JP2003161730A - Chip for detecting base sequence, hybridization monitoring system, and hybridization monitoring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To optimize hybridization conditions. <P>SOLUTION: The chip for detecting a base sequence comprises a DNA chip 1 having a substrate 11, and a plurality of quasi-complementary probes 122a that are arranged on the substrate 11, have a quasi-complementary base sequence having a low complementary property to a standard base sequence than a base sequence that is complemental to a target base sequence to be detected while the number of non-complementary bases without any complementarity with the standard base sequence in the quasi-complementary base sequence mutually differs, a signal-processing section 2 for processing a signal by detecting the occurrence rate of the hybridization reaction between the quasi-complementary probe 122a in the DNA chip 1 and a target DNA, and a display section 3 for displaying the occurrence rate obtained by the signal- processing section 2. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a nucleotide sequence detecting chip, a hybridization monitoring system, and a hybridization monitoring method used for genetic testing and the like.

[0002]

2. Description of the Related Art In recent years, genetic testing technology using a DNA chip as a DNA detection sensor has been attracting attention. In the inspection using this DNA chip, DNA probes are arranged in a plurality of matrix-shaped arrays provided on the DNA chip. Then, by detecting the presence or absence of a hybridization reaction between the sample DNA and the DNA probe,
Information such as the base sequence of the sample DNA can be acquired.

If the reaction conditions are not optimal when performing hybridization on a DNA chip, the probability that the sample DNA will erroneously hybridize, that is, bind to an irrelevant DNA probe will increase.

[0004]

However, since there is no quantitative method for determining the hybridization conditions, it is necessary to carry out many experiments under different hybridization conditions. Therefore, there is a problem that the development cost of the DNA chip becomes high.

There is also a problem that the development cost of the DNA chip is further increased when the optimum hybridization conditions differ depending on the types of DNA to be detected and probe DNA.

[0006] The present invention has been made to solve the above problems, and an object of the present invention is to detect a nucleotide sequence for optimizing hybridization conditions,
To provide a hybridization monitoring system and a hybridization monitoring method.

[0007]

According to one aspect of the present invention, a substrate and a target base sequence that is immobilized on the substrate and has a target complementary base sequence that is complementary to the target base sequence to be detected. Has a plurality of types of first quasi-complementary probes having a quasi-complementary base sequence having low complementarity with respect to each other. There is provided a base sequence detection chip comprising the first quasi-complementary probe group.

Further, according to another aspect of the present invention, the substrate and the target base sequence immobilized on the substrate are more complementary to the target base sequence than the target complementary base sequence complementary to the target base sequence to be detected. A plurality of types of first quasi-complementary probes having quasi-complementary base sequences having low complementarity are included, and the plurality of types of first quasi-complementary probes differ from each other in the number of non-complementary bases having no complementarity with the target base sequence. A chip for detecting a base sequence provided with a group of 1 quasi-complementary probes, a detecting means for detecting an incidence of a hybridization reaction between each first quasi-complementary probe and a sample base sequence in the chip for detecting a base sequence, and a detecting means. And a means for outputting the reaction occurrence rate detected by the hybridization monitoring system.

According to this structure, the occurrence rate of erroneous hybridization reaction can be quantitatively monitored in association with the hybridization reaction condition during the hybridization reaction. The condition can be easily obtained.

According to one embodiment of the present invention, it further comprises a storage means for storing the incidence of the hybridization reaction detected by the detection means in association with the hybridization reaction condition.

According to another embodiment of the present invention, the target base sequence is more complementary than the target complementary base sequence immobilized on the substrate and complementary to the target base sequence. A plurality of second quasi-complementary probes having a low quasi-complementary base sequence are provided, and the plurality of second quasi-complementary probes have a second quasi-complementary probe group having the same number of non-complementary bases and different base sequences from each other. . As a result, it is not necessary to select a quasi-complementary probe that is optimal for monitoring, and it is possible to reduce measurement variations such as a decrease in measurement accuracy due to erroneous selection of a quasi-complementary probe.

According to another embodiment of the present invention, the solvent is spread on the substrate by one dropping of the solvent of the spotting device, and the solvent is dropped by another dropping of the solvent. Has a plurality of regions that do not have an overlapping portion with other regions that spread on the substrate and that minimizes the distance between the solvent dropping positions, and a plurality of types of quasi-complementary probes are formed in each region. As a result, the quasi-complementary probes can be mounted at a density higher than the resolution of the spotting device, and the base sequence detection chip can be miniaturized. Also,
By performing statistical processing and the like using output results obtained from a plurality of probes, it can be used for error testing.

According to another embodiment of the present invention, the number of non-complementary bases is the same among the first and second quasi-complementary probe groups based on the occurrence rate detected by the detecting means. A first statistical process for calculating an average value of incidences of hybridization reactions in a plurality of quasi-complementary probes having mutually different base sequences, and excluding reaction incidences that deviate from the average value by a predetermined threshold value φ or more, Second statistical processing for calculating the representative value by adding the occurrence rates of hybridization reactions in a plurality of quasi-complementary probes having the same number of complementary bases and different base sequences from each other. Result obtained by the first statistical processing At least one of the third statistical processing for calculating the average value of the reaction occurrence rates, and the fourth statistical processing for calculating the variance value of the reaction occurrence rates obtained as a result of being excluded by the first statistical processing Further comprising a statistical processing means for executing. Thereby, the accuracy of monitoring the hybridization state can be improved.

Further, according to another embodiment of the present invention, the detecting means is further provided with an immobilizing electrode disposed on the substrate for immobilizing the first or second quasi-complementary probe, and a counter electrode. Calculates the occurrence rate of the hybridization reaction based on the amount of charge flowing from the immobilized electrode. According to another embodiment of the present invention, an immobilization electrode disposed on the substrate for immobilizing the first or second quasi-complementary probe,
The detection unit further includes a counter electrode, and the detection unit calculates the rate of occurrence of the hybridization reaction based on the conductance obtained based on the voltage or current between the fixed electrode and the counter electrode. According to another embodiment of the present invention,
An imaging unit for imaging the first or second quasi-complementary probe of the nucleotide sequence detection chip after the hybridization reaction is further provided, and the detection unit is based on the brightness of the fluorescence imaged by the imaging unit, and the occurrence rate of the hybridization reaction. To calculate.

Further, the present invention relating to the apparatus is also realized as an invention of a method realized by the apparatus.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings.

(First Embodiment) FIG. 1 is a diagram showing the overall configuration of a hybridization state monitoring system according to a first embodiment of the present invention. As shown in Figure 1,
DNA chip 1 as a base sequence detection chip, and DN
A signal processing unit 2 that performs signal processing based on the output of the A chip 1
And a display unit 3 for displaying a signal processed by the signal processing unit 2 and an electrode control for controlling a reaction such as a hybridization reaction in each electrode by controlling voltage application to each electrode of the DNA chip 1. It is composed of a unit 4. Further, the signal processing unit 2 has a storage unit 2a, and the storage unit 2a stores data such as the hybridization rate and the hybridization reaction condition obtained by signal processing in the signal processing unit 2.

FIG. 2 is a diagram showing a detailed structure of the DNA chip 1. As shown in FIG. 2, the substrate 11 is provided with a plurality of probe electrodes 121 and 122. 121 is a complementary probe electrode and 122 is a quasi-complementary probe electrode. Further, a counter electrode (not shown) is provided on the substrate 11 or other than the substrate 11.

