JP2002281978A - Method for non-label detection of target dna using dna probe array - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new detection method that permits the quantitative detection of a target nucleic acid molecule without beforehand labelling the target nucleic acid per se when the target nucleic acid molecule is detected by forming a hybrid with a DNA probe using a DNA array. SOLUTION: The presence or absence of the formation of a hybrid in a DNA probe is detected by utilizing such a phenomenon that, when binding a fluorescein dye to a terminal of the DNA probe, a difference in fluorescent property is caused between the dye in associated state and that in dissociated state, and so, when the hybrid is formed from the DNA probe and a target nucleic acid, the association state is cancelled ant it causes the difference in fluorescent property and which is measured with an optical means.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for detecting a target DNA utilizing the formation of a hybrid with a DNA probe, and a DNA probe used therefor.
More specifically, using a DNA array in which a plurality of types of DNA probes are immobilized in an array on a solid phase, a nucleic acid having a specific base sequence (DNA Or RNA), or a highly sensitive target DNA used to identify its base sequence or to test for the presence or absence of various mutations in the base sequence.
The detection method.

[0002]

2. Description of the Related Art At present, a worldwide project for decoding the entire nucleotide sequence of the human genome and elucidating its genetic information is being conducted. As part of this, the identification of genes associated with diseases has been promoted, and the results have been reported one after another. Furthermore, using the obtained results,
There is a movement to detect the presence or absence of genes associated with various diseases and use them for diagnosis and prediction of diseases. At that time, for each gene of the item you want to examine,
Instead of the conventional method of examining the presence or absence using R or hybridization method, a method of simultaneously examining the presence / absence (mutation) of multiple items of genes is being studied.
Specifically, many types of DNA probes are regularly arranged on the same substrate, and hybridization with nucleic acid molecules such as multiple types of gene DNA contained in the same sample is performed at once. Attempts have been made to quickly make judgments on multiple items, such as the presence or absence of gene mutations in
Various DNA arrays used for the purpose have been developed.

Have reported a method of synthesizing a DNA probe on a glass substrate and preparing probes having different base sequences one by one on the same substrate (Nu).
cleic Acids Research, 22
Vol., P. 1368, (1994)). Affymetrix also claims that 400,000 types of D on a 1/2 inch square substrate
The NA probe is arrayed to detect the gene (Science, vol. 274, p610, (19)
96)). They applied the method of photolithography and repeated the reaction of linking nucleotides on a substrate, and arranged ones having various base sequences arranged in an array.
We are synthesizing 5-base length DNA. For example, means for forming a DNA array on a solid phase such as a glass substrate are different from each other, and although each has its own characteristics, the detection method is basically based on the DNA hybridization method.

[0004] In a hybridization reaction, a probe DNA and a target DNA form a hybrid by hydrogen bonding at a base sequence complementary to each other. In the conventional method, usually, a label is attached to a target DNA, a hybrid is formed with a DNA probe having a specific base sequence, and the hybrid is used for detection of the immobilized target DNA. As a method of applying the label, a radioactive substance
Alternatively, a method of attaching a fluorescent dye as a label, or using a primer labeled with a fluorescent dye or isotope during amplification by PCR, or a PCR amplification product incorporating an isotope-labeled base And further, based on the PCR amplification product, a single-stranded R
In the process of biosynthesizing NA, there are various methods such as a method of incorporating a base serving as an isotope label as a label.

[0005]

The above-mentioned means for labeling a target nucleic acid molecule such as a target DNA has advantages and disadvantages in each method. For example, in the end labeling method, since one label is produced from one target DNA, the quantitative property can be maintained, but sufficient sensitivity cannot be achieved when the amount of the target DNA present in the sample sample is small. On the other hand, in the PCR method and the method of incorporating a base serving as an isotope label as a label at the time of producing single-stranded RNA, a plurality of labeled substances are produced from the initial target DNA serving as a template, but the sensitivity is increased. Between the number of labeled DNAs forming a hybrid and the label (the number of bases to be an isotope label) contained therein,
Since there is not always a one-to-one correspondence, it lacks quantitativeness. For example, when performing a quantitative comparison between the amount of a target DNA of a single base mismatch and the amount of a target DNA of a perfect match based on the intensity of the signal derived from the isotope labeling, the determination may be made in some cases. It also becomes an obstacle such as difficulty.

[0006] In addition, amplification by the PCR method itself has an essential problem that it does not necessarily reflect the amount of target DNA in an initial state. That is, when amplifying by a PCR reaction, there are a base sequence having a high amplification efficiency and a base sequence having a low amplification efficiency, and it is necessary to compensate for the difference in the amplification efficiency and to compare the two. It is necessary to carry out a control experiment using a standard gene as a reference in parallel with the experiment.

Further, when detecting a large number of specimens, from the viewpoint of operation, a PCR reaction or a reaction for labeling an end takes a considerable amount of time including a purification step. When using isotope labels, the label density changes over time during that time.
Practical issues remain.

