JP2001116721A - Method for detecting partial complementary nucleic acid fragment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting a partial complementary nucleic acid which is useful to detect a genetic polymorphism formed of a single-strand normal gene fragment and a single-strand abnormal gene fragment. SOLUTION: The method for detecting whether or not a sample nucleic acid fragment is partially identical to a complementary nucleic acid fragment in terms of a base sequence includes a process 1 in which a sample solution having the sample nucleic acid fragment dissolved or dispersed is brought in contact with a DNA analysis element having a DNA fragment immobilized at its one end to a surface of a conductive substrate, a potential is impressed to the analysis element in the presence of an electrochemical activity-embedded intercalater and a value of a current flowing between the intercalater and the analysis element is measured, and a process 2 in which the current value measured in the process 1 is compared with a standard current value of the complementary nucleic acid fragment obtained in the same operation as in the operation of the process 1 with the use of a nucleic acid fragment complementary to the DNA fragment immobilized to the DNA analysis element.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting a gene polymorphism and a method for analyzing a mutation in an abnormal gene fragment, which are important as one of means for genetic diagnosis.

[0002]

2. Description of the Related Art In general, diseases are thought to be caused by environmental factors acting on hereditary predisposition. However, with the development of genetic technology, many diseases are accompanied by hereditary or exogenous genetic abnormalities. It has been elucidated. What is genetic diagnosis?
Abnormalities (base substitution, base deletion,
Base insertions, base point mutations, translocations, etc.), not only for hereditary diseases in which germline genes are abnormal, but also in somatic cell genes caused by external action Also includes diseases caused by abnormalities.

The direction of research in the field of genetic diagnosis is as follows.
One is the search and identification of unknown abnormal genes. Positional cloning (a method of extracting the DNA of a patient's family and determining the degree of abnormality by linkage analysis to determine the chromosomal locus of the mutated gene) was established,
At present, reliable identification of etiological genes has become possible, and not only hereditary diseases with a clear family history, but also diseases (environmental factors such as diabetes, hypertension, etc.) that have been thought to act more on environmental factors than on heredity. ) Genetic diagnosis is possible. Another is the development of a rapid and accurate diagnostic method for a large number of patients having a known abnormal gene.
Currently, more than 2000 types of genetic diseases are known, among them, childhood hyperuricemia, sickle cell anemia,
Phenylketonuria is a typical disease. For this type of disease, mass screening methods for newborns and fetuses have been established, and early detection has increased,
The development of effective diagnostic methods for future gene therapy is important.

[0004] As a genetic diagnosis method, SSCP (single-stranded conformation pol) for detecting a mutation in a gene fragment is used.
Various mutation analysis methods are known, including the ymorphism (single-stranded DNA higher-order structural polymorphism) method. FIG. 1 shows a conventional SSCP method. Double-stranded normal gene fragment (1
When 1a) and the double-stranded abnormal gene fragment (11b) are denatured (31), both gene fragments are dissociated, and the single-stranded normal gene fragment (21a) and the single-stranded abnormal gene fragment ( 21b) (FIG. 1A). The mark “x” in the figure indicates the position of the mutated base. Abnormal gene fragment (11b) means a DNA fragment obtained from a patient with a specific genetic abnormality. These gene fragments (21a
And 21b) were subjected to electrophoresis in a polyacrylamide gel. Since each gene fragment had a unique higher-order structure depending on its base sequence, different mobilities depending on the difference in the higher-order structure. (FIG. 1 (B)). Therefore, by detecting this mobility, a subtle mutation of the gene can be confirmed.

However, in the SSCP method, a change in the base of a single-stranded DNA fragment may not always be linked to a change in the mobility. Particularly, when the base change occurs near the end of the DNA fragment, the change in the higher order structure is small, and thus the mobility of the DNA fragment without base change and the DNA fragment with any base change As a result, there is a problem that it is difficult to distinguish between these two DNA fragments.

[0006] As a method other than the SSCP method, a gene change is determined by hybridization of a single-stranded normal gene fragment with a single-stranded abnormal gene fragment.
Several methods are known for confirmation by detecting a double-stranded DNA fragment having an NA structure. Tokiohei 9-5
No. 01561 discloses a method for detecting a double-stranded DNA fragment having a special DNA structure by using an external fluorescence microscope. A method is also known in which the double-stranded DNA fragment is detected by temperature gradient gel electrophoresis using a difference in mobility due to a difference in stability of the double-stranded DNA. here,
The term “special DNA structure” means that a normal DNA structure is a “full match structure” (a structure forming a complete double strand), whereas a “mismatch structure” (a normal gene fragment and an abnormal gene fragment are two). Structure, but the double strand has one non-base pairing portion), "bulge structure" (these gene fragments form a double strand, A structure in which the double strand has a plurality of non-base pair-forming portions). A structure that does not form a complete double strand can be broadly referred to as a mismatch structure, but will be divided as described above in this specification.

However, the above-mentioned method using an extra-fluorescence microscope has a problem that a fluorescent substance such as fluorescein used as a label is easily discolored. In the method using electrophoresis, conditions must be set for each measurement, and there is a problem that the method lacks in speed. The radioisotope method has been used, but it still has problems in terms of safety. Therefore, in order to easily and highly sensitively detect any change in a gene, a double-stranded DNA having a special DNA structure is required.
There is a need to develop a method that can rapidly and highly sensitively detect NA fragments.

Japanese Patent Application Laid-Open No. 9-288080 discloses a method for detecting a double-stranded DNA fragment having a “full match structure” using an electrochemical method. In this method, a sample DNA fragment having perfect complementarity to a known DNA fragment is detected by using a DNA probe in which a known DNA fragment is immobilized on a solid support and an electrochemically active sewn intercalator. be able to. This method is excellent in that it does not have the fading seen in the fluorescence method, can eliminate the safety problem in the radioisotope method, and does not require labeling of the sample DNA fragment.

Japanese Patent Application Laid-Open No. Hei 11-236396 describes that D
Although NA is a molecule having a negative charge, PNA (peptide nucleic acid), which is a molecule having no negative charge and imitating DNA (or RNA), is disclosed in place of the DNA.

[0010] PNA is known as one of so-called antisense molecules. Antisense molecules bind to a single-stranded DNA base sequence transcription region or RNA base sequence translation region in a single-stranded manner when a gene is expressed, so as to restrict the function of the region. It is a working molecule and is known as an antisense drug in the field of gene therapy. PNA has a nucleobase in its molecule like a nucleic acid, and specifically hybridizes to a nucleic acid having a complementary base sequence to form a double strand (P.
E. Nielsen et al., Science, 254, 1497-1500 (199
1)). PNA is functionally similar to nucleic acid, but its structure is quite different, and N- (2-aminoethyl)
The basic skeleton is a polyamide containing glycine as a unit, and the molecule does not contain sugar and phosphoric acid. The nucleobase is attached to the polyamide backbone, usually via a methylene carbonyl group. The typical structure of PNA is shown below together with the structure of DNA.

