JP4361768B2 - Hybridization method, base sequence detection method, and base sequence detection kit - Google Patents

Description

  The present invention relates to a nucleic acid hybridization method, a base sequence detection method, and a base sequence detection kit.

  Gene / nucleic acid analysis methods using solid-liquid interface fields are attracting attention in array analysis and biosensors (for example, see Non-Patent Document 1 below).

In nucleic acid analysis, probe molecules complementary to the base sequence of the nucleic acid to be detected are immobilized on the solid surface, and specific hybridization reactions between the immobilized molecule and the detection target sequence are measured by various methods. It is basic. In this method, quartz glass in which an amino group is introduced (aminosilanized) on the surface by an amino group-containing silane coupling agent is widely used as a solid-liquid interface field.
LeProust, E.; Zhang, H.; Yu, P.; Zhou, X.; Gao, X.Nucleic Acids Res. 2001, 29, 2171-2180

  However, the probe nucleic acid immobilization method based on amino silanization reported so far and the nucleic acid analysis method based thereon have the following problems.

  That is, in the conventional nucleic acid analysis method, the concentration of the target sequence detected by the hybridization reaction is relatively high (in many cases, nM or more), and the amount of sample used for detection must be increased. Therefore, there is a problem that nucleic acid analysis cannot be performed when a sufficient sample amount cannot be secured. In particular, in array analysis in which many detection target sequences are detected at once, such a problem has been a fatal drawback.

  Accordingly, an object of the present invention is a hybridization method using a probe immobilized on a solid phase carrier, and has high selectivity and accuracy even when the concentration of the target nucleic acid (target base sequence) is extremely low. A hybridization method capable of hybridizing a probe and a target nucleic acid. Another object of the present invention is to provide a base sequence detection method using this hybridization method and a base sequence detection kit therefor.

  In order to achieve the above object, the present invention provides a solution containing a peptide nucleic acid probe immobilized on a solid phase carrier and a target nucleic acid complementary to the peptide nucleic acid probe, which is 1.0% on the basis of the solution. The peptide nucleic acid probe and the nucleic acid are hybridized by contacting with a solution further containing a ~ 3.0 mol / L hydrophilic organic solvent and having a pH adjusted to 9.1 to 10.6. A featured hybridization method is provided.

  Thus, in the hybridization method of the present invention, a peptide nucleic acid probe is used as a probe immobilized on a solid phase carrier, and the target nucleic acid to be detected is used at a predetermined pH together with a predetermined concentration of a hydrophilic organic solvent. It is a feature. With such a configuration, even when the concentration of the target nucleic acid is extremely low, the probe and the target nucleic acid can be hybridized with high selectivity and accuracy.

  When the concentration of the hydrophilic organic solvent is lower than 1.0 mol / L or exceeds 3.0 mol / L, hybridization does not occur or is significantly inhibited. On the other hand, when the pH is less than 9.1, although hybridization occurs, non-specific adsorption of the target nucleic acid becomes remarkable, and when the pH exceeds 11, the hybridization is inhibited.

  As the hydrophilic organic solvent, at least one selected from the group consisting of formamide, dimethylformamide, diethylformamide, acetamide, dimethylacetamide, propionamide, benzamide, dimethyl sulfoxide, acetone, methanol, ethanol, butanol, ethylene glycol and glycerol is used. It may be used, and the solution containing the target nucleic acid preferably further contains polyvinyl alcohol. With such a configuration, non-specific adsorption is more reliably suppressed, and hybridization occurs with higher selectivity and accuracy.

  Since the same effect is produced, it is preferable that the solid phase carrier on which the peptide nucleic acid is immobilized has the following configuration. That is, the solid phase carrier is a solid phase carrier having an amino group on the surface, the peptide nucleic acid probe is a peptide nucleic acid probe having a cysteine residue introduced at the end thereof, and the peptide nucleic acid probe is The solid phase carrier may be immobilized by a linker having an amino group reactive group and a mercapto group reactive group. The peptide nucleic acid probe preferably has a length of 8 to 20 mer (more preferably 10 to 18 mer). If the peptide nucleic acid probe is smaller than 8 mer, the target nucleic acid detection specificity cannot be exhibited, and if it is larger than 20 mer, synthesis of the peptide nucleic acid probe itself becomes difficult.