The complementary probe electrode 121 has a base sequence complementary to the target base sequence 120 (target complementary base sequence).
The complementary probe 121a is fixed. There are a plurality of quasi-complementary probe electrodes 122, each of which has a complementary probe 12.
The quasi-complementary probe 122a having lower complementarity with the target base sequence 120 than 1a is fixed. The complementary probe electrode 121, the quasi-complementary probe electrode 122, and the counter electrode are connected to the signal processing unit 2, respectively. Probe electrode 12
The charges flowing out to the probe electrodes 121 and 122 when a voltage is applied between the counter electrodes 1 and 122 and the counter electrode are generated by the signal processing unit 2.
And the signal processing unit 2 detects the amount of the electric charge, so that the hybridization rate (the rate at which the reaction between the probe and the sample base sequence is bound) can be measured.

In applying a voltage to each probe electrode, a three-electrode system may be used in which a reference electrode is used in addition to the counter electrode.

The target base sequence is a base sequence targeted for detection. The sample base sequence is a sample base sequence to be tested, and the hybridization reaction is monitored by hybridizing this sample base sequence with a complementary probe or a quasi-complementary probe.

The complementary probe is a probe used for the purpose of detecting whether or not the sample base sequence contains the target base sequence, and has a base sequence complementary to the target base sequence (target complementary base sequence). Have. In addition, in order to compare the hybridization reaction of the complementary probe with the hybridization reaction of the quasi-complementary probe, it should be used not only for detecting whether the sample base sequence has the target base sequence but also for monitoring the hybridization state. You can also

The quasi-complementary probe is a probe used for optimizing the hybridization reaction conditions by monitoring the hybridization state, and has a base sequence complementary to the target base sequence (target complementary base sequence). ), At least one (any one or more of the bases from the starting point base to the ending point base) is replaced with another base.

In the following embodiments, the hybridization reaction conditions include not only the conditions for causing a hybridization reaction between a certain base sequence and another base sequence but also the double strands in which this hybridization reaction has occurred. It also includes the processing conditions in the steps required before.

Quasi-complementary probe 122a and target base sequence 1
A conceptual diagram of 20 is shown in FIG. As shown in FIG. 3, the target base sequence 120 is CAG from the starting base to the ending base.
In the case of -A-T-C-A, the base sequence having complementarity with the target base sequence 120 is GT-CT-A from the starting point to the ending point due to the complementarity of the bases. -G
-T. Therefore, the complementary probe 121a becomes a probe having this base sequence "GTCTTTAGT".

There are plural kinds of quasi-complementary probes 122a, and these plural kinds of quasi-complementary probes are referred to as a first quasi-complementary probe group. Each first constituting the first quasi-complementary probe group
As the quasi-complementary probe, a first quasi-complementary probe having a base sequence complementary to the target base sequence 120 (target complementary base sequence) and a base sequence of GT-CTTTGT that differs by one base G different in two bases from probe (1) 1221
The first quasi-complementary probe (2) 1222 having a base sequence of -T-C-T-T-C-T and G- differing in three bases.
As in the case of the first quasi-complementary probe (3) 1223 having the base sequence of T-C-T-T-C-A, the target complementary base sequence and the number of bases (the number of non-complementary bases) are i (i is a natural number). And is equal to or less than the number of bases in the target base sequence)
First quasi-complementary probe (i) 12 having different base sequence
Have 2i.

In the first quasi-complementary probe (2) 1222, a base adjacent to a base different from the target complementary base sequence in the first quasi-complementary probe (1) 1221 becomes a base different from the target complementary base sequence. It has such a base sequence. Therefore, the first quasi-complementary probe (2) 1222 has a base sequence obtained by replacing two bases of the target complementary base sequence in the complementary probe 121a with another base, and the two bases are adjacent to each other.

The first quasi-complementary probe (3) 1223
Has a base sequence such that one base adjacent to two bases different from the target complementary base sequence in the first quasi-complementary probe (2) 1222 becomes a base different from the target complementary base sequence. Therefore, the first quasi-complementary probe (3) 1223 has a base sequence in which three bases of the target complementary base sequence in the complementary probe 121a are replaced with other bases, and the three bases are adjacent to each other.

Similarly, the first quasi-complementary probe (i) 122
i is the first quasi-complementary probe (i-1) 122 (i-1)
Has a base sequence such that one base adjacent to (i-1) bases different from the target complementary sequence becomes a base different from the target complementary base sequence. Each probe of the first quasi-complementary probe group is immobilized on a different electrode.

The first quasi-complementary probe (1) 1 shown in FIG.
221-1st quasi-complementary probe (3) 1223 is an example. First quasi-complementary probe (i) 122i
In the case of, the number of bases different from the target complementary base sequence is i, which i base is to be selected in the base sequence, and the i bases are selected as the target complementary base sequence A , G, C, T, the three bases other than the specific base are freely defined. Each electrode 12 of the DNA chip 1 for all of these possible types and multiple types
2 may be fixed, or only one representative type may be fixed to each electrode 122 of the DNA chip 1. Further, the number i of different bases may be any number as long as it is 2 or more.

In the present embodiment, for convenience of description, a representative base sequence for each of the first quasi-complementary probe (1) 1221, the first quasi-complementary probe (2) 1222, and the first quasi-complementary probe (3) 1223. Only one type will be described.

Next, the first method for monitoring the hybridization state using the hybridization state monitoring system shown in FIGS. 1 to 3 will be described.

First, a test solution containing a sample base sequence is treated with DNA.
The electrodes 121 and 122 in the chip 1 are injected and brought into contact with each other, and a hybridization reaction is caused between the sample base sequence contained in the injected test solution and the complementary probe 121a and the quasi-complementary probe 122a. Here, for convenience of explanation, it is assumed that the sample base sequence has the same base sequence as the target base sequence. The solvent of the reaction solution has a predetermined pH.
Buffer solution and the like are used. In addition, the reaction is carried out with the test liquid at a predetermined temperature, for example, for 1 minute or longer. In the hybridization reaction, by applying a predetermined voltage to the fixed electrodes and the counter electrodes of the probe electrodes 121 and 122 by the electrode control unit 4, the reaction that was conventionally required for several hours to several days can be performed for several minutes. It can be shortened to the extent.

When the hybridization reaction is completed, the probe electrodes 121 and 122 are washed with a predetermined buffer solution, and the double-stranded portion (complementary probe 1) formed on the electrode surface is washed.
21a, the quasi-complementary probe 122a and an intercalating agent that selectively binds to the double-stranded portion of the sample base sequence).
Then, the probe electrodes 121 and 122 acted by the intercalating agent.
A predetermined voltage is applied to the fixed electrode and the counter electrode of, and the signal processing unit 2 measures the charge amount of each of the electrodes 121 and 122 derived from the intercalating agent. The obtained measurement result is displayed on the display unit 3 as the hybridization rate.

