JP2007295872A - Method for designing probe nucleic acid sequence, probe nucleic acid, detection surface, program, information-memorizing medium, and system for designing probe nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To consider the interaction of a double strand nucleic acid with an intercalator to improve the detection accuracy, quantitativity, and the like of the double strand nucleic acid on a bioassay or the like at a step for designing a probe nucleic acid. <P>SOLUTION: This method for designing the probe nucleic acid, comprising selecting a sequence used for the probe nucleic acid from a base sequence obtained as gene information, is characterized by including a process for removing a sequence region having a prescribed base sequence from the selected range on the basis of profile data related from the double strand nucleic acid with the intercalator, or a process for weighting the sequence region. Herein, the profile data is data for each base sequence, related to the fluorescence intensity of the intercalator, the interaction quantity of the double strand nucleic acid with the intercalator, and the like. The detection accuracy, quantitativity, and the like of the double strand nucleic acid on a bioassay or the like can be improved by designing the probe nucleic acid on the basis of the present invention. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a probe nucleic acid design method for selecting a sequence to be used for a probe nucleic acid from a base sequence acquired as genetic information based on profile data relating to the interaction between a double-stranded nucleic acid and an intercalator, and related to them The present invention relates to a nucleic acid probe, a detection surface, a DNA chip, a program, an information storage medium, a probe nucleic acid design system, and the like.

  In recent years, techniques using probe nucleic acids have been widely used as gene analysis means in the fields of medicine and physiology.

  For example, a probe nucleic acid is immobilized on a detection surface (electrode surface, etc.) of a QCM (quartz crystal microbalance) device or SPR (surface plasmon resonance biosensor), and the binding (hybridization) between the probe nucleic acid and the target nucleic acid is performed. Some technologies for detecting the change in weight have been put into practical use.

  In addition, for example, a technique for comprehensively detecting hybridization between a probe nucleic acid and a target nucleic acid by immobilizing a large number of probe nucleic acids on a solid phase substrate such as a DNA chip has been put into practical use. ing.

  For example, when a target nucleic acid is detected using a probe nucleic acid, it is preferable that each probe nucleic acid has a specific sequence and the reactivity between each probe nucleic acid and the corresponding target nucleic acid is uniform. Therefore, the design of probe nucleic acid generally involves the melting temperature of double-stranded nucleic acid formed by hybridization of probe nucleic acid and target nucleic acid, the length of probe nucleic acid, the presence or absence of homologous sequences in other probe nucleic acids, etc. Take this into consideration. That is, in the design stage of the probe nucleic acid, a sequence that matches those conditions is selected from the entire base sequence of the target gene, and the sequence is used as the probe nucleic acid.

  Here, an intercalator will be described below as a matter related to the present invention. An intercalator is a substance that emits fluorescence by binding to a predetermined site of a double-stranded nucleic acid. In recent years, a part of means for detecting a double-stranded nucleic acid formed by hybridization of a probe nucleic acid and a target nucleic acid using an intercalator has been put into practical use.

For example, Patent Document 1 discloses a probe design method including a step of calculating a difference in melting temperature of oligonucleotide sequences and selecting a probe having the difference of 0 to 7 ° C. A probe sequencing system for the array is each described.
JP 2005-318895 A JP 2003-52385 A

  In designing the probe nucleic acid, little consideration is given to the interaction between the double-stranded nucleic acid and the intercalator. Therefore, the present invention improves the detection accuracy and quantification of double-stranded nucleic acid during bioassay, etc. by considering the interaction between double-stranded nucleic acid and intercalator at the design stage of probe nucleic acid. Is the main purpose.

  As for the interaction between the double-stranded nucleic acid and the intercalator, the present inventor has determined the intercalator fluorescence sensitivity, the amount of interaction between the double-stranded nucleic acid and the intercalator, the binding constant between the double-stranded nucleic acid and the intercalator, etc. Have newly found that there are different characteristics depending on the sequence of the double-stranded nucleic acid.

  Therefore, in the present invention, there is provided a probe nucleic acid design method for selecting a sequence to be used for a probe nucleic acid from a base sequence acquired as gene information, which is based on profile data relating to the interaction between a double-stranded nucleic acid and an intercalator. There is provided a probe nucleic acid design method comprising a step of excluding a sequence region having a nucleotide sequence of from the selection range or a step of weighting the sequence region.

  Here, the profile data is data for each base sequence regarding the fluorescence intensity of the intercalator, the amount of interaction between the double-stranded nucleic acid and the intercalator, the binding constant between the double-stranded nucleic acid and the intercalator, and the like.

