JP4957063B2 - Method for immobilizing probe nucleic acid on detection surface - Google Patents

Description

  The present invention relates to a detection surface used for detecting a double-stranded nucleic acid moiety formed by hybridization of a probe and a target nucleic acid with an intercalator, a double-stranded nucleic acid moiety and an intercalator using the detection surface. The present invention relates to an interaction promotion method that promotes the interaction between the probe and a hybridization detection method that detects hybridization between a probe immobilized on the detection surface and a target nucleic acid.

  Methods using probe nucleic acids have begun to be put into practical use as gene analysis techniques in fields such as medicine and physiology. For example, in the case of a DNA chip or the like, various kinds of probe nucleic acids for detection are immobilized on a solid phase substrate or the like, and target nucleic acids that hybridize with the probe nucleic acids for detection are comprehensively analyzed.

  At present, as one of means for quantitatively and highly accurately detecting hybridization between a probe nucleic acid for detection and a target nucleic acid, means using an intercalator is being studied. An intercalator is a fluorescent substance having the property of entering between double-stranded nucleic acids. For example, a double-stranded nucleic acid portion formed by hybridization of a probe nucleic acid for detection and a target nucleic acid interacts with an intercalator, and the intensity of fluorescence by the intercalator is detected to quantitatively increase the nucleic acid. There is a possibility that it can be analyzed accurately.

For example, Patent Document 1 and Patent Document 2 disclose a technique for detecting a double-stranded nucleic acid using an intercalator in a DNA chip or the like.
JP 2006-10621 A JP 2006-10582 A

  When detecting hybridization between a probe nucleic acid for detection immobilized on a solid phase substrate and the target nucleic acid using an intercalator, compared to detecting hybridization in a homogeneous liquid phase, etc. Strict condition setting is required.

  For example, when detecting a double-stranded nucleic acid using a detection probe nucleic acid immobilized on a solid phase substrate or the like, the performance and sensitivity of the intercalator itself is in a uniform liquid phase due to the interaction inhibition due to steric hindrance, etc. There is a possibility of lower than.

  In addition, when detecting a double-stranded nucleic acid using a detection probe nucleic acid immobilized on a solid phase substrate or the like, it may be assumed that a three-dimensional structure abnormality occurs in the double-stranded nucleic acid part due to steric hindrance or the like. . In that case, even if a double-stranded nucleic acid is formed, there is an abnormality in its three-dimensional structure, and a part of the intercalator cannot interact with the double-stranded nucleic acid part, so that the accuracy and quantitativeness of hybridization detection are reduced. there is a possibility.

  On the other hand, suitable conditions for the interaction between the double-stranded nucleic acid formed on the solid phase substrate and the intercalator have not been sufficiently studied.

  Therefore, the present invention provides suitable conditions for the interaction between the double-stranded nucleic acid formed on the solid-phase substrate and the intercalator, thereby allowing the double-stranded nucleic acid formed on the solid-phase substrate or the like to The main purpose is to improve the accuracy and quantitativeness when detecting using an intercalator.

  The present inventor has examined in detail, for example, suitable conditions for the interaction between a double-stranded nucleic acid formed on a solid phase substrate and the intercalator. Then, by detecting a probe nucleic acid for detection via a linker having a predetermined length and adjusting the fixing interval of the probe nucleic acid, detection accuracy and quantification of the double-stranded nucleic acid formed on the solid phase substrate I found out that I can improve.

Therefore, in the present invention, a detection probe nucleic acid having a predetermined length is formed on a detection surface used when detecting a double-stranded nucleic acid portion formed by hybridization of a detection probe nucleic acid and a target nucleic acid with an intercalator. A method of immobilizing via a linker, wherein the immobilization density of the probe nucleic acid is determined by calculating the chain length of the double-stranded nucleic acid part and the chain length of the linker when the double-stranded nucleic acid part is formed by hybridization. Provided is a probe nucleic acid immobilization method that is determined based on the combined length. In this probe nucleic acid immobilization method, the probe nucleic acid immobilization interval is longer than the sum of the chain length of the double-stranded nucleic acid portion and the chain length of the linker.

  By fixing the probe nucleic acid for detection via a linker of a predetermined length and setting the fixing interval of the probe nucleic acid for detection as described above, the double-stranded nucleic acid formed on the detection surface and the intercalator The influence of steric hindrance and the like on the interaction can be eliminated as much as possible, and the amount of interaction can be made the same as in the uniform liquid phase.