[0008] On the other hand, when a fluorescent dye is used, the above-mentioned problem that the label density changes over time is avoided, but another obstacle due to the binding of the fluorescent dye to the target DNA is a concern. Specifically, a fluorescent dye is generally a molecule that is hydrophobic and easily interacts with a base portion of a target DNA. Depending on each probe base sequence, the property may cause variation in the efficiency of the hybridization reaction. In addition, fluorescent dyes
Due to its hydrophobicity, it also has the property of easily causing adsorption on the surface of the DNA array substrate. This adsorption, when a fluorescent dye is bound to the target nucleic acid and used as a label, in addition to immobilization via hybridization with a DNA probe,
The effect of immobilization due to the adsorption of the fluorescent dye itself to the substrate surface is brought about. The immobilization by the adsorption of the fluorescent dye itself gives background fluorescence upon detection, lowering the ratio to the fluorescence signal due to the immobilization through the formation of the original hybrid, ie, the S / N ratio. This causes a problem of lowering the detection sensitivity.

[0009] The present invention solves the above-mentioned problems, and an object of the present invention is to use a DNA array in which a plurality of types of DNA probes are bonded in an array on a substrate.
To provide a novel detection method capable of quantitatively detecting a nucleic acid molecule to be detected without forming a label in advance when detecting a nucleic acid in a sample by forming a hybrid with a probe. It is in. More specifically, an object of the present invention is to provide a method for detecting nucleic acid molecules in a sample.
Labeling the NA probe and reflecting the presence or absence of the formation of a hybrid between the nucleic acid molecule having a complementary base sequence and the DNA probe, the difference in the fluorescent properties of the fluorescent dye used for the labeling is determined as the target. It is an object of the present invention to provide a method capable of quantitatively detecting the formation of a hybrid between a nucleic acid molecule and a DNA probe, and a labeled DNA probe used for such a purpose.

[0010]

Means for Solving the Problems The present inventor has conducted research and studies to solve the above-mentioned problems. As a result, when a nucleic acid molecule in a sample and a DNA probe form a hybrid, the DNA probe itself becomes Due to the formation of such a double-stranded structure, the conventional flexibility is lost, and for example, attention is paid to the fact that it is no longer possible to interact with an adjacent DNA probe of the same kind. It is possible to detect the formation of a hybrid of a target nucleic acid molecule with a DNA probe of the same kind by utilizing the phenomenon that it is impossible to interact with the same type of DNA probe from the state where it was possible to interact with the DNA probe. I thought of being. When the idea is put into practice, further investigations and studies are made on more efficient means, and the same fluorescent dye is bonded to the end of the same type of DNA probe, and this fluorescent dye can form an association state with each other. However, under certain circumstances, more specifically, when a hybrid is formed in a DNA probe, one that eliminates the associated state is selected, and the associated state and the associated state are determined. It was confirmed that in the state where the fluorescence properties were eliminated, it was possible to detect the presence or absence of the formation of a hybrid in the DNA probe by optical means by utilizing the difference in the fluorescence characteristics. Based on the above findings, the present inventor
When using a DNA array constituted by immobilizing probes in an array, a fluorescent dye capable of associating with each other is covalently bonded to the end of the probe DNA strand, and is used as a label. The present inventors have demonstrated that it is possible to detect the presence or absence of the formation of a hybrid using a highly sensitive fluorescence detection method without labeling the molecule itself, and have completed the present invention.

That is, in the method for detecting a target DNA non-label, a target DNA capable of forming a hybrid with the DNA probe is detected by using a DNA probe array in which a plurality of types of DNA probes are bonded in an array on a substrate. A method, wherein said DNA is bound on a substrate
The probe covalently binds a fluorescent dye to the end of the DNA, and the DNA probe can form a hybrid with a target DNA having a base sequence complementary to the base sequence in a state of being fixed on a substrate. In the state where such a hybrid is not formed, the fluorescent dye covalently bonded to the end of the DNA takes an associated state, and in the state where such a hybrid is formed, the state of association of the fluorescent dye with each other The fluorescent dye covalently bonded to the end of the DNA exhibits different fluorescence characteristics between the associated state and the state after the association state has been eliminated, and the presence or absence of the change in the fluorescent characteristic is observed. By doing so, each of a plurality of types of DNA probes constituting the DNA array is hybridized to the specific DNA probe. And detecting the presence or absence of the target DNA to form a body, a non-labeled method of detecting a target DNA.

[0012] In the detection method of the present invention, the fluorescent dye is used, and its fluorescence characteristics are in a quenched state when mutually associated, and in a state in which the associated state is eliminated.
It is preferable to use a fluorescent dye that emits fluorescence.

At this time, a coumarin derivative can be used as the fluorescent dye. Further, a fluorescein derivative can be used as the fluorescent dye.