  By using such a hybridization method, a very small amount of target nucleic acid can be detected. That is, a base sequence detection method for detecting a minute amount of a target nucleic acid, comprising at least a hybridization step for carrying out the hybridization method of the present invention and a detection step for detecting a hybridized target nucleic acid. A characteristic base sequence detection method is provided.

  From the viewpoint of detection accuracy, detection is preferably performed using a fluorescent dye. That is, (1) in the hybridization step, the target nucleic acid labeled with a fluorescent dye is hybridized with the peptide nucleic acid probe, and this fluorescent dye is detected in the detection step, or (2) in the hybridization step, the peptide nucleic acid probe and Detection with a fluorescent dye labeled by hybridizing a target nucleic acid partially having a complementary sequence region with a peptide nucleic acid probe and further having a sequence complementary to at least a part of a sequence other than the sequence region in the target nucleic acid It is preferable to hybridize the base sequence and detect this fluorescent dye in the detection step.

  For such detection, a solution to which a solid phase in which a peptide nucleic acid probe is immobilized on a solid phase carrier and a target nucleic acid having a sequence complementary to the peptide nucleic acid probe are to be added, A base sequence detection kit containing a solution containing 1.0 to 3.0 mol / L of a hydrophilic organic solvent and having a pH adjusted to 9.1 to 10.6 is applicable.

  A hybridization method using a probe immobilized on a solid phase carrier, which hybridizes the probe and the target nucleic acid with high selectivity and accuracy even when the concentration of the target nucleic acid (target base sequence) is extremely low. A hybridization method is provided. Also provided are a base sequence detection method using this hybridization method and a base sequence detection kit therefor.

  DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, preferred embodiments of the invention will be described with reference to the drawings. In addition, the same code | symbol is attached | subjected to the same element and the overlapping description is abbreviate | omitted.

(Solid phase)
FIG. 1 is a schematic diagram showing a first embodiment of a solid phase applicable in the present invention. A solid phase 1 shown in FIG. 1 includes a solid phase carrier 2, a peptide nucleic acid probe 4, and a linker 6 for fixing the peptide nucleic acid probe 4 to the solid phase carrier 2. A target nucleic acid (target base sequence) having a complementary sequence to this hybridizes.

  FIG. 2 is a schematic diagram showing a second embodiment of a solid phase applicable in the present invention. A solid phase 1 shown in FIG. 2 includes a solid phase carrier 2, peptide nucleic acid probes 4a to 4e having different sequences, and a linker 6 for fixing the peptide nucleic acid probes 4a to 4e to the solid phase carrier 2. The target nucleic acid (target base sequence) having a complementary sequence is hybridized to each of the peptide nucleic acid probes 4a to 4e. Thus, since the solid phase 1 shown in FIG. 2 includes different peptide nucleic acid probes, it can be applied to array analysis for detecting many detection targets at once.

Examples of the solid phase carrier 2 include glass (quartz glass and the like), nylon (registered trademark) membrane (nylon ^TM membrane), controlled-pore glass, acrylamide gel, polystyrene matrix, activated dextran (activated dextran). ), Avidin-coated polystyrene beads, and metals such as gold (Au). A current detection type DNA chip is known as one that uses metal as a solid phase carrier. Among these, it is preferable to use glass because it is transparent and has few impurities and is strong and inexpensive.

  Such a solid phase carrier 2 is preferably provided with an amino group on the surface, and this amino group is preferably used for binding to the linker 6. For example, when the solid phase carrier 2 is glass, the amino group can be introduced by reacting the surface of the solid phase carrier 2 with a silane coupling agent.