FIG. 4 is a diagram showing an example of measurement results displayed on the display unit 3. RTO ₀ is the complementary probe electrode 12
Hybridization rates obtained based on the amount of charges detected for ₁ and RTO ₁ , RTO ₂ , and RTO ₃ are hybridization rates obtained based on the amount of charges detected for the quasi-complementary probe electrode 122. RTO
₁ is the hybridization rate of the first quasi-complementary probe (1) 1221, RTO ₂ is the first quasi-complementary probe (2) 1
222 is the hybridization rate, RTO ₃ is the hybridization rate of the first quasi-complementary probe (3) 1223.

FIG. 4 (a) and FIG. 4 (b) show measurement results under different hybridization reaction conditions. The different hybridization reaction conditions include, for example, the reaction temperature, the base sequence of the quasi-complementary probe to be reacted, the ionic strength and pH of the test solution, the type and conditions of the hybridization promoter, the conditions such as stirring and shaking, and the reaction time. , Type of intercalating agent, concentration, ionic strength of intercalating agent buffer, pH
And its various conditions.

As shown in FIG. 4 (a), the hybridization rate decreases with the number of bases that do not match the complementary base sequence of the target base sequence. Therefore, it can be seen that the hybridization reaction is normally performed. On the other hand, also in FIG. 4B, the number of bases that do not match the complementary base sequence of the target base sequence (the number of non-complementary bases) as in FIG. 4A.
Accordingly, the hybridization rate is decreasing. Therefore, it can be seen that the hybridization reaction is normally performed.

Comparing FIG. 4 (a) and FIG. 4 (b), the decrease rate of the hybridization rate with the increase in the number of mismatched bases (the number of non-complementary bases) in FIG. 4 (a) is shown in FIG.
It is smaller than the measurement result of (b). Therefore, the hybridization reaction of FIG.
It can be seen that it is more optimized than the hybridization reaction of FIG.

5 (a) and 5 (b) show examples of measurement results obtained under hybridization reaction conditions different from those shown in FIGS. 4 (a) and 4 (b). In both cases of FIG. 5A and FIG. 5B, RTO ₀ >> R
The relationship of TO ₁ > RTO ₂ > RTO ₃ is not established, and there is no causal relationship between the number of bases that do not match the complementary base sequence of the target base sequence (the number of non-complementary bases) and the hybridization rate. The value of RTO ₀ is the hybridization rate when it indicates that a normal hybridization reaction is occurring. Therefore, RTO ₁ , R
When the values of TO ₂ and RTO ₃ are large, it can be seen that at least an abnormal hybridization reaction has occurred. The cause of the abnormal hybridization reaction is considered to be the problem that the hybridization conditions such as contamination of impurities and the temperature of the hybridization reaction are too low.

6 (a) and 6 (b) show examples of measurement results obtained under hybridization reaction conditions different from those in FIGS. 4 and 5. In the case of FIG. 6A, the relationship of RTO ₀ ≈RTO ₁ >>> RTO ₂ > RTO ₃ is established. In the case of FIG. 6B, RTO ₀ ≈RTO ₁ ≈R
The relationship of TO ₂ ≈ RTO ₃ is established. In either case, there is no causal relationship between the hybridization rate and the number of mismatched bases (the number of non-complementary bases) as in the case of FIG. 4A or FIG. It can be seen that an abnormal hybridization reaction has occurred. As in the case of FIG. 5 (a) and FIG. 5 (b), the cause of the abnormal hybridization reaction may be the problem that the hybridization conditions such as contamination of impurities and the temperature of the hybridization reaction are too low. To be

Next, the second technique for monitoring the hybridization state will be described. Detailed description of the devices used and parts common to the first method is omitted.

The difference from the first method for monitoring the hybridization state is that this second method is used for measuring the hybridization rate using a plurality of first quasi-complementary probes having different mismatched base numbers (non-complementary base numbers). ,
This is a point of analysis in which the hybridization rate according to the number of non-complementary bases is associated with temperature conditions.

First, a test solution containing a sample base sequence is treated with DNA.
It is injected into each electrode 121, 122 in the chip 1. next,
Target nucleic acid chain and complementary probe 1 contained in injected test solution
21a and the quasi-complementary probe 122a cause a hybridization reaction. Here, for convenience of explanation, it is assumed that the sample base sequence has the same base sequence as the target base sequence. As the reaction solution, a buffer solution having a predetermined pH is used. In addition, the reaction is performed with a test solution for, for example, 1 minute or more. Also, during this hybridization reaction,
A predetermined voltage is applied to the probe electrodes 121 and 122 by the electrode control unit 4. Further, the other measurement conditions are made the same, and a plurality of reactions with different temperature conditions are caused.

Upon completion of the hybridization reaction, the probe electrodes 121 and 122 are washed with a predetermined buffer solution to form a double-stranded portion (complementary probe 1) formed on the electrode surface.
21a, 122a and the sample base sequence) are caused to act on the intercalating agent. Then, a predetermined voltage is applied to the probe electrodes 121 and 122 on which the intercalating agent acts, and the charge amount of each of the electrodes 121 and 122 derived from the intercalating agent is determined by the signal processing unit 2.
To measure. The obtained measurement result is displayed on the display unit 3 as the hybridization rate. In the second method, the other measurement conditions are made the same and a plurality of reactions with different temperature conditions are caused to occur, so that a plurality of measurement results of reaction under different temperature conditions can be obtained.

FIG. 7 is a diagram showing an example of measurement results displayed on the display unit 3. RTO ₀ is the complementary probe electrode 12
The hybridization ratios RTO ₁ , RTO ₂ , and RTO ₃ obtained based on the amount of charges detected for 1 are the hybridization ratios obtained based on the amount of charges detected for the quasi-complementary probe electrode 122, and RTO.
₁ is the hybridization rate of the first quasi-complementary probe (1) 1221, RTO ₂ is the first quasi-complementary probe (2) 1
222 is the hybridization rate, RTO ₃ is the hybridization rate of the first quasi-complementary probe (3) 1223. FIG. 7 (a) and FIG. 7 (b) are measurement results obtained by causing a hybridization reaction under different temperature conditions. Specifically, FIG. 7B corresponds to the case where the hybridization reaction is caused to occur at a temperature lower than that in FIG. 7A.

As shown in FIG. 7 (a), the hybridization rate decreases according to the number of bases that do not match the complementary base sequence of the target base sequence (the number of non-complementary bases). Therefore, it can be seen that the hybridization reaction is normally performed. On the other hand, in FIG. 7B, the hybridization rate decreases according to the number of bases that do not match the complementary base sequence of the target base sequence (the number of non-complementary bases), as in FIG. 7A. Therefore, it can be seen that the hybridization reaction is normally performed. 7 (a) and 7
Comparing (b), the decrease rate of the hybridization rate with the increase in the number of mismatched bases (the number of non-complementary bases) in the case of FIG. 7 (b) is smaller than that in the measurement result of FIG. 7 (a). ing. Therefore, it can be seen that the hybridization reaction of FIG. 7 (b) is more optimized than the hybridization reaction of FIG. 7 (a). These Figure 7
From the measurement results of (a) and (b), it is understood that the hybridization reaction can be optimized by lowering the hybridization reaction temperature.