  For example, when detecting hybridization by the fluorescence intensity of the intercalator at the time of bioassay, etc., in the probe nucleic acid design stage, a sequence region having a predetermined base sequence based on profile data for each base sequence relating to the fluorescence intensity of the intercalator Is excluded from the selection. This makes it possible to design a probe nucleic acid with uniform fluorescence sensitivity when a double-stranded nucleic acid interacts with an intercalator. By using the probe nucleic acid, highly accurate and quantitative detection can be performed during bioassays. It can be carried out.

  For example, when detecting hybridization or the like based on the amount of interaction between a double-stranded nucleic acid and an intercalator at the time of a bioassay, etc., the base sequence relating to the amount of interaction between the double-stranded nucleic acid and the intercalator at the probe nucleic acid design stage A sequence region having a predetermined base sequence is excluded from the selection range based on the profile data for each. As a result, it is possible to design a probe nucleic acid in which the interaction amount between the double-stranded nucleic acid and the intercalator is uniform. By using the probe nucleic acid, highly accurate and quantitative detection can be performed at the time of bioassay or the like. .

  For example, when detecting hybridization by the binding constant between a double-stranded nucleic acid and an intercalator at the time of bioassay, etc., for each base sequence related to the binding constant between the double-stranded nucleic acid and the intercalator at the probe nucleic acid design stage A sequence region having a predetermined base sequence is excluded from the selection range based on the profile data. This makes it possible to design a probe nucleic acid that has a uniform binding constant when the double-stranded nucleic acid interacts with the intercalator. Therefore, by using the probe nucleic acid, highly accurate and quantitative detection can be performed during bioassays. It can be performed.

  Hereinafter, technical terms according to the present invention will be described.

  “Nucleic acid” refers to a polymer (nucleotide chain) of a nucleoside phosphate ester in which a purine or pyrimidine base and a sugar are glycosidically linked, and an oligonucleotide, polynucleotide, purine nucleotide and pyrimidine nucleotide containing a nucleic acid probe are polymerized. Widely includes DNA (full length or a fragment thereof), cDNA obtained by reverse transcription (cDNA probe), RNA, polyamide nucleotide derivative (PNA) and the like.

  “Double-stranded nucleic acid” includes all nucleic acids in which complementary nucleic acids are hybridized. For example, a double-stranded nucleic acid or a double-stranded nucleic acid part formed by hybridization of a probe nucleic acid and a target nucleic acid is also included in the double-stranded nucleic acid according to the present invention. Here, the “probe nucleic acid” is a nucleic acid (nucleotide chain) that functions as a detector for detecting a target nucleic acid, and is fixed on a detection surface or the like. The “target nucleic acid” is a nucleic acid having a sequence complementary to the base sequence of the nucleic acid probe for detection over the entire length or a part thereof, and is dropped or supplied to the reaction field when detecting hybridization.

  An “intercalator” is a substance that emits fluorescence by binding to a predetermined site of a double-stranded nucleic acid.

  “Interaction” is the binding of two substances to each other.

  “Hybridization” is a complementary bond between nucleic acids (nucleotide strands), and is a polymer-polymer, polymer-small molecule, small molecule-small molecule specific bond, DNA-DNA, DNA-RNA, RNA -Widely includes specific binding between RNA and the like.

  “Profile data” is information acquired and stored in advance regarding a specific matter.

  By designing the probe nucleic acid based on the present invention, it is possible to improve the detection accuracy and quantitativeness of the double-stranded nucleic acid during bioassay.

<About the probe nucleic acid design method according to the present invention>
First, an example of the probe nucleic acid design method according to the present invention will be described below with reference to FIGS. The probe nucleic acid design method according to the present invention includes at least a step of excluding a sequence region having a predetermined base sequence from a selection range based on profile data relating to the interaction between a double-stranded nucleic acid and an intercalator. Includes all. Therefore, the present invention is not limited to a narrow range depending on whether or not other steps shown below are included, for example.

  FIG. 1A and FIG. 1B are flowcharts showing an example of general processing steps of probe nucleic acid design. In addition, since this figure is a flowchart which shows the outline | summary of a process process, the conditional branch etc. in each process are abbreviate | omitted in this figure.

  First, gene information is acquired from a database of gene information (reference S1) and stored in the information storage medium A or the like. As a database of gene information, for example, a public database such as Gen Bank can be used. The acquired gene information is preferably described in a data format that can be cross-referenced, such as Fasta header information.

  Next, a base sequence of a specific gene is extracted from the stored gene information (reference S3), and filter processing (reference S4, S5), motif processing (reference S6), and the like are performed on the base sequence.