  Therefore, by using this detection surface, the interaction between the double-stranded nucleic acid moiety and the intercalator can be promoted, so the detection accuracy and quantitativeness of hybridization between the probe nucleic acid for detection and the target nucleic acid are improved. Can be made.

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

  The “detection surface” is a portion of a surface such as a solid phase substrate that faces a reaction field such as hybridization.

  “Nucleic acid” means 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 probe nucleic acid 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.

  A “probe nucleic acid” is a nucleic acid (nucleotide chain) that functions as a detector for detecting a target nucleic acid, and is immobilized on a detection surface or the like.

  A “target nucleic acid” is a nucleic acid having a sequence complementary to the base sequence of a probe nucleic acid for detection over its entire length or a part, and is dropped or supplied to a reaction field when detecting hybridization.

  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.

  An “intercalator” is a substance that emits fluorescence by binding to a double-stranded nucleic acid moiety. In addition, the binding mode of the intercalator to the double-stranded nucleic acid site is not particularly limited.

  “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.

  According to the present invention, in detecting a double-stranded nucleic acid moiety formed by hybridization of a probe nucleic acid for detection and a target nucleic acid with an intercalator, the detection accuracy and quantitativeness of hybridization can be improved.

<About the detection surface according to the present invention>
First, an example of the detection surface according to the present invention will be described below with reference to FIG.

  In FIG. 1, a reaction field B is formed on the detection surface A, and a detection probe nucleic acid 2 is fixed to the detection surface A via a linker 1 having a predetermined length. As illustrated in FIG. 1, the probe nucleic acid 2 for detection and the target nucleic acid 3 are hybridized to form a double-stranded nucleic acid portion 4. And when the intercalator 5 couple | bonds with the double stranded nucleic acid part 4 (refer code | symbol X), the intercalator 5 will carry out fluorescence excitation. By detecting the fluorescence, hybridization can be detected quantitatively and with high accuracy.

  A material such as a solid phase substrate including the detection surface A is not particularly limited, but a material capable of transmitting excitation light (for example, fluorescence excitation light) having a predetermined wavelength can be excited from the substrate side when detecting an interaction. From the viewpoint of the above, it is preferable. For example, a substrate similar to a known optical information recording medium such as CD (compact disc), DVD (digital versatile disc), MD (mini disc) can be used, and materials such as glass such as quartz, polycarbonate, polystyrene, etc. Is preferred.

  The reaction field B is a hybridization field between the probe nucleic acid 2 for detection and the target nucleic acid 3. In the reaction field B, a sample (including the target nucleic acid 3 and the like), a medium (such as a buffer solution), a reagent (including the intercalator 5 and the like), and the like are supplied and stored.

  The linker 1 may be any molecule that has a predetermined chain length, can link the detection surface A and the probe nucleic acid 2 and does not interact with nucleic acids other than the linked probe nucleic acid or intercalator in principle. A known spacer molecule can be used for the linker 1. For example, a chain molecule composed of carbon (C), oxygen (O), nitrogen (N), phosphorus (P), etc. and having a chain length of about 40 to 50 mm is suitable.

  As the probe nucleic acid 2 for detection, one having a base sequence complementary to all or part of the base sequence of the nucleic acid to be detected is used. The probe nucleic acid 2 is not narrowly limited depending on whether it is a synthetic nucleic acid. The chain length (mer number) can be appropriately set according to the purpose.

  A known method can be used as the means for immobilizing the linker 1 and the probe nucleic acid 2 for detection, and is not particularly limited. For example, the detection surface A may be treated with avidin, one end of the linker 1 may be biotinylated, and the probe nucleic acid 2 may be bound to the other end of the linker 1. In this case, the probe nucleic acid 2 can be relatively easily fixed to the detection surface A in the order of detection surface A-linker 1-probe nucleic acid 2 by the avidin-biotin bond.

  In the present invention, the fixed length (fixed density) n of the probe nucleic acid 2 for detection is assumed to be the length of the double-stranded nucleic acid portion (symbol m) when it is assumed that the double-stranded nucleic acid portion is formed by hybridization, for example. ) And the chain length (sign 1) of the linker. As a result, the influence of steric hindrance etc. on the interaction between the double-stranded nucleic acid formed on the detection surface and the intercalator can be eliminated as much as possible. Therefore, the detection accuracy and quantification of the hybridization between the probe nucleic acid for detection and the target nucleic acid Can be improved.