8 (a) and 8 (b) show examples of measurement results obtained under hybridization reaction conditions different from those in FIGS. 7 (a) and 7 (b). Figure 8
In both cases (a) and (b), RTO ₀ >> RT
The relationship of O ₁ > RTO ₂ > RTO ₃ is not established, and there is no causal relationship between the number of bases mismatching with the complementary base sequence of the target base sequence and the hybridization rate. RTO ₀
The value of is the hybridization rate when it indicates that a normal hybridization reaction is occurring. Therefore, when the values of RTO ₁ , RTO ₂ , and RTO ₃ are larger than this, it can be seen that at least an abnormal hybridization reaction occurs. The cause of the abnormal hybridization reaction may be a problem such as the temperature of the hybridization reaction being too low. Therefore, it is possible to take measures such as increasing the temperature of the hybridization reaction.

9 (a) and 9 (b) show examples of measurement results obtained under hybridization reaction conditions different from those in FIGS. 7 and 8. In the case of FIG. 9A, the relationship of RTO ₀ ≈RTO ₁ >> RTO ₂ > RTO ₃ is established. In the case of FIG. 9B, RTO ₀ ≈RTO ₁ ≈R
The relationship of TO ₂ ≈ RTO ₃ is established. In either case, as in the case of FIG. 7 (a) or FIG. 7 (b), there is no causal relationship between the hybridization rate and the number of non-matching bases (the number of non-complementary bases), or it is not so often observed. It can be seen that an abnormal hybridization reaction has occurred. As in the case of FIGS. 8A and 8B, the cause of the abnormal hybridization reaction may be a problem such as the hybridization reaction temperature being too low. Therefore, it may be necessary to increase the hybridization reaction temperature. To be

Next, a third method for monitoring the hybridization state will be described.

In the third method, first, a test solution containing a sample base sequence is injected into each of the electrodes 121 and 122 in the DNA chip 1 to bring the sample base sequence into contact with the probe. Next, a hybridization reaction occurs between the sample base sequence contained in the injected test solution and the complementary probe 121a and the quasi-complementary probe 122a. Here, for convenience of explanation, it is assumed that the sample base sequence has the same base sequence as the target base sequence. As the reaction solution, a buffer solution having a predetermined pH is used. In addition, the reaction is carried out with the test liquid at a predetermined temperature, for example, for 1 minute or longer. During the hybridization reaction, the electrode control unit 4 causes the probe electrode 12
By applying a predetermined voltage to 1,122, it is possible to shorten the reaction that was conventionally required from several hours to several days to about several minutes.

Upon completion of the hybridization reaction, the probe electrodes 121 and 122 are washed with a predetermined buffer solution, and an intercalating agent that selectively binds to the double-stranded portion formed on the electrode surface is made to act. Then, a predetermined voltage is applied to the probe electrodes 121 and 122 acted by the intercalating agent. The signal processing unit 2 measures the charge amount of each of the electrodes 121 and 122 derived from the intercalating agent at each temperature at which the charge changes. The obtained measurement result is displayed on the display unit 3 as the hybridization rate.

FIG. 10 is a diagram showing the concept of the hybridization state monitoring of the obtained third method and the measurement results. FIG. 10 (a) is a conceptual diagram of the hybridization state monitoring of the third method, FIG. 10 (b)-
(E) is a measurement result.

FIG. 10 (a) is a conceptual diagram of the reaction when the temperature becomes lower as going to the uppermost stage, the uppermost stage 2, the uppermost stage 3, the uppermost stage, and the lowermost stage. Further, the left end is a schematic diagram of the hybridization reaction between the complementary probe 121a and the sample base sequence, the second from the left end is a schematic diagram of the hybridization reaction between the first quasi-complementary probe (1) 1221 and the sample base sequence, and the third from the left end is Schematic diagram of the hybridization reaction between the first quasi-complementary probe (2) 1222 and the sample base sequence, the right end being the first quasi-complementary probe (3) 1
FIG. 3 is a schematic diagram of a hybridization reaction between 223 and a sample base sequence. From FIG. 10A, it can be seen that the hybridization reaction with the complementary probe 121a or the quasi-complementary probe 122a is more likely to occur due to the action of the intercalating agent when the temperature is lower than when the temperature is high. . Further, a probe having a large number of mismatched bases (the number of non-complementary bases) with respect to the base sequence complementary to the target base sequence is less likely to cause a hybridization reaction, and a probe having a smaller number of the mismatched bases (the number of non-complementary bases) is It can be seen that the hybridization reaction is likely to occur.

The measurement results obtained under the respective measurement conditions shown in FIG. 10 (a) are shown in FIGS. 10 (b)-(d).

As shown in FIGS. 10 (b) to 10 (d), in the measurement result of the highest temperature (FIG. 10 (b)) with the highest temperature, the hybridization rate was low overall, and the hybridization between the probes was carried out. It is difficult to compare the ratios, and there is no causal relationship between the number of mismatched bases (the number of non-complementary bases) in the target base sequence and the complementary base sequence and the hybridization rate.

In the second highest measurement result from FIG. 10 (a) (FIG. 10 (c)) at the next highest temperature, the complementary probe 121a has the highest hybridization rate and is complementary to the target base sequence. The hybridization rate decreases significantly as the number of mismatched bases (number of non-complementary bases) with different base sequences increases, and the decrease rate of hybridization rate increases with the increase of the number of mismatched bases (number of non-complementary bases). is decreasing. From this, a causal relationship between the number of mismatched bases (the number of non-complementary bases) and the hybridization rate is extremely clear.

When the temperature is next high, in the measurement result of the third stage from the top in FIG. 10 (a) (FIG. 10 (d)), the complementary probe 121a has the highest hybridization rate and is complementary to the target base sequence. As the number of non-complementary bases (number of non-complementary bases) with such a base sequence increases, the hybridization rate greatly decreases.
It is the same as the measurement result of the second stage from the top of (a). The difference from the measurement result of the second highest temperature is that the decrease rate of the hybridization rate does not change much with the increase of the number of mismatched bases (the number of non-complementary bases). It can be said that a causal relationship between the hybridization rate and the number of mismatched bases (the number of non-complementary bases) is clearly shown in the second stage as compared with the measurement result in the second stage.

In the measurement result at the bottom of FIG. 10 (a) at the lowest temperature (FIG. 10 (e)), there is no causal relationship between the number of mismatched bases (the number of non-complementary bases) and the hybridization rate. In some cases, the hybridization rate of the quasi-complementary probe 122a is higher than that of the complementary probe 121a.

Of the obtained measurement results, RTO ₀ >> R
The temperature at which the relationship of TO ₁ > RTO ₂ > RTO ₃ holds is the case of the temperature at the second stage from the top. Therefore, the temperature condition at which the measurement result of the second stage from the top is obtained is detected as the optimum hybridization temperature.

FIG. 11 shows a detailed structure of the DNA chip 1 for realizing the hybridization state monitoring of this embodiment. As shown in FIG. 11, a plurality of spot blocks 91 are arranged in a matrix on the substrate 11. A spot block is a region that includes at least a probe electrode that fixes one type of probe, and further that includes an electrode set that includes at least one of a reference electrode and a counter electrode, and the regions of each block do not have overlapping regions. The region corresponds to the unit processed by the spotting device for dropping the mixture containing the sample base sequence and the intercalating agent to each electrode as one point. Therefore, in the spot block, the distance between the blocks is determined according to the minimum resolution of the spotting device, and a spot block has a spotting device.
The mixture containing the nucleotide sequence of the inspection body and the intercalating agent was dropped, and another spot block was covered with another spotting device.
A mixture containing the base sequence of the inspection body and the intercalating agent is dropped. The minimum resolution of the spotting device is a region where the solvent spreads on the substrate by one drop of the solvent of the spotting device, and has a portion which overlaps with another region where the solvent spreads on the substrate by another dropping of the solvent. In other words, it refers to a region in which the distance between the respective solvent dropping positions is minimized, and is, for example, a region of 100 μm square or less or a region of 100 μm diameter or less.