[0011]

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PNA is electrically neutral and does not charge even under salt-free conditions. Although the functions of PNA are almost the same as those of nucleic acids,
The double strand formed by DNA and nucleic acid is more stable than the double strand formed by DNA and nucleic acid, and has the feature of being able to recognize the nucleotide sequence of nucleic acid more strictly (Naomi Sugimoto, The Chemical Society of Japan) 74th Spring Meeting Abstracts, p. 1287). Therefore, PNA is a molecule that is expected to be applied to various diagnostic agents other than antisense drugs (Japanese Patent Application Laid-Open No. Hei 6-509).
063).

Japanese Patent Application Laid-Open No. H11-332595 discloses a PN having a PNA fragment immobilized at one end thereof on the surface of a solid support.
A method for detecting a DNA fragment using the A probe and the PNA probe is disclosed. This PNA probe is
A PNA fragment is immobilized on a measurement chip for a surface plasmon resonance biosensor by the avidin-biotin method. Detection of a DNA fragment using a PNA probe is performed by measuring a surface plasmon resonance signal. A method of detecting a sample DNA fragment having complementarity using a PNA probe and a sample DNA fragment labeled with a radioisotope is also known (PENielsen et al., Science, 254, 1497-1500).
(1991)). In the avidin-biotin method and the radioisotope method, both the PNA fragment and the sample D were used.
Because the electric repulsion generated between the NA fragments is reduced,
Detection sensitivity can be improved.

[0014]

DISCLOSURE OF THE INVENTION The present invention provides a method for detecting a partially complementary nucleic acid fragment useful for detecting a gene polymorphism formed between a single-stranded normal gene fragment and a single-stranded abnormal gene fragment. It is to provide a way to do it. Another object of the present invention is to provide a method for detecting the above-described partially complementary nucleic acid fragment by utilizing a property unique to PNA.

[0015]

According to the present invention, there is provided (1) a DNA substrate comprising a conductive substrate and a DNA fragment fixed at one end to the surface thereof.
The sample solution obtained by dissolving or dispersing the sample nucleic acid fragment is brought into contact with the NA analysis element, and a potential is applied to the analysis element in the presence of the electrochemically active sewn-in intercalator, whereby the intercalator is A step including an operation of measuring a value of a current flowing between the analytical element and (2)
The current value measured in the above step (1) is converted to the above DNA
Using a nucleic acid fragment having complementarity to the DNA fragment fixed to the analysis element, and collating with a standard current value of the complementary nucleic acid fragment obtained by the same operation as the above (1). And a method for detecting whether or not the sample nucleic acid fragment used in the step (1) is partially identical to the complementary nucleic acid fragment with respect to the base sequence.

According to the present invention, there is also provided (1) a method in which a sample solution obtained by dissolving or dispersing a sample nucleic acid fragment is brought into contact with a DNA analysis element having a DNA fragment fixed to one end of the surface of a conductive substrate. By applying a potential to the analytical element in the presence of an active sewn intercalator,
(2) contacting an aqueous liquid not containing a sample nucleic acid fragment with a DNA analysis element having the same configuration as described above, (3) a step of measuring a background current value flowing between the intercalator and the analysis element by applying a potential to the analysis element in the presence of the active threaded intercalator; ) The current value measured in the above step (1) is compared with the complementation obtained by the same operation as the above (1) using a nucleic acid fragment having complementarity to the DNA fragment fixed to the DNA analysis element. Against the standard current value of the nucleic acid fragment,
And a step in which the sample nucleic acid fragment used in step (1) is partially identical to the complementary nucleic acid fragment with respect to the nucleotide sequence, including a step of comparing the current value measured in step (2). There is also a method of detecting whether or not.

Preferred embodiments of the detection method using a DNA analysis element of the present invention are as follows. (A) As the DNA analysis element, a DNA analysis element is used which has been subjected to a masking treatment on the surface other than the fixing site of the DNA fragment to prevent the electrochemically active sewn intercalator from contacting the surface of the conductive substrate. (B) The mask treatment was performed by a method in which a DNA fragment was fixed to the surface of a conductive substrate at one end, and then surface-treated with 2-mercaptoethanol. (C) As the DNA analysis element, a DNA analysis element having a DNA fragment having a known base sequence bonded at one end is used. (D) A ferrocene-modified electrochemically active stitched intercalator having redox activity is used as the electrochemically active stitched intercalator. (E) The current value in each step is measured by differential pulse voltammography.

The present invention further provides (1) a method in which a sample solution obtained by dissolving or dispersing a sample nucleic acid fragment is brought into contact with a PNA analysis element in which a PNA fragment is fixed to one end of the surface of a conductive substrate; A step including an operation of measuring a current value flowing between the intercalator and the analysis element by applying a potential to the analysis element in the presence of the active stitching type intercalator; Using the nucleic acid fragment having complementarity to the PNA fragment immobilized on the PNA analysis element, the current value measured in step (1) of step (1) is used to obtain a complementary nucleic acid obtained by the same operation as (1) above. Checking whether or not the sample nucleic acid fragment used in step (1) is partially identical to the complementary nucleic acid fragment with respect to the base sequence. Inspection To a method for certain.

According to the present invention, there is further provided (1) a method in which a sample solution in which a sample nucleic acid fragment is dissolved or dispersed is brought into contact with a PNA analysis element in which a PNA fragment is fixed at one end to the surface of a conductive substrate; A step including applying a potential to the analysis element in the presence of the chemically active embroidery intercalator to measure a current value flowing between the intercalator and the analysis element; An aqueous liquid not containing a sample nucleic acid fragment is brought into contact with a PNA analytical element having the same configuration as that described above, and a potential is applied to the analytical element in the presence of an electrochemically active sewn-in intercalator. A step of measuring a background current value flowing between the PNA analysis element and the analysis element; and (3) converting the current value measured in the step (1) into the PNA analysis element. Using a nucleic acid fragment having complementarity to the immobilized PNA fragment, comparing with the standard current value of the complementary nucleic acid fragment obtained by the same operation as the above (1), and measuring the current measured in the step (2) A method for detecting whether or not the sample nucleic acid fragment used in step (1) is partially identical to the complementary nucleic acid fragment with respect to the base sequence, comprising a step of comparing the value with the complementary nucleic acid fragment. There is also.