In addition, as the above-mentioned fluorescent dye, the following general formula (I):

[0015]

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(Wherein, R represents a substituted alkyl group having a functional group for binding to the end of DNA as a substituent). Similarly, as the fluorescent dye, the following general formula (I
I):

[0017]

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(Wherein R represents a functional group for binding to the end of DNA, or a substituted alkyl group having such a functional group as a substituent). Further, as the fluorescent dye, the following general formula (III):

[0019]

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(Wherein, R represents a functional group for binding to the end of DNA, or a substituted alkyl group having such a functional group as a substituent).

In addition, the present invention provides a method for detecting non-labeled target DNA of the present invention having the above-mentioned structure.
An NA probe is also provided, ie, the DN of the present invention.
The A probe is a DNA used for producing a DNA array formed by binding a plurality of types of DNA probes in an array on a substrate.
A probe, wherein the DNA probe can be fixed by binding to a DNA array substrate, and a fluorescent dye is covalently bound to an end of a DNA portion of the DNA probe;
The DNA probe has a target DN having a base sequence complementary to its base sequence when immobilized on a substrate.
A is capable of forming a hybrid with A, and in a state where such a hybrid is not formed, the fluorescent dye covalently bonded to the end of the DNA assumes a state in which the fluorescent dyes are associated with each other to form such a hybrid. Then, the association state between the fluorescent dyes is eliminated, and the D
The fluorescent dye covalently bonded to the end of NA is a probe characterized by exhibiting different fluorescence characteristics in an associated state and in a state in which the associated state has been eliminated.

In the DNA probe of the present invention, the above-mentioned fluorescent dye is used as the above-mentioned fluorescent dye, and has a fluorescence characteristic in a quenched state when it is associated with each other, and a fluorescent state when the association state is eliminated. It is preferable to use a certain fluorescent dye.

At this time, a coumarin derivative can be used as the fluorescent dye. In addition, a fluorescein derivative can be used as the fluorescent dye. The probe according to claim 9, which is used.

In addition, as the above-mentioned fluorescent dye, the following general formula (I):

[0025]

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(Wherein, R represents a substituted alkyl group having as a substituent a functional group for binding to the end of DNA). Similarly, as the fluorescent dye, the following general formula (I
I):

[0027]

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(Wherein, R represents a functional group for binding to the end of DNA or a substituted alkyl group having such a functional group as a substituent). Further, as the fluorescent dye, the following general formula (III):

[0029]

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(Wherein, R represents a functional group for binding to the end of DNA, or a substituted alkyl group having such a functional group as a substituent).

[0031]

In the detection method of the present invention, an associative fluorescent dye having the above-mentioned constitution is covalently bound to its terminal,
By using a DNA array comprising a labeled DNA probe, an operation of labeling a target nucleic acid molecule to be detected, which has a base sequence complementary to the DNA probe and forms a hybrid, becomes unnecessary. Therefore, instead of the step of applying a different label to each nucleic acid molecule to be detected contained in the specimen sample in advance, a DNA probe previously labeled with a fluorescent dye is synthesized at a time, and a DNA array prepared using the same is synthesized. By using each time, practically,
The detection time can be significantly reduced. In addition, when labeling a nucleic acid molecule to be detected with a fluorescent dye, for example, non-specific adsorption due to the hydrophobicity of the fluorescent dye itself, or the labeled nucleic acid on the surface of the peptide array substrate When non-specific adsorption by the molecule itself occurs, regardless of the hybrid formation by these DNA probes,
The fluorescence from the fluorescent dye immobilized on the surface of the DNA array causes an increase in the background fluorescence, and the problem that causes a decrease in quantitative performance and the like is also essentially solved.

In addition, in the detection method of the present invention, since the operation of labeling the target nucleic acid molecule becomes unnecessary, a means for labeling by using a labeled primer by PCR is used. When used, it is also effective in eliminating the effect of the difference in PCR efficiency, which has been a problem of quantitative performance.

That is, in the present invention, D
Since the end of the NA probe is fluorescently labeled and a DNA array prepared using the fluorescent label is used, it is not necessary to label each of the nucleic acid molecules as a sample. The fluorescent dye used in this method is a dye that causes fluorescence quenching by association. Therefore, DN
When the DNA probe molecules are arranged at a high density as in the A-array, the fluorescent dyes bonded to their ends can be brought into a state extremely close to each other. Changes in properties occur, for example, quenching of fluorescence. Furthermore, secondary problems such as secondary structure formation due to the interaction between the nucleobase of the probe DNA and the fluorescent dye molecule can be avoided before the association between the fluorescent dyes.

Compared with the association of the fluorescent dye, the hybridization reaction (hybrid formation) is based on a dense interaction between mutually complementary base sequences, and the binding constant is extremely large. The association between the fluorescent dye attached to the end of the DNA probe
By the intervention of the target DNA hybridized with the probe, the association state is eliminated, and the original fluorescent properties of the fluorescent dye are restored. As a result, the fluorescence intensity corresponding to the number of hybrids formed is observed only in the portion of the DNA probe that has been hybridized with the target DNA.

