, 20,... In the same row.
[0052]
On the probe electrodes 35, 35,..., N types of spots 60 are fixed in a row for each probe electrode 35, and a total of (m × n) types of spots 60 are arranged in a matrix. Have been. Then, one type of spot 60 is arranged for one photosensor element 20 so that one type of spot 60 overlaps one photosensor element 20 in plan view. One spot 60 is a crowd of many single-stranded probe DNA fragments 61. Many single-stranded probe DNA fragments 61 contained in one spot 60 have the same base sequence (nucleotide sequence). The base sequence of the single-stranded probe DNA fragment 61 is different for each. Each of the spots 60 has a known base sequence.
[0053]
Further, the n types of spots 60 in the same row are composed of single-stranded probe DNA fragments 61 having different base sequences, but are single-stranded DNA fragments 61 having substantially the same number of bases. That is, the single-stranded DNA fragments 61 of the n types of spots 60 in the same row all have substantially the same number of bases. Although the number of bases of the single-stranded probe DNA fragment 61 of the spot 60 may be significantly different from row to row, some adjacent rows of the m rows have the same number of bases as the single-stranded DNA fragment 61 of the same base. It may be. A row set consisting of one row or several adjacent rows among the m rows of spots 60, 60,... Is a single-stranded DNA fragment 61 having substantially the same number of bases, and a single-stranded DNA fragment 61 having substantially the same number of bases. There are several sets of strands designated as strand DNA fragments 61. The set of single-stranded probe DNA fragments 61 having substantially the same number of bases is arranged such that the number of bases increases toward the left, and the set of single-stranded probe DNA fragments 61 having substantially the same number of bases is shifted to the right. Are arranged such that the number of bases decreases as approaching.
Therefore, the plurality of probe electrodes 35 are also classified according to the number of bases of the probe DNA fragment 61. That is, the plurality of probe electrodes 35, 35,... Are divided into one probe electrode 35 or an electrode set composed of several adjacent probe electrodes 35, and the spots 60, 60,. The number of bases of the single-stranded probe DNA fragment 61 is almost the same, but the base sequence is different. Here, “substantially the same” means that the number of bases of one probe DNA fragment is the same among a plurality of spots arranged in a predetermined electrode set among the plurality of electrode sets, or Means that they are closer to each other than the number of bases of one probe DNA fragment of the spots arranged on the probe electrodes other than the above electrode set.
[0054]
In the following, in order to simplify the description, 81 photosensor elements 20, 20,... Are arranged in a matrix of 9 rows and 9 columns, and 9 columns of probe electrodes 35, 35,. It is assumed that nine types of spots 60 are arranged on each probe electrode 35. Also, the probe DNA fragments 61 of the spots 60, 60,... Formed on the electrode set composed of the three rows of probe electrodes 35, 35, 35 have different base sequences for each spot 60, but are substantially the same. The number of bases. The probe DNA fragments 61 of the spots 60, 60,... Formed on the electrode set composed of the three rows of probe electrodes 35, 35, 35 have base sequences different from each other for each spot 60, but have substantially the same number of bases. It is. The probe DNA fragments 61 of the spots 60, 60,... Formed on the electrode set composed of the three rows of probe electrodes 35, 35, 35 have different base sequences for each spot 60, but have substantially the same number of bases. It is. Further, the number of bases of the probe DNA fragment 61 of the spots 60, 60,... Formed on the electrode set composed of the three rows of probe electrodes 35, 35, 35 on the left side is the same as that of the center three rows of probe electrodes 35, 35, 35. , Spots 60, 60,... Formed on the electrode set composed of the three rows of probe electrodes 35, 35, 35 in the center, which are larger than the number of bases of the probe DNA fragment 61 of the spots 60, 60,. The number of bases of the probe DNA fragments 61 of 60,... Is larger than the number of bases of the probe DNA fragments 61 of spots 60, 60,. . Also, the leftmost column probe electrode 35 is the first column, the rightmost column probe electrode 35 is the ninth column, and the order of the probe electrodes 35 is from left to right. That is, the first to third rows form one common electrode set, the fourth to sixth rows form one common electrode set, and the seventh to ninth rows form one common electrode set. It becomes a common electrode set, and in each electrode set, the number of bases of the single-stranded probe DNA fragment 61 is the same or the number of bases of one probe DNA fragment of a spot arranged in an electrode set other than the electrode set Than each other.
[0055]
The spots 60, 60,... Are fixed to the probe electrode 35 by dispensing a probe DNA fragment prepared in advance to the probe electrode 35 surface-treated with a polycation (poly-L-lysine, polyethyleneimine, etc.). And a method of applying electrostatic charge to the surface of the photodetection device 2 using the charge of DNA is applied.
As another fixing method, a method using a silane coupling agent having an amino group, an aldehyde group, an epoxy group or the like is also used. In this case, since amino groups, aldehyde groups, and the like are introduced into the surface of the probe electrode 35 by covalent bonds, they are stably present on the probe electrode 35 as compared with the case of using polycations.
As another fixing method, there is a method in which an oligonucleotide having a reactive group introduced therein is synthesized, the oligonucleotide is spotted on the surface-treated probe electrode 35, and covalently bonded.
[0056]
The DNA sensor 1 as described above is detachable from the bottom of the bathtub 71 of the DNA reader 100 with the spots 60, 60,. Note that the depth direction of the bathtub 71 is orthogonal to the horizontal direction.
[0057]
The bathtub 71 is open at the top so that an electrophoretic medium 82 having a predetermined viscosity can be injected into the bathtub 71. Examples of the electrophoretic medium 82 include a colloid solution (sol) in which particles are dispersed in a dispersion medium, an electrolytic solution containing gel, and other electrolytic solutions.
[0058]
The bathtub 71 is mounted on a shaker 83, and the electrophoresis medium 82 is stirred by shaking the bathtub 71 by the shaker 83. The shaker 83 may be one that shakes the bath tub 71 by ultrasonic waves, one that shakes the bath tub 71 in a rotary manner, or one that shakes the bath tub 71 in a reciprocating manner. There may be.
[0059]
When the DNA sensor 1 is mounted on the bottom of the bathtub 71, when the bathtub 71 is viewed from above, a plurality of probe electrodes 35, 35,... Are arranged between the first electrode 74 and the second electrode 75, Is located closer to the first electrode 74 formed on the left wall of the bathtub 71, and the rightmost probe electrode 35 is located closer to the second electrode 75 formed on the right wall of the bathtub 71.
[0060]
In the bathtub 71 and on the front wall surface of the bathtub 71, one temperature control element 72 for controlling the temperature is provided for each probe electrode 35. When the DNA sensor 1 is mounted on the bottom of the bathtub 71, the temperature control elements 72, 72,... Are provided so as to come into contact with the ends of the respective probe electrodes 35, 35,. The temperature of the spots 60, 60,... Is adjusted via the electrodes 35, 35,. Examples of the temperature control element 72 include a Peltier element capable of performing heating and cooling, and a combination of a heater capable of performing heating and a heat sink capable of performing cooling. However, the probe electrodes 35 are selectively heated by heat generation. , And the temperature of the probe electrodes 35, 35,... And the vicinity thereof can be adjusted by selectively cooling the probe electrodes 35, 35,. Note that the temperature control element 72 may be one that can only perform heating, such as a heating resistor or another heating element.
[0061]
A plurality of terminals 73a, 73a,... Of a voltage control circuit 73 (shown in FIG. 5) for performing voltage control are provided in the bathtub 71 and on the rear wall surface of the bathtub 71. Are provided so as to come into contact with the probe electrodes 35, 35, respectively when the DNA sensor 1 is mounted on the bottom of the bathtub 71. The voltage control circuit 73 supplies the terminals 73a, 73a,. , 35, 35,... Are individually applied with voltages.
[0062]
In the bathtub 71, a plurality of terminals of a driver circuit 76 (shown in FIG. 5) for driving the light detection device 2 are provided. As shown in FIG. 6, the driver circuit 76 includes a top gate driver 76A, a bottom gate driver 76B, and a drain driver 76C. When the DNA sensor 1 is mounted on the bottom of the bathtub 71, the terminals of the top gate driver 76A, the bottom gate driver 76B, and the drain driver 76C are connected to the top gate lines 44, 44,. , And the drain lines 43, 43,..., Respectively, and the voltage is applied by the driver circuit 76 to the top gate lines 44, 44,. .. Are appropriately applied. The source lines 42 are all held at the potential Vss, and when the DNA sensor 1 is mounted on the bottom of the bathtub 71, the potentials Vss of the source lines 42, 42,... And the electromagnetic shield layer 32 may be the ground potential.
[0063]
By applying a voltage to the top gate lines 44, 44,..., The bottom gate lines 41, 41,. You. When the light detection device 2 is driven, the amount of light incident on each of the photo sensor elements 20 is converted into an electric signal, and the electric signal is detected by the driver circuit 76 so that imaging is performed.
[0064]
The drive control method of the photo sensor element 20 described above will be described with reference to the drawings. FIG. 7 is a timing chart illustrating an example of a basic drive control method of the photosensor element 20, FIG. 8 is a conceptual diagram of the operation of the photosensor element 20, and FIG. FIG. 4 is a diagram showing the optical response characteristics of the optical element. Here, a description will be given with reference to the configuration of the photosensor element 20 (FIGS. 1 and 4) as appropriate.
[0065]
First, in the reset operation (initialization operation), as shown in FIG. 7 and FIG. 8A, under the control of the controller 78, through the top gate line 44 of an arbitrary i-th column (1 ≦ i ≦ m). Then, a pulse voltage (hereinafter, referred to as a “reset pulse”; for example, a high level of Vtg = + 15 V) φTi is applied to the top gate electrode 30 of the photosensor element 20 in the i-th column, and the semiconductor layer 23 and the channel Carriers (here, holes) accumulated near the interface with the semiconductor layer 23 in the protective film 24 are released (reset period Trst).
[0066]
Next, in the light accumulation operation (charge accumulation operation), as shown in FIGS. 7 and 8B, the top gate electrode of the photosensor element 20 in the i-th column is connected via the top gate line 44 in the i-th column. By applying a low-level (for example, Vtg = −15 V) bias voltage φTi to 30, the reset operation ends, and the light accumulation period Ta by the carrier accumulation operation starts. In the light accumulation period Ta, electron-hole pairs are generated in the incident effective region of the semiconductor layer 23, that is, the carrier generation region, according to the amount of light incident from the top gate electrode 21 side, and the generated electron-hole pairs are generated. Are accumulated near the interface between the semiconductor layer 23 and the semiconductor layer 23 in the channel protective film 24, that is, around the channel region.