  Examples of silane coupling agents include 3-aminopropyltriethoxysilane (APTES), N- (2-aminoethyl) -3-aminopropyltriethoxysilane (N- (2-aminoethyl) -3-aminopropyltriethoxysilane). , EDA), trimethoxysilylpropyldiethylenetriamine (DETA), m, p- (aminoethyl-aminomethyl) phenethyltrimethoxysilane (PEDA), N- (2- An example is aminodecanoyl) -3-aminopropyltriethoxysilane (N- (2-aminodecanoyl) -3-aminopropyltriethoxysilane).

  Peptide nucleic acids are schematically shown in FIG. B in the peptide nucleic acid 10 shown in FIG. 3 represents a base (B may be different from each other), and is designed to be complementary to the target nucleic acid to be detected. N determines the length of the peptide nucleic acid, and is preferably 8 to 20 (more preferably 10 to 18).

  The peptide nucleic acid shown in FIG. 3 can itself be used as the peptide nucleic acid probe 4, but preferably has a cysteine residue at its end from the viewpoint of the binding property to the linker 6. Here, the terminal for introducing the cysteine residue may be the N terminal or the C terminal. In addition, it is preferable from the viewpoint of ease of hybridization to introduce a spacer (divalent group) as shown in FIG. 4 between the peptide nucleic acid 10 shown in FIG. 3 and the cysteine residue.

FIG. 5 is a diagram showing an embodiment of the peptide nucleic acid probe 4 having a spacer having a cysteine residue introduced at the C-terminus. In FIG. 5, the spacer is shown as “spacer”, and the bond (divalent group) as shown in FIG. 4 is applied. Note that the -COOH group of the cysteine residue is changed to -CONH ₂ group by the probe synthesis reaction.

  The linker 6 is a polyfunctional compound having a functional group reactive with the functional group on the surface of the solid phase carrier 2 and a functional group reactive with the terminal functional group of the peptide nucleic acid probe. In the case where an amino group is present on the surface of the solid phase carrier 2, a reactive functional group (amino group-reactive group) includes thiocyanate group, aldehyde group, succinimidyl group, cyanuric chloride (cyanuric chloride). ) And epoxy groups. When a mercapto group is present at the end of the peptide nucleic acid probe, examples of the functional group reactive with the mercapto group (mercapto group-reactive group) include an epoxy group, an isocyanate group, and a maleimide group.

  Examples of the linker 6 having an amino group reactive group and a mercapto group reactive group include SMCC (succinimidyl-4- (N-maleimiomethyl) cyclohexane-1-carboxylate) and sSMCC (4- (N-maleimidomethyl) cyclohexane. -1-carboxylic-3-sulfo-n-hydroxysuccineimide) and LC-SMCC (succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxy- (6-amido-caproate)).

  FIG. 6 shows a solid phase when glass is used as the solid phase carrier 2, APTES is used as the silane coupling agent for introducing an amino group on the glass surface, LC-SMCC is used as the linker 6, and the peptide nucleic acid probe of FIG. FIG. 3 is a schematic diagram showing phase 1; Note that B and spacer in FIG. 6 are the same as those in FIG.

  In the solid phase 1 as shown in FIGS. 1, 2 and 6, the linker 6 is bound to the peptide nucleic acid probe 4 and then bound to the solid phase carrier 2 (preferably a solid phase carrier having an amino group on the surface). Or a method in which the linker 6 is bound to the solid phase carrier 2 (preferably a solid phase carrier having an amino group on the surface) and the linker 6 and the peptide nucleic acid probe 4 are bound to each other.

