JP3887212B2 - Base sequence detection chip, hybridization monitoring system, and hybridization monitoring method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a base sequence detection chip, a hybridization monitoring system, and a hybridization monitoring method used for genetic testing and the like.
[0002]
[Prior art]
In recent years, gene testing technology using a DNA chip as a DNA detection sensor has attracted attention. In the inspection using this DNA chip, DNA probes are arranged in a plurality of matrix 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.
[0003]
When hybridization is performed on a DNA chip, if the reaction conditions are not optimum, the probability that the sample DNA will hybridize incorrectly, that is, bind to an irrelevant DNA probe increases.
[0004]
[Problems to be solved by the invention]
However, since there is no quantitative method for determining hybridization conditions, it is necessary to perform many experiments with different hybridization conditions. For this reason, there has been a problem that the development cost of the DNA chip becomes high.
[0005]
In addition, when the optimal hybridization conditions differ depending on the type of DNA or probe DNA to be detected, there has been a problem that the development cost of the DNA chip is further increased.
[0006]
The present invention has been made to solve the above-mentioned problems, and an object thereof is to provide a base sequence detection chip, a hybridization monitoring system, and a hybridization monitoring method for optimizing hybridization conditions. It is in.
[0007]
[Means for Solving the Problems]
  According to one aspect of the invention,A plurality of first complementary base sequences having a substrate and a quasi-complementary base sequence that is immobilized on the substrate and is less 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, a base sequence detection chip comprising a first quasi-complementary probe group having different numbers of non-complementary bases that are not complementary to the target base sequence; , Detecting means for detecting the rate of occurrence of the hybridization reaction between each first quasi-complementary probe and the sample base sequence in the base sequence detection chip, changing means for changing the reaction conditions of the hybridization reaction, and detection by the detecting means A storage means for storing the occurrence rate of the generated hybridization reaction in association with the reaction condition, and based on the occurrence rate of the hybridization reaction in each reaction condition. Hybridization monitoring system comprising a detection means for detecting an optimal reaction conditions haveIs provided.
[0009]
According to such a configuration, the occurrence rate of an erroneous hybridization reaction can be quantitatively monitored during the hybridization reaction in association with the hybridization reaction condition, so that the optimal hybridization reaction condition can be easily achieved by a small number of hybridization experiments. Can be requested.
[0011]
In addition, according to another embodiment of the present invention, the quasi-complementary that is less complementary to the target base sequence than the target complementary base sequence that is immobilized on the substrate and complementary to the target base sequence. The plurality of second quasi-complementary probes each having a base sequence includes a second quasi-complementary probe group having the same number of non-complementary bases and different base sequences. Thereby, 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.
[0012]
According to another embodiment of the present invention, the solvent spreads on the substrate by one dropping of the solvent of the spotting device on the substrate, and the solvent is dropped on the substrate by another dropping of the solvent. There are a plurality of regions that do not overlap with other regions that spread out and minimize the distance between each solvent dropping position, and a plurality of types of semi-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, so that the base sequence detection chip can be miniaturized. Moreover, it can use for an error test by performing a statistical process etc. using the output result obtained from a some probe.
[0013]
According to another embodiment of the present invention, based on the occurrence rate detected by the detection means, the first and second quasi-complementary probe groups have the same number of non-complementary bases and the base sequences of each other. A first statistical process for calculating an average value of the occurrence rate of hybridization reactions in a plurality of quasi-complementary probes having different values and excluding a reaction rate that deviates from the average value by a predetermined threshold value φ or more, the number of non-complementary bases Second statistical processing for calculating a representative value by adding the occurrence rates of hybridization reactions in a plurality of quasi-complementary probes having the same base sequence and different base sequences, and the occurrence of a reaction obtained as a result of being excluded by the first statistical processing A third statistical process for calculating an average value of the rates, and a fourth statistical process for calculating a variance value of the reaction rate obtained as a result of being excluded by the first statistical process. Further comprising a processing means. Thereby, the monitoring accuracy of the hybridization state can be improved.
[0014]
According to another embodiment of the present invention, the detector further includes an immobilization electrode disposed on the substrate and immobilizing the first or second quasi-complementary probe, and a counter electrode. The occurrence rate of the hybridization reaction is calculated based on the amount of charge flowing from the activating electrode. According to another embodiment of the present invention, the detector further includes an immobilization electrode disposed on the substrate and immobilizing the first or second quasi-complementary probe, and a counter electrode. The rate of occurrence of the hybridization reaction is calculated based on the conductance obtained based on the voltage or current between the counter electrode and the counter electrode. According to another embodiment of the present invention, the imaging device further includes imaging means for imaging the first or second quasi-complementary probe of the base sequence detection chip after the hybridization reaction, and the detection means includes the imaging means. The occurrence rate of the hybridization reaction is calculated based on the brightness of the fluorescence imaged.
[0015]
Further, the present invention relating to an apparatus is also established as an invention of a method realized by the apparatus.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0017]
(First embodiment)
FIG. 1 is a diagram showing an overall configuration of a hybridization state monitoring system according to the first embodiment of the present invention. As shown in FIG. 1, a DNA chip 1 as a base sequence detection chip, a signal processing unit 2 that performs signal processing based on the output of the DNA chip 1, and a display unit that displays a signal processed by the signal processing unit 2 3 and an electrode control unit 4 that controls the application of a voltage to each electrode of the DNA chip 1 and controls a reaction such as a hybridization reaction at each electrode. Further, the signal processing unit 2 has a storage unit 2a, and data such as a hybridization rate and a hybridization reaction condition obtained by signal processing by the signal processing unit 2 are stored in the storage unit 2a.
[0018]
FIG. 2 is a diagram showing a detailed configuration of the DNA chip 1. As shown in FIG. 2, a plurality of probe electrodes 121 and 122 are provided on the substrate 11. 121 is a complementary probe electrode, and 122 is a semi-complementary probe electrode. A counter electrode (not shown) is provided on the substrate 11 or other than on the substrate 11.