[0067]
In the precharge operation, as shown in FIGS. 7 and 8C, the drain electrode 28 of the photosensor element 20 in the i-th column is set in parallel with the light accumulation period Ta based on the precharge signal φpg. In order to apply a predetermined voltage (precharge voltage) Vpg, electric charges are held in each drain line 43 (precharge period Tprch). Next, in the read operation, as shown in FIGS. 7 and 8D, after the precharge period Tprch has elapsed, a high-level (for example, Vbg = + 10 V) bias voltage (read select signal) is applied to the bottom gate electrode 21. The photosensor element 20 is turned on by applying φBi (selected state) (reading period Tread).
[0068]
Here, in the read period Tread, carriers (holes) accumulated in the channel region of the photosensor element 20 into which light is incident relax Vtg (−15 V) applied to the top gate electrode 30 having the opposite polarity. 9A, an n-channel is formed by Vbg (+15 V) of the bottom gate electrode 21, and the voltage (drain voltage) VD of the drain electrode 28 and the drain line 43 according to the drain current is as shown in FIG. 2 shows a tendency to gradually decrease from the precharge voltage Vpg over time.
[0069]
That is, when the light accumulation state in the light accumulation period Ta is a bright state, carriers (holes) corresponding to the amount of incident light are captured in the channel region as shown in FIG. It acts to cancel the negative bias of the electrode 30, and the photosensor element 20 is turned ON by the positive bias of the bottom gate electrode 21 by the amount of the cancellation. Then, according to the ON resistance according to the amount of incident light, the drain voltage VD decreases as shown in FIG.
[0070]
On the other hand, when the light accumulation state is a dark state and no carriers (holes) are accumulated in the channel region, the bottom gate electrode 21 is turned off by the negative bias of the top gate electrode 30 as shown in FIG. Is canceled, the photosensor element 20 is turned off, and the drain voltage VD is maintained almost as it is, as shown in FIG. 9A.
[0071]
Therefore, as shown in FIG. 9A, the change tendency of the drain voltage VD is such that the read pulse φBi is applied to the bottom gate electrode 21 from the end of the reset operation by the application of the reset pulse φTi to the top gate electrode 30. Time (light accumulation period Ta), it is closely related to the amount of light received, when there are many accumulated carriers (bright state), it tends to decrease sharply, and when there are few accumulated carriers. (Dark state) shows a tendency to decrease gradually. Therefore, by detecting the drain voltage VD (= Vrd) after a lapse of a predetermined time from the start of the readout period Tread, or by setting a predetermined threshold voltage as a reference, the time required to reach the voltage is detected. By detecting, the amount of light (irradiation light) incident on the photosensor element 20 is converted.
[0072]
In the timing chart shown in FIG. 7, after the precharge period Tprch has elapsed, a low level (for example, Vbg = 0 V) is applied to the bottom gate electrode 21 as shown in FIGS. 8 (f) and 8 (g). When the state (non-selection state) is continued, the photosensor element 20 keeps the OFF state, and as shown in FIG. 9B, the drain voltage VD holds a voltage close to the precharge voltage Vpg. In this manner, a selection function of selecting the read state of the photosensor element 20 and switching the read state to the non-selection state according to the state of application of the voltage to the bottom gate electrode 21 is realized.
[0073]
A first electrode 74 is provided in the upper part of the left wall in the bathtub 71, and a second electrode 75 is provided in the upper part of the right wall. The electrodes 74 and 75 are exposed inside the bathtub 71. ing. Further, the electrodes 74, 75 are located above the probe electrodes 35, 35, ... located at the bottom of the bathtub 71. Voltages are individually applied to the electrodes 74 and 75 by the voltage control circuit 73.
[0074]
When the DNA sensor 1 is attached to the bottom of the bathtub 71, when the bathtub 71 is viewed from above (when viewed in the height direction of the bathtub 71), the arrangement position of the light detection device 2 is the first electrode 74 and the second electrode 74. Between the electrode 75. When the DNA sensor 1 is mounted on the bottom of the bathtub 71, when the bathtub 71 is viewed from the right or left (when viewed in the width direction of the bathtub 71), the arrangement height of the light detection device 2 is the first electrode 74 and the height. It is lower than the arrangement height of the second electrode 75.
[0075]
Above the bathtub 71, an ultraviolet irradiator 77 for irradiating light downward is provided. The light emitted from the ultraviolet irradiator 77 includes the ultraviolet wavelength range and hardly includes the fluorescent wavelength range.
[0076]
Next, a control configuration of the DNA identification device 100 will be described with reference to FIG.
This DNA identification device 100 includes a display device 81, a controller 78 for controlling the whole, an irradiation drive circuit 79 for driving an ultraviolet irradiator 77, and a temperature for driving the temperature control elements 72, 72,. And a control circuit 80.
[0077]
The controller 78 is a dedicated logic circuit or an arithmetic processing unit having a CPU (central processing unit) and the like. The voltage control circuit 73, the driver circuit 76, the irradiation drive circuit 79, the temperature control circuit 80, a display device 81 and a shaker 83 are operated.
[0078]
As described with reference to FIGS. 6 to 9, the driver circuit 76 drives the light detection device 2 in accordance with an instruction from the controller 78 and causes the light detection device 2 to perform an imaging operation. The image captured by the light detection device 2 is A / D-converted by the driver circuit 76 and is output from the driver circuit 76 to the controller 78 as image data.
[0079]
The irradiation drive circuit 79 drives the ultraviolet irradiator 77 in accordance with an instruction from the controller 78, and causes the ultraviolet irradiator 77 to perform a light emitting operation.
[0080]
The temperature control circuit 80 drives the temperature adjusting elements 72, 72,... In accordance with an instruction from the controller 78, and causes the temperature adjusting elements 72, 72,.
[0081]
The voltage control circuit 73 individually applies a voltage to the terminals 73 a, 73 a,..., The first electrode 74, and the second electrode 75 in accordance with an instruction from the controller 78.
[0082]
The display device 81 displays an image captured by the light detection device 2 according to an instruction from the controller 78.
[0083]
Next, the operation of the DNA identification device 100 will be described with reference to FIGS. Here, FIGS. 10 to 12 are flowcharts showing the flow of the operation of the DNA identification device 100.
[0084]
First, DNA is collected from a sample and denatured to obtain a plurality of types of single-stranded DNA fragments having different numbers of bases. However, even if a plurality of types of single-stranded DNA fragments having the same number of bases and different base sequences are contained, good. A fluorescent substance is bound to the single-stranded DNA fragment, and the single-stranded DNA fragment is labeled with the fluorescent substance. The obtained single-stranded DNA fragment is called a sample DNA fragment. Examples of the fluorescent substance for labeling the sample DNA fragment include CyDye Cy2 and Cy3 (manufactured by Amersham). As the fluorescent substance, a substance that is excited by the wavelength of the ultraviolet light emitted from the ultraviolet irradiator 77 of the DNA identification device 100 is selected. The fluorescent substance emits fluorescent light when excited by ultraviolet light, and the wavelength of the fluorescent light is a wavelength that generates carriers in the semiconductor film 23 of the photosensor element 20, and a light ray having a longer wavelength than the ultraviolet light is preferable.
[0085]
Next, the DNA sensor 1 is mounted on the bottom of the bathtub 71, and the electrophoresis medium 82 is injected into the bathtub 71.
Next, the power of the DNA identification device 100 is turned on, and the DNA identification device 100 is activated.
[0086]
Then, the controller 78 of the DNA identification device 100 controls the temperature control circuit 80, whereby the temperature control circuit 80 drives the temperature control elements 72, 72,. To perform a heating operation. , Generate heat, and the heat of the temperature control elements 72, 72,... Is conducted to the probe electrodes 35, 35,. When the probe electrodes 35 are heated by the temperature control elements 72, 72,..., The electrophoretic medium 82 in the bathtub 71 is also heated to 95 ° C. or higher. Thereafter, the temperature control circuit 80 controls the temperature adjusting element 72 according to the instruction of the controller 78, so that the temperature of the electrophoretic medium 82 is maintained at 95 ° C. or higher. When the electrophoretic medium 82 is heated to 95 ° C. or higher, the probe DNA fragments 61 in one spot 60 do not partially hybridize.
[0087]
Note that the temperature at which the electrophoretic medium 82 is kept is not limited to 95 ° C. or higher, and may be any temperature at which the single-stranded DNA fragments maintain a single strand without complementary binding. Further, a temperature detector for detecting the temperature of the electrophoretic medium 82 is provided in the bathtub 71, the temperature detected by the temperature detector is fed back to the controller 78, and the controller 78 controls the temperature control circuit 80 according to the detected temperature. The temperature control elements 80 may be controlled so that the temperature control circuit 80 keeps the temperature of the electrophoretic medium 82 at 95 ° C. or higher.
[0088]
Then, the plurality of types of single-stranded sample DNA fragments obtained as described above are put into the bathtub 71 from above the bathtub 71. However, instead of putting the sample DNA fragment in the entire bathtub 71, the sample DNA fragment is put in a portion where there is no probe electrode 35, 35,... In plan view near the first electrode 74 (see FIGS. 1 and 2). 3) In particular, gently insert the sample DNA fragment into the portion away from the first electrode 74 so as not to diffuse. At this time, the single-stranded sample DNA fragment may be injected into the bath 71 together with a solution partially containing a reagent used for PCR amplification. In FIG. 1, among a plurality of types of single-stranded sample DNA fragments, a single-stranded sample DNA fragment 151 having the smallest number of bases, a single-stranded sample DNA fragment 153 having the largest number of bases, and a single-stranded sample DNA fragment A single-stranded sample DNA fragment 152 having a larger number of bases than 151 and a smaller number of bases than a single-stranded sample DNA fragment 153 is shown.
[0089]
Since the electrophoretic medium 82 is heated to 95 ° C. or higher by the heat generated by the temperature control elements 72, 72,..., Even if some of the sample DNA fragments have a complementary base sequence in part. In addition, the sample DNA fragments do not partially hybridize and bind. Therefore, the sample DNA fragments are electrophoresed in the electrophoresis medium 82 in a completely fragmented state.