Preferred embodiments of the detection method using a PNA analysis element of the present invention are as follows. (A) As the PNA analysis element, a PNA analysis element whose surface other than the PNA fragment fixing site is subjected to a mask treatment for preventing the electrochemically active sewn intercalator from contacting the surface of the conductive substrate is used. (B) The mask treatment was performed by a method in which a PNA fragment was fixed to the surface of a conductive substrate at one end, and then the surface treatment was performed with 2-mercaptoethanol. (C) As the PNA analysis element, a PNA analysis element having a PNA fragment having a known base sequence bound at one end is used. (D) A ferrocene-modified electrochemically active stitched intercalator having redox activity is used as the electrochemically active stitched intercalator. (E) The current value in each step is measured by differential pulse voltammography.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION In the method for detecting a partially complementary nucleic acid fragment of the present invention, an electrode type is used for detecting a hybrid DNA obtained by hybridization between a single-stranded normal gene fragment and a single-stranded abnormal gene fragment. The sensor method (Japanese Unexamined Patent Publication No. 9-288080 and the 57th Analytical Chemistry Symposium Proceedings, pp. 137-138 (1996)) is used. This electrode type sensor method has an output terminal and a DNA
In this method, a sample nucleic acid fragment is brought into contact with an electrode to which the fragment is immobilized in the presence of an electrochemically active sewn intercalator, and a current value is measured to detect a complementary sample nucleic acid fragment.

Hereinafter, a detection method using a DNA analysis element will be mainly described, but the same applies to a case using a PNA analysis element unless otherwise specified. In this specification,
It is defined as follows. The “PNA fragment” does not include a fragment of PNA obtained by synthesis such as cleavage. "DNA analysis element" refers to an analysis element in which a large number of DNA fragments are fixed at one end to the surface of a conductive substrate. “Mask treatment” means that a solution containing a compound for mask treatment is dropped on the entire surface of an untreated DNA analysis element, dried if necessary, and fixed. “Hybrid DNA” refers to a double-stranded fragment formed from a DNA fragment fixed on a DNA analysis element and a sample nucleic acid fragment, and “hybrid PNA” refers to a PNA fragment fixed on a PNA analysis element. A double-stranded fragment formed with a sample nucleic acid fragment. “Binding” refers to a state in which the hybrid DNA-binding electrochemically active molecule is inserted into the hybrid DNA structure, or a state in which the electrochemically active molecule is electrostatically interacting with the hybrid DNA outside the hybrid DNA structure. A "partially complementary nucleic acid fragment" is a nucleic acid fragment that exhibits partial complementarity to a base sequence of a DNA fragment immobilized on a DNA analysis element and that exhibits partial non-complementarity. Say. Therefore, a nucleic acid fragment that shows perfect complementarity and a nucleic acid fragment that does not show perfect complementarity are not targets of partially complementary nucleic acid fragments.

FIG. 2 schematically shows a typical example of the method for detecting a partially complementary nucleic acid fragment of the present invention. Hereinafter, the detection method of the present invention will be described in detail focusing on the case where a DNA analysis element is used, but the same applies to the case where a PNA analysis element is used, unless otherwise specified. The detection method of the present invention comprises the following three steps. Step A: A single-stranded DNA fragment (single-stranded normal gene fragment) (51) is immobilized on the surface of a conductive substrate (41), and DN
A analysis element (61). Here, as a sample nucleic acid fragment, an abnormal gene fragment (71b) having a base sequence containing a base that forms a non-base pair with a specific base of the normal gene fragment (71a), or a normal gene fragment (71a).
An abnormal gene fragment (7) having a base sequence containing bases forming non-base pairs with respect to the specific two bases of (a), respectively.
1c or 71d) is brought into contact with a sample solution prepared by dissolving or dispersing, and
When (71c) and (71d) (the mark “x” indicates the position of the mutated base) were respectively bound, the hybrid DNA (91b) having a mismatch structure and the hybrid DNA (91c or 91c) having a bulge structure were combined.
d) is obtained respectively. (91c) and (91d)
Have two non-base pairing moieties. In these,
When the electrochemically active stitched intercalator (100) is contacted and bonded, (91b), (91)
Complexes (101b), (101c), and (101d) in which (100) is bonded to c) or (91d), respectively.
Is obtained. Next, a potential is applied to these, and the amount of current flowing between the intercalator and the conductive substrate is measured. Step (B): A normal gene fragment (71
By performing the same operation as in step (A) using a), a hybrid DNA (91
a) is obtained. Here, the intercalator (10
0) is contacted and bound, and the complex (101
a) is obtained. The current value is measured in the same manner as in the step (A). Step (C): The current value measured in the step (A) is compared with the current value measured in the step (B). Therefore, by the steps (A), (B) or (C), the abnormal gene fragment (71b) (or (71c) or (71d))
Whether the nucleotide sequence is partially identical to the normal gene fragment (71a) can be detected. FIG.
Shows the case where the abnormal gene fragment has a base substitution, but the same detection method can be used also in the case of a base change such as base deletion or base insertion.

Further, the detection method of the present invention includes a detection method including a step (D) in addition to the above steps (A) to (C). Step (D) is a step of obtaining a background current value by contacting only the intercalator with the DNA analysis element without contacting the sample nucleic acid fragment. That is, the current values obtained in the steps (A) and (B) are current values that have changed with respect to the background current value, respectively.

[Conductive Substrate] A conductive substrate is formed of a hydrophobic (or low hydrophilic) conductive substrate or a plurality of hydrophobic (or low hydrophilic) electrically insulating carriers on a hydrophobic (or low hydrophilic) electrically insulating carrier. It is preferable that the substrate is provided with a (hydrophilic) conductive substrate. Both the conductive hydrophobic substrate and the electrically insulating hydrophobic carrier can be preferably used even if their surfaces have irregularities and low flatness. As the material of the electrically insulating carrier, glass, cement, ceramics such as ceramics or new ceramics, polyethylene terephthalate, cellulose acetate, bisphenol A polycarbonate, polystyrene, polymethyl methacrylate and other polymers, silicon, activated carbon, porous Glass, porous ceramics, porous silicon, porous activated carbon, woven / knitted fabric, nonwoven fabric, filter paper,
Examples of the material include a porous material such as a short fiber and a membrane filter, and it is particularly preferable to use various polymers, glass, or silicon. This is due to the ease of surface treatment and the ease of analysis by electrochemical methods. The thickness of the electrically insulating carrier is not particularly limited, but is preferably in the range of 100 to 10000 μm in the case of a plate shape. As the hydrophobic conductive substrate, an electrode, an optical fiber, a photodiode, a thermistor, a piezo element, a surface acoustic wave element, and the like can be preferably used, but the use of an electrode is particularly preferable. Examples of the material of the electrode include a carbon electrode such as graphite and glassy carbon, a noble metal electrode such as platinum, gold, palladium, and rhodium; an oxide electrode such as titanium oxide, tin oxide, manganese oxide, and lead oxide; Si, Ge, and ZnO. And a semiconductor electrode such as CdS, and an electron conductor such as titanium. It is particularly preferable to use gold or glassy carbon. These electron conductors may be covered with a conductive polymer or may be covered with a monomolecular film.