[0019]
A complementary probe 121 a having a base sequence complementary to the target base sequence 120 (target complementary base sequence) is fixed to the complementary probe electrode 121. There are a plurality of quasi-complementary probe electrodes 122, and a quasi-complementary probe 122a having a lower complementarity with the target base sequence 120 than the complementary probe 121a is fixed to each. The complementary probe electrode 121, the quasi-complementary probe electrode 122, and the counter electrode are each connected to the signal processing unit 2. The charges that flow out to the probe electrodes 121 and 122 when a voltage is applied between the probe electrodes 121 and 122 and the counter electrode are output to the signal processing unit 2, and the signal processing unit 2 detects the amount of charges to perform hybridization. The rate (the rate at which the reaction in which the probe and the sample base sequence bind to each other) can be measured.
[0020]
In applying a voltage to each probe electrode, a three-electrode system using a reference electrode in addition to the counter electrode may be used.
[0021]
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 the sample base sequence with a complementary probe or semi-complementary probe.
[0022]
A complementary probe is a probe used for detecting whether or not a sample base sequence includes a target base sequence, and has a base sequence complementary to the target base sequence (target complementary base sequence). In addition, in order to compare the hybridization reaction of the complementary probe with that of the semi-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 status. You can also.
[0023]
The quasi-complementary probe is a probe used for optimizing the hybridization reaction conditions by monitoring the hybridization state, and is at least a base sequence complementary to the target base sequence (target complementary base sequence). It has a base sequence in which one base (any one or more of bases from the start base to the end base) is replaced with another base.
[0024]
In the following embodiments, the hybridization reaction conditions include not only conditions for causing a hybridization reaction between a certain nucleotide sequence and another nucleotide sequence, but also the detection of a double strand in which this hybridization reaction has occurred. Processing conditions in necessary steps are also included.
[0025]
A conceptual diagram of the quasi-complementary probe 122a and the target base sequence 120 is shown in FIG. As shown in FIG. 3, when the target base sequence 120 is C-A-G-A-T-C-A from the start base to the end base, the base that is complementary to the target base sequence 120 The sequence is GTCTCTAGT from the start base to the end base due to base complementarity. Therefore, the complementary probe 121a is a probe having this base sequence “GTCCTAGT”.
[0026]
There are a plurality of types of semi-complementary probes 122a, and these plural types of semi-complementary probes are used as a first semi-complementary probe group. As each first quasi-complementary probe constituting the first quasi-complementary probe group, a base sequence complementary to the target base sequence 120 (target complementary base sequence) and one base different in GTCTCTT The first quasi-complementary probe (1) 1221 having a base sequence of GT and the first quasi-complementary probe (2) having a base sequence of GT-CT-TCT having two different bases The target complementary base sequence and the number of bases (non-complementary), such as 1222, and a first quasi-complementary probe (3) 1223 having three different bases of GTCTCTTCA The first quasi-complementary probe (i) 122i having a different base sequence (i) (i is a natural number and is equal to or less than the base number of the target base sequence) is included.
[0027]
The first quasi-complementary probe (2) 1222 is a base in which a base adjacent to a base different from the target complementary base sequence in the first quasi-complementary probe (1) 1221 is a base different from the target complementary base sequence. Has an array. Therefore, the first quasi-complementary probe (2) 1222 has a base sequence in which two bases in the target complementary base sequence in the complementary probe 121a are replaced with other bases, and the two bases are adjacent to each other.
[0028]
The first quasi-complementary probe (3) 1223 is different from the target-complementary base sequence in one base adjacent to two bases different from the target complementary base sequence in the first quasi-complementary probe (2) 1222. It has a base sequence that becomes a base. Therefore, the first quasi-complementary probe (3) 1223 has a base sequence in which three bases in the target complementary base sequence in the complementary probe 121a are replaced with other bases, and the three bases are adjacent to each other.
[0029]
Similarly, the first quasi-complementary probe (i) 122i is adjacent to (i-1) bases different from the target complementary sequence of the first quasi-complementary probes (i-1) 122 (i-1). One base has a base sequence that is different from the target complementary base sequence. Each probe of the first quasi-complementary probe group is immobilized on a different electrode.
[0030]
The first quasi-complementary probe (1) 1221 to the first quasi-complementary probe (3) 1223 shown in FIG. 3 show an example. In the case of the first quasi-complementary probe (i) 122i, there are i bases different from the target complementary base sequence, and this i base is any of the base sequences, and the i bases are the target. It is freely determined from three bases other than a specific base among A, G, C, and T selected as complementary base sequences. All of these possible types or a plurality of types may be fixed to each electrode 122 of the DNA chip 1, or only one representative type may be fixed to each electrode 122 of the DNA chip 1. Further, the number i of the different bases may be any number as long as it is 2 or more.
[0031]
In this embodiment, for convenience of explanation, each of the first quasi-complementary probe (1) 1221, the first quasi-complementary probe (2) 1222, and the first quasi-complementary probe (3) 1223 has a representative base sequence 1 Explanation will be made using only seeds.
[0032]
Next, a first technique for monitoring the hybridization state using the hybridization state monitoring system shown in FIGS. 1 to 3 will be described.
[0033]
First, a test solution containing a sample base sequence is injected and brought into contact with each of the electrodes 121 and 122 in the DNA chip 1, and the sample base sequence contained in the injected test solution is hybridized with the complementary probe 121a and the semi-complementary probe 122a. Cause a 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 solvent of the reaction solution, a buffer solution having a predetermined pH is used. In addition, the reaction is performed at a predetermined temperature, for example, for 1 minute or longer. In this hybridization reaction, the electrode control unit 4 applies a predetermined voltage to the fixed electrode and the counter electrode of the probe electrodes 121 and 122, so that the reaction that has conventionally required several hours to several days can be performed for several minutes. It can be shortened to the extent.
[0034]
When the hybridization reaction is completed, the probe electrodes 121 and 122 are washed with a predetermined buffer solution, and double-stranded portions formed on the electrode surface (double-stranded portions based on the complementary probe 121a, the semi-complementary probe 122a and the sample base sequence) An intercalating agent that selectively binds to an activator. Then, a predetermined voltage is applied to the fixed electrode and the counter electrode of the probe electrodes 121 and 122 on which the intercalating agent has acted, and the charge amount of each of the electrodes 121 and 122 derived from the intercalating agent is measured by the signal processing unit 2. The obtained measurement result is displayed on the display unit 3 as a hybridization rate.