  For example, for the solid phase shown in FIG. 6, after immersing quartz glass in an APTES solution, the quartz glass is held for a predetermined time at room temperature or under heating, and -Si- between the alkoxy group of APTES and the surface silanol group of quartz glass. O-bonds are generated and amino groups are introduced on the surface of quartz glass. Next, the silica glass into which an amino group has been introduced is treated with LC-SMCC, which is a linker, so that the linker is bound to the quartz glass surface via the APTES residue. Furthermore, the amino group which did not react with LC-SMCC is capped with an acetyl group by treating with acetic anhydride. Then, this is immersed in a solution of a peptide nucleic acid probe in which a cysteine residue is introduced into the C-terminus via a spacer and reacted for a predetermined time, whereby the mercapto group-reactive group of LC-SMCC becomes a cysteine residue. By reacting with a mercapto group, a peptide nucleic acid probe is immobilized on the surface of quartz glass, which is a solid phase carrier.

(solution)
The solution to be brought into contact with the peptide nucleic acid probe immobilized on the solid phase carrier is a solution containing a target nucleic acid having a base sequence complementary to the peptide nucleic acid probe, and 1.0 to 3.0 mol / mol based on this solution. L is a solution containing a hydrophilic organic solvent and having a pH of 9.1 to 10.6.

  As the hydrophilic organic solvent, those listed above are preferable, and dimethylformamide (DMF) and diethylformamide (DEF) are particularly preferable. The concentration of the solution is more preferably 1.9 to 2.6 mol / L. Here, 1.9 mol / L and 2.6 mol / L correspond to 15% and 20% of DMF, respectively. The pH of the solution is particularly preferably 9.4-10. By setting the lower limit of pH to 9.4, nonspecific adsorption can be almost completely prevented.

  The solution contains a target nucleic acid (target base sequence) and a hydrophilic organic solvent, and the pH may be in the above range. Besides these, a buffer such as a phosphate ester buffer, a diethanolamine buffer, or a sodium bicarbonate buffer may be used. It is preferable to contain about 10 mM. Moreover, nonspecific adsorption | suction can be suppressed effectively by containing polyvinyl alcohol about 0.5% (w / v). Other components that can be included include NaCl, KCl, and the like.

(Hybridization method and nucleotide sequence detection method)
A solution (1.0-3.0 mol / L hydrophilic) containing a target nucleic acid (target base sequence) having a base sequence complementary to this peptide nucleic acid on a solid phase in which a peptide nucleic acid probe is immobilized on a solid phase carrier In addition, an organic solvent is further contained, and the pH is adjusted to 9.1 to 10.6.), Thereby bringing about hybridization between the peptide nucleic acid probe and the target nucleic acid. Specifically, the solid phase is immersed in the solution containing the target nucleic acid (target base sequence), and is preferably held or shaken and stirred at room temperature for 8 to 12 hours.

  FIG. 7 is a schematic diagram showing a state in which a target nucleic acid 8 labeled with a fluorescent dye 10 is hybridized to a peptide nucleic acid probe 4 immobilized on a solid phase carrier 2 with a linker 6. After such hybridization, the target base sequence can be detected by detecting the fluorescent dye 10 of the target nucleic acid 8 using a known detection means. In this case, since the hybridization is performed using the above-described solution, the probe and the target nucleic acid are hybridized with high selectivity and accuracy even when the concentration of the target nucleic acid is extremely low. Even a specimen containing only a small amount of (target base sequence) can be detected with high accuracy.

  FIG. 8 shows that the target nucleic acid 8 is hybridized to the peptide nucleic acid probe 4 immobilized on the solid phase carrier 2 with the linker 6, and the detection base sequence 12 labeled with a fluorescent dye is further hybridized to the target nucleic acid 8. It is a schematic diagram of a state.

  Here, the target nucleic acid 8 partially has a sequence region complementary to the peptide nucleic acid probe 4, and the detection base sequence 12 is a sequence complementary to a part of the sequence other than the sequence region in the target nucleic acid 8. have. Also by causing such hybridization, high-precision detection can be performed on an analyte containing only a very small amount of target nucleic acid (target base sequence).