As the conductive substrate, it is particularly preferable to use a substrate in which a plurality of hydrophobic conductive substrates are provided on a hydrophobic electrically insulating carrier. At this time, it is preferable that the conductive substrates are regularly arranged on an electrically insulating carrier so as not to contact each other. Before providing the conductive substrate on the electrically insulating carrier, a layer made of a charged hydrophilic polymer substance or a layer made of a crosslinking agent may be provided on the electrically insulating carrier. By providing such a layer, unevenness of the carrier can be reduced.
In addition, depending on the type of the carrier, it is possible to include a hydrophilic polymer substance having a charge in the carrier, and a carrier that has been subjected to such a treatment can also be preferably used.

[0027] As an example in which a plurality of electrodes are arranged on an electrically insulating carrier, reference is made to a document (Sosnowski, RG et al., Pro.
Acad. Sci. USA, 94, 1119-1123 (1997)), a silicon chip on which nucleic acids are not immobilized can also be preferably used. Also, like printed wiring conductive substrates,
The electrodes may be printed on a conductive substrate.

[DNA Fragment] The detection method of the present invention
Hybrid DN formed by fragment A and sample nucleic acid fragment
This is a primary screening method whose primary purpose is to detect A (gene polymorphism) and further analyze the mode of mutation of a sample nucleic acid fragment. Therefore, it is preferable that one of the DNA fragment and the sample nucleic acid fragment immobilized on the DNA analysis element is a normal gene fragment and the other is an abnormal gene fragment. Either the DNA fragment fixed to the DNA analysis element or the sample nucleic acid fragment may be a normal gene fragment, but it is preferable to use a normal gene fragment as the DNA fragment.

As the normal gene fragment, DNA having no base mutation extracted from a biological sample, DNA having no base mutation produced by genetic manipulation or the like is cut with a restriction enzyme and then separated by electrophoresis. It is preferable to use a DNA fragment purified in the above. These DNA
The fragments are dissociated into single-stranded DNA fragments before or after the above separation operation by heat treatment, alkali treatment, or the like.
In addition, a single-stranded oligonucleotide having the same base sequence as a single-stranded DNA fragment obtained from DNA having no base mutation extracted from a biological sample is chemically synthesized,
This can also be used.

The base sequence of the DNA fragment is preferably determined in advance by a general base sequence determination method, and its base type is also preferably known. D
The NA fragment is preferably a 3- to 50-mer,
It is particularly preferred that it is a 0 to 25 mer. As a DNA fragment, in order to immobilize DNA fragments having different base sequences on each of a plurality of conductive substrates arranged at regular intervals on an electrically insulating carrier, such a plurality of types of DNA fragments are used. It is also preferable to use them.

As a method for immobilizing a DNA fragment, a known method can be used. An appropriate method for fixing the DNA fragment to the conductive substrate can be selected according to the type of the DNA fragment and the type of the conductive substrate (protein / protein).
Nucleic acid / enzyme, Vol. 43, No. 13, 2004-2011 (1998)). When immobilizing a synthetic nucleotide, a method of directly synthesizing on a conductive substrate, or a method of synthesizing an oligomer in which a reactive group for covalent bonding has been introduced into a terminal in advance and covalently bonding to a surface-treated conductive substrate Can be used. For example, when the surface of the conductive substrate is subjected to vapor deposition treatment with gold, a mercapto group is introduced into the 5 ′ or 3 ′ end of the DNA fragment,
The DNA fragment is fixed on a conductive substrate. A method for introducing a mercapto group into the DNA fragment is described in the literature (M. Maeda et al.,
Chem. Lett., 1805-1808 (1994) and BA Connolly, Nuc
leic Acids Res., 13, 4484 (1985)). When the surface of the conductive substrate is coated with glassy carbon, the glassy carbon is oxidized with potassium permanganate to introduce a carboxylic acid group on the surface of the conductive substrate, and the DNA fragment is removed. It can be fixed to the surface of the conductive substrate by an amide bond. The actual immobilization method is described in the literature (KMMillan et al., Analyti
cal Chemistry, 65, 2317-2323 (1993)). The DNA fragment may be a hybrid DNA prepared in advance. When the hybrid DNA is immobilized on a conductive substrate that has been vapor-deposited with gold, a mercapto group is introduced into the 5 'or 3' end (preferably, the 5 'end) of one strand of the hybrid DNA. Then, after fixing the hybrid DNA, the double strand is dissociated,
Single strands can also be immobilized. Examples of the reactive group for the covalent bond include an amino group, an aldehyde group, a mercapto group, and biotin. When glass or silicon is used as the conductive substrate, a known silane coupling agent is preferably used for the surface treatment.

The DNA fragments are fixed by spotting an aqueous liquid obtained by dissolving or dispersing the DNA fragments on each of a plurality of conductive substrates arranged at regular intervals on an electrically insulating carrier. It is preferable to carry out. The concentration of the DNA fragment to be spotted is preferably in the range of several pM to several mM, and particularly preferably in the range of several pM to several nM. The spotted amount is preferably in the range of 1 to 100 nL, particularly preferably in the range of 1 to 10 nL. The aqueous liquid containing the DNA fragment may contain an additive for increasing the viscosity of the aqueous liquid. Such additives include sucrose, polyethylene glycol, glycerol and the like. Dots are
Although it can be performed by manual operation, it is also preferable to use a spotter provided in a commonly used DNA chip manufacturing apparatus. After the spotting, the DNA fragments are left at a predetermined temperature for several hours to fix the DNA fragments on a plurality of conductive substrates. After spotting, it is also preferable to carry out incubation. After the incubation, it is preferable to wash and remove unspotted DNA fragments. The amount of the DNA fragment fixed by ^spotting is preferably in the range of 10 ^{-20 to} 10 ^-12 mol per 1 mm ^{2 of the} surface of the conductive substrate. The fixed amount of the DNA fragment can be determined by instrumental analysis such as HPLC (high performance liquid chromatography).

[PNA Fragment] The PNA fragment preferably used in the present invention is a compound represented by the following formula (I).