[0035]
FIG. 4 is a diagram illustrating an example of measurement results displayed on the display unit 3. RTO₀Is the hybridization rate obtained based on the amount of charge detected for the complementary probe electrode 121, RTO₁, RTO₂, RTO_ThreeIs the hybridization rate obtained based on the amount of charge detected for the quasi-complementary probe electrode 122. RTO₁Is the hybridization rate of the first quasi-complementary probe (1) 1221, RTO₂Is the hybridization rate of the first quasi-complementary probe (2) 1222, RTO_ThreeIs the hybridization rate of the first quasi-complementary probe (3) 1223.
[0036]
FIG. 4 (a) and FIG. 4 (b) are measurement results under different hybridization reaction conditions. 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 accelerator, the conditions such as stirring and shaking, and the reaction time. These include the type and concentration of the intercalating agent, the ionic strength of the intercalating agent buffer, pH, and various conditions thereof.
[0037]
As shown in FIG. 4 (a), the hybridization rate decreases according to 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, in FIG. 4B as well, 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.
[0038]
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. 4 (b). It is smaller than the measurement result. Therefore, it can be seen that the hybridization reaction in FIG. 4B is optimized more than the hybridization reaction in FIG.
[0039]
5A and 5B show examples of measurement results obtained under further different hybridization reaction conditions than those in FIGS. 4A and 4B. In both cases of FIGS. 5A and 5B, RTO₀>> RTO₁> RTO₂> RTO_ThreeThis relationship is not established, and a 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 is not observed. RTO₀Is the hybridization rate when a normal hybridization reaction is occurring. Therefore, RTO than this₁, RTO₂, RTO_ThreeWhen the value of is large, it can be seen that at least an abnormal hybridization reaction has occurred. The cause of the abnormal hybridization reaction may be a problem that the hybridization conditions such as contamination of impurities and the hybridization reaction temperature are too low.
[0040]
Examples of measurement results obtained under further different hybridization reaction conditions than those in FIGS. 4 and 5 are shown in FIGS. In the case of FIG. 6A, RTO₀≒ RTO₁>> RTO₂> RTO_ThreeThe relationship is established. In the case of FIG. 6B, RTO₀≒ RTO₁≒ RTO₂≒ RTO_ThreeThe relationship is established. In either case, there is no causal relationship between the hybridization rate and the number of mismatched bases (number of non-complementary bases) as in the case of FIG. 4 (a) or FIG. 4 (b). It can be seen that an abnormal hybridization reaction has occurred. As in FIGS. 5 (a) and 5 (b), abnormal hybridization reactions may be caused by problems such as contamination of impurities and hybridization conditions such as the hybridization reaction temperature being too low. It is done.
[0041]
Next, a second technique for monitoring the hybridization state will be described. Detailed description of the apparatus used and parts common to the first method will be omitted.
[0042]
Unlike the first method for monitoring the hybridization state, this second method is non-complementary in measuring the hybridization rate using a plurality of first quasi-complementary probes having different number of mismatched bases (number of non-complementary bases). This is a point of analyzing the hybridization rate according to the number of bases in association with the temperature condition.
[0043]
First, a test solution containing the sample base sequence is injected into each of the electrodes 121 and 122 in the DNA chip 1. Next, a hybridization reaction is caused between the target nucleic acid chain contained in the injected test solution, the complementary probe 121a, and the semi-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 solution for 1 minute or more, for example. Further, during this hybridization reaction, a predetermined voltage is applied to the probe electrodes 121 and 122 by the electrode controller 4. Moreover, the other measurement conditions are made the same, and a plurality of reactions in which the temperature conditions are changed are generated.
[0044]
When the hybridization reaction is completed, the probe electrodes 121 and 122 are washed with a predetermined buffer, and an intercalating agent that selectively binds to the double-stranded portion (complementary probes 121a and 122a and the sample base sequence) formed on the electrode surface. Act. Then, a predetermined voltage is applied to the probe electrodes 121 and 122 on which the intercalating agent has acted, and the signal processing unit 2 measures the charge amounts of the electrodes 121 and 122 derived from the intercalating agent. The obtained measurement result is displayed on the display unit 3 as a hybridization rate. In the second method, other measurement conditions are made the same, and a plurality of reactions in which the temperature conditions are changed are generated. Therefore, a plurality of measurement results reacted under different temperature conditions can be obtained.
[0045]
FIG. 7 is a diagram illustrating an example of measurement results displayed on the display unit 3. RTO₀Is the hybridization rate obtained based on the amount of charge detected for the complementary probe electrode 121, RTO₁, RTO₂, RTO_ThreeIs the hybridization rate obtained based on the amount of charge detected for the quasi-complementary probe electrode 122 and RTO₁Is the hybridization rate of the first quasi-complementary probe (1) 1221, RTO₂Is the hybridization rate of the first quasi-complementary probe (2) 1222, RTO_ThreeIs the hybridization rate of the first quasi-complementary probe (3) 1223. FIG. 7 (a) and FIG. 7 (b) are measurement results in which a hybridization reaction was caused under different temperature conditions. Specifically, FIG. 7B corresponds to the case where the hybridization reaction is caused at a lower temperature than FIG. 7A.
[0046]
As shown in FIG. 7A, 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, as in FIG. 7A, the hybridization rate in FIG. 7B 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. Comparing FIG. 7 (a) and FIG. 7 (b), the decrease rate of the hybridization rate accompanying the increase in the number of mismatched bases (the number of non-complementary bases) in FIG. 7 (b) is shown in FIG. 7 (a). It is smaller than the measurement result. Therefore, it can be seen that the hybridization reaction in FIG. 7B is optimized more than the hybridization reaction in FIG. From the measurement results of FIGS. 7A and 7B, it can be seen that the hybridization reaction can be optimized by lowering the hybridization reaction temperature.
[0047]
8A and 8B show examples of measurement results obtained under further different hybridization reaction conditions than those in FIGS. 7A and 7B. In both cases of FIGS. 8A and 8B, RTO₀>> RTO₁> RTO₂> RTO_ThreeIs not established, and a causal relationship between the number of bases that do not match the complementary base sequence of the target base sequence and the hybridization rate is not observed. RTO₀Is the hybridization rate when a normal hybridization reaction is occurring. Therefore, RTO than this₁, RTO₂, RTO_ThreeWhen the value of is large, it can be seen that at least an abnormal hybridization reaction has occurred. The cause of the abnormal hybridization reaction may be a problem such as the hybridization reaction temperature being too low, and a countermeasure for increasing the hybridization reaction temperature may be considered.