By arranging the spot blocks corresponding to this minimum resolution, the mixture is dropped onto a certain spot block, and the mixture is not dropped onto another spot block. That is, an independent dropping operation can be performed for each spot block so that the admixtures do not mix with each other.

In each spot block 91, either the fixed electrode only for fixing the same type of probe, the combination of the fixed electrode and the reference electrode, or the combination of the fixed electrode, the reference electrode and the counter electrode is used. The set is placed. The immobilized electrode is a complementary probe 121a.
And an electrode for immobilizing the quasi-complementary probe 122a and hybridizing with the sample base sequence. The counter electrode is an electrode arranged to cause a redox reaction or the like by applying a predetermined voltage between the fixed electrode and the fixed electrode by using the electrode control unit 4. The reference electrode is an electrode serving as a reference for the fixed electrode and the counter electrode. For example, by using the reference electrode as a reference, the potential of the fixed electrode with respect to the reference electrode and the potential of the counter electrode are measured.

In FIG. 11A, a plurality of fixed electrodes 91 are provided.
The electrode set 921 composed of 1 is arranged in a matrix of p × q in some blocks of the plurality of spot blocks 91. The plurality of fixed electrodes 911 are
Since it corresponds to the minimum resolution of the spotting function of the spotting device as described above, the mixed solution is dropped onto all the fixed electrodes 911 in this one spot block 91 by one spotting.

In the case of the example of FIG. 11A, the electrode set 92
When the mixed material is dropped on the spot block 91 in which 1 is arranged by the spotting device, the mixed material is collectively supplied to the p × q fixed electrodes 911.

The blocks other than the spot block 91 in which the electrode set 921 consisting of only the fixed electrode 911 is arranged are the blocks in which the electrode set 922 including the combination of the fixed electrode 911 and the reference electrode 912 is arranged, This is a block in which an electrode set 923 including a combination of a fixed electrode 911, a reference electrode 912, and a counter electrode 913 is arranged. The electrode set 922 is arranged in a block arranged in the outermost region of the plurality of spot blocks 91 on the DNA chip 1 (for example, the rightmost column of the matrix-shaped spot blocks in FIG. 11A). .

The electrode set 923 is arranged in the innermost block of the plurality of spot blocks 91 on the DNA chip 1. Electrode set 92
1 is arranged in a block other than the electrode set 922 and the electrode set 923.

Note that the spot block 91 in which the electrode set 922 of FIG. 11A is arranged is replaced with an electrode set including only the reference electrode 912, and the spot block 91 in which the electrode set 923 is arranged is formed from only the counter electrode 913. The electrode set may be replaced.

FIG. 11B shows another arrangement example of the electrode set. The fixed electrode 911, the reference electrode 912, and the counter electrode 9 are provided in all the spot blocks 91.
The same electrode set 923 consisting of 13 is arranged respectively. In each spot block 91, a counter electrode 913 is arranged in the innermost area in the block.
In addition, the reference electrode 9 surrounds the counter electrode 913.
12 are arranged. Then, the fixed electrode 911 is arranged in the outermost region.

As shown in FIGS. 11A and 11B, spot blocks are arranged in correspondence with the minimum resolution of the spotting device, and an electrode set consisting of a plurality of electrodes is arranged in the spot block. Thus, plural kinds of base sequence probes can be arranged in each spotting region. Therefore, the base sequence probes are mounted at a density higher than the resolution of the spotter to downsize the DNA chip. In the above embodiment, the sample nucleic acid chain and the intercalating agent are supplied on the DNA chip 1 in the form of a mixed agent, but they may be supplied by different solvents.

The arrangement of the electrode sets shown in FIGS. 11A and 11B is merely an example. For example, the counter electrodes 913 may be arranged in the peripheral region on the substrate 11, and then the spot blocks 91 composed of the electrode set 921 may be arranged in a matrix in the region surrounded by the counter electrodes 913. Further, the spot blocks 91a for arranging the electrode sets 913 and the spot blocks 91b for arranging the electrode sets 911 may be alternately arranged for each column. In addition, the arrangement of the electrode set can be variously changed.

In the spot block 91 shown above, a voltage is applied between the fixed electrode 911 and the counter electrode 913, and a reaction such as electrolysis caused by the application of the voltage causes a hybridization reaction. Different magnitudes of current flow between these electrodes between the probe and the non-reactive probe. The signal processing unit 2 can confirm the hybridization rate by converting the current into an electric signal and displaying the electric signal on the display unit 3. In order to obtain the signal necessary for calculating the hybridization rate, changes in redox current, changes in redox potential, changes in capacitance, changes in conductance, and changes in electrochemiluminescence can be used as indexes. For example, in order to obtain a change in conductance, the fixed electrode 911 and the counter electrode 913 are used.
It is obtained by measuring the amount of current obtained by applying a constant voltage during the period, or by measuring the voltage obtained by passing a constant current.

Among the obtained hybridization rates, the complementary probe 121a under the hybridization reaction condition obtained as a result of the measurement has a clear causal relationship with the number of mismatched bases (the number of non-complementary bases) with respect to the target complementary base sequence. Can be used as a reliable measurement result.

An example of a method for improving the measurement accuracy of the above hybridization rate measurement by using statistical processing will be described below.

A monitoring system using statistical processing is shown in FIG. Although the basic configuration is the same as that of the monitoring system of FIG. 1, the signal processing unit 2 and the statistical processing unit 5 are connected, and the statistical processing unit 5 is connected to the display unit 3. The hybridization rate obtained by the signal processing in the signal processing unit 2 is output to the statistical processing unit 5.
The statistical processing unit 5 performs statistical processing based on the obtained hybridization rate, derives a test result, and displays the result on the display unit 3. Further, the statistical processing unit 5 stores in the storage unit 5 the statistical processing result calculated by the statistical processing unit 5.
a.

In the method using statistical processing, first, a first quasi-complementary probe group consisting of a first quasi-complementary probe RT _i (i is a natural number of 1 to n) having i bases which are not complementary to the target base sequence. The electrodes on which the respective probes are immobilized and the electrodes on which the second quasi-complementary probes of the second quasi-complementary probe group are immobilized are arranged on the DNA chip 1.

The plurality of second quasi-complementary probes forming the second quasi-complementary probe group have a quasi-complementary base sequence having low complementarity to the target base sequence. Further, the number of bases having no complementarity with the target base sequence (the number of non-complementary bases) in the base sequence is the same as that of at least one of the first quasi-complementary probes. In addition, the plurality of types of second quasi-complementary probes forming the second quasi-complementary probe group have different base sequences.