[0090]
Next, as shown in step S1 (first voltage application step) of FIG. 10, the controller 78 controls the voltage control circuit 73, whereby the voltage control circuit 73 controls the first electrode 74 according to the instruction of the controller 78. A voltage is applied between the first electrode 74 and the second electrode 75 so that the potential is lower than the potential of the second electrode 75. At the same time, the voltage control circuit 73 operates according to the instruction of the controller 78 so that the potentials of all the probe electrodes 35, 35,... Become lower than the potential of the second electrode 75, preferably lower than the potential of the first electrode 74. A voltage is applied to the electrodes 35. As a result, the first electrode 74 becomes a cathode, the second electrode 75 becomes an anode, and all the probe electrodes 35, 35,... Have a negative voltage or an equal voltage with respect to the second electrode 75. Here, the potential of the first electrode 74 is desirably set to the ground potential, the voltage of all the probe electrodes 35, 35,... With respect to the first electrode 74 is desirably the same voltage or a negative voltage. The voltage is higher than the voltage required for separating the mishybridized sample DNA fragment from the probe DNA fragment, and is lower than the voltage at which the sample DNA fragment completely hybridized to the probe DNA fragment is not separated from the probe DNA fragment. The term “mishybridization” as used herein means partial hybridization, in which a part of the base sequence of one of the sample DNA fragment and the probe DNA fragment is complementary to a part or the whole of the other base sequence. In this case, the sample DNA fragment and the probe DNA fragment are partially bound, and complete hybridization means that all the bases of the sample DNA fragment are complementary to all the bases of the probe DNA fragment. Means to be done.
[0091]
As a result, a plurality of types of sample DNA fragments migrate in the electrophoresis medium 82, particularly toward the surface layer of the electrophoresis medium 82 toward the second electrode 75. Here, the voltage control circuit 73 maintains the above voltage state for a predetermined time, but the application time and the applied voltage are substantially the same as the number of bases in the shortest probe DNA fragment 61 in the seventh to ninth columns. For this reason, a relatively small volume of the sample DNA fragment having the same volume as the probe DNA fragment 61 in the seventh to ninth columns migrates onto the probe electrode 35 in the seventh column. . Therefore, as shown in FIG. 13, if the sample DNA fragment 151 having the least number of bases has substantially the same number of bases as the probe DNA fragment 61 in the seventh to ninth columns, the sample DNA fragment 151 Electrophoresis is performed on the probe electrodes 35 in the row. However, since the sample DNA fragments 152 and 153 have a larger volume than the sample DNA fragment 151, the fluid resistance during electrophoresis is larger than that of the sample DNA fragment 151, and the sample DNA fragments 152 and 153 do not migrate above the probe electrodes 35 in the seventh column.
Here, even if a voltage is applied to the probe electrodes 35, 35,..., The grounded electromagnetic shield layer 32 is between the probe electrode 35 and the photosensor element 20, so that the electric field of the probe electrodes 35, 35,. Does not adversely affect the operation of the photosensor element 20.
[0092]
After a lapse of a predetermined time after a sample DNA fragment having the same number of bases as the probe DNA fragments 61 in the seventh to ninth columns is expected to reach the electrophoretic medium 82 above the probe DNA fragments 61 in the seventh column, The controller 78 controls the voltage control circuit 73, and the voltage control circuit 73 cancels the voltage application state to make the first electrode 74, the second electrode 75 and all the probe electrodes 35, 35,. Then, the controller 78 controls the driver circuit 76 and the irradiation drive circuit 79, and the driver circuit 76 drives the light detection device 2 according to the instruction of the controller 78 according to the drive control method of the photosensor element 20 and the irradiation drive circuit. 79 drives the ultraviolet irradiator 77 according to the instruction of the controller 78 (step S2).
[0093]
As a result, the ultraviolet irradiator 77 emits light, and the ultraviolet light emitted from the ultraviolet irradiator 77 is irradiated onto the surface of the electrophoretic medium 82 in a planar manner. As a result, the ultraviolet light is incident on the fluorescent substance attached to the sample DNA fragment floating in the electrophoretic medium 82 above the probe DNA fragments 61 in the seventh to ninth columns, so that the fluorescent substance emits fluorescence. The light is transmitted through the ultraviolet shielding layer 34, the overcoat layer 33, the electromagnetic shielding layer 32, the protective insulating film 31, the top gate electrode 30, the top gate insulating film 29, and the channel protective film 24 and enters the semiconductor film 23. By performing photo-electric conversion of the photo sensor elements 20, 20,... Into electric signals in accordance with the intensity or light amount of the fluorescence incident on the respective semiconductor films 23, the light detection device 2 allows the respective photo sensor elements 20, 20,. To detect the fluorescence intensity or light amount, and acquire a two-dimensional fluorescence intensity distribution as a two-dimensional image. The image acquired by the light detection device 2 is A / D-converted by the driver circuit 76 and output from the driver circuit 76 to the controller 78 as image data.
Here, since the ultraviolet shielding layer 34 is provided, the ultraviolet rays emitted from the ultraviolet irradiator 77 do not enter the photosensor elements 20, 20,..., And the photosensor elements 20, 20,. do not do.
[0094]
Then, the controller 78 determines whether or not the seventh row of photosensor elements 20, 20,... Has detected fluorescence based on the image data input from the driver circuit 76 in step S2 (step S3).
[0095]
Here, when there are sample DNA fragments having substantially the same number of bases as the probe DNA fragments 61 in the seventh to ninth columns among a plurality of types of sample DNA fragments, the relative number of bases having the same number of bases is relatively large. As shown in FIG. 13, the sample DNA fragment 151 having a small volume has migrated to the position above the probe electrode 35 in the seventh column in step S1. Therefore, when there is a sample DNA fragment having substantially the same number of bases as the probe DNA fragments 61 in the seventh to ninth columns among a plurality of types of sample DNA fragments, as shown in FIG. Since the fluorescent light emitted from the light source enters the semiconductor film 23 of the photosensor elements 20, 20,... Of the seventh column with high intensity, the controller 78 determines in step S3 that the photosensor elements 20, 20,. Is determined to have detected fluorescence (step S3: Yes), and the process of the controller 78 proceeds to step S4. On the other hand, when there is no sample DNA fragment having substantially the same number of bases as the probe DNA fragments 61 in the seventh to ninth columns among a plurality of types of sample DNA fragments, the relative number of bases is relatively small because of the substantially same number of bases Since there is almost no small sample DNA fragment 151 of small volume, the fluorescent light enters the semiconductor film 23 of the photosensor elements 20, 20,. , No fluorescence was detected by the photosensor elements 20, 20,... In the seventh column, so that there was no sample DNA fragment having substantially the same number of bases as the probe DNA fragments 61 in the seventh to ninth columns. Is determined (step S3: No), and the process of the controller 78 proceeds to step S6.
FIG. 13 shows a case where the sample DNA fragment 151 has substantially the same number of bases as the probe DNA fragments 61 in the seventh to ninth columns and has a relatively small volume of the sample DNA fragment in the step S1. It is a drawing showing the migration position of 151. FIG. 14 shows that a relatively small volume of sample DNA fragment 151 having substantially the same number of bases as the probe DNA fragments 61 in the seventh to ninth columns is used as the probe DNA fragment 61 in the seventh to ninth columns. 11 is a graph illustrating a fluorescence intensity distribution of an image acquired by the light detection device 2 when electrophoresis is performed up to the vicinity of the seventh column.
[0096]
In step S4, the controller 78 controls the voltage control circuit 73, whereby the voltage control circuit 73 changes the potential of the probe electrode 35 in the eighth column to the other probe electrodes 35, 35,. A voltage is applied so as to be higher than the first electrode 74. Here, the probe electrodes 35, 35,... And the first electrode 74 other than the probe electrodes 35 in the eighth column are set to the ground potential, and the probe electrodes 35 in the eighth column are set to the positive voltage. With respect to the potentials of the probe electrodes 35 other than the probe electrodes 35 in the eighth column, 35,. With such a higher voltage, an electrode set including the probe electrodes 35, 35, 35 in the seventh to ninth columns is selected, and the voltage between the first electrode 74 and the second electrode 75 after this is selected. Regardless, a relatively small volume of the sample DNA fragment 151 having substantially the same number of bases as the probe DNA fragments 61 in the seventh to ninth columns is placed near the probe electrode 35 in the eighth column, that is, in the seventh column. To the ninth row of probe DNA fragments 61. In step S4 and step S5 described later, it is preferable that the second electrode 75 is also at the same potential as the first electrode 74 because the sample DNA fragment does not move in the direction from the first electrode 74 to the second electrode 75.
[0097]
Next, the controller 78 controls the driver circuit 76, whereby the driver circuit 76 drives the light detection device 2, and the light detection device 2 acquires an image representing the fluorescence intensity distribution during emission of the ultraviolet irradiator 77, The image data is output from the driver circuit 76 to the controller 78. Then, the controller 78 determines whether or not fluorescence has been detected by the photosensor elements 20, 20,... In the eighth column based on the image data (step S5).
[0098]
Here, in step S4, when a voltage higher than the voltage of the first electrode 74 is applied to the eighth column of the probe electrodes 35, the number of bases substantially equal to that of the probe DNA fragments 61 in the seventh to ninth columns As shown in FIG. 15, the sample DNA fragment migrates from above the probe electrodes 35 in the seventh column toward the probe electrodes 35 in the eighth column. Accordingly, when a relatively small volume of the sample DNA fragment 151 having substantially the same number of bases as the probe DNA fragments 61 in the seventh to ninth columns migrates to the vicinity of the probe electrode 35 in the eighth column, it is shown in FIG. As described above, since the fluorescence emitted from the sample DNA fragment is incident on the semiconductor 23 of the photosensor elements 20, 20,... In the eighth row with high intensity, the controller 78 sets the photosensor elements in the eighth row in the above step S5. At 20, 20,..., It is determined that fluorescence has been detected (step S5: Yes), and the process of the controller 78 proceeds to step S6. In step S4, the sample DNA fragment 151 having a relatively small volume due to substantially the same number of bases as the probe DNA fragments 61 in the seventh to ninth columns is held near the probe electrode 35 in the eighth column. At this stage, the probe DNA fragments 61 in the seventh to ninth columns do not necessarily have to hybridize with the sample DNA fragment 151 having a relatively small volume. On the other hand, if a sample DNA fragment having substantially the same number of bases as the probe DNA fragments 61 in the seventh to ninth columns has not migrated to the probe electrode 35 in the eighth column, the controller 78 proceeds to step S5. It is determined that the photosensor elements 20, 20,... In the eighth row do not detect fluorescence (step S5: No), and the process of the controller 78 proceeds to step S4. By repeating the processing of the controller 78 from step S5: No to step S4, the probe electrodes 35 in the eighth column are moved until the relatively small volume of the sample DNA fragment 151 migrates to the vicinity of the probe electrodes 35 in the eighth column. , A voltage higher than the voltage of the first electrode 74 is continuously applied.