  The filtering process is a process of masking a sequence region that is not suitable as a probe nucleic acid in the base sequence of a specific gene and excluding it from the selection range. By excluding a sequence region that is not suitable as a sequence used for the probe nucleic acid from the entire base sequence in advance, the search process can be omitted, so that the calculation time can be shortened.

  In FIG. 1A, the filter process 1 (reference S4) is a filter process for a repeated arrangement. There are many repetitive sequences in genetic information. Since repeated sequences are widely present in many genes and the like, they are not suitable for sequences used for probe nucleic acids. Therefore, in the filter processing step, the search process is omitted by excluding sequences that repeatedly appear from the selection range.

  In FIG. 1A, filter process 2 (reference S5) is a filter process for an array region having a high appearance frequency. Since a sequence region having a high appearance frequency is not a specific sequence, it is not suitable as a sequence used for a probe nucleic acid. Therefore, in the filter processing step, the search process is omitted by excluding array regions with high appearance frequency from the selection range.

  The motif process (reference S6) is a process for marking a region to be selected (motif region). For example, when designing a probe nucleic acid to be used for identification of a template for a certain disease group, diagnosis of invasion / subtype in malignant tumors, etc., a sequence containing a specific gene mutation site (splicing abnormality site, insertion / deletion site, etc.) You need to select a region. Therefore, a sequence including the essential sequence region can be selected by marking the essential sequence region by motif processing.

  Note that whether or not to perform each of these processes and the order in which these processes are performed can be arbitrarily set according to the purpose.

  Next, a sequence to be used for the probe nucleic acid is selected from sequence regions determined to be selectable by filter processing (reference S4, S5) and motif processing (reference S6) (reference S7 to S15).

  A sequence selection procedure to be used for the probe nucleic acid is determined (reference S8), and one candidate sequence is obtained from the sequence region determined to be selectable based on the selection procedure (reference S9). The candidate sequence is subjected to inspection whether the melting temperature (Tm) is within a predetermined range, whether it is a stable secondary structure, homology search, etc., and conforms to these conditions. Things are obtained as sequences used for probe nucleic acids. On the other hand, if the candidate sequence does not meet these conditions, the process returns to step 9 to acquire another candidate sequence, and the same processing is performed thereafter. By looping the above procedure (reference numerals S7 to S15), one or a plurality of sequences that can be used for the probe nucleic acid can be obtained from a specific base sequence.

  The melting temperature inspection routine (reference S10) is a processing step for inspecting whether or not the melting temperature (Tm) is within a predetermined range when a probe nucleic acid is prepared with the obtained candidate sequence. By this processing step, the melting temperature of each probe nucleic acid can be made uniform, so that comprehensive and quantitative analysis becomes possible at the time of double-stranded nucleic acid analysis and the like.

  The secondary structure inspection routine (reference S11) is a processing step for inspecting whether a candidate sequence forms a stable secondary structure. By this processing step, the self-loop formation of the designed probe nucleic acid can be eliminated in advance, so that the detection accuracy at the time of double-stranded nucleic acid analysis can be increased.

  The homology search 1 (reference S12) is a process of searching for homology between the entire base sequence of the specific gene extracted in the processing step of reference S3 and the candidate sequence of the probe nucleic acid. For example, in the case of a bioassay using a probe nucleic acid, if there are a plurality of portions having homology with the sequence of the probe nucleic acid in the base sequence of the target gene, the accuracy in detecting the hybridization, Quantitative performance decreases. On the other hand, in this processing step, by removing sequences having homology with a plurality of positions of the base sequence of a specific gene from the probe nucleic acid candidates, those adverse effects can be eliminated.

  The homology search 2 (reference S13) is a step of searching for homology between the genetic information obtained in the step S1 and the candidate sequence of the probe nucleic acid. For example, if the candidate sequence has homology with the base sequence of another gene, they may hybridize. Therefore, by this processing step, sequences having homology with the base sequences of other genes are excluded from probe nucleic acid candidates.

  As described above, by looping the steps S3 to S16, one or a plurality of sequences that can be used for the probe nucleic acid can be obtained for each of the base sequences of the gene.

  In addition, a well-known thing can be used for the algorithm of the arithmetic processing in each process.

  FIG. 2 is a diagram showing a process of selecting a bioassay method to be employed in the probe nucleic acid design method according to the present invention.

  Examples of a bioassay method for quantitatively detecting hybridization between a probe nucleic acid and a target nucleic acid using an intercalator include, for example, (a) a method for obtaining a hybridization amount based on fluorescence intensity from an intercalator, (b) There are a method for obtaining the amount of hybridization by obtaining the amount of interaction of the intercalator for the double-stranded nucleic acid, and a method for obtaining the amount of hybridization by (c) thermodynamic measurement of the intercalation reaction.