In the present invention, as a fluorescent dye to be attached to a DNA probe as a label, a fluorescent dye which easily forms an aggregate is used.
For example, a system such as coumarin or fluorescein that emits fluorescence with a monomer but easily forms an aggregate, and self-quenching and quenching of the aggregate can be used.

Such a property of the fluorescent dye can be examined by plotting the fluorescence intensity observed in a solution prepared by changing the concentration of the fluorescent dye variously against the solution concentration. When the fluorescence of a fluorescent dye that produces strong fluorescence in a dilute solution decreases with increasing concentration, it can be determined that the change in the fluorescence characteristics is due to the formation of an aggregate and the accompanying fluorescence quenching. It should be noted that it is not so-called concentration quenching, which shows a similar decrease in fluorescence intensity,
It needs to be verified. In concentration quenching, the absorption intensity of the fluorescent dye itself is proportional to each concentration, but in the formation of aggregates, the absorption spectrum changes with the formation of the aggregates, resulting in a change in the absorption peak wavelength of the fluorescent dye itself. A decrease in absorption intensity is observed. In some cases, the peak of the absorption spectrum derived from the aggregate can be clearly separated and observed at a longer wavelength portion than the absorption peak wavelength of the fluorescent dye itself. Therefore, by verifying the above-mentioned phenomenon, it is possible to easily confirm not the so-called concentration quenching, but the re-confirmation of the formation of the aggregate and the accompanying fluorescence quenching.

Examples of the fluorescent dye having the property of the formation of the aggregate and the quenching of the fluorescent light due to the formation of the aggregate include pyrylium dyes in addition to the well-known dyes such as coumarin and fluorescein. The present inventor has found that a pyrylium dye having the following structure easily forms an aggregate and is quenched with the formation of the aggregate, and in the present invention, like coumarin and fluorescein, for labeling a DNA probe. Used as a fluorescent dye.

The present inventors have confirmed that the pyrylium dye has excellent properties of causing self-association and causing fluorescence quenching. The following formulas (IV), (V) and (V)
A pyrylium dye derivative having the structure shown in I) can be mentioned.

[0039]

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(Wherein, R represents a carboxyl group or a straight-chain alkyl group substituted with a carboxyl group at the terminal)

[0041]

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(Wherein, R represents a carboxyl group or a linear alkyl group substituted with a carboxyl group at the terminal)

[0043]

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(In the formula, R represents a carboxyl group or a linear alkyl group substituted with a carboxyl group at the terminal.) These pyrylium dye derivative compounds have a phenyl group at two positions 2, 4, and 6 of the pyrylium ring. Is substituted, and the remaining one has structural similarity substituted by an alkyl group or a 4-alkyl-substituted phenyl group. Examples of such pyrylium dye derivative compounds include compounds represented by the following formulas (VII), (VIII) and (IX).

[0045]

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[0046]

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[0047]

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In practice, the above general formulas (IV), (V),
The pyrylium dye derivative having the structure shown in (VI) uses an alkyl group represented by R, for example, a methyl group portion to give DN.
The DNA probe is bound to the terminal of the DNA probe in a form in which a functional group useful for binding to A is introduced as a substituent at the terminal of the alkyl group.

The pyrylium dye derivatives represented by the above general formulas (I), (II) and (III) which can be used in the present invention are represented by the above formulas (VII), (VIII) and (IX) Methods for synthesizing compounds, for example, W. Foerst et al.
`` New Methods of Preparative Organic Chemistr
y ", Acad. Press (1964), and R. Wizinger
See, for example, the synthesis procedures described in Helv. Chim. Acta, 39, 217 (1956), and any of them can be synthesized according to the synthesis methods disclosed in these documents.

In the present invention, the fluorescent dye to be bonded to the end of the DNA probe is in a state where an aggregate is formed and a state where the aggregate is eliminated, such as a property of self-association and fluorescence quenching. It is not particularly limited as long as it has a property that makes a distinct difference in the fluorescent characteristics.

The method for producing the DNA array is not particularly limited. When the DNA probe is synthesized on the substrate by the photolithography method, a step of binding a fluorescent dye may be added at the last stage of the synthesis. In addition, DN
In the case where the A probe is synthesized and then a DNA probe solution is supplied by a pin or ink jet method or the like, the end (3 ′ end) opposite to the end (usually 5 ′ end) to be bonded to the substrate during the DNA synthesis. Then, a fluorescent dye may be introduced by a covalent bond.

As a method for introducing this fluorescent dye, DNA
There is no particular limitation as long as it is linked to the moiety by a covalent bond. For example, using a commonly used amino linker reagent, an amino group is previously introduced into the 3 ′ end of the oligonucleotide of the DNA probe, and the terminal amino group is chemically reacted with the carboxyl group introduced into the fluorescent dye, Examples of the method include an amide bond.