  The fluorescent dye was evanescently excited, and the emitted fluorescence was expressed as "Sutherland, RM; Daehne, C. In Biosensors: Fundamentals and Applications; Turner, APF; Karube, I .; Wilson, GS Eds .; Oxford University Press: Oxford, 1987. , pp 655-678. ”, measurement and quantification are possible.

(Nucleic acid detection kit)
In order to simply carry out the hybridization method and the base sequence detection method described above, a base sequence detection kit containing the following (a) and (b) can be used.
(A) a solid phase in which a peptide nucleic acid probe is immobilized on a solid phase carrier;
(B) a solution to which a target nucleic acid having a base sequence complementary to the peptide nucleic acid probe is to be added, containing 1.0 to 3.0 mol / L of a hydrophilic organic solvent based on the solution, and having a pH of Solution adjusted to 9.1 to 10.6.

  The hydrophilic organic solvent in the solution of (b) is selected from the group consisting of formamide, dimethylformamide, diethylformamide, acetamide, dimethylacetamide, propionamide, benzamide, dimethyl sulfoxide, acetone, methanol, ethanol, butanol, ethylene glycol and glycerol. At least one hydrophilic organic solvent selected is preferable, and dimethylformamide (DMF) and diethylformamide (DEF) are particularly preferable. The concentration of the solution is more preferably 1.9 to 2.6 mol / L. The pH of the solution is particularly preferably 9.4-10.

  The solution (b) can contain about 50 mM of the above-described buffer, and can contain about 0.5% (w / v) polyvinyl alcohol. Other components that can be included include NaCl, KCl, and the like.

  EXAMPLES Hereinafter, although the preferable Example of this invention is described in detail, this invention is not limited to these Examples.

(Production of solid phase)
Quartz glass is used as a solid support (18 x 18 x 0.15 mm), and the above solid support is immersed in 1% 3-aminopropyltriethoxysilane (APTES) in 95% acetone / water at room temperature in order to introduce amino groups. I let you. After holding for 1 hour, it was washed with a sufficient amount of acetone and dried. Then, in order to stabilize aminosilanization, the obtained solid phase carrier was heated at 110 ° C. for 2 hours. Next, the substrate was immersed in a dimethylsulfoxide (DMSO) solution of 1 mM LC-SMCC (succcinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxy- (6-amidocaproate)) and treated at room temperature for 2 hours.

  Thereafter, the substrate was washed with a sufficient amount of acetone, dried, and further immersed in a chloroform solution of 1% acetic anhydride (acetic anhydride) and treated at room temperature for 2 hours. Then, it was washed with a sufficient amount of acetone and then dried. As a result, most of the remaining amino groups were capped with acetyl groups after LC-SMCC treatment.

  A 0.5 mM to 1.0 mM solution of a peptide nucleic acid probe (16mer, sequence is ACG CCA TTG TAG CAC G from the N-terminal side to the C-terminal side) with a cysteine residue introduced at the C-terminus (the solvent is 10 mM) MOPS, 5 mM EDTA, 0.1 M NaCl buffer, pH 7.2) was immersed in the solid phase carrier obtained above and kept overnight at room temperature. As a result, the peptide nucleic acid probe was introduced into the maleimide group of LC-SMCC. Finally, it was thoroughly washed with the same buffer, further washed with water, dried and stored in a desiccator.

  Thus, a solid phase as shown in FIG. 1 was produced. FIG. 9 shows an absorption spectrum of quartz glass into which the peptide nucleic acid (PNA) probe has been introduced in the gas phase (PNA-glass in air). In addition, the absorption spectrum of the peptide nucleic acid probe in neutral aqueous solution (PNA in soln) is shown.