  Therefore, in order to design a probe nucleic acid suitable for the bioassay method to be employed, in the probe nucleic acid design method according to the present invention, as shown in FIG. 2, the step (i) or (ii) in FIG. And a step of selecting a bioassay method to be adopted is added.

  FIG. 3 is a diagram showing an example of an additional flow in the case of designing a probe nucleic acid used in a bioassay method for obtaining a hybridization amount based on fluorescence intensity from an intercalator (when (a) is selected in FIG. 2). .

  When (a) is selected in FIG. 2, the flow shown in FIG. 3 is added to any one of steps (vii), (viii), and (ix) in FIG.

  For example, when “SYBR Green I (Molecular Probes, USA)” is used as an intercalator during bioassay, the fluorescence intensity in the interaction between the double-stranded nucleic acid and the intercalator is the GC content of the double-stranded nucleic acid. Is high in a portion where GC is relatively high, and a portion where the GC pair and AT pair are randomly arranged, and low in a portion where the AT content is relatively high.

  Therefore, as shown in FIG. 3, among candidate sequences remaining in the previous processing steps, those having a GC content within a predetermined range and not having a predetermined number or more of AT sites are left as probe nucleic acid candidate sequences. For those candidate sequences, the subsequent processing steps are continued. On the other hand, when those conditions are not satisfied, the sequence is excluded from the candidate sequences of the probe nucleic acid, and the next candidate sequence is obtained by returning to the step of obtaining the candidate sequence (symbol S9 in FIG. 1), Each processing step is performed on the candidate sequence. In addition, in this processing step, the array region may be weighted and the following processing steps may be continued.

  The numerical values (X1, X2) relating to the GC content and the number of consecutive AT sites (mer number) are set based on profile data relating to the interaction between the double-stranded nucleic acid and the intercalator obtained in advance.

  FIG. 4 shows an additional flow when designing a probe nucleic acid to be used in a method for obtaining the amount of hybridization by obtaining the amount of interaction of an intercalator with a double-stranded nucleic acid (when (b) is selected in FIG. 2). It is a figure which shows the example of.

  When (b) is selected in FIG. 2, the flow shown in FIG. 4 is added to any one of steps (vii), (viii), and (ix) in FIG.

  For example, when “SYBR Green I (Molecular Probes, USA)” is used as an intercalator during bioassay, the interaction amount (binding amount) between a double-stranded nucleic acid and an intercalator is There are few GC pairs in a continuous part, a part having a relatively high AT content, and a part in which a GC pair and an AT pair are randomly arranged.

  Therefore, as shown in FIG. 4, among candidate sequences remaining in the previous processing steps, the AT content is within a predetermined range and the AT sites do not continue for a predetermined number or more are left as probe nucleic acid candidate sequences, For those candidate sequences, the subsequent processing steps are continued. On the other hand, if those conditions are not satisfied, the sequence is excluded from the probe nucleic acid sequence candidates as described above, and the next candidate sequence is returned to the step of obtaining the candidate sequence (symbol S9 in FIG. 1). Obtaining and performing each processing step on the candidate sequence. In addition, as described above, in this processing step, the array region may be weighted and the following processing steps may be continued.

  The numerical values (X3, X4) regarding the AT content and the number of consecutive AT sites (mer number) are based on the profile data regarding the interaction between the double-stranded nucleic acid and the intercalator obtained in the same manner as described above. To set.

  FIG. 5 is a diagram showing an example of an additional flow when designing a probe nucleic acid to be used in a method for obtaining a hybridization amount by thermodynamic measurement of an intercalation reaction (when (c) is selected in FIG. 2). is there.

  When (c) is selected in FIG. 2, the flow shown in FIG. 5 is added to any one of steps (iii), (iv), (v), and (vi) in FIG. It is most preferable to add the flow shown in FIG. 5 to the stage (vi), and it is preferable to add it to the stage (v) from the viewpoints of calculation time, arithmetic processing algorithm, and the like. Note that the calculation processing efficiency and the like when the flow shown in FIG. 5 is added to the stage (iii) or (iv) are substantially the same.

  For example, when “SYBR Green I (Molecular Probes, USA)” is used as an intercalator during bioassay, the binding constant between a double-stranded nucleic acid and an intercalator includes a GC pair in the double-stranded nucleic acid. It is high when a minor groove and a minor groove consisting only of an AT pair (but not poly A or poly T) are included, and low at a minor groove including an AT pair and a poly A or poly T sequence site.