Here, the first quasi-complementary probe RT _i (i
Is a natural number of 1 to n) n species and each first quasi-complementary probe RT
The second quasi-complementary probe having the same number of non-complementary bases as _i and a different base sequence is immobilized on m types including the first quasi-complementary probe having a different base sequence for a total of m × n quasi-complementary probes The prepared electrode was placed on the DNA chip 1. Also,
A complementary probe having a base sequence complementary to the target base sequence (target complementary base sequence) is also immobilized.

Next, supply of the above-mentioned sample base sequence, hybridization reaction, dropping of intercalating agent, signal processing unit 2 to the DNA chip 1 on which these m × n quasi-complementary probes and the complementary probe are arranged. Signal is detected. Here, for convenience of explanation, the sample base sequence and the target base sequence are the same.

The obtained signal processing result is output to the statistical processing section 5 as the hybridization rate. Hereinafter, the hybridization rate of the j-th probe RT _ij out of the m probes having i bases that are not complementary to the target base sequence is RTO _ij .

Specifically, there is no complementarity with the target base sequence.
Complementary probe RT having 0 bases _0jThe hybridise
The solution rate is RTO₀₀, No complementarity with target base sequence
Quasi-complementary probe RT having one part_1jHybridi
The zation rate is RTO₁₁~ RTO_1m, The target base sequence
Quasi-complementary probe RT having n non-complementary portions_njof
Hybridization rate is RTO_n1~ RTO_nmGiven by
available.

The statistical processing unit 5 performs the following statistical processing method (1) based on the obtained hybridization rate RTO _ij.
At least one of (4) is performed.

In the statistical processing method (1), the hybridization rate RTO _ij (RTO _i1
~ RTO _im ) is calculated. Then, the hybridization rate RTO _ij deviated from the average value by a predetermined threshold value φ or more is excluded, and only the hybridization rate RTO _ij within the range of the threshold value φ is extracted.
The threshold value φ may be set according to the number of mismatched bases, or may be commonly given to all the numbers of mismatched bases. Then, only the extracted hybridization rate RTO _ij is displayed on the display unit 3 for monitoring.

As a result, the abnormal hybridization reaction can be excluded, and the reliability of the chip output value can be improved.

In the statistical processing method (2), the hybridization rates RTO _{i1 to} RTO _{im with the same} number of mismatched bases are used.
Are added to calculate the total value S S = RTO _i1 + RTO _i2 + ... + RTO _im of the hybridization rates with the same number of mismatched bases. Then, the obtained total value S is monitored as a representative value of the hybridization rate for the number of mismatched bases. As a result, highly reliable monitoring is possible even when each hybridization rate is minute.

In the statistical processing method (3), the hybridization rate RTO _ij (RTO _i1
~ RTO _im ) is calculated. Then, the hybridization rate RTO _ij deviating from the average value by a predetermined threshold value φ or more is excluded, and only the hybridization rate RTO _ij within the range of the threshold value φ is extracted. The threshold value φ may be determined according to the number of non-matching bases (the number of non-complementary bases), or may be commonly given to all the number of non-matching bases (the number of non-complementary bases). Good.
Then, the extracted hybridization rate RT
Among the O _ij ′, hybridization rates (RTO _i0 ′ -RT) with the same number of non-matching bases (the number of non-complementary bases)
The average value (extracted average value) of O _im ') is calculated. And
The extracted average value is the number of mismatched bases (the number of non-complementary bases)
Is monitored as a representative value of the hybridization rate for This makes it possible to average hybridization rates with the same number of non-matching bases (the number of non-complementary bases), which enables highly reliable monitoring.

In the statistical processing method (4), the hybridization rate RTO _ij (RTO _i1
~ RTO _im ) is calculated. Then, a hybridization rate R deviating by more than a predetermined threshold value φ
TO _ij is excluded and only the hybridization rate RTO _ij within the range of the threshold value φ is extracted. The threshold value φ may be set according to the number of mismatched bases, or may be commonly given to all the numbers of mismatched bases (the number of non-complementary bases). Then, among the extracted hybridization rates RTO _ij ′, the hybridization rates (RTO _ij ) in which the number of mismatched bases (the number of non-complementary bases) is equal.
Calculate the average value (extracted average value) of _i1 'to RTO _im '). Then, using the obtained extraction average value, the variance value s _i of the hybridization rate in which the number of mismatched bases (the number of non-complementary bases) is equal is calculated. The variance value s _i is calculated by the following formula.

The RTO _i _'is calculated after excluding hybridization rates that deviate by more than a predetermined threshold value φ, and the hybridization rates RTO _ij are equal in the number of mismatched bases (the number of non-complementary bases) i. '(RT
O _i1 '-RTO _im ')
_i _ '= (RTO _i1 ' + RTO _i2 '+ ... + RTO _im ')
/ M.

S _i = {(RTO _i1 '-RTO _i _') ² +
(RTO _i2 '-RTO _i _') ² + ... + (RTO _im '-R
TO _{i —} ') ² } / (m−1) Then, the extracted variance value s _i is tested to determine whether or not each of the obtained hybridization rates is within a certain range. If it is determined that the hybridization reaction conditions are within the certain range, it means that the hybridization reaction conditions can be adopted. If it is determined that the hybridization reaction conditions are not within the certain range, the hybridization reaction conditions cannot be adopted. It is shown that.

The statistical processing methods (1) to (4) may be separately applied to the present invention, or a plurality of them may be applied in combination. In addition, statistical processing method (1)
In (4), only one complementary probe was used, and
Although the case where the statistical processing is performed using the hybridization rate RTO ₀₀ of the species probe is shown, the present invention is not limited to this. For example, although there are 0 bases that are not complementary to the target base sequence, m complementary probes of different types may be prepared in the same manner as the second quasi-complementary probe, and statistical processing may be performed. In the above statistical processing methods (1) to (4), the value of i indicating the number of bases that are not complementary to the target base sequence was calculated as 1 to n, but when using m kinds of complementary probes,
The value of i may be calculated as 0 to n.

As described above, according to this embodiment,
At the time of hybridization reaction, while changing at least one hybridization reaction condition in real time,
Since it can be quantitatively monitored in association with changing hybridization reaction conditions, the optimum hybridization reaction conditions can be easily determined by a small number of hybridization experiments. As a result, DNA
Chip development costs can be reduced.

In the above embodiment, the fixed electrode 91 is used.
An example has been shown in which the hybridization rate is detected based on the voltage between the counter electrode 913 and the counter electrode 913, the amount of charge flowing in the fixed electrode 911, the current, etc., but the invention is not limited to this. For example, the monitoring system shown in FIG. 12 and the signal processing unit 2 may not be directly connected, but the imaging unit 6 may be separately connected to the signal processing unit 2. In order to detect the hybridization state using the imaging means, a sample base sequence labeled with a fluorescent substance or the like is used so that the probe in which hybridization has occurred emits fluorescence. An example of the hybridization state monitoring system is shown in FIG.

As shown in FIG. 13, the probes 121a and 122 of the DNA chip 1 imaged by the imaging means 6 are taken.
A signal indicating the brightness of fluorescence generated by the hybridization reaction of a or image data obtained by imaging the brightness of fluorescence is output to the signal processing unit 2. Then, the signal processing unit 2
The signal processing of the received signal or image data makes it possible to calculate the hybridization rate for each probe 121a or 122a. Of course, this imaging means 6 can also be applied to the monitoring system shown in FIG.