FIG. 15 shows that the sample DNA fragment 151 having a relatively small volume due to substantially the same number of bases as the probe DNA fragments 61 in the seventh to ninth columns is the same as the sample DNA fragment 151 in step S4. It is a drawing showing an electrophoresis position. FIG. 16 is a graph showing the fluorescence intensity distribution of an image acquired by the light detection device 2 when the sample DNA fragment 151 has substantially the same number of bases as the probe DNA fragments 61 in the seventh to ninth columns. .
[0099]
In step S6 (second voltage application step), the voltage control circuit 73 causes the first voltage 74 and the second voltage 75 so that the potential of the first electrode 74 becomes lower than the potential of the second electrode 75 in accordance with an instruction from the controller 78. And a voltage is applied. At the same time, according to the instruction from the controller 78, the voltage control circuit 73 sets the potentials of the probe electrodes 35, 35,... Apply a voltage to the probe electrodes 35, 35,... So as to be equal to or lower than the potential of the first electrode 74. Until immediately before step S16, the potential of the probe electrodes 35, 35,. The applied voltage is set to be equal to or higher than the applied voltage of the second electrode 75, but is preferably equal to the second electrode 75 from the viewpoint of holding the sample DNA fragment 151.
[0100]
As a result, a relatively medium volume of the sample DNA 152 and a relatively large volume of the sample DNA fragment 153 on the surface of the electrophoresis medium migrate through the electrophoresis medium 82 toward the second electrode 75. Since the medium-sized sample DNA fragment 152 has relatively lower fluid resistance than the relatively large-volume sample DNA fragment 153, it migrates at a higher speed than the relatively large-volume sample DNA fragment 153. . Here, the voltage control circuit 73 maintains the above voltage state for a predetermined time, but the application time and the applied voltage are substantially the same as the number of bases of the probe DNA fragment 61 in the fourth to sixth columns. This is such that a relatively medium volume of the sample DNA fragment 152 migrates onto the probe electrode 35 in the fourth row. At this time, the relatively large volume of the sample DNA fragment 153 has not reached above the probe electrodes 35 in the fourth row. Therefore, as shown in FIG. 17, assuming that the sample DNA fragment 152 has substantially the same number of bases as the probe DNA fragments 61 in the fourth to sixth columns, the sample DNA fragment 152 extends to the top of the fourth column. Run electrophoresis. Further, the relatively small volume sample DNA fragment 151 is held near the eighth row of probe electrodes 35 by the electric field of the eighth row of probe electrodes 35 so as to sink to the bottom of the bathtub 71 in the previous step S4. Therefore, almost no electrophoresis occurs in step S6. When the process proceeds from the step S5 to the step S6, the voltage higher than the voltage of the first electrode 74 for holding the sample DNA fragment 151 is applied to the probe electrode 35 in the eighth column from the step S6 to the step S15. It continues to be applied by the circuit 73.
[0101]
After a predetermined period of time when a relatively medium volume of the sample DNA fragment is expected to reach the electrophoretic medium 82 above the fourth-row probe DNA fragment 61, the voltage control circuit 73 sets the voltage in accordance with the instruction of the controller 78. The application state is released and the first electrode 74, the second electrode 75, and the probe electrodes 35, 35,... (However, when the process proceeds from step S5 to step S6, the probe electrode 35 in the eighth column is excluded). Make the voltage equal. Then, the controller 78 controls the driver circuit 76 and the irradiation drive circuit 79, and the driver circuit 76 drives the light detecting device 2 according to the instruction of the controller 78, and the irradiation drive circuit 79 drives the ultraviolet irradiator 77 according to the instruction of the controller 78. (Step S7). As a result, the driver circuit 76 drives the light detection device 2, the light detection device 2 acquires an image representing the fluorescence intensity distribution during emission of the ultraviolet light irradiator 77, and the image data is output from the driver circuit 76 to the controller 78. Is done.
[0102]
Then, based on the image data, the controller 78 determines whether or not the fourth-row photosensor elements 20, 20,... Have detected the fluorescence of the sample DNA fragment 152 having a relatively medium volume (step). S8).
[0103]
Here, when there is a relatively medium volume of sample DNA 152 having substantially the same number of bases as the probe DNA fragments 61 in the fourth to sixth rows on the surface layer of the electrophoresis medium 82, the bases having substantially the same number of bases are used. As shown in FIG. 17, the number of sample DNA fragments has migrated to above the fourth row of probe electrodes 35 in step S6. Therefore, when the sample DNA fragments of a plurality of types include a relatively medium volume of sample DNA 152 having substantially the same number of bases as the probe DNA fragments 61 in the fourth to sixth columns, FIG. As shown, since the fluorescence emitted from the sample DNA fragment is incident with high intensity on the semiconductors 23 of the photosensor elements 20, 20,... In the fourth row, the controller 78 determines in step S8 that the photosensors in the fourth row It is determined that fluorescence has been detected by the elements 20, 20,... (Step S8: Yes), and the processing of the controller 78 proceeds to step S9. On the other hand, when there is no sample DNA fragment 152 having substantially the same number of bases as the probe DNA fragments 61 in the fourth to sixth columns among the plurality of types of sample DNA fragments, the photosensor element 20 in the fourth column is used. , 20,..., The fluorescent light enters at low intensity or hardly enters the semiconductor film 23, and thus the controller 78 does not detect the fluorescent light in the photosensor elements 20, 20,. Is determined (step S8: No), the process of the controller 78 proceeds to step S11.
FIG. 17 shows the migration position of the sample DNA fragment 152 in the above step S6 when the sample DNA fragment 152 has substantially the same number of bases as the probe DNA fragments 61 in the fourth to sixth columns. It is a drawing. FIG. 18 is a graph showing a fluorescence intensity distribution of an image acquired by the light detection device 2 when the sample DNA fragment 152 has substantially the same number of bases as the fourth to sixth column probe DNA fragments 61. . 17 and 18, it is assumed that the sample DNA fragment 151 having the smallest number of bases and relatively small volume has substantially the same number of bases as the probe DNA fragments 61 in the seventh to ninth columns.
[0104]
In step S9, the voltage control circuit 73 changes the potential of the probe electrode 35 in the fifth column to the other probe electrodes 35, 35,... (Provided that the process has shifted from step S5 to step S6) in accordance with the instruction from the controller 78. , The potential of the probe electrode 35 in the fifth column and the potential of the probe electrode 35 in the eighth column in step S9 and step S10 to be described later are equal to each other and different from the potential of the probe electrode 35 in the fifth column.) A voltage is applied so as to be higher than the first electrode 74. Here, the probe electrodes 35, 35,... Other than the probe electrode 35 in the fifth column (excluding the probe electrode 35 in the eighth column when the process proceeds from step S5 to step S6), and the first The electrode 74 is set to the ground potential, and the probe electrodes 35 in the fifth column are set to a voltage higher than the ground potential. At this time, it is desirable that the second electrode 75 has the same potential as the first electrode 74, but may be higher than the voltage of the first electrode 74. As a result, an electrode set composed of the fourth to sixth rows of probe electrodes 35, 35, 35 is selected, and regardless of the voltage between the subsequent first electrode 74 and second electrode 75, A relatively medium volume sample DNA fragment 152 having approximately the same number of bases as the probe DNA fragments 61 in the seventh to ninth columns is placed near the probe electrode 35 in the fifth column, that is, in the fourth to fifth columns. It can be held near the probe DNA fragment 61 in the sixth row. In step S9 and step S10 to be described later, it is preferable that the second electrode 75 is also at the same potential as the first electrode 74 because the sample DNA fragment does not move in the direction from the first electrode 74 to the second electrode 75.
[0105]
Next, the driver circuit 76 drives the light detection device 2 in accordance with an instruction from the controller 78, and the light detection device 2 acquires an image representing the fluorescence intensity distribution during emission of the ultraviolet irradiator 77, and the image data is stored in the driver circuit 76. To the controller 78. Then, the controller 78 determines whether or not fluorescence has been detected by the photosensor elements 20, 20,... In the fifth column based on the image data (step S10).
[0106]
Here, when a voltage higher than that of the first electrode 75 is applied to the fifth row of probe electrodes 35 in step S9, the sample having substantially the same number of bases as the fourth to sixth row of probe DNA fragments 61 is obtained. As shown in FIG. 19, the DNA fragments migrate so as to sink from above the probe electrodes 35 in the fourth row toward the probe electrodes 35 in the fifth row. When a sample DNA fragment having substantially the same number of bases as the probe DNA fragments 61 in the fourth to sixth columns migrates to the vicinity of the probe electrode 35 in the fifth column and is retained, as shown in FIG. Since the fluorescence emitted from the DNA fragment is incident on the semiconductor 23 of the fifth row of photosensor elements 20, 20,... With high intensity, the controller 78 in step S10 causes the fifth row of photosensor elements 20, 20,. Are determined to have detected fluorescence (step S10: Yes), and the process of the controller 78 proceeds to step S11. On the other hand, if a sample DNA fragment having substantially the same number of bases as the probe DNA fragments 61 in the fourth to sixth columns has not migrated to the probe electrode 35 in the fifth column, the controller 78 proceeds to step S10. It is determined that fluorescence is not detected by the photosensor elements 20, 20,... In the fifth column (step S10: No), and the process of the controller 78 proceeds to step S9. By repeating the processing of the controller 78 in step S9: No → step S10, the probe in the fifth column is moved until the relatively medium volume of the sample DNA fragment 152 migrates to the vicinity of the probe electrode 35 in the fifth column. A voltage higher than the first electrode 74 is continuously applied to the electrode 35.
FIG. 19 shows the migration position of the sample DNA fragment 152 in the above step S9 when the sample DNA fragment 152 has substantially the same number of bases as the fourth to sixth column probe DNA fragments 61. It is a drawing. FIG. 20 is a graph showing the fluorescence intensity distribution of an image acquired by the light detection device 2 when the sample DNA fragment 152 has substantially the same number of bases as the fourth to sixth column probe DNA fragments 61. .
[0107]
In step S11 (third voltage application step), the voltage control circuit 73 causes the first voltage 74 and the second voltage 75 so that the potential of the first electrode 74 becomes lower than the potential of the second electrode 75 in accordance with an instruction from the controller 78. And a voltage is applied. At the same time, the voltage control circuit 73 maintains the potentials of the probe electrodes 35, 35,... In the fifth and eighth columns at a voltage that the sample DNA fragment can hold, in accordance with an instruction from the controller 78. Are applied to the probe electrodes 35, 35,... So that the potentials of the probe electrodes 35, 35,. The potential of the probe electrodes 35, 35,... In the fifth and eighth columns is set to be higher than the applied voltage of the first electrode 74 and lower than the applied voltage of the second electrode 75, respectively. It is desirable that the second electrode 75 has the same potential as the DNA fragment 152 from the viewpoint of retention.