  Therefore, as shown in FIG. 5, among the sequence regions not excluded from the selection range in the previous processing step, the number of GC pairs in the adjacent 10 base pairs is 2 or more, and contains GC pairs. The region in which the two regions of 10 base pairs are not separated by 5 base pairs or more is set as a region that can be selected as a candidate sequence of the probe nucleic acid, and the subsequent processing steps are continued. On the other hand, those that do not satisfy these conditions are excluded from the selection range (masked), and the subsequent processing steps are continued. In addition, as described above, in this processing step, the array region may be weighted and the following processing steps may be continued.

  Through the above steps, a sequence that can be used for the probe nucleic acid, in particular, a sequence that takes into account the interaction between the double-stranded nucleic acid and the intercalator can be obtained. A probe nucleic acid is designed using the obtained sequence.

<About probe nucleic acid, detection surface, DNA chip, etc. according to the present invention>
The probe nucleic acid, detection surface, DNA chip and the like according to the present invention will be described below.

  The probe nucleic acid can be synthesized by a known method based on the above-described probe nucleic acid design method. Further, depending on the purpose and application, the synthesized probe nucleic acid may be subjected to a predetermined treatment such as biotinylation or linker modification. The linker is a spacer molecule that has a predetermined chain length and connects the detection surface and the probe nucleic acid, and in principle, is a molecule that does not interact with nucleic acids other than the linked probe nucleic acid or an intercalator.

  These probe nucleic acids can be applied to sensor chips, DNA chips, etc. by holding or fixing them on a predetermined detection surface.

  For example, after immersing the probe nucleic acid on the detection surface (electrode surface, etc.) of a QCM (quartz crystal microbalance) device or SPR (surface plasmon resonance biosensor) and soaking in the reaction region, the target nucleic acid and intercalator are Drop sequentially into the reaction zone. Thereby, the hybridization between the probe nucleic acid and the target nucleic acid, and the interaction between the double-stranded nucleic acid formed by the hybridization and the intercalator can be detected as a change in weight.

  In addition, for example, various or many probe nucleic acids can be used as a DNA chip by accumulating and fixing on each detection surface on a solid phase substrate.

  A known means can be used to hold or fix the probe nucleic acid to the detection surface, and is not particularly limited. For example, it is possible to employ means for treating the detection surface with avidin, biotinylating one end of the probe nucleic acid, and holding or immobilizing the probe nucleic acid on the detection surface by an avidin-biotin bond.

<About programs, information storage media, systems, etc. according to the present invention>
The program, information storage medium, system and the like according to the present invention will be described below.

  The probe nucleic acid design method described above can be automatically processed by describing it as a program. For example, a probe nucleic acid design system can be constructed by storing the program in an information storage medium.

  FIG. 6 is a diagram showing an example of a probe nucleic acid design system according to the present invention.

  The probe nucleic acid design system shown in FIG. 6 includes a gene information storage unit 1 that stores gene information acquired from a gene information database, and a probe nucleic acid setting item storage that stores input setting items (such as the chain length of the probe nucleic acid to be designed). Unit 11, target gene base sequence storage unit 21 that stores the base sequence of a specific gene extracted from the gene information, repetitive sequence filter unit 22 that performs a filtering process on the repetitive sequence, filter for a sequence region having a high appearance frequency An appearance frequency filter unit 23 that performs processing, a motif processing unit 24 that performs motif processing, a selection procedure determination unit 31 that determines a sequence selection procedure used for probe nucleic acid, a melting temperature condition inspection unit 32 that performs a melting temperature inspection routine, Secondary structure condition inspection unit 33 for performing secondary structure inspection routine, homology search It comprises such homology search processing unit 34 that performs.

  In addition, the present system includes a bioassay method selection unit 41 that selects a bioassay method to be employed, and an interaction condition filter unit 42 that performs filtering based on profile data relating to the interaction between a single-stranded nucleic acid and an intercalator. You may make it the structure provided with the interaction condition test | inspection part 43 etc. which perform the test | inspection routine of a sequence | arrangement based on the profile data regarding interaction with a double stranded nucleic acid and an intercalator. This makes it possible to design a probe nucleic acid that is compatible with the bioassay method to be employed, and to design a probe nucleic acid in consideration of the interaction between the double-stranded nucleic acid and the intercalator. The interaction condition filter unit 42 is an interaction condition processing unit that executes the flow of (c) in FIG. 2, and the interaction condition inspection unit 43 is (a) or (b) in FIG. It is an interaction condition processing unit that executes a flow such as.

  In addition, this system includes a CPU (medium arithmetic processing device) 51 that executes each program and controls the device, a RAM 52 that stores data necessary for arithmetic processing, an input device 53, an output device 54, the Internet, and the like. The modem 55 is configured to be connected to a public database.