[0048]
Examples of measurement results obtained under further different hybridization reaction conditions than those in FIGS. 7 and 8 are shown in FIGS. 9 (a) and 9 (b). In the case of FIG. 9A, RTO₀≒ RTO₁>> RTO₂> RTO_ThreeThe relationship is established. In the case of FIG. 9B, RTO₀≒ RTO₁≒ RTO₂≒ RTO_ThreeThe relationship 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. 7 (a) or FIG. 7 (b). 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. It is done.
[0049]
Next, a third technique for monitoring the hybridization state will be described.
[0050]
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, and the sample base sequence and the probe are brought into contact with each other. Next, a hybridization reaction is caused between the sample base sequence contained in the injected test solution, the complementary probe 121a, and the semi-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 performed at a predetermined temperature, for example, for 1 minute or longer. In addition, by applying a predetermined voltage to the probe electrodes 121 and 122 by the electrode controller 4 at the time of this hybridization reaction, the reaction that conventionally required several hours to several days can be shortened to several minutes. .
[0051]
When the hybridization reaction is completed, the probe electrodes 121 and 122 are washed with a predetermined buffer, and an intercalating agent that selectively binds to the double-stranded portion formed on the electrode surface is allowed to act. Then, a predetermined voltage is applied to the probe electrodes 121 and 122 on which the intercalating agent has acted. 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 that changes. The obtained measurement result is displayed on the display unit 3 as a hybridization rate.
[0052]
FIG. 10 is a diagram showing the concept of hybridization state monitoring and measurement results of the obtained third method. FIG. 10A is a conceptual diagram of the third state of hybridization state monitoring, and FIGS. 10B to 10E are measurement results.
[0053]
FIG. 10A is a conceptual diagram of the reaction when the temperature decreases as it goes from the top, the top two, the top three, and the bottom in FIG. 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 the 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. The schematic diagram of the hybridization reaction between the first quasi-complementary probe (2) 1222 and the sample base sequence, and the right end is the schematic diagram of the hybridization reaction between the first quasi-complementary probe (3) 1223 and the 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 when the temperature is lower than when the temperature is higher due to the action of the intercalating agent. . A probe with a larger number of mismatched bases (non-complementary bases) than a base sequence complementary to the base sequence of the target base sequence is less likely to cause a hybridization reaction, and a probe with a lower number of mismatched bases (non-complementary bases). It can be seen that hybridization reaction is likely to occur.
[0054]
The measurement results obtained under the measurement conditions shown in FIG. 10A are shown in FIGS. 10B to 10D.
[0055]
As shown in FIGS. 10 (b) to 10 (d), the uppermost measurement result (FIG. 10 (b)) with the highest temperature has a low overall hybridization rate, and the comparison of the hybridization rates between the probes. In addition, there is no causal relationship between the number of mismatched bases (number of non-complementary bases) between the target base sequence and the complementary base sequence and the hybridization rate.
[0056]
Next, in the measurement result in the second stage from the top in FIG. 10A when the temperature is high (FIG. 10C), the hybridization rate of the complementary probe 121a is the highest, and the base sequence complementary to the target base sequence As the number of mismatched bases (the number of non-complementary bases) increases, the hybridization rate decreases greatly. The decrease rate of the hybridization rate decreases as the number of mismatched bases (the number of non-complementary bases) increases. Yes. From this, the causal relationship between the number of mismatched bases (the number of non-complementary bases) and the hybridization rate appears very clearly.
[0057]
Next, in the measurement result in the third stage from the top in FIG. 10A when the temperature is high (FIG. 10D), the complementary probe 121a has the highest hybridization rate, and the base sequence complementary to the target base sequence. As the number of mismatched bases (the number of non-complementary bases) increases, the hybridization rate greatly decreases, which is the same as the measurement result in the second row from the top in FIG. is there. The difference from the measurement result with the second highest temperature is that the decrease rate of the hybridization rate does not change much as the number of mismatched bases (the number of non-complementary bases) increases. It can be said that the causal relationship between the hybridization rate and the number of mismatched bases (number of non-complementary bases) clearly appears in the second stage compared with the measurement results in the second stage.
[0058]
In the measurement result at the bottom of FIG. 10A when the temperature is the lowest (FIG. 10E), there is no causal relationship between the number of mismatched bases (number of non-complementary bases) and the hybridization rate. In some cases, the hybridization rate of the semi-complementary probe 122a is higher than the hybridization rate of 121a.
[0059]
Of the obtained measurement results, RTO₀>> RTO₁> RTO₂> RTO_ThreeThe temperature at which this relationship is established is the temperature at the second stage from the top. Therefore, the temperature condition at which the measurement result in the second stage from the top is obtained is detected as the optimum hybridization temperature.
[0060]
FIG. 11 shows a detailed structure of the DNA chip 1 for realizing the hybridization state monitoring of the present embodiment. As shown in FIG. 11, a plurality of spot blocks 91 are arranged in a matrix on the substrate 11. A spot block includes at least a probe electrode for fixing one kind of probe, and further includes an electrode set including at least one of a reference electrode and a counter electrode, and the areas of the blocks do not overlap each other. A spotting device for dropping a mixture containing a specimen base sequence and an intercalating agent onto each electrode corresponds to a unit that is treated as one point. Therefore, the distance between each block of the spot block is determined according to the minimum resolution of the spotting device, and a mixture containing one base structure and an intercalating agent is dropped onto the spot block by the spotting device. The other spot block is dropped with a mixture containing another base body sequence and an insertion agent by a spotting device. The minimum resolution of the spotting device is a region where the solvent spreads on the substrate by one dropping of the solvent of the spotting device and has an overlapping portion with another region where the solvent spreads on the substrate by another dropping of the solvent. First, it refers to a region that minimizes the distance between the solvent dropping positions, for example, a region having a diameter of 100 μm or less or a region having a diameter of 100 μm or less.
[0061]
By arranging the spot blocks corresponding to this minimum resolution, the mixture is not dropped on other spot blocks due to the drop of the mixture on one spot block. That is, an independent dropping operation can be performed for each spot block so that the mixed agents are not mixed with each other.
[0062]
Each spot block 91 is provided with an electrode set of either an immobilization electrode for immobilizing the same type of probe, a combination of an immobilization electrode and a reference electrode, or a combination of an immobilization electrode, a reference electrode and a counter electrode Is done. The immobilized electrode is an electrode for immobilizing the complementary probe 121a or the semi-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 to the fixed electrode 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 or the potential of the counter electrode is measured with respect to the reference electrode.