[0108]
Accordingly, a relatively large volume of the sample DNA fragment 153 remaining on the surface layer of the electrophoresis medium without being retained migrates in the electrophoresis medium 82 toward the second electrode 75. Here, the voltage control circuit 73 maintains the above voltage state for a predetermined time, but the applied time and the applied voltage are relatively the same as the number of bases of the probe DNA fragment 61 in the first to third columns. In this case, a large volume of the sample DNA fragment migrates onto the probe electrode 35 in the first row. Therefore, as shown in FIG. 21, if the sample DNA fragment 153 has approximately the same number of bases as the probe DNA fragments 61 in the first to third columns, the sample DNA fragment 153 extends to the top of the first column. Run electrophoresis. However, since the sample DNA fragments 151 and 152 are held so that the voltage for holding the sample DNA fragments is applied to the probe electrode 35 and settled at the bottom of the bathtub 71 after the previous steps S4 and S9, respectively. There is almost no migration in step S11 without being affected by the electric field between the first electrode 74 and the second electrode 75 located above. When the process proceeds from step S10 to step S11, a voltage relatively higher than the first electrode 74 is continuously applied to the fifth row of probe electrodes 35 by the voltage control circuit 73 from step S11 to step S15.
[0109]
After a lapse of a predetermined time during which a relatively large volume of the sample DNA fragment is expected to reach the electrophoretic medium 82 above the first-row probe DNA fragment 61, the voltage control circuit 73 changes the voltage applied state according to the instruction of the controller 78. , And the first electrode 74 and the probe electrodes 35, 35,... (However, when the process proceeds from step S5 to step S6, except for the probe electrode 35 in the eighth column, the process proceeds from step S10 to step S11). In this case, the probe electrodes 35 in the fifth column are excluded). It is desirable that the second electrode 75 has the same voltage as the first electrode 74. Then, the controller 78 controls the driver circuit 76 and the irradiation drive circuit 79, and the driver circuit 76 drives the light detecting device 2 according to the instruction of the controller 78, and the irradiation drive circuit 79 drives the ultraviolet irradiator 77 according to the instruction of the controller 78. (Step S12). As a result, the driver circuit 76 drives the light detection device 2, the light detection device 2 acquires an image representing the fluorescence intensity distribution during emission of the ultraviolet light irradiator 77, and the image data is output from the driver circuit 76 to the controller 78. Is done.
[0110]
Then, the controller 78 determines whether or not fluorescence has been detected by the photosensor elements 20, 20,... In the fourth column based on the image data (step S13).
[0111]
Here, when there is a sample DNA fragment having substantially the same number of bases as the probe DNA fragments 61 in the first to third rows on the surface layer of the electrophoresis medium 82, the sample DNA fragment having substantially the same number of bases is used. As shown in FIG. 21, the electrophoresis has been performed on the probe electrodes 35 in the first row in the step S11. Accordingly, when a sample DNA fragment having substantially the same number of bases as the probe DNA fragments 61 in the first to third rows is included in a plurality of types of sample DNA fragments, as shown in FIG. Since the fluorescent light emitted from the first row of the photosensor elements 20, 20,... Is incident on the semiconductor 23 of the first row of photosensor elements 20, 20,. It is determined that fluorescence has been detected (step S13: Yes), and the process of the controller 78 proceeds to step S14. On the other hand, when there is no sample DNA fragment having substantially the same number of bases as the probe DNA fragments 61 in the first to third columns in the plurality of types of sample DNA fragments 153, the photosensor element 20 in the first column , 20,..., The fluorescent light enters at low intensity or hardly enters the semiconductor film 23, and thus the controller 78 does not detect the fluorescent light in the first row of photosensor elements 20, 20,. Is determined (step S13: No), the process of the controller 78 proceeds to the confirmation step SC1.
FIG. 21 shows the migration position of the sample DNA fragment 152 in the above step S11 when the sample DNA fragment 153 has substantially the same number of bases as the probe DNA fragments 61 in the first to third columns. It is a drawing. FIG. 22 is a graph showing a fluorescence intensity distribution of an image acquired by the light detection device 2 when the sample DNA fragment 153 has substantially the same number of bases as the first to third column probe DNA fragments 61. . 21 and 22, it is assumed that the sample DNA fragment 151 has substantially the same number of bases as the probe DNA fragments 61 in the seventh to ninth columns, and the sample DNA fragment 152 is in the fourth to sixth columns. Has the same number of bases as the probe DNA fragment 61.
[0112]
In step S14, the voltage control circuit 73 changes the potential of the probe electrode 35 in the second column to the other probe electrodes 35, 35,... (Provided that the process has shifted from step S5 to step S6) in accordance with the instruction from the controller 78. When the process proceeds from step S10 to step S11 except for the probe electrode 35 in the eighth column, the probe electrode 35 in the fifth column is excluded.) And a voltage is applied so as to be higher than the first electrode 74. . Here, the probe electrodes 35, 35,... And the first electrode 74 other than the probe electrodes 35 in the second column are set to the ground potential, and the probe electrodes 35 in the second column are set to a voltage higher than the ground potential. . As a result, an electrode set including the first to third rows of probe electrodes 35, 35, 35 is selected, and regardless of the voltage between the first electrode 74 and the second electrode 75 thereafter, A relatively large volume of the sample DNA fragment 153 having substantially the same number of bases as the probe DNA fragments 61 in the first to third rows is placed near the probe electrode 35 in the second row, that is, in the first to third rows. It can be retained near the eye probe DNA fragment 61. In step S14 and step S15 described later, it is preferable that the second electrode 75 is also at the same potential as the first electrode 74 because the sample DNA fragment does not move in the direction from the first electrode 74 to the second electrode 75.
[0113]
Next, the driver circuit 76 drives the light detection device 2 in accordance with an instruction from the controller 78, and the light detection device 2 acquires an image representing the fluorescence intensity distribution during emission of the ultraviolet irradiator 77, and the image data is stored in the driver circuit 76. To the controller 78. Then, the controller 78 determines whether or not fluorescence has been detected by the photosensor elements 20, 20,... In the second column based on the image data (step S15).
[0114]
Here, when a voltage higher than the voltage applied to the first electrode 74 is applied to the probe electrodes 35 in the second column in the above step S14, the probe DNA fragments 61 in the first to third columns are applied. As shown in FIG. 23, the sample DNA fragment having substantially the same number of bases migrates so as to sink from above the probe electrodes 35 in the first row toward the probe electrodes 35 in the second row. When a sample DNA fragment having substantially the same number of bases as the probe DNA fragments 61 in the first to third columns is held on the probe electrode 35 in the second column, the sample DNA fragments are generated from the sample DNA fragments as shown in FIG. The high-intensity fluorescent light is incident on the semiconductors 23 of the photosensor elements 20, 20,... In the first row, so that the controller 78 causes the fifth row of photosensor elements 20, 20,. It is determined that it has been detected (step S15: Yes), and the process of the controller 78 shifts to the confirmation step SC1. On the other hand, if the sample DNA fragment having substantially the same number of bases as the probe DNA fragments 61 in the first to third columns has not migrated to the probe electrode 35 in the second column, the controller 78 proceeds to step S15. It is determined that fluorescence is not detected by the photosensor elements 20, 20,... In the second row (Step S15: No), and the process of the controller 78 proceeds to Step S14. By repeating the processing of the controller 78 from Step S15: No → Step S14, the positive voltage is continuously applied to the probe electrodes 35 in the second column until the sample DNA fragment migrates to the probe electrodes 35 in the second column.
FIG. 23 shows the case where the sample DNA fragment 153 having a relatively large volume has substantially the same number of bases as the probe DNA fragments 61 in the first to third rows, and It is a drawing showing the migration position of 153. FIG. 24 is a graph showing a fluorescence intensity distribution of an image obtained by the light detection device 2 when the sample DNA fragment 153 has substantially the same number of bases as the first to third column probe DNA fragments 61. .
In confirmation step SC1, controller 78 confirms whether fluorescence has been detected in the vicinity of probe electrode 35 in the seventh column in step S3, or whether fluorescence has been detected in the vicinity of probe electrode 35 in the eighth column in step S5. . Here, if it is confirmed that the fluorescence has been detected, the process proceeds to step S16, and if it is confirmed that the fluorescence has not been detected, the process proceeds to a confirmation step SC2.
[0115]
In step S16, the controller 78 activates the shaker 83, and the shaker 83 sets the bathtub at an optimum intensity for a sample DNA fragment having substantially the same number of bases as the probe DNA fragments 61 in the seventh to ninth columns. The electrophoretic medium 82 in 71 is agitated. At this time, the voltage control circuit 73 sets the voltages of all the probe electrodes 35, 35,..., The first electrode 74 and the second electrode 75 to the ground potential according to the instruction of the controller 78, and may set all the voltages to the same voltage. If the agitation disperses the sample DNA fragments more than necessary, the probe electrodes 35, 35,... In the seventh to ninth columns may be set to a higher voltage than the first electrode 74.
[0116]
Next, the driver circuit 76 drives the light detection device 2 in accordance with an instruction from the controller 78, and the light detection device 2 acquires an image representing the fluorescence intensity distribution during emission of the ultraviolet irradiator 77, and the image data is stored in the driver circuit 76. Is output to the controller 78 (step S17). Then, the controller 78 determines whether or not fluorescence has been detected by the photosensor elements 20, 20,... In the seventh to ninth columns based on the image data (step S18).
[0117]
Here, when the electrophoretic medium 82 is agitated in the step S16, the sample DNA fragments migrated to the probe electrodes 35 in the eighth row move to the probe electrodes 35 in the seventh row and the ninth row. spread. When the sample DNA fragment spreads to the probe electrodes 35 in the seventh column and the probe electrode 35 in the ninth column, as shown in FIG. 25, the fluorescence emitted from the sample DNA fragment becomes the photo in the seventh to ninth columns. .. Are incident on the semiconductor 23 of the sensor elements 20, 20,... At a high intensity, it is determined in step S18 that the controller 78 has detected fluorescence with the photosensor elements 20, 20,. (Step S18: Yes), and the process of the controller 78 shifts to the confirmation step SC2. On the other hand, if the sample DNA fragment has not spread to the probe electrodes 35 in the seventh row and the ninth row, the controller 78 proceeds to step S18 and the controller 78 sets the photosensor elements 20 and 20 in the ninth row. ,... (Step S18: No), the process of the controller 78 proceeds to Step S16. By repeating the processing of the controller 78 in step S18: No → step S16 → step S17, the shaker 83 continues the stirring operation until the sample DNA fragment spreads to the probe electrodes 35 in the seventh and ninth rows.