  This system can be mounted on a personal computer, a workstation, or the like.

  In Example 1, the relationship between the base sequence of the double-stranded nucleic acid and the fluorescence intensity of the intercalator was verified.

  First, double-stranded nucleic acid was prepared. First, oligonucleotides having the base sequences shown in SEQ ID NO: 1 to SEQ ID NO: 4 and their complementary strand oligonucleotides were prepared. In this example, the synthesis of both oligonucleotides was commissioned to Tsukuba Oligo Service Co., Ltd. Next, both oligonucleotides were dissolved in 0.1 M phosphate buffer (containing 200 mM NaCl, pH 7.4) so as to be 100 nM each. Next, the solution was heated to 95 ° C. and then cooled to 0 ° C. to anneal both nucleotides to prepare double-stranded nucleic acid.

  In this example, the base sequence shown in SEQ ID NO: 1 was designed assuming a sequence containing poly A or poly T. Similarly, the base sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 4 assuming a sequence containing many AT pairs, and the base sequence shown in SEQ ID NO: 3 is shown assuming a sequence containing many GC pairs. The base sequences were designed assuming a sequence in which GC pairs and AT pairs are randomly arranged.

  Subsequently, an intercalator was added to the double-stranded nucleic acid solution at a final concentration of 1.96 μM. In this example, “SYBR Green I (US Molecular Probes Co., Ltd., hereinafter the same)” was used as an intercalator. Then, after adding SYBR Green I, the solution was incubated at 25 ° C. for 10 minutes.

  Subsequently, the fluorescence spectrum was measured with a fluorometer. A solution to which double-stranded nucleic acid and SYBR Green I were added was placed in the sample chamber of the apparatus, the excitation wavelength was set to 497 nm, and the fluorescence intensity was measured at 25 ° C.

  The results are shown in FIG. FIG. 7 is a graph showing the fluorescence intensity at the peak by a fluorometer.

  In FIG. 7, the horizontal axis represents the sequence number of the double-stranded nucleic acid used, and the vertical axis represents the peak fluorescence intensity (maximum fluorescence intensity).

  As shown in FIG. 7, the fluorescence intensity was low in the double-stranded nucleic acid having the base sequence shown in SEQ ID NO: 2, whereas the double-stranded nucleic acid having the base sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 was fluorescent. The strength was high. As a result, the fluorescence intensity in the interaction between the double-stranded nucleic acid and the intercalator is high in the portion where the GC content of the double-stranded nucleic acid is relatively high, and the portion where the GC pair and AT pair are randomly arranged, and the AT content. Indicates that it is low at a relatively high part.

  In Example 2, the relationship between the base sequence of the double-stranded nucleic acid and the interaction between the double-stranded nucleic acid and the intercalator was verified using a QCM (quartz crystal microbalance) apparatus.

In this embodiment, “AffinixQ (“ Affinix ”is a registered trademark, manufactured by Initium Co., Ltd.)” was used as the QCM device. A sensor chip attached to this device includes an Au electrode (diameter 2.5 mm) and a crystal oscillator (diameter 8 mm). When a substance adheres to the Au electrode, the oscillation frequency of the crystal oscillator changes in proportion to the weight of the substance attached. Therefore, the weight of the substance attached to the Au electrode can be measured by measuring the change in oscillation frequency (ΔF) with a QCM device. In the present embodiment, a change in the oscillation frequency of 1 Hz corresponds to a weight change of 0.62 ng / cm ² .

  The outline of the experimental procedure is shown below.

  First, double-stranded nucleic acids having the base sequences shown in SEQ ID NO: 2 to SEQ ID NO: 4 were prepared. Preparation of double-stranded nucleic acid was carried out in the same manner as in Example 1. Each double-stranded nucleic acid was biotinylated at one end according to the attached protocol.

  Subsequently, the double-stranded nucleic acid was immobilized on an Au electrode substrate. First, the Au surface of the sensor chip was coated with avidin in advance according to the protocol attached to the apparatus. Avidin manufactured by Wako Pure Chemical Industries, Ltd. (product name “Avidin”) was used. In addition, the prepared double-stranded nucleic acid buffer solution was placed in a reaction tank in the apparatus. Then, the sensor chip was immersed in the reaction vessel, and the double-stranded nucleic acid was bound on the Au electrode substrate by an avidin-biotin bond and fixed.

  Subsequently, the reaction vessel was replaced with one containing an intercalator solution, and a sensor chip was immersed therein, and fluctuations in oscillation frequency were measured with a QCM device. As in Example 1, SYBR Green I was used as the intercalator.

  Then, the interaction amount between the double-stranded nucleic acid and the intercalator and the binding constant Ka thereof were acquired from the acquired measurement value (change in oscillation frequency).