[0063]
In FIG. 11A, an electrode set 921 composed of a plurality of fixed electrodes 911 is arranged in a matrix of p × q in some of the plurality of spot blocks 91. Since the plurality of fixed electrodes 911 correspond to the minimum resolution of the spotting function of the spotting device as described above, the mixed solution is applied to all the fixed electrodes 911 in the one spot block 91 by one spotting. It is dripped.
[0064]
In the case of the example in FIG. 11A, when the mixed agent is dropped onto the spot block 91 in which the electrode set 921 is arranged by the spotting device, the mixed agent is supplied to the p × q fixed electrodes 911 at once.
[0065]
Further, blocks other than the spot block 91 in which the electrode set 921 including only the fixed electrode 911 is disposed include a block in which an electrode set 922 including a combination of the fixed electrode 911 and the reference electrode 912 is disposed, and a fixed electrode. 911, a reference electrode 912, and an electrode set 923 composed of a combination of a counter electrode 913 is a block. The electrode set 922 is arranged in a block (for example, the rightmost column of the matrix-like spot block in FIG. 11A) among the plurality of spot blocks 91 on the DNA chip 1. .
[0066]
The electrode set 923 is arranged in a block arranged in the innermost region among the plurality of spot blocks 91 on the DNA chip 1. The electrode set 921 is disposed in a block other than where the electrode set 922 and the electrode set 923 are disposed.
[0067]
In addition, the spot block 91 in which the electrode set 922 in FIG. 11A is disposed is replaced with an electrode set including only the reference electrode 912, and the spot block 91 including the electrode set 923 is replaced with an electrode set including only the counter electrode 913. May be substituted.
[0068]
FIG. 11B shows another arrangement example of the electrode set. In all the spot blocks 91, the same electrode set 923 including the fixed electrode 911, the reference electrode 912, and the counter electrode 913 is disposed. In each spot block 91, a counter electrode 913 is arranged in the innermost region in the block. Further, a reference electrode 912 is disposed so as to surround the counter electrode 913. And the fixed electrode 911 is arrange | positioned in the outermost area | region.
[0069]
As shown in FIGS. 11 (a) and 11 (b), each spot block is arranged corresponding to the minimum resolution of the spotting device, and an electrode set composed of a plurality of electrodes is arranged in the spot block. Plural kinds of base sequence probes can be arranged in the spotting region. Therefore, the base sequence probe is mounted at a density higher than the resolution of the spotter, and the DNA chip is miniaturized. 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 these may of course be supplied in separate solvents.
[0070]
In addition, arrangement | positioning of the electrode set shown to Fig.11 (a) and (b) is an example to the last. For example, after the counter electrode 913 is arranged in the peripheral region on the substrate 11, the spot block 91 including the electrode set 921 may be arranged in a matrix form in the region surrounded by the counter electrode 913. Moreover, it is good also as a structure which arrange | positioned alternately the spot block 91a which arrange | positions the electrode set 913, and the spot block 91b which arrange | positions the electrode set 911 for every row | line | column. In addition, the arrangement of the electrode set can be variously changed.
[0071]
Among the spot blocks 91 shown above, a voltage is applied between the fixed electrode 911 and the counter electrode 913, and the reaction with the probe that has caused a hybridization reaction due to a reaction such as electrolysis caused by the application of the voltage. A current of a different magnitude flows between these electrodes in a probe that does not generate a current. The signal processing unit 2 can check the hybridization rate by converting the current into an electric signal and displaying it on the display unit 3. In order to obtain a signal necessary for calculating the hybridization rate, a change in redox current, a change in redox potential, a change in capacitance, a change in conductance, and a change in electrochemiluminescence can be used as an index. For example, in order to obtain a change in conductance, it is obtained by measuring the amount of current obtained by applying a constant voltage between the fixed electrode 911 and the counter electrode 913 or by measuring the voltage obtained by passing a constant current. It is done.
[0072]
Then, of the obtained hybridization rates, the hybridization of the complementary probe 121a under the hybridization reaction conditions obtained as a measurement result clearly showing the causal relationship with the number of mismatched bases (the number of non-complementary bases) relative to the target complementary base sequence. The rate can be used as a reliable measurement result.
[0073]
An example of a method for improving the measurement accuracy of the above hybridization rate measurement using statistical processing will be described below.
[0074]
A monitoring system using statistical processing is shown in FIG. The basic configuration is the same as that of the monitoring system of FIG. 1, but the signal processing unit 2 and the statistical processing unit 5 are connected, and the display unit 3 is connected to the statistical processing unit 5. 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 causes the display unit 3 to display the result. In addition, the statistical processing unit 5 includes a storage unit 5 a that stores the statistical processing result calculated by the statistical processing unit 5.
[0075]
In the technique using statistical processing, first, the first quasi-complementary probe RT having i bases that are not complementary to the target base sequence_iThe DNA chip 1 includes an electrode on which each probe of the first quasi-complementary probe group consisting of (i is a natural number of 1 to n) and an electrode on which each second quasi-complementary probe group of the second quasi-complementary probe group is immobilized. To place.
[0076]
The plurality of second quasi-complementary probes constituting the second quasi-complementary probe group have quasi-complementary base sequences having low complementarity to the target base sequence. Furthermore, the number of bases that are not complementary to the target base sequence (the number of non-complementary bases) is the same as that of at least one of the first quasi-complementary probes. Further, the plurality of types of second quasi-complementary probes constituting the second quasi-complementary probe group have different base sequences.
[0077]
Here, the first quasi-complementary probe RT_i(I is a natural number of 1 to n) n types and each first quasi-complementary probe RT_iThe second quasi-complementary probes with the same number of non-complementary bases and different base sequences were immobilized in a total of mxn types of quasi-complementary probes, each of which is different from the first quasi-complementary probe in different types of base sequences. The electrode was placed on the DNA chip 1. A complementary probe having a base sequence complementary to the target base sequence (target complementary base sequence) is also immobilized.
[0078]
Next, supply of the above-described sample base sequence, hybridization reaction, dropping of the intercalating agent, signal in the signal processing unit 2 to the DNA chip 1 on which these m × n types of quasi-complementary probes and complementary probes are arranged. Perform detection. Here, for convenience of explanation, the sample base sequence and the target base sequence are the same.