In the confirmation step SC2, the controller 78 confirms whether fluorescence has been detected in the vicinity of the fourth row of the probe electrodes 35 in step S8 or has been detected in the vicinity of the fifth row of the probe electrodes 35 in step S10. . Here, if it is confirmed that the fluorescence has been detected, the process proceeds to step S19, and if it is confirmed that the fluorescence has not been detected, the process proceeds to a confirmation step SC3.
[0118]
In step S19, the controller 78 activates the shaker 83, and the shaker 83 stirs the electrophoretic medium 82 in the bathtub 71 at an optimum intensity for the fourth to sixth rows of the probe DNA fragments 61. I do. At this time, the voltage control circuit 73 changes the potentials of the probe electrodes 35, 35, 35 in the seventh to ninth columns according to instructions from the controller 78, and sets the potentials of the other probe electrodes 35, 35,. The voltage is applied so as to be higher than the potential of the second electrode 75. In particular, the probe electrodes 35, 35,... Other than the probe electrodes 35, 35, 35 in the seventh to ninth columns, the first electrode 74 and The second electrode 75 is set to the ground potential.
[0119]
Next, the driver circuit 76 drives the light detection device 2 in accordance with an instruction from the controller 78, and the light detection device 2 acquires an image representing the fluorescence intensity distribution during emission of the ultraviolet irradiator 77, and the image data is stored in the driver circuit 76. Is output to the controller 78 (step S20). Then, the controller 78 determines whether or not fluorescence has been detected by the photosensor elements 20, 20,... In the fourth to sixth columns based on the image data (step S21).
[0120]
Here, when the electrophoretic medium 82 is agitated in the step S19, the sample DNA fragments migrated to the probe electrodes 35 in the fifth row move to the probe electrodes 35 in the fourth row and the probe electrodes 35 in the sixth row. spread. When the sample DNA fragment spreads to the probe electrodes 35 in the fourth row and the probe electrodes 35 in the sixth row, as shown in FIG. .. Are incident on the semiconductor 23 of the sensor elements 20, 20,... At a high intensity, it is determined in step S21 that the controller 78 has detected fluorescence with the photosensor elements 20, 20,. (Step S21: Yes), and the process of the controller 78 shifts to the confirmation step SC3. On the other hand, if the sample DNA fragment has not spread to the probe electrodes 35 in the fourth row and the probe electrodes 35 in the sixth row, the controller 78 sets the fourth and sixth photosensor elements 20 and 20 in step S21. ,... (Step S21: No), the process of the controller 78 proceeds to Step S19. By repeating the processing of the controller 78 in step S21: No → step S19 → step S20, the shaker 83 continues the stirring operation until the sample DNA fragment spreads to the probe electrodes 35 in the fourth and sixth rows. At the time of stirring by the shaker 83, a higher voltage than the first electrode 74 is applied to the probe electrodes 35, 35, 35 in the seventh to ninth columns, so that the seventh to ninth columns are used. The sample DNA fragment migrated and diffused to the probe electrodes 35, 35, 35 does not spread far away from the probe electrodes 35, 35, 35 in the seventh to ninth columns.
In the confirmation step SC3, the controller 78 confirms whether fluorescence has been detected in the vicinity of the first row of probe electrodes 35 in step S13 or whether fluorescence has been detected in the vicinity of the second row of probe electrodes 35 in step S15. . If it is confirmed that the fluorescence has been detected, the process proceeds to step S22. If it is confirmed that the fluorescence has not been detected, the process proceeds to a confirmation step SC4.
[0121]
In step S22, the controller 78 activates the shaker 83, and the shaker 83 generates a relatively large sample DNA fragment having substantially the same number of bases as the probe DNA fragments 61 in the first to third rows. The electrophoretic medium 82 in the bathtub 71 is agitated with the optimum strength. At this time, the voltage control circuit 73 changes the potentials of the fourth to ninth column probe electrodes 35, 35,... In the first to third column probe electrodes 35, 35 in accordance with instructions from the controller 78. , 35, the first electrode 74 and the second electrode 75 are applied with a voltage higher than the potential of the probe electrodes 35, 35, 35, the first electrode 74 and the The two electrodes 75 are set to the ground potential.
[0122]
Next, the driver circuit 76 drives the light detection device 2 in accordance with an instruction from the controller 78, and the light detection device 2 acquires an image representing the fluorescence intensity distribution during emission of the ultraviolet irradiator 77, and the image data is stored in the driver circuit 76. Is output to the controller 78 (step S23). Then, the controller 78 determines whether or not fluorescence has been detected by the photosensor elements 20, 20,... In the first to third columns based on the image data (step S24).
[0123]
Here, when the electrophoretic medium 82 is stirred in the step S22, the sample DNA fragments migrated to the probe electrodes 35 in the second row are moved to the probe electrodes 35 in the first row and the probe electrodes 35 in the third row. spread. When the sample DNA fragment spreads to the first row of probe electrodes 35 and the third row of probe electrodes 35, as shown in FIG. 27, the fluorescence emitted from the sample DNA fragments becomes the first row to the third row of photons. .. Are incident on the semiconductor 23 of the sensor elements 20, 20,... At a high intensity, it is determined that the controller 78 has detected the fluorescence in the photosensor elements 20, 20,. (Step S24: Yes), and the process of the controller 78 proceeds to Step S25. On the other hand, if the sample DNA fragment has not spread to the first row of probe electrodes 35 and the third row of probe electrodes 35, the controller 78 sets the first and third row of photosensor elements 20 and 20 in step S24. ,... (Step S24: No), the process of the controller 78 proceeds to Step S22. By repeating the processing of the controller 78 in step S24: No → step S22 → step S23, the shaker 83 continues the stirring operation until the sample DNA fragment spreads to the first and third row of probe electrodes 35. Since a higher voltage than the first electrode 74 is applied to the probe electrodes 35, 35,... In the fourth to ninth columns during the stirring by the shaker 83, the fourth to ninth columns are used. , The sample DNA fragments migrated and diffused to the probe electrodes 35, 35,... Do not spread far away from the probe electrodes 35, 35, 35 in the fourth to ninth rows.
In the confirmation step SC4, it is confirmed whether or not the controller 78 has confirmed that fluorescence has been detected in at least one of the confirmation steps SC1 to SC3. Here, if it is confirmed that the detection of the fluorescence has been confirmed, the process proceeds to step S25, and if it is confirmed that the detection of the fluorescence has not been confirmed, the operation is terminated.
[0124]
As described above, when the processing of the controller 78 shifts to step S25, the sample DNA fragments having substantially the same number of bases as the respective probe DNA fragments 61 have reached the spots 60, 60,. A plurality of types of sample DNA fragments are sorted according to the number of bases. That is, as shown in FIG. 28, for example, when the number of bases of the sample DNA fragment 151 is the same as the number of bases of the probe DNA fragment 61 of the spots 60, 60,. The sample DNA fragment 151 reaches the spots 60, 60,... In the seventh to ninth columns at a high concentration, and the number of bases in the sample DNA fragment 152 is the spot 60 in the fourth to sixth columns. , 60,..., The sample DNA fragment 152 reaches the spots 60, 60,. When the number of bases of the sample DNA fragment 153 is the same as the number of bases of the probe DNA fragment 61 in the spots 60, 60,. Spot in the third column 0, 60, has been reached at a high concentration up to ....
[0125]
In step S25, the stirring by the shaker 83 ends, and the voltage control circuit 73 sets all the probe electrodes 35, 35,..., The first electrode 74, and the second electrode 75 to the same voltage according to the instruction of the controller 78. . In particular, the voltage control circuit 73 preferably sets the probe electrodes 35, 35,..., The first electrode 74, and the second electrode 75 to the ground potential. Then, the temperature control circuit 80 drives the temperature adjusting elements 72, 72,... According to the instruction of the controller 78, and causes the temperature adjusting elements 72, 72,. As a result, the temperature control elements 72, 72,... Absorb heat, heat is conducted from the probe electrodes 35, 35,... To the temperature control elements 72, 72,. Cooled. If the temperature control elements 72, 72,... Can only perform heating, the temperature control circuit 80 stops the heat generation by the temperature control elements 72, 72,. Is done.
[0126]
When the electrophoretic medium 82 in the bathtub 71 is cooled, if the probe DNA fragment 61 of the spot 60 where the sample DNA fragment has reached is complementary to the sample DNA fragment, the sample DNA fragment is added to the probe DNA fragment 61. Hybridize. On the other hand, when the probe DNA fragment 61 in the spot 60 where the sample DNA fragment has reached is not complementary to the sample DNA fragment, the sample DNA fragment does not hybridize to the probe DNA fragment 61.
[0127]
After a lapse of a predetermined time, the voltage control circuit 73 operates according to an instruction from the controller 78 so that the potential of the first electrode 74 and the potential of the second electrode 75 become higher than the potentials of all the probe electrodes 35, 35,. A voltage is applied (step S26). In particular, the voltage control circuit 73 preferably sets the first electrode 74 and the second electrode 75 to the ground potential, and sets the probe electrodes 35, 35,. Here, the voltage of each of the probe electrodes 35, 35,... With respect to the first electrode 74 is equal to or higher than the voltage at which the sample DNA fragment mishybridized to the probe DNA fragment is separated from the probe DNA fragment and hybridized to the probe DNA fragment. The voltage is lower than the voltage at which the sample DNA fragment is not separated from the probe DNA fragment.
[0128]
When a negative voltage is applied to the probe electrodes 35, 35,..., The sample DNA fragment or probe DNA mishybridized with the probe DNA fragment 61 of the spots 60, 60,. Since the sample DNA fragment not hybridized with the fragment 61 is not complementary to the probe DNA fragment 61, it is separated from the probe DNA fragment 61 and migrates to the first electrode 74 and the second electrode 75. On the other hand, the sample DNA fragment hybridized with the probe DNA fragments of the spots 60, 60,... Among the plurality of types of sample DNA fragments is complementary to the probe DNA fragment 61 and is not separated from the probe DNA fragment 61. Maintain the state of being connected to
[0129]
Therefore, after step S26, each of the plural types of sample DNA fragments remains only in the spot 60 composed of the complementary probe DNA fragments 61 having the same number of bases. Each of the plurality of types of sample DNA fragments does not remain on the spot 60 composed of the probe DNA fragments 61 having different numbers of bases, nor does it remain on the spot 60 composed of the non-complementary probe DNA fragments 61. For example, as shown in FIG. 29, when the sample DNA fragment 151 has the same number of bases and is complementary to the probe DNA fragment 61 of any of the spots 60 in the eighth column, the sample DNA fragment 151 It remains at any spot 60 in the eye. Similarly, when the sample DNA fragment 152 has the same number of bases and is complementary to the probe DNA fragment 61 of any spot 60 in the fifth column, the sample DNA fragment 152 It remains at the spot 60. If the sample DNA fragment 153 has the same number of bases and is complementary to the probe DNA fragment 61 of any of the spots 60 in the second column, the sample DNA fragment 153 is placed in any of the spots 60 in the second column. Remains. When the sample DNA fragments 151, 152, and 153 are distributed as shown in FIG. 29, the fluorescence intensity distribution along the row direction in the image becomes the fluorescence intensity distribution as shown in FIG.