In the DNA probe according to the present invention, the probe base length of the DNA portion is not particularly limited, but is usually selected from the range of 8 to 20 bases as the probe base length of the DNA portion. Is preferred. However, if the probe base length is too long, the DNA of the probe itself may form a secondary structure in some cases. In that case, the efficiency of the formation of aggregates between the fluorescent dyes at the ends of the probes that are adjacent at high density,
Since there is a possibility that the length of the probe itself decreases with the formation of the secondary structure, it is more preferable to prevent the base length from being excessively long according to the base sequence. When selecting a probe base length that is too short as the probe base length of the DNA portion, for example, when trying to detect a target DNA molecule having a single base misfit, the desired binding stability may not be achieved. . That is, when used for diagnosis of a disease that causes a genetic disease by mutating one base compared to the base sequence of a normal gene, in addition to normal gene DNA, However, in order to enable detection of a gene DNA having a mutation, it is desirable to use, for example, a DNA probe having a length of 8 bases or more.

In addition, in order to bind and immobilize the DNA probe on the substrate, in addition to the base sequence involved in the hybridization reaction, an addition used for immobilization depends on the method of binding and immobilization on the substrate. It is possible to appropriately select the entire base length as long as a basic DNA portion is added and the base length does not become excessively long.

[0055]

The present invention will be described below more specifically with reference to examples. Although these examples are examples of the best mode of the present invention, the present invention is not limited by these examples.

Example 1 1) Synthesis of Oligonucleotide for DNA Probe An 18-mer oligonucleotide having an amino linker at the 3 ′ end and having the following base sequence was synthesized by an automatic DNA synthesizer. The introduction of an amino group to the 3 ′ end was performed using a commercially available reagent kit, 3′-amino modifier C3 (manufactured by Glen Research). 5 'GTTGTAAAACGACGGCCAGT-N
The H ₂ 3 ′ synthesized DNA was cut out from the CPG support, and deprotected and subjected to high performance liquid chromatography (HP
LC).

2) Coupling of Coumarin 330 μg of the above oligonucleotide was dissolved in 100 μl of pure water, and 1M phosphate buffer (pH 7.0) 16
μl was added. Then, 7-hydroxycoumarin-3-
60 μl of a 60 mM acetonitrile solution of carboxylic acid (manufactured by Sigma) was added and reacted at 40 ° C. for 24 hours. After completion of the reaction, a gel filtration column NA for DNA manufactured by Pharmacia
In P-25, crude purification was performed to remove unreacted substances,
Further purification was performed by HPLC. After purification by HPLC, desalting was performed using the gel filtration column, and 7
-A DNA probe labeled with hydroxycoumarin was obtained.

The fluorescence characteristics of the obtained DNA probe were measured. Fluorescence was measured with a Hitachi F-4500 fluorescence spectrophotometer. The probe solution adjusted to a concentration of 8 μM contains 386 n
When excited with m, fluorescence from the excimer was emitted at 448 nm.

3) Binding of Labeled DNA Probe to Substrate Poly-L-lysine-coated slide (manufactured by Sigma) was ligated with the above-mentioned coumarin-bound oligonucleotide to a Telech
Stamping was performed using a 16-pin type stamper manufactured by Em Corporation. Then, after placing on a hot plate at 95 ° C. and drying, the oligonucleotides were cross-linked by a UV cross-linking machine (UV irradiation intensity was set to 60 mJ).

As a result of observation with a fluorescence microscope, the fluorescence observed in the solution had disappeared. This is considered to be the result of quenching caused by the self-association of the coumarin molecules at the ends of adjacent probes.

4) Hybridization reaction of target DNA with M13mp18 Using a commercially available M13mp18 phage vector solution as a target DNA, a hybridization reaction was carried out by a conventional method. This gene DNA has a partial base sequence complementary to the base sequence of the above-mentioned DNA probe fixed on the substrate. Hybrid chamber (Tel
chem) (100 mM NaCl containing M13mp18 on the edge of the cover glass).
The solution was placed on the slide glass, and the slide glass was covered with a cover glass while taking care not to cause bubbles. In that state, 6
The hybridization reaction was performed at 5 ° C. for 6 hours.

Then, as a result of observation with a fluorescence microscope, DN
Blue fluorescence was observed at the portion where the A probe was fixed. Since this fluorescence has the same color as the fluorescence observed in the solution before being fixed to the substrate, the DNA probe and M13m
Upon hybridization with p18 target DNA, M13
It is determined that the mp18 target DNA intervenes between the coumarin molecules bound to the DNA probe ends, the state of association of the labeled fluorescent dye coumarin is eliminated, and the original fluorescent property is emitted.