[0079]
The obtained signal processing result is output to the statistical processing unit 5 as a hybridization rate. Hereinafter, j-th probe RT out of m probes having i bases that are not complementary to the target base sequence_ijHybridization rate of RTO_ijAnd
[0080]
Specifically, a complementary probe RT having zero bases that are not complementary to the target base sequence_0jHybridization rate of RTO₀₀Quasi-complementary probe RT having one part that is not complementary to the target base sequence_1jHybridization rate of RTO₁₁~ RTO_1mQuasi-complementary probe RT having n portions not complementary to the target base sequence_njHybridization rate of RTO_n1~ RTO_nmGiven in.
[0081]
The statistical processing unit 5 obtains the obtained hybridization rate RTO._ijBased on the above, at least one of the following statistical processing methods (1) to (4) is performed.
[0082]
In statistical processing method (1), hybridization rate RTO with the same number of mismatched bases_ij(RTO_i1~ RTO_im) Is calculated. The hybridization rate RTO deviates from the average value by a predetermined threshold value φ or more._ijHybridization rate RTO within the range of the threshold φ_ijExtract only. The threshold value φ may be determined according to the number of mismatched bases, or may be given in common to all the number of mismatched bases. And the extracted hybridization rate RTO_ijIs displayed on the display unit 3 for monitoring.
[0083]
Thereby, an abnormal hybridization reaction can be excluded and the reliability of the chip output value can be improved.
[0084]
In statistical processing method (2), hybridization rate RTO with the same number of mismatched bases_i1~ RTO_imAnd the total value S of the hybridization rates with the same number of mismatched bases.
S = RTO_i1+ RTO_i2+ ... + RTO_im
Is calculated. Then, the obtained total value S is monitored as a representative value of the hybridization rate for the number of mismatched bases. This enables highly reliable monitoring even when the hybridization rate is very small.
[0085]
In statistical processing method (3), hybridization rate RTO with the same number of mismatched bases_ij(RTO_i1~ RTO_im) Is calculated. The hybridization rate RTO deviates from the average value by a predetermined threshold value φ or more._ijHybridization rate RTO within the range of the threshold φ_ijExtract only. The threshold value φ may be determined according to the number of mismatched bases (number of non-complementary bases), or may be given in common to all the number of mismatched bases (number of non-complementary bases). Good. And the extracted hybridization rate RTO_ijHybridization rate (RTO) with the same number of mismatched bases (number of non-complementary bases)_i0'~ RTO_im′) Average value (extraction average value) is calculated. The obtained extraction average value is monitored as a representative value of the hybridization rate with respect to the number of mismatched bases (the number of non-complementary bases). As a result, hybridization rates with the same number of mismatched bases (the number of non-complementary bases) can be averaged, and highly reliable monitoring is possible.
[0086]
In statistical processing method (4), hybridization rate RTO with the same number of mismatched bases_ij(RTO_i1~ RTO_im) Is calculated. The hybridization rate RTO deviates by more than a predetermined threshold φ_ijHybridization rate RTO within the range of the threshold φ_ijExtract only. The threshold value φ may be determined according to the number of mismatched bases, or may be given in common to all the number of mismatched bases (number of non-complementary bases). And the extracted hybridization rate RTO_ijHybridization rate (RTO) with the same number of mismatched bases (number of non-complementary bases)_i1'~ RTO_im′) Average value (extraction average value) is calculated. Then, using the obtained extracted average value, the variance value s of hybridization rates with the same number of mismatched bases (number of non-complementary bases)_iIs calculated. Variance value s_iIs calculated by the following equation.
[0087]
RTO_i_ 'Is calculated after excluding a hybridization rate that deviates by more than a predetermined threshold value φ, and a hybridization rate RTO with the same number of mismatched bases (number of non-complementary bases) i_ij’(RTO_i1'~ RTO_im′) Extracted average value and RTO_i_ ’= (RTO_i1'+ RTO_i2’+… + RTO_im′) / M.
[0088]
s_i= {(RTO_i1'-RTO_i_ ’)²+ (RTO_i2'-RTO_i_ ’)²+ ... + (RTO_im'-RTO_i_ ’)²} / (M-1)
And the extracted variance value s_iTo determine whether each obtained hybridization rate is within a certain range. If it is determined that it falls within a certain range, it indicates that the hybridization reaction condition can be adopted, and if it is judged that it does not fall within a certain range, the hybridization reaction condition cannot be adopted. It is shown that.
[0089]
The statistical processing methods (1) to (4) may be applied separately to the present invention, or may be applied in combination with each other. In statistical processing methods (1) to (4), only one type of complementary probe is used, and the hybridization rate RTO of the one type of probe is used.₀₀Although the case where statistical processing is performed using is shown, it is not limited to this. For example, although there are 0 bases that are not complementary to the target base sequence, m different types of complementary probes may be prepared and statistical processing may be performed in the same manner as the second quasi-complementary probe. In the statistical processing methods (1) to (4), the value of i indicating the number of bases not complementary to the target base sequence is calculated as 1 to n. However, when m kinds of complementary probes are used, i The value may be calculated as 0 to n.
[0090]
As described above, according to the present embodiment, at the time of the hybridization reaction, at least one hybridization reaction condition is changed in real time, and the occurrence rate of an erroneous hybridization reaction is correlated with the changed hybridization reaction condition. Since the monitoring can be performed quantitatively, the optimal hybridization reaction conditions can be easily obtained by a small number of hybridization experiments. As a result, the development cost of the DNA chip can be reduced.
[0091]
In the above embodiment, the example in which the hybridization rate is detected based on the voltage between the fixed electrode 911 and the counter electrode 913, the amount of charge flowing in the fixed electrode 911, the current, and the like has been described. Is not to be done. For example, the imaging system 6 may be separately connected to the signal processing unit 2 without directly connecting the monitoring system and the signal processing unit 2 shown in FIG. 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, and fluorescence is generated in the probe in which hybridization has occurred. An example of a hybridization state monitoring system is shown in FIG.
[0092]
As shown in FIG. 13, a signal processing unit 2 uses a signal indicating the brightness of fluorescence generated by the hybridization reaction of the probes 121a and 122a of the DNA chip 1 imaged by the imaging means 6 or image data obtained by imaging the brightness of fluorescence. Output to. The signal processing unit 2 can calculate the hybridization rate for each of the probes 121a and 122a by processing the received signal and image data. Of course, the imaging means 6 can also be applied to the monitoring system shown in FIG.