(Hybridization and nucleotide sequence detection)
1. Hybridization selectivity A strand of DNA complementary to the peptide nucleic acid probe (16mer, the sequence described in SEQ ID NO: 1 in the sequence listing), which is labeled with FITC (hereinafter referred to as "FITC-Tgt"). Was added to 2.0 ml of a basic solution (50 mM sodium carbonate-sodium bicarbonate buffer, pH 9.5 containing 0.2 M NaCl, 0.5% (w / v) polyvinylalcohol (PVA), 20% (v / v) DMF). After immersing one solid phase prepared above and shaking and stirring overnight (over 8 hours) at room temperature, the fluorescence spectrum of the external liquid is obtained and compared with the fluorescence spectrum before immersion. The adsorption amount of FITC-Tgt on quartz glass was determined.

  Further, it is a 16-mer deoxyoligonucleotide that is not complementary to the peptide nucleic acid probe (sequence described in SEQ ID NO: 2 in the Sequence Listing) and is labeled with FITC (hereinafter referred to as “FITC-unrelated sequence”). The above DNA strand not labeled with FITC (sequence described in SEQ ID NO: 1 in the sequence listing; hereinafter referred to as “Tgt”) and the peptide nucleic acid probe itself (hereinafter referred to as “capture probe”) are also as described above. Hybridization was attempted in the same manner. The results are shown in Table 1.

表１から明らかなように、ペプチド核酸プローブと相補的なFITC-Tgtは固相に吸着される（Assay No.1）。しかし、相補性のないFITC-unrelated sequenceはまったく固相表面に吸着されない（Assay No.5）。FITC-Tgt存在下、Tgt同じ配列を持つオリゴヌクレオチド（FITCでラベルされていない）を共存させるとFITC-Tgt吸着を競争的に阻害する（Assay No.2 & 3）。もちろん、このような競争阻害をFITC-unrelated sequenceは起こさせない（Assay No.4）。

As is apparent from Table 1, FITC-Tgt complementary to the peptide nucleic acid probe is adsorbed to the solid phase (Assay No. 1). However, non-complementary FITC-unrelated sequences are not adsorbed on the solid surface at all (Assay No. 5). In the presence of FITC-Tgt, the presence of an oligonucleotide having the same sequence as Tgt (not labeled with FITC) competitively inhibits FITC-Tgt adsorption (Assay No. 2 & 3). Of course, FITC-unrelated sequence does not cause such competition inhibition (Assay No. 4).

2. Effect of pH on hybridization 1
FITC-unrelated sequence, 2.0 ml of basic solution with various pH (50 mM sodium carbonate-sodium bicarbonate buffer, containing 0.2 M NaCl, 0.5% (w / v) polyvinylalcohol (PVA), 20% (v / v) DMF) Added to. After immersing one solid phase prepared above and shaking and stirring overnight (over 8 hours) at room temperature, the fluorescence spectrum of the external liquid is obtained and compared with the fluorescence spectrum before immersion. The amount of FITC-unrelated sequence adsorbed on quartz glass was determined. The results are shown in Table 2.

表２から明らかなように、ｐＨが中性条件では，非特異吸着が起る。しかし，ｐＨを９．４にすることでほぼ完全に非特異吸着が抑制される。ただし，より完全な抑制には，0.5% PVAまたは20% DMF添加が好ましいことが表からわかる。非特異吸着抑制のｐＨに対する依存性をより詳細に検討した結果を図１０に示す。図１０より基本溶液のｐＨは9.1以上，好ましくは９．４以上が必要であることが判明した。

As is apparent from Table 2, nonspecific adsorption occurs when the pH is neutral. However, nonspecific adsorption is suppressed almost completely by adjusting the pH to 9.4. However, it can be seen from the table that 0.5% PVA or 20% DMF is preferred for more complete suppression. The result of examining the dependence of nonspecific adsorption inhibition on pH in more detail is shown in FIG. From FIG. 10, it was found that the pH of the basic solution is 9.1 or more, preferably 9.4 or more.