The results are shown in Table 1.

  As shown in Table 1, in the double-stranded nucleic acid having the base sequence shown in SEQ ID NO: 2, the interaction amount between the double-stranded nucleic acid and the intercalator and the binding constant thereof were large, whereas SEQ ID NO: 3 and SEQ ID NO: In the double-stranded nucleic acid having the base sequence shown in 4, the interaction amount and its binding constant were small. These results show that the interaction amount and binding constant between the double-stranded nucleic acid and the intercalator are large in the portion where the AT content of the double-stranded nucleic acid is relatively high, the portion where the GC content is relatively high, and the GC pair. Indicates that the AT pair is small in the randomly arranged part.

  In Example 3, the relationship between the base sequence of the double-stranded nucleic acid and the interaction between the double-stranded nucleic acid and the intercalator is thermodynamically determined by ITC (Isothermal Titration Calorimetry; the same shall apply hereinafter). Verified.

  In the case of reaction between substances, heat is generated or absorbed. By measuring the amount of heat, it is possible to obtain knowledge relating to the coupling constant, coupling ratio, enthalpy change, entropy change, etc. of the reaction between substances. Therefore, a thermal profile regarding the interaction between the double-stranded nucleic acid and the intercalator was obtained by ITC, and the contents were verified.

  In this example, “VP-ITC (manufactured by MicroCal, USA)” was used as the ITC apparatus, and “Origin ver 7.0 (manufactured by MicroCal, USA)” was used for control and data analysis of the apparatus.

  The outline of the procedure is shown below.

  First, double-stranded nucleic acids having the base sequences shown in SEQ ID NO: 1 to SEQ ID NO: 4 were prepared. Preparation of double-stranded nucleic acid was carried out in the same manner as in Example 1.

  Next, 9 μM of double-stranded nucleic acid was placed in 0.1 M phosphate buffer (containing 200 mM NaCl, pH 7.4), DMSO was mixed therein to a concentration of 8.75%, and the solution was mixed with ITC. Placed in device cell.

  Next, after dissolving the intercalator in 0.1M phosphate buffer (containing 200 mM NaCl, pH 7.4) to 1.75 mM, 6 μL of the intercalator solution is obtained every 6 minutes using a syringe. It was dripped at the cell of the ITC apparatus. As the intercalator, SYBR Green I was used as in Example 1.

  And the change of calorie | heat amount (reaction heat) was measured using the ITC apparatus, dripping the intercalator solution. In this example, the measurement by ITC was performed under stirring conditions of 25 ° C. and 300 rpm.

  As a control, the heat of dilution was measured. DMSO was mixed with 0.1 M phosphate buffer (containing 200 mM NaCl, pH 7.4) to a concentration of 8.75%, and the control solution was dropped into the cell of the ITC device instead of the intercalator solution. . And the value which remove | divided the calorie | heat amount (dilution heat) at the time of dripping the control solution from the calorie | heat amount (reaction heat) at the time of dripping the intercalator solution was used for the data analysis etc.

  And about the measurement result obtained by ITC, it analyzed using the two site model (two phase model), and acquired the thermodynamic profile.

The results are shown in Table 2 and FIG. 8 (FIGS. 8A to 8D).

In Table 2, “First Step Reaction” and “Second Step Reaction” represent the analysis results of the first reaction and the second reaction in the two-phase model, respectively. In the table, “n” represents the number of intercalator bindings of each phase to the double-stranded nucleic acid, and “Ka (× 10 ⁶ M)” represents the binding constant of each phase in the interaction between the double-stranded nucleic acid and the intercalator. , “ΔH (kcal / mol)” represents the enthalpy change of each phase, “TΔS (kcal / mol)” represents the entropy change, and “ΔG (kcal / mol)” represents the free energy change of each phase.

  8A to 8D are graphs showing measurement results by ITC. FIG. 8A shows the measurement results when using the double-stranded nucleic acid having the base sequence shown in SEQ ID NO: 1, and FIG. 8B shows the measurement results when using the double-stranded nucleic acid having the base sequence shown in SEQ ID NO: 2. FIG. 8C shows the measurement results when the double-stranded nucleic acid having the base sequence shown in SEQ ID NO: 3 is used, and FIG. 8D shows the measurement results when the double-stranded nucleic acid having the base sequence shown in SEQ ID NO: 4 is used. Each is shown.

  In each of FIGS. 8A to 8D, the upper graph represents a change in the amount of heat (reaction heat) when the intercalator solution is dropped. The horizontal axis of the graph represents the time (min) during which the intercalator solution was dropped, and the vertical axis represents the change in heat (reaction heat) for 1 second (μcal / sec). An endothermic reaction occurs when the value decreases with time, and an exothermic reaction occurs when the value increases.