[0034]

Embedded image

In the above formula, B¹¹Is a ligand,
Natural nucleobases (A, T, C, G, I or U) or
Represents a base analog. B¹¹Is a position found in nature,
That is, a preform containing adenine, guanine or inosine
Nin, thymine, uracil or
For pyrimidines including cytosine
Are combined. Base analogs are non-naturally occurring nucleic acids
An organic base similar to a base, such as a purine or pyrimidine ring
Where part of is replaced by C to N or N to C
Compound or a part of the purine or pyrimidine ring.
Decorated compounds (sulfhydryl groups and halogen atoms
Is a compound into which is introduced). Also, B ¹¹Is the nucleic acid base
Aromatic moieties containing no carbon atoms, alka having 1 to 4 carbon atoms
It may be a noyl group, a hydroxyl group or a hydrogen atom. salt
Group analogs include 7-deazaadenine, 6-azaura
Sil and 5-azacytosine can be mentioned. Scripture
Type nucleobase ligands and exemplary synthetic ligands
Are shown in WO92 / 20702,
Propylthymine and 3-deazauracil are used for DNA cleavage.
It is known to increase the binding affinity to
Japanese Unexamined Patent Publication No. Hei 11-236396). Other useful naturally occurring
6-thioguanine and pyrazolo
[4,3d] -pyrimidines are useful (International Application PC
T / US92 / 04795). Further, B¹¹Is DNA
Intercalators, reporter ligands (eg, full
Orophore), hapten and biotin protein labeling,
It may be a spin label or a radioactive label. B¹¹
Is a nucleic acid base (A, T, C, G or U)
Is particularly preferred.

R ¹¹ represents a hydrogen atom or a group representing a side chain of a natural α-amino acid. Examples of the group representing the side chain of the natural α-amino acid include an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and a carbon atom containing an alkyl group having 1 to 6 carbon atoms. Number 7 to 26
An aralkyl group, a heteroaryl group having 6 to 20 carbon atoms, a hydroxyl group, an alkoxy group having 1 to 6 carbon atoms, an alkylthio group having 1 to 6 carbon atoms, -NR ¹³
It is preferably a group selected from the group consisting of R ¹⁴ groups, mercapto groups, and alkyl groups having 1 to 6 carbon atoms. An alkyl group having 1 to 6 carbon atoms further includes
It may be substituted with a hydroxyl group, an alkoxy group having 1 to 6 carbon atoms or an alkylthio group having 1 to 6 carbon atoms. R ¹³ and R ¹⁴ each independently comprise a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, an alkylthio group having 1 to 3 carbon atoms, and a hydroxyl group. Represents an atom or a hydroxyl group selected from the group. The group representing the side chain of the natural α-amino acid may form an alicyclic ring or a heterocyclic ring together with the hydrogen atom of the carbon atom to which R ¹¹ is bonded.

L ¹¹ represents a linking group, preferably a —CO— group or a —CO—NR ¹² — group,
Particularly preferred is a group. R ¹² represents an atom or a group selected from the group consisting of a hydrogen atom, an alkylene group having 1 to 4 carbon atoms, a hydroxyl group, an alkoxy group having 1 to 4 carbon atoms and an amino group. The alkylene group having 1 to 4 carbon atoms, the alkoxy group having 1 to 4 carbon atoms and the amino group are all an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms or It may be substituted with a hydroxyl group.

D represents an integer of 1 to 60. d is 1
It is preferably an integer of from 40 to 40. a, b and c
Represents an integer of 0 to 5 each independently. It is preferable that a, b and c are all 1.

The PNA fragment used in the present invention has the following general formula:
The compound represented by the formula (II) is particularly preferred. In the formula, B ¹¹ and d each represent B
¹¹ and d.

[0040]

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The PNA fragment can be synthesized by a liquid phase method or a solid phase method similarly to the peptide, and the synthesized one is also commercially available. The method of synthesizing PNA is described in JP-T-6-509063, US Pat. No. 2,758,988.
Issue specification, PENielsen etal., Journal of American
Chemical Society, 114, 1895-1987 (1992), PENiels
en et al., Journal of American Chemical Society, 1
14, 9677-9678 (1992) and the like.

The amount of the DNA fragments fixed to the DNA analysis element ranges from 10 ^{-15 to} 10 per 1 mm ^{2 of the} conductive substrate.
Preferably, it is in the range of ^-10 moles. This is because, even when an aqueous liquid formed by dissolving or dispersing a DNA fragment is spotted on the surface of the conductive substrate, the spotted amount usually differs from the actually fixed amount.

When the DNA fragments are immobilized on the conductive substrate in an amount of 10 ^-10 mol or more, the DNA fragments are densely packed on the conductive substrate, and when less than 10 ^-12 mol, It is presumed that a portion where the DNA fragment is not bonded to the surface of the conductive substrate, that is, a gap portion is formed.
Due to the formation of this gap, many of the DNA fragments
It is considered that they are bonded on the surface of the conductive substrate in a falling state. For efficient hybridization between the DNA fragment and the sample nucleic acid fragment, it is desirable that the DNA fragment be fixed in a state of being extended in the vertical direction as much as possible with respect to the surface of the conductive substrate. In addition, it is presumed that the electrochemically active stitched intercalator comes into contact with the gap portion and non-specifically adsorbs, thereby increasing the background current value. Therefore, the DNA analysis element used in the present invention is preferably subjected to the following mask treatment.

[Mask Treatment] The mask treatment can be carried out by spotting a solution containing the compound for mask treatment on the surface of the DNA analysis element. The masking compound is preferably used in the range of 0.5 to 2 mM, particularly preferably in the range of 0.8 to 1.5 mM. After spotting the compound for mask treatment, it is preferable to leave the compound at a predetermined temperature (preferably room temperature) for several hours. After spotting, incubation may be performed if necessary. The masked DNA analysis element can be stored for several days, and can be used repeatedly several times.

The masking compound is added to a chain molecule having a hydrophilic group at one end or in the vicinity thereof, which may have a cyclic group in the middle, and a hydrophilic group side end of the chain molecule. Is a compound in which a reactive group capable of bonding to the surface of the conductive substrate is bonded to the opposite end or its vicinity. The chain molecule is immobilized on the surface of the conductive substrate via a reactive group capable of binding to the surface of the conductive substrate. Having a hydrophilic group at one end or in the vicinity thereof indicates that the hydrophilic group does not necessarily have to be located at the end of the chain molecule, but preferably has a hydrophilic group at the end. . The number of hydrophilic groups may be one or plural. It is preferable that the reactive group capable of binding to the surface of the conductive substrate be bound to the end of the chain molecule opposite to the end on the side of the hydrophilic group. The chain molecule which may have a cyclic group in the middle is a molecule derived from the masking compound. As the masking compound, D is first fixed to a conductive substrate.
A compound having a linear length shorter than that of an NA fragment and having a hydrophilic group at one end of a linear alkylene group having 1 to 6 carbon atoms and bonding to a conductive substrate at the other end. Preferably, the compound has a reactive group.