3. Effect of pH on hybridization 2
FITC-Tgt was added to a basic solution of various pH 2.0 ml (0.1 M sodium carbonate-sodium bicarbonate buffer, containing 0.2 M NaCl, 0.5% (w / v) polyvinylalcohol (PVA), 15% (v / v) DMF. In addition, 0.1M Na ₂ HPO ₄ / NaOH buffer was used for the preparation of pH 11.4. After immersing one solid phase prepared above and shaking and stirring overnight (over 8 hours) at room temperature, the fluorescence spectrum of the external liquid is obtained and compared with the fluorescence spectrum before immersion. The adsorption amount of FITC-Tgt on quartz glass was determined. The results are shown in FIG. From FIG. 11, it was found that the pH of the basic solution must be 9.1 or more and 10.6 or less.

4). Study of hydrophilic organic solvent 1
FITC-Tgt, 2.0 ml of basic solution with various DMF concentrations (v / v) (50 mM Na ₂ CO ₃ / NaHCO ₃ buffer, pH 9.5 containing 0.2 M NaCl, 0.5% (w / v) polyvinylalcohol (PVA)) (FITC-Tgt 10 nM). After immersing one solid phase prepared above and shaking and stirring overnight (over 8 hours) at room temperature, the fluorescence spectrum of the external liquid is obtained and compared with the fluorescence spectrum before immersion. The adsorption amount of FITC-Tgt on quartz glass was determined. The results are shown in FIG. From FIG. 12, it is judged that 15% or more, preferably 15 to 20% of DMF is added, and therefore 1.0 to 3.0 mol / L, more preferably 1.9 to 2.6 mol / L of DMF is necessary. It was. A 15% solution of DMF is 1.9 M in terms of molar concentration.

5. Study of hydrophilic organic solvent 2
In place of DMF in “4. Study of hydrophilic organic solvent 1”, various hydrophilic organic solvents were used so as to obtain the same molar concentration, and the adsorption amount of FITC-Tgt was determined in the same manner as described above. As a result, it was found that the following hydrophilic organic solvents can induce adsorption equivalent to or higher than DMF: formamide, dimethylformamide, diethylformamide, dimethylsulfoxide, acetone, methanol, ethanol, ethylene glycol, acetamide, dimethylacetamide.

6). Hybridization concentration limit
FITC-Tgt in various concentrations to 2.0 ml basic solution (50 mM sodium carbonate-sodium bicarbonate buffer, containing 0.2 M NaCl, 0.5% (w / v) polyvinylalcohol (PVA), 20% (v / v) DMF) Added. By immersing one solid phase prepared above in this and shaking and stirring overnight (over 8 hours) at room temperature, the fluorescence spectrum of the external liquid is obtained and compared with the fluorescence spectrum before immersion. The adsorption amount of FITC-Tgt on quartz glass was determined. The results are shown in FIG. FIG. 13 shows that specific adsorption occurs up to FITC-Tgt of 50 pM. In this assay method (that is, the amount of adsorption of FITC-Tgt is determined from the decrease in the fluorescence intensity of FITC-Tgt), it is close to the detection limit of FITC and 50 Concentrations below pM could not be examined.

It is a schematic diagram which shows 1st Embodiment of the solid-phase applicable in this invention. It is a schematic diagram which shows 2nd Embodiment of the solid-phase applicable in this invention. It is a figure which shows a peptide nucleic acid typically. It is a figure which shows a spacer. It is a figure which shows embodiment of the peptide nucleic acid probe provided with the spacer in which the cysteine residue was introduce | transduced into C terminal. 5 is a schematic diagram showing a solid phase when glass is used as a solid phase carrier, APTES is used as a silane coupling agent for introducing an amino group on the glass surface, LC-SMCC is used as a linker, and the peptide nucleic acid probe of FIG. 5 is used as a peptide nucleic acid probe. is there. FIG. 3 is a schematic diagram showing a state in which a target base sequence labeled with a fluorescent dye is hybridized to a peptide nucleic acid probe fixed to a solid phase carrier by a linker. It is a schematic diagram of a state in which a target base sequence is hybridized to a peptide nucleic acid probe fixed to a solid phase carrier by a linker, and a detection base sequence labeled with a fluorescent dye is further hybridized to the target base sequence. It is the absorption spectrum in the gaseous phase of the quartz glass which introduce | transduced the peptide nucleic acid probe. It is a figure which shows the dependence with respect to pH of nonspecific adsorption | suction suppression. It is a figure which shows the dependence with respect to pH of nonspecific adsorption | suction suppression. It is a figure which shows the dependence with respect to the hydrophilic organic solvent of nonspecific adsorption | suction suppression. It is a figure which shows the detection density | concentration limit of a target base sequence.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Solid phase, 2 ... Solid phase support | carrier, 4, 4a, 4b, 4c, 4d, 4e ... Peptide nucleic acid probe, 6 ... Linker, 8 ... Target base sequence, 10 ... Fluorescent dye, 12 ... Base sequence for detection.