Example 2 Detection of Target DNA Using Pyrylium Dye (1) as Labeling Fluorescent Dye of DNA Probe 1) Synthesis of 2- (3-carboxypropyl) -4,6-diphenylpyrylium iodide Preparation of a DNA probe bound to the pyrylium dye (1) 163 mg of acetophenone and 456 mg of glutaric anhydride were added to 3 ml of concentrated sulfuric acid in an ice bath and completely dissolved. Subsequently, the solution was heated to 120 ° C. in an oil bath, stirred for about 3 hours, and allowed to cool at room temperature. This reaction solution was added to 100 ml of water and stirred, and then 60 ml of chloroform was added, and the mixture was washed by an extraction operation, and an aqueous layer was collected. This washing with chloroform extraction was repeated a total of four times to remove unreacted acetophenone. Next, 1.50 g of sodium iodide was added to the aqueous layer, stirred, and cooled in a refrigerator overnight.
The deposited yellow precipitate was separated by filtration and collected to obtain a crude crystal. The obtained crude crystals were recrystallized with an appropriate amount of water to obtain 66 mg of 2- (3-carboxypropyl) -4,6-diphenylpyrylium iodide as crystals.

The obtained pyrylium compound (40 mg) and N-
46 mg of hydroxysuccinimide was dissolved in 1 ml of dry DMF. After confirming complete dissolution, the mixture was transferred to an ice bath and cooled. Next, 80 mg of N, N'-dicyclohexylcarbodiimide (DCC) was added, and the mixture was stirred in a dark place for 24 hours. The mixture was stirred in an ice bath for the first 2 hours, and thereafter, stirred at room temperature. Thereafter, the floating DCC urea was removed with a membrane filter, and then dropped into about 50 ml of diethyl ether. The operation of collecting the precipitated precipitate and washing again with 60 ml of diethyl ether was repeated several times, and the obtained powder was dried with a vacuum pump.

In the same process as in Example 1, 18m having the above base sequence and having an amino linker bonded to the 3 ′ end was used.
er oligonucleotide was synthesized with a DNA automatic synthesizer. The synthesized oligonucleotide 330 μm
g was dissolved in 100 μl of pure water, and 16 μl of 1M phosphate buffer (pH 7.0) was added. Then, 2- (3
-Carboxypropyl) -4,6-diphenylpyrylium 60 μl of a 50 mM acetonitrile solution of a succinimide derivative of iodide was added and reacted at 40 ° C. for 24 hours. After completion of the reaction, crude purification was performed using a gel filtration column for DNA manufactured by Pharmacia NAP-25 to remove unreacted substances, and further purification was performed by HPLC. After purification by HPLC, desalting was performed using the gel filtration column, and 3 ′
A DNA probe was obtained in which 2- (3-carboxypropyl) -4,6-diphenylpyrylium iodide was bound to the terminal via an amide bond. Its structure is as follows.

[0066]

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The absorption spectrum of the obtained DNA probe was measured. An absorption corresponding to the absorption spectrum of the pyrylium dye alone was observed at around 370 nm, and an absorption near 260 nm corresponding to the absorption spectrum of DNA itself (nucleic acid base) was also observed on the shorter wavelength side. Since the absorption intensities of both of them almost match the absorption intensities expected from the molar absorption and extinction coefficients of the fluorescent dye and the nucleic acid base alone, the target DNA probe in which the fluorescent dye and DNA are bound is synthesized. It was determined.

When the fluorescence of the pyrylium dye-binding probe was measured, strong fluorescence was shown at 440 nm by excitation at 370 nm corresponding to the absorption derived from the pyrylium dye.

2) Binding of DNA Probe Binding to Pyrylium Dye to Substrate In the same manner as in Example 1, the DNA probe was spotted on the surface of the poly-L-lysine-coated substrate, and then immobilized by UV crosslink. In this state, the fluorescence derived from the pyrylium dye in the portion where the DNA probe was fixed on the substrate surface was quenched.

3) Hybridization reaction of target DNA with M13mp18 Under the same conditions and procedures as in Example 1, M
A hybridization reaction with the DNA probe was performed using a 13mp18 phage vector. After the reaction, for the region where the DNA probe is fixed,
When the fluorescence was observed, fluorescence derived from the pyrylium dye of the label was detected in the wavelength region observed in the solution.

Example 3 Detection of Target DNA Using Pyrylium Dye (2) as Labeling Fluorescent Dye of DNA Probe 1) 2,6-Diphenyl-4-carboxypyrylium
Synthesis of iodide and pyrylium dye (2)
Preparation of bound DNA probe 6.53 mg of acetophenone and 1.84 of glyoxalic acid
mg was placed in a 100 ml flask and dissolved in 15 ml of concentrated sulfuric acid under ice-cooling, followed by stirring at 50 ° C. for 2 hours. To this reaction solution, 10 g of sodium iodide dissolved in 300 ml of water was added, and the mixture was stirred at 90 ° C. for 30 minutes, then returned to room temperature, and the deposited precipitate was filtered. The obtained powder was recrystallized and purified with a mixed solvent of acetophenone and benzene.