[0093]
Further, the configuration 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 in FIG.
[0094]
The base sequence of the first quasi-complementary probe (2 ′) 1222 ′ is a base that is not adjacent to a base different from the target-complementary base sequence of the first quasi-complementary probe (1) 1221, but is different from the target complementary base sequence. The base sequence is as follows. Therefore, the first quasi-complementary probe (2 ′) 1222 ′ has a base sequence in which two bases in the target complementary base sequence in the complementary probe 121a are replaced with other bases, and these two bases are adjacent to each other. Not done.
[0095]
The base sequence of the first quasi-complementary probe (3 ′) 1223 ′ is such that one base that is not adjacent to two bases different from the target-complementary base sequence of the first quasi-complementary probe (2 ′) 1222 ′ It has a base sequence that is different from the base sequence. Therefore, the first quasi-complementary probe (3 ′) 1223 ′ has a base sequence in which three bases in the target complementary base sequence in the complementary probe 121a are replaced with other bases, and the three bases are Not adjacent.
[0096]
Thus, the first quasi-complementary probe (i ′) 122i is different from the first quasi-complementary probe ((i + 1) ′) 122 (i + 1) in the number of mismatched bases with respect to the target complementary base sequence, and the mismatched The bases may not be adjacent to each other in the base sequence.
[0097]
(Second Embodiment)
This embodiment relates to a modification of the first embodiment. In the first embodiment, an example in which single nucleotide polymorphism (SNP) is not taken into consideration has been shown, but this embodiment relates to an embodiment in which the target nucleotide sequence has a single nucleotide polymorphism. Descriptions of parts common to the first embodiment are omitted.
[0098]
FIG. 15 is a diagram showing a detailed configuration of the DNA chip 1 of the present embodiment. As shown in FIG. 15, a plurality of probe electrodes 201 and 202 are provided on the substrate 11. The probe electrode 201 is a complementary probe electrode, and four 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 a quasi-complementary probe 202a is fixed. The complementary probe electrodes 201a to 201d are a base A detection complementary probe electrode 201a, a base T detection complementary probe electrode 201b, a base C detection complementary probe electrode 201c, and a base G detection complementary probe electrode 201d.
[0099]
The base A detection complementary probe 2011a is fixed to the base A detection complementary probe electrode 201a, the base T detection complementary probe 2011b is fixed to the base T detection complementary probe electrode 201b, and the base C detection complementary probe electrode 201c. The base C detection complementary probe 2011c is fixed to the base G detection complementary probe 2011d, and the base G detection complementary probe 2011d is fixed to the base G detection complementary probe 201d.
[0100]
FIG. 16 is a conceptual diagram of the base sequences of the complementary probes 2011a to 2011d, the semi-complementary probe 202a, and the target base sequence 200 of this embodiment. The target base sequence 200 has SNPs base 2001.
[0101]
The base A detection complementary probe 2011a is a base sequence complementary to the target base sequence when the SNPs base 2001 of the target base sequence 200 is set to A (target complementary base sequence: GTCG AAG). -T). The complementary probe 2011b for detecting the base T has a base sequence complementary to the target base sequence when the SNPs base 2001 of the target base sequence is set to T (target complementary base sequence: GTCTGA-G- T). The complementary probe 2011c for detecting base C is a base sequence complementary to the target base sequence when the SNPs base 2001 of the target base sequence is C (target complementary base sequence: GTCC-A-G- T). The base G detection complementary probe 2011d 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: GTCGAGG). T).
[0102]
The quasi-complementary probe 202a is a base sequence in which one base is substituted with another base in the target complementary base sequence when the SNPs base 2001 is T in the target base sequence 200 (G-T-C-T- The first quasi-complementary probe (1) 2021, having a TGT), and the first quasi-complement having a base sequence (GTCCTT) having two bases substituted with other bases Complementary probe (2) 2022, first quasi-complementary probe (3) 2023 having a base sequence (GTCCTTCA) in which three bases are substituted with other bases Has a first quasi-complementary probe (i) 202i having i different base sequences.
[0103]
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 this embodiment, the base T detection complementary probe when the hybridization rate of the base T detection complementary probe 2011b is maximized. The hybridization rate of 2011b is TO, and the hybridization rate of quasi-complementary probe 202i is RTO._iTO and RTO_iBy measuring the relationship, it is possible to obtain an abnormality in the hybridization reaction and an appropriate indicator of the hybridization reaction temperature.
[0104]
Thus, 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.
[0105]
The quasi-complementary probe 202a has been described with respect to the case where the base corresponding to the SNPs base 2001 of the target base sequence 200 is T. However, when the base is replaced with another base A, C, or G, the same is set separately. . Further, four types of T, A, C and G corresponding to the SNP base 2001 of the target base sequence 200 are set, and the semi-complementary probes 202a to 202d are fixed to the semi-complementary probe electrode 202 for monitoring. Also good.
[0106]
The present invention is not limited to the above embodiment.
[0107]
In the above embodiment, an example in which the hybridization reaction is optimized by detecting the base sequence of DNA as the sample base sequence has been described. However, the present invention can be widely applied to base sequences such as other RNAs in addition to DNA. .
[0108]
Moreover, although the example which displays a measurement result on the display part 3 was shown as an example of an output means, it is not limited to this, You may notify optimal hybridization reaction conditions with an audio | voice.
[0109]
In addition, although the detection of the occurrence rate of the hybridization reaction has been illustrated by performing charge amount calculation, conductance calculation, fluorescence imaging, and the like, it is not limited thereto.
[0110]
Further, in the detailed configuration example of the DNA chip 1 illustrated in FIG. 11, an example in which three types of electrodes, the fixed electrode 911, the reference electrode 912, and the counter electrode 913 are arranged on the substrate 11, is illustrated. Of course, the counter electrode 913 may be omitted as appropriate according to the measurement method and the like, and the present invention can be applied even if only the fixed electrode 911 is disposed. When the reference electrode 912 and the counter electrode 913 are omitted, the reference electrode 912 and the counter electrode 913 may be disposed on a substrate different from the substrate on which the fixed electrode 911 is disposed.