  As the target nucleic acid 3, for example, a nucleic acid extracted, prepared, or synthesized from a biological sample collected from a human body or the like by a known method can be used. In that case, for example, nucleic acid may be extracted from the target sample and used, or after extraction of the nucleic acid from the target sample, cDNA or the like may be synthesized and used by a known method.

  As the intercalator 5, a known one can be used. Examples of the intercalator include POPO-1, TOTO-3, SYBR Green I, PicoGreen (manufactured by Molecular Probes, USA), Hoechst 33258, and the like.

<About the method according to the present invention>
Next, the interaction promotion method and hybridization detection method according to the present invention will be described below.

  By using the detection surface according to the present invention, the influence of steric hindrance etc. on the interaction between the double-stranded nucleic acid formed on the detection surface and the intercalator can be eliminated as much as possible. Interaction can be promoted. Therefore, the detection accuracy and quantitativeness of hybridization between the probe nucleic acid for detection and the target nucleic acid can be improved.

  For example, first, the reaction field on the detection surface on which the detection probe nucleic acid is immobilized is filled with a buffer solution, and the sample nucleic acid is dropped into the sample to add a high concentration of the detection probe nucleic acid and the target nucleic acid. Allow hybridization to form. Next, an intercalator is dropped into the reaction field, and hybridization is detected by fluorescence excitation. The dripping means and timing of the target nucleic acid and intercalator can be appropriately changed.

As the buffer solution, a known buffer solution such as a phosphate buffer solution, PBS (phosphate buffered physiological saline), a HEPES buffer solution, or a Tris buffer solution can be used. By adjusting the concentration of the buffer salt (NaCl, MgCl _2, etc.), non-specific adsorption of nucleic acid or the like to the substrate surface can be prevented. In addition, when a salt-containing HEPES buffer or the like is used as the buffer, nonspecific adsorption of nucleic acid or the like to the substrate surface can be prevented.

  In Example 1, the influence of the immobilization density of the probe nucleic acid for detection on the interaction between the double-stranded nucleic acid moiety formed by hybridization of the probe nucleic acid and the target nucleic acid and the intercalator was examined using QCM (quartz crystal oscillation). This was verified using a (child microbalance) device.

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 acid was prepared. A probe nucleic acid (an oligonucleotide having a base sequence shown in SEQ ID NO: 1) and an oligonucleotide of its complementary strand (an oligonucleotide having a base sequence shown in SEQ ID NO: 2) are prepared, and a linker is provided at the 5 ′ end of the probe nucleic acid. After modification and biotinylation, the probe nucleic acid and its complementary strand were annealed to prepare a double-stranded nucleic acid.

The modification and biotinylation of the linker was carried out by 1-Dimethylityroxy-3-O- (N-biotinyl-3-aminopropyl) -triethyleneglycyl-glyceryl-2-O- (2-cyanophytyl)-(N, N-diisotropy)-(N, N-diisotropy) Glen Research Corporation) and 18-O-Dimethyltriethylhexylethyleneglycol, 1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite (Glen Research) At the 5 ′ end, a linker and Chin went to bind in the order of "oligonucleotide - biotin - linker". In this example, the synthesis of both oligonucleotides, linker modification of the probe nucleic acid and biotinylation were entrusted to Tsukuba Oligo Service Co., Ltd. The chemical structural formula of the portion from biotin to the linker in the probe nucleic acid is shown in “Chemical Formula 1”.

  In this example, both synthetic oligonucleotides purchased from the same company were purified by reverse phase HPLC and used in experiments. The column was purified using a “SunFire 3.5 μm Analytical Column (C18, 4.6 × 150 mm, manufactured by Waters Corporation)” with a gradient of 50 mM ammonium formate and acetonitrile at 60 ° C. After purification, the oligonucleotide was dissolved in 0.1 M HEPES buffer (containing 20 mM magnesium chloride, pH 7.7), and P1 nuclease (manufactured by Bio Medicals, Germany), alkaline phosphatase (manufactured by Takara Bio Inc.) by enzymatic degradation. ) Was used to determine the concentration.

  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.