By the same operation as in Example 1, the obtained pyrylium compound was converted to a succinimide derivative. Utilizing this succinimide derivative of a pyrylium compound, a binding reaction was performed with an oligonucleotide having the above-mentioned base sequence and having an amino group introduced at the 3 ′ end, as in Example 1.
A probe DNA was obtained in which the 2,6-diphenyl-4-carboxypyrylium iodide dye was bound to the 3 ′ end via an amide bond. Its structure is as follows.

[0073]

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When the fluorescence of the aqueous solution of the DNA probe was observed, when it was excited at 380 nm, which corresponds to the absorption derived from the pyrylium dye, it emitted blue fluorescence at 445 nm.

2) Binding of the pyrylium dye-bound DNA probe to the substrate and hybridization reaction of the target DNA with M13mp18 The same procedure as in Example 1 was used to fix the pyrilium dye-bound DNA probe on the substrate in the form of a spot. . In this state, the blue fluorescence derived from the pyrylium dye in the portion where the DNA probe was fixed on the substrate surface was quenched.

Under the same conditions and procedures as in Example 1, target DN
As A, using a M13mp18 phage vector,
A hybridization reaction with the DNA probe was performed. After the reaction, the fluorescence of the region where the DNA probe was immobilized was observed. As a result, blue fluorescence derived from the labeled pyrylium dye was detected in the wavelength region observed in the solution.

Example 4 Detection of Target DNA Using Pyrylium Dye (3) as Labeling Fluorescent Dye of DNA Probe 1) Synthesis of 2,6-diphenyl-4- (4-carboxyphenyl) pyrylium iodide, and Preparation of DNA Probe Binding to Pyrylium Dye (2) 1 g of P-formylbenzoic acid and 2.2 mg of acetophenone
Was dissolved in 8.3 ml of concentrated sulfuric acid and stirred at 90 ° C. for 2 hours. To this reaction solution, 10 g of sodium iodide dissolved in 300 ml of water was added, and the mixture was stirred at 90 ° C. for 30 minutes, returned to room temperature, and the precipitated precipitate was filtered. The obtained powder was recrystallized and purified with a mixed solvent of acetophenone and benzene.

By the same operation as in Example 1, the obtained pyrylium compound was converted to a succinimide derivative. Utilizing this succinimide derivative of a pyrylium compound, a binding reaction was performed with an oligonucleotide having the above-mentioned base sequence and having an amino group introduced at the 3 ′ end, as in Example 1.
2,6-diphenyl-4- (4-carboxyphenyl)
A probe DNA was obtained in which the pyrylium iodide dye was linked via an amide bond to the 3 ′ end. Its structure is as follows.

[0079]

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When the fluorescence of the aqueous solution of the DNA probe was observed, it was excited at 380 nm, which corresponds to the absorption derived from the pyrylium dye, and emitted blue fluorescence at 462 nm.

2) Binding of the pyrylium dye-bound DNA probe to the substrate and hybridization reaction of target DNA with M13mp18 The same procedure as in Example 1 was used to fix the pyrilium dye-bound DNA probe on the substrate in the form of spots. . In this state, the blue fluorescence derived from the pyrylium dye in the portion where the DNA probe was fixed on the substrate surface was quenched.

Under the same conditions and procedures as in Example 1, target DN
As A, using a M13mp18 phage vector,
A hybridization reaction with the DNA probe was performed. After the reaction, the fluorescence of the region where the DNA probe was immobilized was observed. As a result, blue fluorescence derived from the labeled pyrylium dye was detected in the wavelength region observed in the solution.

[0083]

According to the detection method of the present invention, instead of labeling a target nucleic acid molecule to be detected, a DNA is selectively hybridized with the target nucleic acid molecule and used for immobilization on a substrate.
A fluorescent dye is covalently bonded to the end of the A probe and used as a label, and a plurality of DNA probes labeled with such a fluorescent dye are used, and by using a DNA array in which each is spotted in an array, the association state of the fluorescent dye and Based on the difference in the fluorescence characteristics existing between the dissociated state and the state, the presence or absence of a hybrid formed by the binding of the target nucleic acid molecule to a DNA probe having a complementary base sequence is quantitatively determined. Can be detected. In addition, nucleic acid molecules such as target DNA to be detected need not be labeled, and are included in a specimen sample in advance.
Instead of a step of applying a different label to each nucleic acid molecule such as a DNA to be detected, DNA previously labeled with a fluorescent dye
DNA synthesized by synthesizing a probe at one time
By using the array each time, practically, the detection time can be significantly reduced. Furthermore, when labeling a nucleic acid molecule such as DNA to be detected with a fluorescent dye, non-specific adsorption or labeling on the surface of the DNA array substrate, for example, due to the hydrophobicity of the fluorescent dye itself, is performed. When non-specific adsorption by the protein itself occurs, these DNs
Irrespective of the selective hybrid formation by the A probe, the fluorescence from the fluorescent dye attached to the DNA immobilized on the surface of the DNA array causes an increase in the background fluorescence and a decrease in the quantitative property. The essential problem is also solved. In addition, in the detection method of the present invention, since the operation of labeling the target nucleic acid molecule becomes unnecessary, the use of a PCR method, the use of labeled primers, Furthermore, the present invention is also effective in eliminating the influence of the difference in PCR efficiency, which has been a problem of quantitativeness.