[0111]
The constituent materials of the complementary probe, semi-complementary probe, and sample base sequence are not particularly limited, but nucleic acids such as DNA, RNA, PNA, methylphosphonate skeleton, and other nucleic acid analogs can be used.
[0112]
【The invention's effect】
As described above in detail, according to the present invention, hybridization conditions can be optimized.
[Brief description of the 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 a DNA chip according to the embodiment.
FIG. 3 is a diagram showing a conceptual diagram of a quasi-complementary probe and a target base sequence according to the embodiment.
FIG. 4 is a view showing an example of a measurement result according to the embodiment.
FIG. 5 is a view showing an example of a measurement result according to the embodiment.
FIG. 6 is a view showing an example of a measurement result according to the embodiment.
FIG. 7 is a view showing an example of a measurement result according to the embodiment.
FIG. 8 is a view showing an example of a measurement result according to the embodiment.
FIG. 9 is a view showing an example of a measurement result according to the embodiment.
FIG. 10 is a view showing a 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 embodiment.
FIG. 12 is a diagram showing an example of a monitoring system using statistical processing according to the embodiment.
FIG. 13 is a view showing a modification of the monitoring system using statistical processing according to the embodiment.
FIG. 14 is a view showing a modification of the quasi-complementary probe according to the 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 the base sequences of the complementary probe, semi-complementary probe 202a, and target nucleic acid chain according to the embodiment;
[Explanation of symbols]
1 ... DNA chip
2 ... Signal processor
3 ... Display section
4 ... Electrode controller
91 ... Spot block
120: Target base sequence
121 ... Complementary probe electrode
121a ... complementary probe
122... Semi-complementary probe electrode
122a ... Semi-complementary probe
921-923 ... Electrode set
1221: First quasi-complementary probe (1)
1222 ... First quasi-complementary probe (2)
1223 ... First quasi-complementary probe (3)

Claims (9)

A substrate,
A plurality of kinds of first bases having a quasi-complementary base sequence which is immobilized on the substrate and has a lower complementarity to the target base sequence than a target complementary base sequence complementary to the target base sequence to be detected A plurality of types of first quasi-complementary probes having a quasi-complementary probe, and a first quasi-complementary probe group having different numbers of non-complementary bases that are not complementary to the target base sequence, Chips,
Detection means for detecting a rate of occurrence of a hybridization reaction between each first quasi-complementary probe and the sample base sequence in the base sequence detection chip;
Changing means for changing reaction conditions of the hybridization reaction;
Storage means for storing the occurrence rate of the hybridization reaction detected by the detection means in association with the reaction conditions;
A hybridization monitoring system comprising detection means for detecting an optimal reaction condition based on the rate of occurrence of the hybridization reaction under each of the reaction conditions. Furthermore, a plurality of types of second quasi-complementary probes having a quasi-complementary base sequence immobilized on the substrate and having a lower complementarity to the target base sequence than a target complementary base sequence complementary to the target base sequence has the plurality of second quasi complementary probes, the hybridization monitoring system of claim 1 non-complementary bases numbers comprising different second semi-complementary probe groups of identical and nucleotide sequence to each other. On the substrate, the region where the solvent spreads on the substrate by one drop of the solvent of the spotting device, and the solvent does not overlap with other regions where the solvent spreads on the substrate by another drop of the solvent, The hybridization monitoring system according to claim 1 , wherein the hybridization monitoring system has a plurality of regions that minimize the distance of each solvent dropping position, and a plurality of types of the semi-complementary probes are formed in each of the regions. Generation of a hybridization reaction in a plurality of semi-complementary probes having the same number of non-complementary bases and different base sequences from each other in the first and second semi-complementary probe groups based on the occurrence rate detected by the detection means A first statistical process for calculating an average value of the rates and excluding the reaction occurrence rate deviating from the average value by a predetermined threshold value φ or more, a plurality of non-complementary base numbers being the same and different base sequences from each other A second statistical process for calculating the representative value by adding the occurrence rates of the hybridization reactions in the quasi-complementary probes, and a third statistical value for calculating the average value of the reaction occurrence rates obtained as a result of exclusion by the first statistical process. Statistical processing means for executing at least one of statistical processing and fourth statistical processing for calculating a variance value of the reaction occurrence rate obtained as a result of being excluded by the first statistical processing is further provided. Hybridization monitoring system according to claim 2, characterized in that it comprises. An immobilized electrode disposed on the substrate and immobilizing the first quasi-complementary probe or the second quasi-complementary probe; and a counter electrode;
Said detecting means, hybridization monitoring system according to claim 1 or 2, characterized in that to calculate the incidence of hybridization reaction based on the amount of charge flowing from the fixed electrode. An immobilized electrode disposed on the substrate and immobilizing the first or second quasi-complementary probe; and a counter electrode;
The hybridization monitoring according to claim 1 or 2 , wherein the detection means calculates a rate of occurrence of a hybridization reaction based on a conductance obtained based on a voltage or a current between the immobilized electrode and the counter electrode. system. An imaging means for imaging the first or second quasi-complementary probe of the base sequence detection chip after the hybridization reaction is further provided, and the detection means is based on the brightness of the fluorescence imaged by the imaging means. hybridization monitoring system according to claim 1 or 2, characterized in that to calculate the incidence of. A plurality of types of substrates having a quasi-complementary base sequence immobilized on the substrate and having a lower complementarity to the target base sequence than a target complementary base sequence complementary to the target base sequence to be detected A first quasi-complementary probe group, wherein the plurality of types of first quasi-complementary probes are arranged on the substrate with a first quasi-complementary probe group having different numbers of non-complementary bases that are not complementary to the target base sequence; The quasi-complementary base sequence and the sample base sequence are arranged by contacting a base sequence detection chip, which is arranged and has an immobilized electrode for immobilizing the first quasi-complementary probe, with a test solution containing the sample base sequence. Hybridization reaction,
Detecting the rate of occurrence of a hybridization reaction between each of the first quasi-complementary probes and the sample base sequence;
A method for monitoring a hybridization reaction, wherein the hybridization reaction is performed under a plurality of reaction conditions, and an optimal reaction condition is detected based on an occurrence rate of the hybridization reaction in each of the reaction conditions. The hybridization reaction monitoring method according to claim 8 , wherein the plurality of reaction conditions are temperature conditions given by changing a temperature of the base sequence detection chip.

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