The structure of the quasi-complementary probe is not limited to that shown in FIG. A modification of the first quasi-complementary probe is shown in FIG. As shown in FIG. 14, the base sequence of the first quasi-complementary probe (1) 1221 is the same as that in FIG.

The base sequence of the first quasi-complementary probe (2 ′) 1222 ′ is different from the target complementary base sequence of the first quasi-complementary probe (1) 1221 and is not adjacent to the target complementary base sequence. Have a base sequence that results in different bases. Therefore, the first quasi-complementary probe (2 ′) 1222 ′ has a base sequence in which two bases of the target complementary base sequence in the complementary probe 121a are replaced with another base, and these two bases are adjacent to each other. I haven't.

The base sequence of the first quasi-complementary probe (3 ′) 1223 ′ has two bases different from the target complementary base sequence of the first quasi-complementary probe (2 ′) 1222 ′ and one base which is not adjacent. , Has a base sequence that makes a base different from the target complementary base sequence. Therefore, complementary probe 1
The base sequence in which three bases of the target complementary base sequence in 21a are replaced with other bases is the first quasi-complementary probe (3 ′).
1223 'has and its three bases are not adjacent to each other.

Thus, the first quasi-complementary probe (i ')
122i is the first quasi-complementary probe ((i + 1) ′) 12
The number of mismatched bases for 2 (i + 1) and the target complementary base sequence differs by one, and the mismatched bases may not be adjacent to each other in the base sequence.

(Second Embodiment) This embodiment relates to a modification of the first embodiment. In the first embodiment, an example in which single nucleotide polymorphism (SNP: Single Nucleotide Polymorphism) is not taken into consideration is shown, but the present embodiment relates to an embodiment in which the target nucleotide sequence has a single nucleotide polymorphism. Descriptions of parts common to the first embodiment will be omitted.

FIG. 15 is a diagram showing a detailed structure of the DNA chip 1 of this embodiment. As shown in FIG. 15, the substrate 1
1 is provided with a plurality of probe electrodes 201 and 202. The probe electrode 201 is a complementary probe electrode, and
Types of complementary probes 2011a to 2011d are fixed to the complementary probe electrodes 201a to 201d, respectively.
The probe electrode 202 is a quasi-complementary probe electrode, and the quasi-complementary probe 202a is fixed. Complementary probe electrode 2
01a to 201d are complementary probe electrodes 2 for detecting base A
01a, a complementary probe electrode 201b for detecting base T, a complementary probe electrode 201c for detecting base C, and a complementary probe electrode 201d for detecting base G.

The base A detecting complementary probe 2011a is fixed to the base A detecting complementary probe electrode 201a, and the base T detecting complementary probe 2011b is fixed to the base T detecting complementary probe electrode 201b. The probe electrode 201c has a complementary probe 2 for detecting base C.
011c is fixed and complementary probe 201 for detecting base G
A complementary probe 2011d for detecting a base G is fixed to d.

FIG. 16 shows the complementary probe 201 of this embodiment.
1a to 2011d, a quasi-complementary probe 202a, and a target base sequence 200 are nucleotide sequences. The target base sequence 200 has SNPs bases 2001.

The complementary probe 2011a for detecting base A is
When the SNPs base 2001 of the target base sequence 200 is A, it has a complementary base sequence (target complementary base sequence: GTC-A-A-G-T). The complementary probe 2011b for detecting base T is S of the target base sequence.
A base sequence complementary to the target base sequence when the NPs base 2001 is T (target complementary base sequence: GT-CT)
-A-G-T). Complementary probe 2 for detecting base C
011c is the SNPs base 2001 of the target base sequence C
Has a base sequence complementary to the target base sequence (target complementary base sequence: GTC-C-A-G-T). The complementary probe 2011d for detecting the base G is a base sequence complementary to the target base sequence when the SNPs base 2001 of the target base sequence is G (target complementary base sequence: GT).
-C-G-A-G-T).

The quasi-complementary probe 202a is a base sequence (GT-C) in which one base is replaced with another base in the target complementary base sequence when the SNPs base 2001 is T in the target base sequence 200. -T-T-G-T) having a first quasi-complementary probe (1) 2021, a base sequence in which two bases are replaced with another base (G-T-C-T-T-C-
First quasi-complementary probe (2) 2022 having T), a base sequence in which three bases are replaced with another base (GTCT)
-T-C-A) first quasi-complementary probe (3) 20
23 has a first quasi-complementary probe (i) 202i having a base sequence in which bases differ by i.

The same processing as in the first embodiment is performed using the DNA chip having the probe and the probe electrode described above. Assuming that the sample base sequence is a sequence in which the SNP of the target base sequence is T, in the case of the present embodiment, when the hybridization rate of the base T detection complementary probe 2011b is maximum, the base T detection complementary probe is obtained. The hybridization rate of 2011b is TO and the hybridization rate of the quasi-complementary probe 202i is RT.
In the case of O _i , by measuring the relationship between TO and RTO _i , it is possible to obtain an indicator of abnormality in the hybridization reaction or an appropriate hybridization reaction temperature.

As described above, according to the present embodiment, even when the target base sequence has SNPs bases, the hybridization reaction can be monitored as in the first embodiment.

The quasi-complementary probe 202a has been described in the case where the base corresponding to the SNPs base 2001 of the target base sequence 200 is T, but the other bases A, C, G.
If replaced with, set separately. In addition, four types of bases T, A, C, and G corresponding to the SNPs base 2001 of the target base sequence 200 are set, and the quasi-complementary probes 202a to 202d are fixed to the quasi-complementary probe electrode 202 and monitored for each. Good.

The present invention is not limited to the above embodiment.

In the above embodiment, the sample base sequence D
Although an example has been shown in which the hybridization reaction is optimized by detecting the base sequence of NA, the present invention can be widely applied to base sequences of RNA other than DNA.

Further, although an example in which the measurement result is displayed on the display unit 3 is shown as an example of the output means, the present invention is not limited to this, and the optimum hybridization reaction condition may be notified by voice.

Further, the detection of the occurrence rate of the hybridization reaction is performed by calculating the charge amount, the conductance, the fluorescence imaging, etc., but the invention is not limited thereto.

In the detailed configuration example of the DNA chip 1 shown in FIG. 11, an example in which three kinds of electrodes of the fixed electrode 911, the reference electrode 912, and the counter electrode 913 are arranged on the substrate 11 is shown. It is needless to say that the present invention can be applied even if the electrodes 912 and the counter electrode 913 are appropriately omitted depending on the measurement method and only the fixed electrode 911 is arranged. When the reference electrode 912 and the counter electrode 913 are omitted, the reference electrode 912 and the counter electrode 913 may be arranged on a substrate different from the substrate on which the fixed electrode 911 is arranged.

The constituent material of the complementary probe, the quasi-complementary probe, and the sample base sequence is not particularly limited,
Nucleic acids such as DNA, RNA, PNA, and methylphosphonate skeleton, and other nucleic acid analogs can be used.

[0112]

As described in detail above, according to the present invention, hybridization conditions can be optimized.

[Brief description of drawings]

FIG. 1 is a diagram showing an overall configuration of a hybridization state monitoring system according to a first embodiment of the present invention.

FIG. 2 is a view showing a detailed configuration of the DNA chip according to the same embodiment.