[0084]

[Sequence List] SEQUENCE LISTING <110> CANON INC. <120> A Method for Detecting a Target DNA molecule Which is not Labelled, Using a DNA probe-array <130> 4026009 <160> 1 <170> Microsoft Word <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DNA probe for hybidizing with a target gene of M13mp18 <400> 1 gttgtaaaac gacggccagt 20

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Claims (14)

[Claims] 1. A target DN capable of forming a hybrid with said DNA probe, using a DNA probe array in which a plurality of types of DNA probes are bonded in an array on a substrate.
A method for detecting A, wherein said DNA probe bound on a substrate comprises
A fluorescent dye is covalently bonded to the end of NA, and the DNA probe is a target DN having a base sequence complementary to its base sequence when fixed on a substrate.
A can form a hybrid with A. In a state where such a hybrid is not formed, the DN
The fluorescent dyes covalently bonded to the terminus of A take a state in which they are associated with each other. In a state where such a hybrid is formed, the state in which the fluorescent dyes are associated with each other is eliminated, and The fluorescent dye covalently bonded to the terminus exhibits different fluorescence characteristics between the associated state and the state in which the associated state has been eliminated. For each of the species DNA probes, for that particular DNA probe,
A non-label detection method for a target DNA, comprising detecting the presence or absence of a target DNA forming a hybrid. 2. A fluorescent dye, wherein the fluorescent dye has a fluorescence characteristic that is in a quenched state when it is associated with each other and emits fluorescence in a state in which the associated state is eliminated. The detection method according to claim 1, wherein: 3. The detection method according to claim 2, wherein a coumarin derivative is used as the fluorescent dye. 4. The detection method according to claim 2, wherein a fluorescein derivative is used as the fluorescent dye. 5. The fluorescent dye of the following general formula (I): The detection method according to claim 2, wherein a pyrylium dye derivative represented by the following formula (wherein, R represents a substituted alkyl group having a functional group for binding to the terminal of DNA as a substituent) is used. 6. The fluorescent dye according to the following general formula (I)
I): (Wherein R is a functional group for binding to the end of DNA,
Or a pyrylium dye derivative represented by the formula (1) or a substituted alkyl group having such a functional group as a substituent). 7. The fluorescent dye of the following general formula (II)
I): (Wherein R is a functional group for binding to the end of DNA,
Or a pyrylium dye derivative represented by the formula (1) or a substituted alkyl group having such a functional group as a substituent). 8. A DNA used for preparing a DNA array in which a plurality of types of DNA probes are bonded in an array on a substrate.
A probe, wherein the DNA probe can be bound and fixed on a substrate for a DNA array, and a fluorescent dye is covalently bound to an end of a DNA portion of the DNA probe, and the DNA probe is fixed on the substrate. The target DN having a base sequence complementary to the base sequence
A can form a hybrid with A. In a state where such a hybrid is not formed, the DN
The fluorescent dyes covalently bonded to the terminus of A take a state in which they are associated with each other. In a state where such a hybrid is formed, the state in which the fluorescent dyes are associated with each other is eliminated, and A probe characterized in that the fluorescent dye covalently bonded to the end shows different fluorescence characteristics between an associated state and a state where the associated state has been eliminated. 9. The fluorescent dye, which has a fluorescence characteristic that is in a quenched state when it is associated with each other and emits fluorescence when its association state is eliminated. The probe according to claim 8, wherein the probe is used. 10. The probe according to claim 9, wherein a coumarin derivative is used as the fluorescent dye. 11. The probe according to claim 9, wherein a fluorescein derivative is used as the fluorescent dye. 12. The fluorescent dye of the following general formula (I): The probe according to claim 9, wherein a pyrylium dye derivative represented by the following formula (wherein, R represents a substituted alkyl group having a functional group for binding to the terminal of DNA as a substituent) is used. . 13. The fluorescent dye according to the following general formula (I)
I): (Wherein R is a functional group for binding to the end of DNA,
Or a pyrylium dye derivative represented by the formula (1) or a substituted alkyl group having such a functional group as a substituent). 14. The fluorescent dye according to the following general formula (II)
I): (Wherein R is a functional group for binding to the end of DNA,
Or a pyrylium dye derivative represented by the formula (1) or a substituted alkyl group having such a functional group as a substituent).