At that time, the double-stranded nucleic acid binds to the Au electrode substrate, whereby the oscillation frequency of the crystal oscillator varies. Therefore, the double-stranded nucleic acid was immobilized using a QCM apparatus while measuring the fluctuation of the oscillation frequency over time. Then, when the fixed amount (fixed density) of the double-stranded nucleic acid reached a predetermined value, the sensor chip was taken out from the reaction vessel and washed to stop the binding reaction between the double-stranded nucleic acid and the Au electrode substrate. Thereby, the fixed density could be adjusted. In this example, the fixed density was set to 10 values between 2.05 and 10.3 × 10 ¹⁰ pieces / mm ² .

  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 the intercalator, “SYBR Green I (Molecular Probes, USA)” was used.

As an example, FIG. 2 shows the fluctuation of the oscillation frequency in each of the above procedures. FIG. 2 is a graph in the case where the fixation density of the double-stranded nucleic acid is set to 5.42 × 10 ¹⁰ / mm ² . The horizontal axis in the graph represents time (min), and the vertical axis represents the oscillation frequency ΔF (Hz) of the crystal resonator.

  In FIG. 2, the time point “1” indicates a stage in which the sensor chip is immersed in a reaction tank containing a buffer solution of double-stranded nucleic acid. The time point “2” indicates a stage where the sensor chip is taken out of the reaction vessel and the fixation of the double-stranded nucleic acid to the Au electrode substrate is stopped. The point “3” indicates a stage in which the sensor chip is immersed in a reaction tank containing an intercalator solution.

  In FIG. 2, at the stage “(a)”, the double-stranded nucleic acid is fixed to the Au electrode substrate with time by the avidin-biotin bond, and the oscillation frequency fluctuates with time. As shown in FIG. 2, when the double-stranded nucleic acid is fixed to the Au electrode substrate, the fluctuation of the oscillation frequency is relatively slow. Therefore, by adjusting the time point “2”, the double-stranded nucleic acid The fixed amount (fixed density) can be adjusted.

  In FIG. 2, at the stage “(b)”, when the sensor chip is immersed in the reaction vessel (at the time “3”), the intercalator binds to the double-stranded nucleic acid, thereby reducing the oscillation frequency. Yes.

The results are shown in Table 1 and FIG.

  Table 1 shows the amount of interaction between the double-stranded nucleic acid and the intercalator (the amount of binding of the intercalator to the double-stranded nucleic acid) when the fixed density of the double-stranded nucleic acid is set to each value. The interaction amount (mol) is a value obtained by converting the fluctuation of the oscillation frequency (ΔF) into the coupling amount of the intercalator.

FIG. 3 is a graph in which the values in Table 1 are semilogarithmically plotted. The horizontal axis of the graph represents the fixed density of double-stranded nucleic acid (× 10 ¹⁰ / mm ² , natural logarithm), and the vertical axis represents the amount of interaction (mol) between the double-stranded nucleic acid and the intercalator. For these values, the linear approximation formula shown in “Expression 1” was obtained.

As shown in Table 1 and FIG. 3, when the fixed density of the double-stranded nucleic acid was 2.99 to 10.3 × 10 ¹⁰ / mm ² , the amount of interaction gradually increased as the fixed density decreased. .

On the other hand, when the fixed density of the double-stranded nucleic acid was 2.05 × 10 ¹⁰ / mm ² , the amount of interaction increased rapidly. The amount of interaction between the double-stranded nucleic acid and the intercalator was about 18 mol.

  In Example 2, the effect of the linker length on the interaction between the double-stranded nucleic acid moiety formed by hybridization of the detection probe nucleic acid and the target nucleic acid and the intercalator was verified using a QCM apparatus. did.

  First, double-stranded nucleic acid was prepared. After preparing a probe nucleic acid having a strand length of 15 mer, 20 mer, 25 mer, and 30 mer and an oligonucleotide of its complementary strand, modification of a short or long linker at the 5 ′ end of the probe nucleic acid and biotinylation, followed by probe nucleic acid And its complementary strand were annealed to prepare a double-stranded nucleic acid.

  The base sequence of the probe nucleic acid when the chain length is 15 mer is shown in SEQ ID NO: 3, the complementary strand oligonucleotide is shown as SEQ ID NO: 4, the base sequence of the probe nucleic acid when the chain length is 20 mer is shown as SEQ ID NO: 5, The complementary strand oligonucleotide is SEQ ID NO: 6, the base sequence of the probe nucleic acid when the chain length is 25 mer is SEQ ID NO: 7, the complementary strand oligonucleotide is the SEQ ID NO: 8, and the base sequence of the probe nucleic acid when the chain length is 30 mer The sequence is shown in SEQ ID NO: 9, and its complementary oligonucleotide is shown in SEQ ID NO: 10.