[0130]
After the elapse of a predetermined time, the voltage control circuit 73 applies the first electrode 74, the second electrode 75, and all the probe electrodes 35, 35,. . If the driver circuit 76 has confirmed the fluorescence in step S3 or step S5 according to the instruction from the controller 78, for example, the photosensor elements 20, 20 in the columns corresponding to the locations, that is, the seventh to ninth columns Are driven, and if fluorescence has been confirmed in all of the steps S3 or S5, S8 or S10, S13 or S15, the photosensor elements 20 in the first to ninth columns , 20,..., The light detection device 2 acquires an image representing the fluorescence intensity distribution during emission of the ultraviolet irradiator 77, and the image data is output from the driver circuit 76 to the controller 78 (step S27). . The temperature control circuit 80 drives the temperature control elements 72, 72,... Until the step S27 in step S25 to cause the temperature control elements 72, 72,. When the operation 2 is completed, the cooling operation by the temperature control elements 72, 72,. Further, a temperature detector for detecting the temperature of the electrophoretic medium 82 is provided in the bathtub 71, the temperature detected by the temperature detector is fed back to the controller 78, and the controller 78 controls the temperature control circuit 80 according to the detected temperature. The temperature control circuit 80 may control the temperature adjusting elements 72, 72,... So that the temperature of the electrophoretic medium 82 is maintained at 45 ° C. or more from step S25 to step S27. When it is determined that the process proceeds to step S25, in step S27, the photosensor elements 20, 20,... In the first to ninth columns are driven without confirming which column has detected the fluorescence. Is also good.
[0131]
Then, the controller 78 outputs the image data to the display device 81, and confirms in which probe DNA fragment 61 a complete hybrid has occurred from the light-detected image data of the photosensor elements 20, 20,. The base sequence of the hybridized sample DNA fragment is identified from the base sequence data of the probe DNA fragment 61 from the base sequence of the probe DNA fragment 61 (step S28), and the operation of the DNA identification device 100 ends. In step S28, the display device 81 may display the base sequence of the hybridized sample DNA fragment in accordance with the controller 78.
[0132]
As described above, in the present embodiment, the spots 60, 60,... Are the electrode sets of the first to third rows of probe electrodes 35, 35, 35, and the fourth to sixth rows of probes. The electrode set is separated into an electrode set of electrodes 35, 35, and 35, and an electrode set of probe electrodes 35, 35, and 35 in the seventh to ninth columns. The spots 60, 60,... Arranged in the common electrode set have the same number of bases but different base sequences. Furthermore, the number of bases in the spot 60 decreases as the electrode set moves toward the second electrode 75.
[0133]
In the above configuration, a sample DNA fragment is injected into the first electrode 74, and a voltage is applied between the second electrode 75 and the first electrode 74 as in steps S1, S6, and S11. Then, the sample DNA fragment migrates, but the sample DNA fragment having a smaller number of bases migrates closer to the second electrode 75 because of its smaller volume and lower fluid resistance. Accordingly, a plurality of types of sample DNA fragments are separated for each base number. Then, after a sample DNA fragment having approximately the same number of bases as the probe DNA fragments 61 in the seventh to ninth columns is separated in step S1 from the plurality of types of sample DNA fragments, the sample DNA fragment is separated in step S4. By electrophoresis in the depth direction toward the probe electrode 35 in the eighth row, the sample DNA fragment having substantially the same number of bases becomes high concentration around the probe electrode 35 in the eighth row. Step S9 is similarly performed after step S6, and step S14 is similarly performed after step S11.
[0134]
Thereafter, in steps S16, S19, and S22, the optimal agitation is performed for each base number, so that the sample DNA fragments having substantially the same base number are uniformly and highly concentrated in each spot 60. Therefore, in step S25, the sample DNA fragment can be hybridized with only the complementary probe DNA fragment 61 having substantially the same base number. Therefore, the base sequences of a plurality of sample DNA fragments having different numbers of bases can be identified almost simultaneously. Therefore, if the present DNA identification device 1 is used, it is not necessary to perform the process of extracting a sample DNA fragment having the same base number from the sample DNA of the specimen, and the structure of the sample DNA can be analyzed more quickly.
[0135]
At the time when the process proceeds to step S25, since the sample DNA fragments having substantially the same number of bases are distributed at a high concentration in each spot 60, the sample that binds to the complementary probe DNA fragment in the subsequent step S25. The number of DNA fragments increases. Therefore, in the subsequent step S27, the fluorescence intensity emitted from the spot 60 complementary to the sample DNA fragment is increased, and the fluorescence distribution can be more efficiently detected by the light detection device 2.
[0136]
Further, since a lower voltage is applied to the probe electrodes 35, 35,... Than the electrodes 74 and 75 in step S26, the sample DNA fragment that has reached the non-complementary spot 60 has a negative polarity. , From the spot 60 to the first electrode 74 and the second electrode 75. In the subsequent step S27, no fluorescence is emitted from the spot 60 that is not complementary to the sample DNA fragment, and fluorescence is emitted from the spot 60 that is complementary to the sample DNA fragment, and the intensity becomes clear. Therefore, the image acquired by the light detection device 2 has high contrast, and it is possible to easily determine which part in the image is bright.
[0137]
Further, in step S26, since the sample DNA fragment leaves the non-complementary spot 60, it is not necessary to wash the surface of the DNA sensor 1 after the hybridization, and the working efficiency is improved. In step S26, a voltage higher than the probe electrodes 35, 35,... Is applied to both the first electrode 74 and the second electrode 75, but the probe electrode 35 is applied to only one of the first electrode 74 and the second electrode 75. , 35,... May be applied.
[0138]
Also, the temperature control circuit 80 controls the temperature control elements 72, 72,... During steps S1 to S24, so that the temperature of the electrophoretic medium 82 becomes about 95 ° C., so that the sample DNA fragments are hybridized with each other. The denatured state can be maintained without the need for electrophoresis.
[0139]
Further, the temperature control circuit 80 controls the temperature control elements 72, 72,..., So that the temperature of the electrophoretic medium 82 becomes approximately 45 ° C. in step S25, so that the sample DNA fragment and the complementary probe DNA fragment 61 Easy to hybridize.
[0140]
Also, since a plurality of types of spots 60, 60,... Are arranged on the surface of the light detection device 2, a clear image can be captured by the light detection device 2 without a lens or a microscope. A two-dimensional image can be taken even without it. Therefore, it is not necessary to provide a lens, a microscope, and a scanning mechanism in the DNA identification device 100, and the size of the DNA identification device 100 can be reduced.
Since the spots 60 are arranged on the surface of the light detecting device 2, the fluorescent light emitted from the complementary spots 60 is incident on the photo sensor element 20 of the light detecting device 2 with almost no attenuation. Therefore, the sensitivity of the light detection device 2 does not need to be high.
[0141]
The present invention is not limited to the above embodiment, and various improvements and design changes may be made without departing from the spirit of the present invention.
For example, in the above embodiment, the probe electrodes 35, 35,... Are divided into three adjacent electrode sets, but may be divided into one electrode set or two electrode sets. They may be divided into sets, or may be divided into four or more electrode sets. Further, the number of probe electrodes 35 may be different for each electrode set. Further, in the above embodiment, one probe electrode 35 is arranged in one vertical column of the photosensor elements 20, 20,..., But two vertical columns of the photosensor elements 20, 20,. One probe electrode 35 may be arranged per row. Further, in the above embodiment, one photosensor element 20 corresponds to one spot 60, but one spot 60 may correspond to two or more adjacent photosensor elements 20. . However, in any case, the spots 60, 60,... Arranged in the same row have the same number of bases in the probe DNA fragment 61, and the base sequence of the probe DNA fragment 61 differs for each spot 60.
In step S4, only the probe electrodes 35, 35,... In the eighth column have a higher potential than the other probe electrodes 35, 35,. The probe electrodes 35, 35,... In the columns 9 to 9 may have a higher potential than the other probe electrodes 35, 35,. In step S9, only the probe electrodes 35, 35,... In the fifth column have a higher potential than the other probe electrodes 35, 35,. The probe electrodes 35, 35,... In the columns from the sixth column to the sixth column may have a higher potential than the other probe electrodes 35, 35,. In step S14, only the probe electrodes 35, 35,... In the second column have a higher potential than the other probe electrodes 35, 35,. The probe electrodes 35, 35,... In the third to third columns may have a higher potential than the other probe electrodes 35, 35,.
[0142]
In the above embodiment, a single-stranded sample DNA fragment having a smaller number of bases is electrophoretically arranged on an electrophoretic medium 82 above the probe electrodes 35, 35,... Provided with a probe DNA fragment 61 having a smaller number of bases. After that, a single-stranded sample DNA fragment having a larger number of bases is electrophoretically arranged on the electrophoresis medium 82 above the probe electrodes 35, 35,... Provided with the probe DNA fragment 61 having a larger number of bases. However, the single-stranded sample DNA fragment having a larger number of bases was electrophoretically arranged on the electrophoretic medium 82 above the probe electrodes 35 provided with the probe DNA fragment 61 having a larger number of bases. After that, the single-stranded DNA having a smaller number of bases is placed on the electrophoresis medium 82 above the probe electrodes 35, 35, ... provided with the probe DNA fragments 61 having a smaller number of bases. Pull DNA fragment to be disposed by electrophoresis, and a position where the plurality of kinds of sample DNA fragments are injected in the initial state, the probe electrode 35, ... and the position of, may be adjusted. In this case, the order is set so that steps S3 to S5 and steps S13 to S15 are interchanged, and steps S16 to S18 and steps S22 to S24 are interchanged. Also, regardless of the number of bases, the sample DNA fragment is almost simultaneously electrophoresed on the electrophoretic medium 82 above the probe electrodes 35, 35,... Provided with the probe DNA fragments 61 having substantially the same number of bases as the sample DNA fragment. It may be. At this time, the controller 78 may substantially synchronize steps S3, S8, and S13, may substantially synchronize steps S4, S9, and S14, and may substantially synchronize steps S5, S10, and S15. May be.