Examples of the reactive group that can be bonded to the surface of the conductive substrate include a group selected from the group consisting of a mercapto group, a sulfide group, a disulfide group, a thiocarbonyl group and a thiocarboxylic acid group. When a treatment for introducing a reactive group such as an amino group, an aldehyde group, or a carboxylic acid group has been previously performed on the surface of the conductive substrate, the reactive group may be an amino group, an imino group, a hydrazine group, or a hydrazide. Group, amide group, carboxylic acid group, aldehyde group,
A group selected from the group consisting of an epoxy group and a peroxyacid group can also be mentioned. As the above reactive group,
It is more preferably a mercapto group, a thiocarbonyl group or a sulfide group, and particularly preferably a mercapto group.

The hydrophilic group is preferably a hydroxyl group, a carboxylic acid group, an amide group, a phosphoric acid group or a sulfonic acid group, and particularly preferably a hydroxyl group. However, the reactive groups that can be bonded to the surface of the conductive substrate are preferably different from each other. The cyclic group is an aryl group having 6 to 12 ring carbon atoms, a cycloalkyl group having 3 to 6 carbon atoms, or 1 to 4 heteroatoms selected from the group consisting of N, S, O and P. It is preferably a heterocyclic group containing 2 to 10 carbon atoms. The cyclic group may be a group in which reactive groups capable of binding to the surface of the conductive substrate cooperate to form a cyclic group. Preferred examples of the latter include an imidazole-2-thione group.

Therefore, as the masking compound, mercaptomethanol, 2-mercaptoethanol,
Mercaptopropanol, 4-mercaptobutanol,
5-mercaptopentanol, 6-mercaptohexanol, N, N'-di (3-hydroxy-n-propyl)
-Imidazole-2-thione or imidazole-2-thione derivatives described in the literature (AJArduengo et al.,
J. Am. Chem. Soc., 1990, 112, 6153-6154), preferably 2-mercaptoethanol, 3-mercaptopropanol, 4-mercaptobutanol, 5-mercaptopentanol, and 6-mercaptohexanol. Is more preferable, and 2-mercaptoethanol is particularly preferable. The masking compound is
Salts such as sodium and potassium may be used, and the alkylene chain may be substituted by a specific substituent. The specific substituent is preferably a hydrocarbon group having 1 to 6 carbon atoms (preferably a methyl group, an ethyl group, a phenyl group, etc.). It is desirable to select a masking compound according to the method of fixing the DNA fragment. Two or more kinds of masking compounds may be used in combination.

[Hybridization] Hybridization is carried out by bringing a sample nucleic acid fragment into contact with a DNA analysis element in the presence of an electrochemically active sewn intercalator. The contact of the sample nucleic acid fragment with the DNA analysis element can be performed by either a method of dropping an aqueous liquid containing the sample nucleic acid fragment on the DNA analysis element, or a method of immersing the DNA analysis element in the aqueous liquid containing the sample nucleic acid fragment. Can be implemented. At this time, an intercalator may be included in the aqueous liquid containing the sample nucleic acid fragment. Alternatively, an intercalator may be brought into contact with the hybrid DNA formed after completion of the hybridization. In this case, it is preferable to remove unreacted sample nucleic acid fragments. In order to avoid non-specific binding of the intercalator to the single-stranded sample nucleic acid fragment or the double-stranded sample nucleic acid fragment contained in the sample, remove the unreacted sample nucleic acid fragment after the completion of hybridization. Therefore, it is desirable to make the intercalator contact. Washing is preferably performed using a mixed solution of a surfactant (preferably sodium dodecyl sulfate) and a buffer (preferably a citrate buffer). Intercalator is 10n
Preferably, it is used in a concentration range of M to 10 mM.

Hybridization is carried out at room temperature to 70
C. and preferably in the range of 0.5 to 20 hours, but DNA immobilized on a conductive substrate
It is desirable to set optimal hybridization conditions according to the fragment chain length, the type of the sample nucleic acid fragment, and the like. For example, when analyzing gene expression,
It is preferable to perform hybridization for a long time so that low-expressed genes can be sufficiently detected, and it is preferable to perform hybridization for a short time for the purpose of detecting a single nucleotide mutation.

The amount of the intercalator that binds to the hybrid DNA varies depending on the specific DNA structure, as shown in FIG. The amount of this binding decreases as the number of non-base-pairing moieties increases in duplex formation.

[Sample Nucleic Acid Fragment] The sample nucleic acid fragment
This is a fragment having a base sequence that forms a non-base pair with at least one base of the DNA fragment fixed to the analysis element. As the sample nucleic acid fragment, it is preferable to use an abnormal gene fragment corresponding to a preferable DNA fragment fixed to the DNA analysis element being a normal gene fragment. An abnormal gene fragment is a fragment having a base sequence that forms a non-base pair with at least one base of the target normal gene fragment. As long as it has such a base sequence, it may involve any type of base mutation (base substitution, base deletion, base insertion, etc.). Examples of the abnormal gene fragment include DNA extracted from a sample of a genetically abnormal organism, DNA having the same base sequence as the abnormal gene described below prepared by genetic manipulation and the like, cut with a restriction enzyme, and then separated by electrophoresis. DNA purified by
Preferably, fragments are used. These DNA fragments are
Before or after the above separation operation, the DNA is dissociated into single-stranded DNA fragments by heat treatment or alkali treatment. Alternatively, a single-stranded oligonucleotide having the same base sequence as the following abnormal gene can be chemically synthesized and used.

The abnormal gene may be any of a hereditary disease, a disease caused by an exogenous genetic abnormality, and a disease derived from a disease in which an environmental factor acts on a hereditary predisposition.
For example, diseases for which environmental factors seemed to act more frequently than hereditary diseases (diabetes, hypertension, etc.), and known genetic diseases (pediatric hyperuricemia, sickle cell anemia, phenylketonuria) Etc.).

For the contact amount of the sample nucleic acid fragment,
Substrate 1mm^Two10 per^-18To 10 ^-TenMolar amount range
Preferably 10^-18To 10^-12Molar amount
It is particularly preferred that it is in the range. Sample used as control
The contact amount of the nucleic acid fragment should be the same as that of the sample nucleic acid fragment.
Is preferred.