Modification of short linkers and biotinylation can be accomplished by 1-Dimethylityloxy-2- (N-biotinyl-4-aminobutyl) -propyl-3-O- (2-cyanethyl)-(N, N-diisopropyl) -phosphoramideite (Glenorporamide) The product was prepared according to the attached protocol of both compounds, and the linker and biotin were bonded to the 5 ′ end in the order of “oligonucleotide-linker-biotin”. The chemical structural formula of the portion from biotin to the linker in these probe chains is shown in “Chemical Formula 2”.

  Modification of the long linker and biotinylation were performed in the same procedure as in Example 1. In addition, synthesis of each oligonucleotide, linker modification of the probe chain oligonucleotide and biotinylation were entrusted to Tsukuba Oligo Service Co., Ltd. as in Example 1. In addition, purification of oligonucleotides used, concentration determination, and the like were performed in the same manner as in Example 1.

  Subsequently, the double-stranded nucleic acid was immobilized on the Au electrode substrate by the same method as in Example 1. In this example, in consideration of the influence of the chain length of the double-stranded nucleic acid, the fixed amount (fixed density) of the double-stranded nucleic acid is 1.0 pmol and 20 mer in the case of a 15-mer double-stranded nucleic acid, respectively. It was set to 0.75 pmol in the case of a strand nucleic acid, 0.60 pmol in the case of a 25-mer double-stranded nucleic acid, and 0.5 pmol in the case of a 30-mer double-stranded nucleic acid.

  Subsequently, in the same procedure as in Example 1, the reaction vessel was replaced with one containing the intercalator solution, the sensor chip was immersed therein, and the fluctuation of the oscillation frequency was measured with a QCM device. As in Example 1, “SYBR Green I (manufactured by Molecular Probes, Inc.)” was used as the intercalator.

The results are shown in Table 2, FIG. 4 and FIG.

  Table 2 shows the amount of interaction between the double-stranded nucleic acid and the intercalator when the short linker or the long linker is modified to a double-stranded nucleic acid (intercalator binding amount to the double-stranded nucleic acid). The interaction amount (mol) is a value obtained by converting the fluctuation of the oscillation frequency (ΔF) into the binding amount of the intercalator and dividing by the fixed amount of the double-stranded nucleic acid.

  FIG. 4 is a graph obtained by semi-log plotting the amount of interaction in Table 2 when a short linker is modified with a double-stranded nucleic acid. FIG. 5 is a graph obtained by semi-log plotting the amount of interaction in Table 2 when a long linker is modified with a double-stranded nucleic acid. The horizontal axis of both graphs represents the chain length of the double-stranded nucleic acid, and the vertical axis represents the amount of interaction (mol) between the double-stranded nucleic acid and the intercalator per mole of the double-stranded nucleic acid.

For the values shown in the graphs of FIGS. 4 and 5, linear approximation expressions shown in “Equation 2” and “Equation 3” were obtained, respectively.

  As shown in Table 2 and FIGS. 4 and 5, when a long linker is modified to a double-stranded nucleic acid, compared to a case where a short linker is modified to a double-stranded nucleic acid, The amount of action increased. This result shows that the interaction reactivity between the double-stranded nucleic acid and the intercalator can be increased by optimizing the length of the linker.

  In Example 3, the interaction between the double-stranded nucleic acid and the intercalator was thermodynamically verified by ITC (Isothermal Titration Calorimetry; the same applies hereinafter).

  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, a double-stranded nucleic acid in which an oligonucleotide having the base sequence shown in SEQ ID NO: 1 and an oligonucleotide having the base sequence shown in SEQ ID NO: 2 were hybridized was prepared. In this example, the synthesis of this double-stranded nucleic acid was commissioned by Tsukuba Oligo Service Co., Ltd. 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 (manufactured by Molecular Probes, Inc.)” was used as in Example 1 and the like.

  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.

  The results are shown in FIG. FIG. 6 is a graph showing the measurement results by ITC.

  In FIG. 6, the upper graph represents the 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.

  In FIG. 6, 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, the double-stranded nucleic acid and the intercalation solution. The amount of heat in the interaction with the calator. 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.