FIG. 3 is a view showing a conceptual diagram of a quasi-complementary probe and a target base sequence according to the same embodiment.

FIG. 4 is a view showing an example of a measurement result according to the same embodiment.

FIG. 5 is a view showing an example of a measurement result according to the same embodiment.

FIG. 6 is a view showing an example of a measurement result according to the same embodiment.

FIG. 7 is a view showing an example of a measurement result according to the same embodiment.

FIG. 8 is a view showing an example of a measurement result according to the same embodiment.

FIG. 9 is a view showing an example of a measurement result according to the same embodiment.

FIG. 10 is a view showing the concept of hybridization state monitoring and measurement results according to the embodiment.

FIG. 11 is a view showing a detailed structure of the DNA chip according to the same embodiment.

FIG. 12 is a diagram showing an example of a monitoring system using statistical processing according to the same embodiment.

FIG. 13 is a view showing a modified example of the monitoring system using the statistical processing according to the same embodiment.

FIG. 14 is a view showing a modified example of the quasi-complementary probe according to the same embodiment.

FIG. 15 is a diagram showing a detailed configuration of a DNA chip according to a second embodiment of the present invention.

FIG. 16 is a conceptual diagram of base sequences of a complementary probe, a quasi-complementary probe 202a, and a target nucleic acid chain according to the same embodiment.

[Explanation of symbols]

1 ... DNA chip 2 ... Signal processing unit 3 ... Display 4 ... Electrode control unit 91 ... Spot block 120 ... Target base sequence 121 ... Complementary probe electrode 121a ... Complementary probe 122 ... Quasi-complementary probe electrode 122a ... Quasi-complementary probe 921-923 ... Electrode set 1221 ... First quasi-complementary probe (1) 1222 ... First quasi-complementary probe (2) 1223 ... First quasi-complementary probe (3)

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 27/416 G01N 37/00 102 27/42 27/46 301M 37/00 102 341M // C12N 15/09 27/30 351 27/46 336M 386G C12N 15/00 FF Term (reference) 2G060 AA06 AC10 AD06 AE40 AF03 AF10 BA09 FA01 HA02 HC07 HC10 HC18 HD01 HD03 HE03 KA05 4B024 AA11 CA04 HA19 4B029 AA07 AA23 FA13 QA42AQ4Q06QA QAQA QR55 QS34

Claims (12)

[Claims] 1. A substrate, and a quasi-complementary base sequence immobilized on the substrate and having a lower complementarity to the target base sequence than the target complementary base sequence complementary to the target base sequence to be detected. A plurality of types of first quasi-complementary probes having a plurality of types of first quasi-complementary probes, and the plurality of types of first quasi-complementary probes differ from each other in the number of non-complementary bases that are not complementary to the target base sequence. A base sequence detection chip comprising: 2. A substrate, and a quasi-complementary base sequence immobilized on the substrate and having a lower complementarity to the target base sequence than the target complementary base sequence complementary to the target base sequence to be detected. A plurality of types of first quasi-complementary probes having a plurality of types of first quasi-complementary probes, and the plurality of types of first quasi-complementary probes differ from each other in the number of non-complementary bases that are not complementary to the target base sequence. A base sequence detection chip comprising: a detection means for detecting an incidence of a hybridization reaction between each first quasi-complementary probe and a sample base sequence in the base sequence detection chip; A hybridization monitoring system, comprising: an output unit that outputs the reaction occurrence rate. 3. The hybridization monitoring system according to claim 2, further comprising a storage unit that stores the occurrence rate of the hybridization reaction detected by the detection unit in association with the hybridization reaction condition. 4. A plurality of types of first groups having a quasi-complementary base sequence immobilized on the substrate and having a lower complementarity to the target base sequence than the target complementary base sequence complementary to the target base sequence. A second quasi-complementary probe,
3. The hybridization monitoring system according to claim 2, wherein the quasi-complementary probes include a second quasi-complementary probe group having the same number of non-complementary bases and different base sequences. 5. A spotting device 1 is provided on the substrate.
This is a region where the solvent spreads on the substrate by one drop of the solvent and does not overlap with another region where the solvent spreads on the substrate by another drop of the solvent. The hybridization monitoring system according to claim 2, wherein the hybridization monitoring system has a plurality of regions, and a plurality of types of the quasi-complementary probes are formed in each of the regions. 6. A plurality of quasi-complementary probes having the same number of non-complementary bases and different base sequences from each other among the first and second quasi-complementary probe groups based on the occurrence rate detected by said detecting means. A first statistical process of calculating an average value of occurrence rates of hybridization reactions and excluding the reaction occurrence rates that deviate from the average value by a predetermined threshold value φ or more, the number of non-complementary bases is the same, and bases are mutually bases. A second statistical process of adding the occurrence rates of hybridization reactions in a plurality of quasi-complementary probes having different sequences to calculate a representative value, and an average value of the reaction occurrence rates obtained as a result of being excluded by the first statistical process. A statistical process for executing at least one of a third statistical process for calculating and a fourth statistical process for calculating a variance value of the reaction occurrence rate obtained as a result of being excluded by the first statistical process. Hybridization monitoring system of claim 4 further comprising a means. 7. An immobilization electrode, which is disposed on the substrate and immobilizes the first quasi-complementary probe or the second quasi-complementary probe, and a counter electrode, further comprising: The hybridization monitoring system according to claim 2 or 4, wherein the generation rate of the hybridization reaction is calculated based on the amount of flowing charges. 8. An immobilization electrode disposed on the substrate for immobilizing the first or second quasi-complementary probe, and a counter electrode, further comprising: the detection means, the immobilization electrode and the counter electrode. The hybridization monitoring system according to claim 2 or 4, wherein the occurrence rate of the hybridization reaction is calculated based on the conductance obtained based on the voltage or the current between them. 9. An image pickup means for picking up an image of the first or second quasi-complementary probe of the base sequence detection chip after the hybridization reaction is further provided, and the detection means has a brightness of fluorescence imaged by the image pickup means. The hybridization monitoring system according to claim 2, wherein the incidence of the hybridization reaction is calculated based on the above. 10. A substrate and a quasi-complementary base sequence that is immobilized on the substrate and has lower complementarity to the target base sequence than the target complementary base sequence that is complementary to the target base sequence to be detected. A plurality of types of first quasi-complementary probes having a plurality of types of first quasi-complementary probes, and the plurality of types of first quasi-complementary probes differ from each other in the number of non-complementary bases that are not complementary to the target base sequence , A quasi-complementary base sequence by contacting a base sequence detection chip, which is disposed on the substrate and has an immobilized electrode for immobilizing the first quasi-complementary probe, with a test solution containing a sample base sequence. A hybridization reaction is performed with the sample base sequence, the occurrence rate of the hybridization reaction between each of the first quasi-complementary probes and the sample base sequence is detected, and the detected hybridisase is detected. Hybridization reaction monitoring method and outputting the incidence of Deployment reaction. 11. The hybridization reaction comprises:
The hybridization reaction monitoring method according to claim 10, wherein the method is performed under at least a plurality of reaction conditions, and the generation rate is output in association with the reaction conditions. 12. The hybridization reaction monitoring method according to claim 11, wherein the plurality of reaction conditions are temperature conditions provided by changing the temperature of the base sequence detection chip.