[Electrochemically active sewn intercalator] As the electrochemically active sewn intercalator, any molecule can be used as long as it intercalates with hybrid DNA and has electrochemical activity. . However, since it is required that the base pair-forming portion of the hybrid DNA can be quantitatively recognized, a groove-binding type of DNA is not preferable.

The sewn intercalator labeled with a conductive group is preferably a substance having a redox activity. The redox-active moiety is preferably a ferrocene compound, a catecholamine compound, a metal bipyridine complex, a metal phenanthroline complex, a viologen compound, or the like, and particularly preferably a ferrocene compound. The intercalator portion is preferably naphthalenediimide, anthracene, anthraquinone, or the like, and particularly preferably naphthalenediimide. Therefore, as the intercalator, a ferrocene naphthalenediimide derivative (S. Takena) represented by the following formula, which is synthesized by reacting ferrocenecarboxylic acid N-hydroxysuccinimide ester with a corresponding amine compound,
Ka et al., J. Chem. Soc., Commun., 1111 (1998)) is particularly preferred.

[0057]

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Further, a ferrocene naphthalenediimide derivative represented by the following formula is also preferably used.

[0059]

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However, X is a ferrocene derivative represented by the following formula.

[0061]

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[0062]

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[0063]

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[0064]

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The sewn intercalator labeled with a conductive group has a linker portion connecting the redox-active portion and the intercalator portion. 1, represented by the above equation
The 4-dipropylpiperazinyl group corresponds to this linker moiety. Instead of this piperazinyl group, a quaternized imino group can also be introduced. The intercalator represented by the following formula into which a quaternized imino group has been introduced becomes cationic regardless of the pH of the reaction system, so that the binding of the hybrid DNA or hybrid PNA becomes stronger. An intercalator in which a quaternized imino group is introduced is particularly effective when a PNA analytical element is used. The group corresponding to the linker moiety is not limited to those described above. For example, instead of a piperazinyl group,
N-alkyl group (an alkyl group having 1 carbon atom
And preferably an alkyl group of 6 to 6, a methyl group,
It is also preferable to introduce an ethyl group or an n-propyl group). The oxidation-reduction potential of the ferrocene molecule differs due to the difference in the structure of the group corresponding to the linker portion.

[0066]

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When the above-described naphthalenediimide derivative is used as the sewn-in intercalator, the naphthalenediimide derivative binds to the hybrid DNA with high specificity, and is arranged every two bases to be saturated with the hybrid DNA. This means that the two ferrocene molecules of the naphthalenediimide derivative each have a hybrid DN
A means a state in which the main groove and the sub groove of A are closely arranged. Therefore, the naphthalenediimide derivative is a hybrid D
The dissociation rate from NA becomes extremely slow, and hybrid D
A stable complex of NA and a naphthalenediimide derivative can be formed. In addition, the fact that the binding of the naphthalenediimide derivative to the hybrid DNA is in the intercalation mode is generally confirmed by a change in viscosity observed when the intercalator is brought into contact with the hybrid DNA. For example, the virus SV4
It is known that the viscosity of the closed circular plasmid represented by 0 changes with the change of the supercoil when intercalation occurs. On the other hand, the intercalator does not bind to the single-stranded DNA fragment, or once bound, dissociates immediately to become a free intercalator.

[Measurement of Response Current] The amount of current is measured by a method capable of measuring the amount of current flowing between the conductive substrate on which the hybrid DNA is fixed and the conductive group of the electrochemically active sewn intercalator. Any method may be used as long as it is provided. Cyclic voltammography (CV),
Differential pulse voltammography (DPV),
It is preferable to use linear sweep voltammography, potentiostat and the like, and it is particularly preferable to use differential pulse voltammography. It is preferable to immerse the counter electrode and the conductive substrate on which the hybrid DNA is fixed in an electrolyte solution to form a pair of electrolytic systems, and measure differential pulse voltammography.

As described above, the detection method of the present invention
Hybrid D formed by NA fragment and sample nucleic acid fragment
This is a primary screening method whose primary purpose is to detect NA (gene polymorphism) and analyze the mode of mutation of a sample nucleic acid fragment. Therefore, if a specific normal gene fragment is used as a DNA fragment and an abnormal gene fragment is used as a sample nucleic acid fragment, the detection method of the present invention can also be used to quantify the base mutation of the abnormal gene fragment. For example, by screening abnormal gene fragments having a known number of base substitutions with a known number of base substitutions for normal gene fragments, the number of substitutions of which is different from three or more abnormal gene fragments, the base substitution degree and the current value Therefore, if an unknown number of abnormal gene fragments having a base substitution are used as a sample, the base substitution degree can be obtained from the current value. It is also possible to predict a gene polymorphism from the degree of base substitution. Detection of a genetic polymorphism indicates the possibility of a genetic risk diagnosis. For example, if chromosomal DNA of a patient having an abnormal gene of primary type IV hyperlipidemia (hyperglyceridemia) is used as a sample nucleic acid fragment, it is possible to diagnose the presence or absence of the disease from the polymorphism. .

In the detection method of the present invention, the rate of change of the current value depends only on the amount of the electrochemically active thread-intercalator bound to the hybrid DNA, and depends on the amount of the hybrid DNA formed on the conductive substrate. That is not the case, it can be confirmed in Example 5 described later.

[0071]

<Example 1> The d (GTCGGGACTGAAGA), which is the sense strand of the normal lipoprotein lipase (LPL) gene used in Examples 2 to 4, is replaced with the oligonucleotide "WT +" and its antisense strand, d. (CAGCCTGACTTCT) was replaced with oligonucleotide "WT-" and d (GTC
GGAG ^* TGAAGA) to oligonucleotide “S4
47X + ”and its antisense strand, d (CA
GCTCC ^* ACTTCT) with oligonucleotide "S
447X- ". However, a base having an asterisk at the upper right means a base that is different from a normal gene in a mutant gene.