  The result of FIG. 6 suggests that there are two types of binding sites in the interaction between the double-stranded nucleic acid and the intercalator. Then, about the measurement result obtained by ITC, it analyzed using the two site model and acquired the thermodynamic profile.

The results are shown in Table 3.

In the table, “Site1” and “Site2” represent two types of predicted binding sites. In the table, “n” indicates the number of intercalator bindings at each binding site present in the double-stranded nucleic acid, and “Ka (× 10 ⁶ M)” indicates the binding constant in the interaction between the double-stranded nucleic acid and the intercalator. , “ΔH (kcal / mol)” represents an enthalpy change, “TΔS (kcal / mol)” represents an entropy change, and “ΔG (kcal / mol)” represents a free energy change.

  The results in Table 3 show that among the binding sites present in the double-stranded nucleic acid, the interaction ratio between the double-stranded nucleic acid and the intercalator is 1: about 6.55 at the first binding site, and the second binding. The site shows 1: 11.7, and the total is 1: about 18.25. That is, both binding sites are combined per mole of double-stranded nucleic acid, indicating that the intercalator is bound by about 18.25 moles.

In Example 1 (Table 1 and FIG. 3), when the fixed density was 2.05 × 10 ¹⁰ pieces / mm ² , the interaction amount between the double-stranded nucleic acid and the intercalator was 18.66 mol. For this fixed density, the distance between adjacent probe nucleic acids is 1.2 × 10 ⁻⁵ mm. On the other hand, the 20mer nucleic acid has a chain length of about 0.714 × 10 ⁻⁵ mm, and the long linker used in Example 2 has a chain length of about 0.5 × 10 ⁻⁵ mm. That is, the distance between adjacent probe nucleic acids is substantially equal to the distance of the probe nucleic acid chain length and the linker chain length.

  Accordingly, the results from Example 1 to Example 3 show that the fixed interval of the probe nucleic acid is equal to or greater than the distance obtained by combining the probe nucleic acid chain length and the linker chain length, so It shows that the interaction with the calator can be promoted, that is, the detection accuracy of hybridization can be improved.

  According to the present invention, since the interaction between the double-stranded nucleic acid moiety and the intercalator can be promoted, the detection accuracy and quantitativeness of hybridization between the probe nucleic acid for detection and the target nucleic acid can be improved. Therefore, for example, in a system for detecting hybridization between a detection probe nucleic acid immobilized on a solid phase substrate or the like and a target nucleic acid (DNA chip or the like), the present invention is used when detecting hybridization using an intercalator. Useful.

The cross-sectional schematic diagram which shows the example of the detection surface which concerns on this invention. In Example 1, the graph which shows the example of the fluctuation | variation of the oscillation frequency in each procedure. In Example 1, the semilogarithm graph which shows the interaction amount of a double stranded nucleic acid and an intercalator in case the fixed density of a double stranded nucleic acid is set to each value. In Example 2, the semilogarithm graph which shows the interaction amount of a double stranded nucleic acid and an intercalator at the time of modifying a short linker into a double stranded nucleic acid. In Example 2, the semi-logarithmic graph which shows the interaction amount of a double stranded nucleic acid and an intercalator when a long linker is modified into a double stranded nucleic acid. In Example 3, the graph showing the measurement result by ITC.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Linker 2 Probe nucleic acid for detection 3 Target nucleic acid 4 Double-stranded nucleic acid part 5 formed by hybridization of detection probe nucleic acid and target nucleic acid Intercalator A Detection surface l Linker chain length m Detection probe nucleic acid Chain length (chain length of the double-stranded nucleic acid part when it is assumed that a double-stranded nucleic acid was formed by hybridization)
n Fixing interval of probe nucleic acid for detection

Claims (2)

The double-stranded nucleic acid portion formed by hybridization between the probe and target nucleic acid for detection, the detection surface for use in detecting at intercalator, the probe nucleic acid in a manner of fixing through a predetermined length of linker There ,
Probe nucleic acid immobilization that determines the immobilization density of the probe nucleic acid based on the sum of the chain length of the double-stranded nucleic acid moiety and the chain length of the linker when a double-stranded nucleic acid moiety is formed by the hybridization Way . The probe nucleic acid immobilization method according to claim 1, wherein the probe nucleic acid immobilization interval is longer than the sum .