Claims (8)

A base sequence detection method for detecting a minute amount of a target nucleic acid,
To a peptide nucleic acid probe having a length of 8 to 20 mer immobilized on a solid phase carrier,
A solution containing a target nucleic acid having a base sequence complementary to the peptide nucleic acid probe, the solution further containing polyvinyl alcohol and a hydrophilic organic solvent of 1.0 to 3.0 mol / L based on the solution The hydrophilic organic solvent is at least one selected from the group consisting of formamide, dimethylformamide, diethylformamide, acetamide, dimethylacetamide, propionamide, benzamide, dimethylsulfoxide, acetone, methanol, ethanol, butanol, ethylene glycol and glycerol. By contacting a solution having a pH adjusted to 9.1 to 10.6,
A hybridization step of performing a hybridization method characterized by hybridizing the peptide nucleic acid probe and the target nucleic acid ;
And a detection step for detecting the hybridized target nucleic acid. 2. The base sequence detection method according to claim 1, wherein the solution further contains a buffer and / or NaCl or KCl. The base sequence detection method according to claim 1 or 2, wherein the detection limit of the target nucleic acid is 50 pM. The solid phase carrier is a solid phase carrier having an amino group on its surface, and the peptide nucleic acid probe is a peptide nucleic acid probe having a cysteine residue introduced at its end,
The peptide nucleic acid probes, at the cysteine residues, by a linker with an amino group-reactive group and a mercapto group-reactive groups, claim 1-3 for, characterized in that it is immobilized on the solid phase support The base sequence detection method according to any one of the above . 5. The method according to claim 1, wherein the target nucleic acid labeled with a fluorescent dye is hybridized with the peptide nucleic acid probe in the hybridization step, and the fluorescent dye is detected in the detection step. The base sequence detection method according to Item . In the hybridization step, the target nucleic acid partially having a sequence region complementary to the peptide nucleic acid probe is hybridized with the peptide nucleic acid probe, and at least a part of the sequence other than the sequence region in the target nucleic acid having a sequence complementary and a detecting nucleotide sequences labeled and hybridized with a fluorescent dye, in the detection step, any one of claims 1 to 4, characterized in that detecting the fluorescent dye nucleotide sequence detecting method according to. A solid phase in which a peptide nucleic acid probe having a length of 8 to 20 mer is immobilized on a solid phase carrier;
A solution to which a target nucleic acid having a base sequence complementary to the peptide nucleic acid probe is to be added, the solution containing polyvinyl alcohol and 1.0 to 3.0 mol / L of a hydrophilic organic solvent based on the solution The hydrophilic organic solvent is at least one selected from the group consisting of formamide, dimethylformamide, diethylformamide, acetamide, dimethylacetamide, propionamide, benzamide, dimethylsulfoxide, acetone, methanol, ethanol, butanol, ethylene glycol and glycerol. And a solution having a pH adjusted to 9.1 to 10.6. The kit for detecting a base sequence according to claim 7, wherein the solution further contains a buffer and / or NaCl or KCl.

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