  8A to 8D, the lower graph shows a value obtained by dividing the amount of heat (reaction heat) when the intercalator solution is dropped from the amount of heat (dilution heat) when the control solution is dropped, that is, It represents the amount of heat in the interaction between the double-stranded nucleic acid and the intercalator. The horizontal axis of the graph represents the total amount of intercalator dropped (Molar Ratio; the molar ratio of double-stranded nucleic acid (quantitative) and intercalator in the sample chamber), and the vertical axis represents the amount of heat in 1 mol of dropped intercalator (kcal / mole of). (injectant) respectively.

  As shown in Table 2 and FIG. 8 (FIGS. 8A to 8D), in the double-stranded nucleic acid having the base sequence shown in SEQ ID NO: 2, the interaction between the double-stranded nucleic acid and the intercalator is a two-step reaction. There were features such as a high binding constant. On the other hand, the double-stranded nucleic acids having the base sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 are characterized in that the interaction between the double-stranded nucleic acid and the intercalator is a one-step reaction and the binding constant is low. It was.

  According to the present invention, for example, in a bioassay system that detects hybridization between a probe nucleic acid and a target nucleic acid with an intercalator, the detection accuracy and quantitativeness can be improved.

The flowchart which shows the example of the general process of probe nucleic acid design. Flow diagram showing examples of general processing steps for probe nucleic acid design (continued). The figure which shows the process of selecting the bioassay method employ | adopted in the probe nucleic acid design method which concerns on this invention. The figure which shows the example of the additional flow in the case of designing the probe nucleic acid used for the bioassay method which acquires the amount of hybridization with the fluorescence intensity from an intercalator (when (a) is selected in FIG. 2). An example of an additional flow in the case of designing a probe nucleic acid to be used in a method for obtaining the amount of hybridization by obtaining the amount of interaction of an intercalator for a double-stranded nucleic acid (when (b) is selected in FIG. 2) is shown. Figure. The figure which shows the example of the additional flow in the case of designing the probe nucleic acid used for the method of acquiring the amount of hybridization by the thermodynamic measurement of intercalation reaction (when (c) is selected in FIG. 2). The figure which shows the example of the probe nucleic acid design system which concerns on this invention. In Example 1, the graph which shows the fluorescence intensity at the time of the peak by a fluorometer. The graph showing the measurement result by ITC at the time of using the double stranded nucleic acid which has a base sequence shown in sequence number 1 in Example 3. FIG. The graph showing the measurement result by ITC at the time of using the double stranded nucleic acid which has a base sequence shown in sequence number 2 in Example 3. FIG. The graph showing the measurement result by ITC at the time of using the double stranded nucleic acid which has a base sequence shown in sequence number 3 in Example 3. FIG. The graph showing the measurement result by ITC at the time of using the double stranded nucleic acid which has a base sequence shown in sequence number 4 in Example 3. FIG.

Claims (8)

A probe nucleic acid design method for selecting a sequence to be used for a probe nucleic acid from a base sequence acquired as genetic information,
A probe nucleic acid design method comprising a step of excluding a sequence region having a predetermined base sequence from a selection range or a step of weighting the sequence region based on profile data relating to the interaction between a double-stranded nucleic acid and an intercalator.   The profile data is data for each base sequence relating to either the fluorescence intensity of the intercalator, the amount of interaction between the double-stranded nucleic acid and the intercalator, or the binding constant between the double-stranded nucleic acid and the intercalator. The method for designing a probe nucleic acid according to claim 1.   A probe nucleic acid designed by the probe nucleic acid sequence design method according to claim 1.   A detection surface on which the probe nucleic acid according to claim 3 is held or fixed.   A DNA chip comprising the detection surface according to claim 4. A probe nucleic acid design program for selecting a sequence to be used for a probe nucleic acid from a base sequence acquired as genetic information,
It relates to a step of excluding a sequence region having a predetermined base sequence from a selection range or a step of weighting the sequence region based on profile data relating to the interaction between a double-stranded nucleic acid and an intercalator acquired in advance. Probe nucleic acid design program including description.   An information storage medium storing at least the probe nucleic acid design program according to claim 6. A probe nucleic acid design system for selecting a sequence to be used for a probe nucleic acid from a base sequence acquired as genetic information,
An interaction that excludes a sequence region having a predetermined base sequence from the selection range based on profile data relating to the interaction between a double-stranded nucleic acid and an intercalator, or weights the sequence region based on the profile data A probe nucleic acid design system comprising at least a condition processing unit.