[0143]
Further, in the above embodiment, the photodetection device 2 using the photosensor elements 20, 20,... As the photoelectric conversion elements of the pixels has been described as an example, but the photodetection device using the photodiode as the photoelectric conversion element (imaging) Device). As a light detecting device using a photodiode, there are a CCD image sensor and a CMOS image sensor other than a double gate transistor if the temperature required for hybridization is within an operable temperature range.
In a CCD image sensor, photodiodes are arranged in a matrix of n rows and m columns on a substrate, and around each photodiode, an electric signal for photoelectrically converting the photodiode is transferred. A vertical CCD and a horizontal CCD are formed. In a CMOS image sensor, photodiodes are arranged in a matrix of n rows and m columns on a substrate. Pixels for amplifying electric signals photoelectrically converted by the photodiodes are arranged around each photodiode. A circuit is provided.
[0144]
In the above embodiment, the temperature control circuit 80 drives the temperature control elements 72, 72,... During the period from the start of the DNA analyzer 100 to the start of step S25, so that the electrophoretic medium 82 is at 95 ° C. Although the temperature is kept as described above, the temperature control elements 72, 72,... May be controlled as in the following (a) to (d).
[0145]
(A) In the case of Yes in step S3, the temperature control circuit 80 drives the eighth row of temperature control elements 72 according to the instruction of the controller 78 until the start of step S4 to step S16, The eye temperature adjusting element 72 is caused to perform a cooling operation. As a result, the temperature adjusting elements 72 in the eighth row absorb heat, the probe electrodes 35 in the eighth row are cooled, and the spots 60 in the eighth row are locally cooled to about 45 ° C. . Thus, the probe DNA fragments 61 of the spots 60, 60,... In the eighth row can be hybridized. Further, in the case of Yes in step S3, the temperature control circuit 80 drives the temperature control elements 72, 72, 72 in the seventh to ninth columns according to the instruction of the controller 78 from the step S19 to the end of step S27. Then, the cooling operation is performed by the temperature control elements 72, 72, 72 in the seventh to ninth columns.
[0146]
(B) In the case of Yes in step S8, the temperature control circuit 80 drives the fifth row of temperature control elements 72 according to the instruction of the controller 78 until the start of step S9 to step S19, and the fifth row The eye temperature adjusting element 72 is caused to perform a cooling operation. As a result, the fifth-row temperature control elements 72 absorb heat, the eighth-row probe electrodes 35 are cooled, and the fifth-row spots 60 are locally cooled to about 45 ° C. . Thus, the probe DNA fragments 61 in the spots 60, 60,... In the fifth row can be hybridized. Further, in the case of Yes in step S8, the temperature control circuit 80 drives the fourth to sixth rows of temperature control elements 72, 72, 72 according to the instruction of the controller 78 from the step S22 to the end of step S27. Then, the cooling operation is performed by the temperature control elements 72, 72, 72 in the fourth to sixth columns.
[0147]
(C) If Yes in step S13, the temperature control circuit 80 drives the second row of temperature control elements 72 according to the instruction of the controller 78 from the step S14 to the start of step S22. The eye temperature adjusting element 72 is caused to perform a cooling operation. As a result, the temperature adjusting elements 72 in the second row absorb heat, the probe electrodes 35 in the second row are cooled, and the spots 60 in the second row are locally cooled to about 45 ° C. . Thus, the probe DNA fragments 61 in the spots 60, 60,... Of the second row can be hybridized. Further, in the case of Yes in step S13, the temperature control circuit 80 drives the temperature adjusting elements 72, 72, 72 in the first to third columns from the step S25 to the end of step S27 according to the instruction of the controller 78. Then, the cooling operation is performed by the temperature control elements 72, 72, 72 in the first to third columns.
[0148]
(D) The temperature control element 72 that is not performing the cooling operation in (a) to (c) is driven by the temperature control circuit 80 to perform the heating operation.
[0149]
【The invention's effect】
According to the present invention, since the temperature adjusting element adjusts the temperature of the plurality of spots via the probe electrode, it is possible to efficiently cool the vicinity of the spot where hybridization is performed by locally cooling the spot.
[Brief description of the drawings]
FIG. 1 is a partially cutaway side view of a DNA identification device to which the present invention is applied.
FIG. 2 is a top view showing the DNA identification device.
FIG. 3 is a plan view showing some pixels of a light detection device provided in the DNA identification device.
FIG. 4A is a plan view showing one pixel of the photodetection device, and FIG. 4B is a cross-sectional view taken along a line II-II.
FIG. 5 is a block diagram showing a control configuration of the DNA identification device.
FIG. 6 is a circuit diagram showing a light detection device and a driver circuit of the DNA identification device.
FIG. 7 is a timing chart illustrating an example of a basic drive control method of a photosensor element.
FIG. 8 is a conceptual diagram of the operation of the photosensor element.
FIG. 9 is a diagram showing a light response characteristic of an output voltage of the photo sensor element.
FIG. 10 is a flowchart showing the operation of the DNA identification device.
FIG. 11 is a flowchart showing a continuation of FIG. 10;
FIG. 12 is a flowchart showing a continuation of FIG. 11;
FIG. 13 is a drawing showing a distribution state of sample DNA fragments in a bathtub during the operation of the DNA identification device, together with the DNA identification device.
FIG. 14 is a graph showing the fluorescence intensity distribution according to the distribution state of the sample DNA fragments in FIG.
FIG. 15 is a drawing showing a distribution state of a sample DNA fragment in a bathtub during the operation of the DNA identification device, together with the DNA identification device.
FIG. 16 is a graph showing the fluorescence intensity distribution according to the distribution state of the sample DNA fragments in FIG.
FIG. 17 is a drawing showing a distribution state of a sample DNA fragment in a bathtub during operation of the DNA identification device, together with the DNA identification device.
18 is a graph showing the fluorescence intensity distribution according to the distribution state of the sample DNA fragments in FIG.
FIG. 19 is a drawing showing a distribution state of sample DNA fragments in a bathtub during the operation of the DNA identification device, together with the DNA identification device.
20 is a graph showing the fluorescence intensity distribution according to the distribution state of the sample DNA fragments in FIG.
FIG. 21 is a drawing showing a distribution state of sample DNA fragments in a bathtub during operation of the DNA identification device, together with the DNA identification device.
FIG. 22 is a graph showing a fluorescence intensity distribution according to a distribution state of the sample DNA fragments in FIG. 21.
FIG. 23 is a drawing showing a distribution state of sample DNA fragments in a bathtub together with the DNA identification device during operation of the DNA identification device.
FIG. 24 is a graph showing a fluorescence intensity distribution according to a distribution state of the sample DNA fragments in FIG. 23.
FIG. 25 is a graph showing a fluorescence intensity distribution according to a distribution state of a sample DNA fragment after the first stirring.
FIG. 26 is a graph showing a fluorescence intensity distribution according to a distribution state of a sample DNA fragment after the second stirring.
FIG. 27 is a graph showing a fluorescence intensity distribution according to a distribution state of a sample DNA fragment after the third stirring.
FIG. 28 is a drawing showing a distribution state of a sample DNA fragment after the third stirring together with the DNA identification device.
FIG. 29 is a drawing showing a distribution state of a sample DNA fragment after drawing an unhybridized sample DNA fragment to a first electrode and a second electrode together with the DNA identification device.
30 is a graph showing the fluorescence intensity distribution according to the distribution state of the sample DNA fragments in FIG. 29.
[Explanation of symbols]
1 DNA sensor
2 Light detection device
20 Photo sensor element
34 UV shielding layer
35 probe electrode
60 spots
61 Probe DNA fragment
71 Bathtub
72 Temperature control element
74 First electrode
75 Second electrode
73 Voltage control circuit
76 Driver circuit
77 UV irradiator
80 Temperature control circuit
82 Electrophoresis medium
83 Shaker
100 DNA analyzer
151, 152, 153 Sample DNA fragment

Claims (10)

A bathtub containing the electrophoresis medium,
A plurality of probe electrodes arranged in the bath,
A plurality of spots composed of a probe DNA fragment having a known base sequence and arranged on the plurality of probe electrodes,
A temperature control element for controlling the temperature of the plurality of spots via the probe electrode;
A DNA analyzer, comprising: 2. The temperature adjusting element abuts on each of the plurality of probe electrodes, and selectively adjusts a temperature of a part of the plurality of spots via a part of the plurality of probe electrodes. 3. The DNA analyzer according to item 1. The DNA analyzer according to claim 1, further comprising a first electrode and a second electrode arranged in the bathtub and opposed to each other in a width direction of the bathtub. The plurality of probe electrodes are divided into a plurality of electrode sets including one probe electrode or several adjacent probe electrodes,
The spots arranged on a predetermined electrode set among the plurality of electrode sets have different base sequences of the probe DNA fragments, and the number of bases of one probe DNA fragment is the same as each other or the predetermined number of bases are different. More closely approximate to each other than the number of bases of one probe DNA fragment of the spot arranged on the probe electrode other than the electrode set,
The DNA analyzer according to claim 3, wherein one probe DNA fragment located on the second electrode side has a smaller number of bases than one probe DNA fragment located on the first electrode side. 4. A voltage control circuit which repeats a voltage application step of applying a voltage for a predetermined time so that the potential of the second electrode is higher than the potential of the first electrode by the number of the electrode sets, wherein a voltage control circuit is provided. 5. The DNA analyzer according to 4. When the voltage application step by the voltage control circuit is repeated, the temperature control element performs a heating operation, and after the repetition of the voltage application step by the voltage control circuit, the heat generation operation of the temperature control element is performed. The DNA analyzer according to claim 5, further comprising a temperature control circuit for terminating the DNA analysis. The voltage control circuit, after each of the voltage application step, selects one of the plurality of electrode sets in order from the second electrode closest to the second electrode, one or more probe electrodes in the selected electrode set The DNA analyzer according to claim 5, wherein a voltage is applied such that a potential of the first electrode and the second electrode is higher than a potential of at least one of the first electrode and the second electrode. The voltage control circuit is characterized in that a voltage is applied such that the potential of at least one of the first electrode and the second electrode is higher than the potentials of the plurality of probe electrodes after selecting all the electrode sets. The DNA analyzer according to claim 7, wherein The DNA analyzer according to any one of claims 1 to 8, further comprising a plurality of photosensor elements provided corresponding to the plurality of spots arranged on the plurality of probe electrodes. An electrophoresis step of applying a voltage to a plurality of probe electrodes provided in a bath containing an electrophoresis medium to attract sample DNA fragments in the electrophoresis medium to the probe DNA fragments provided in the probe electrodes;
A temperature adjusting step of adjusting the temperature of the probe DNA fragment and the surrounding electrophoretic medium through the probe electrode,
An analysis method comprising:

Images