Example 1 Detection of Partially Complementary DNA Fragment [Detection of Partially Complementary DNA Fragment Using Model Compound as Sample DNA Fragment (1A)] (1) Preparation of DNA Analysis Element A gold electrode having an area of 2 mm ² Immersed in a 2N aqueous sodium hydroxide solution for 1 hour, washed with ultrapure water, immersed in concentrated nitric acid,
Stir for 5 minutes. After washing the gold electrode with ultrapure water, the gold electrode surface, oligonucleotides (HS-dA ₂₀₎ that the 5 'end mercaptohexyl group of the sample DNA fragments were bound 20-mer of adenine (10 pmol / ΜL)
Of dA ₂₀ and HS-dA ₂₀ washed with ultrapure water after spotting 1.0 μL of an aqueous solution of
JP-A-9-288080 and BAConnolly, Nuc
Leic Acids Res., 13, 4484 (1985). An aqueous solution of spotting previous HS-dA ₂₀ for aqueous solutions and spotted HS-dA ₂₀ recovered after subjected to high performance liquid chromatography (HPLC), determines a fixed amount of the gold electrode from the variation of the peak chromatographic did. HS-
dA ₂₀ was almost quantitatively modified on the gold electrode. (2) Electrode mask treatment 1 μL of an aqueous solution of 2-mercaptoethanol (1 mM) was dropped on the surface of the DNA analysis element obtained in (1), and the electrode was capped and left for 2 hours. Then, it was washed with ultrapure water. (3) Preparation of Sample DNA Fragment The sample DNA fragment dT ₉ dAdT ₁₀ represented by the following formula (III): 5′-TTTTTTTTTTATTTTTTTTTT-3 ′ was synthesized according to the method described in the above publication. (4) Synthesis of ferrocene-stitched intercalator Ferrocene-naphthalenediimide represented by the following formula was also synthesized according to the method described in the above publication.

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(5) Hybridization between DNA analysis element and sample DNA fragment 1 μL of the aqueous solution of the sample DNA fragment dT ₉ dAdT ₁₀ (20 pmol / μL) of the above (3) was dropped on the DNA analysis element, Let stand for minutes. (6) Measurement of the amount of electric current An electrolytic solution (0.1 M acetic acid / potassium acetate aqueous solution (pH 5.6)) containing the ferrocene naphthalenediimide (50 μM) of the above (4) was maintained at 20 ° C. using a thermostatic cell.
(A mixture of −0.1 M aqueous potassium chloride solution) in the hybridization electrode (working electrode), platinum (counter electrode) and silver / silver chloride reference electrode obtained in (5) above to form three electrodes. At 42 ° C., differential pulse voltammography (DPV2) was measured (− in FIG. 3).
□-). The DPV1 was measured in the same manner as described above except that no sample DNA fragment was present (-O in FIG. 3).
−). As a result, the peak current value of DPV2 at 460 mV was higher than the peak current value of DPV1 at the same potential, and the amount of change was 22.2%.

[Detection of Partially Complementary DNA Fragment Using Model Compound as Sample DNA Fragment (1B)] Sample DNA
Example 1 except that the fragment was changed to dT ₈ dA ₄ dT ₈ represented by the following formula (IV): 5′-TTTTTTTTAAAAATTTTTTTT-3 ′
Hybridization of the DNA analysis element and the DNA fragment was carried out in the same manner as described above, and its DPV4 was measured (-□-in FIG. 4). dT ₈ dA ₄ dT ₈ was synthesized according to the method described in the above publication. DPV3 was measured in the same manner as above except that no sample DNA fragment was present (-O- in FIG. 4). As a result, the peak current value of DPV4 at 460 mV was changed by 15.1% as compared with the peak current value of DPV3 at the same potential.

[Detection of Partially Complementary DNA Fragment Using Model Compound as Sample DNA Fragment (1C)] Sample DNA
Except that the fragment was changed to a thymine 20-mer (dT ₂₀ )
As in the detection of the partially complementary DNA fragment (1A),
Hybridization between the NA analysis element and the DNA fragment was performed, and the DPV6 was measured (-□ in FIG. 5).
-). However, dT ₂₀ was synthesized according to the method described in the publication. The DPV5 was measured in the same manner as above except that no sample DNA fragment was present (-O in FIG. 5).
-). As a result, the peak current value of DPV6 at 460 mV changed by 37.3% compared to the peak current value of DPV5 at the same potential. The rate of change of the current value in the detection of the partially complementary DNA fragment (1C) (detection of the full-match structure) is the same as that in the above-described detection (1A) (detection of the mismatch structure) and (1B) (detection of the bulge structure). It is a control value for the rate of value change.

According to this example, as the sample DNA fragment, the compounds represented by the above formulas (III, (IV)) and dT ₂₀
It can be seen that as long as is used, the rate of change in the current value decreases with an increase in the non-base pairing portion (increase in the degree of base substitution).

Example 2 Detection of Partial Complementary DNA Fragment [Detection of Partial Complementary DNA Fragment Using Model Compound of LPL Abnormal Gene as Sample DNA Fragment (2A)] (1) Preparation of DNA Analysis Element DNA Fragment As HS-d (C) having a mercaptohexyl group at the 5 ′ end of the oligonucleotide “WT-”
A DNA analysis element was prepared in the same manner as in Example 1, except that an aqueous solution of AGCCTGACTTCT (10 picomoles) was used. Oligonucleotide "WT-" was synthesized according to the method described in the above publication. (2) Preparation of sample DNA fragment As a model gene of a patient having an LPL abnormal gene,
"S447X +" was synthesized according to the method described in the above publication. (3) Hybridization and measurement of current amount The same operation as in Example 1 was carried out except that the DNA analysis element obtained in the above (1) and the sample DNA fragment of the above (2) were used. The DPV was measured (D
PV8). The DPV was measured in the same manner as above except that no sample DNA fragment was present (DPV
7). As a result, the peak current value of DPV8 at 460 mV was changed by 6.3% compared to the peak current value of DPV7 at the same potential.

[Detection of Partially Complementary DNA Fragment Using Model Compound of Normal LPL Gene as Sample DNA Fragment (2C)] Except for using oligonucleotide “WT +” as a sample DNA fragment, the same procedure as in Example 4 was carried out. The DPV was measured (DPV10). Oligonucleotide "WT +" was synthesized according to the method described in the above publication. The DPV was measured in the same manner as above except that no sample DNA fragment was present (DPV9). As a result, the peak current value of DPV10 at 460 mV is 6 times smaller than the peak current value of DPV9 at the same potential.
It changed by 5.4%. The rate of this change corresponds to the control value for detection (2A) described above.

From Example 2, it can be seen that when the double-stranded DNA fragment has a mismatch structure, the rate of change in the current value is significantly reduced.

Example 3 Detection of Partial Complementary DNA Fragment [Detection of Partial Complementary DNA Fragment Using Model Compound of LPL Abnormal Gene as Sample DNA Fragment (3A)] (1) Preparation of DNA Analysis Element Oligonucleotide HS-d (CAGCCTCA) having a mercaptohexyl group at the 5 ′ end of “S447X-”
A DNA analysis element was prepared in the same manner as in Example 1 except that (CTTCT) (10 picomoles / μL) was used. Oligonucleotide "S447X-" was synthesized according to the method described in the above publication. (2) Hybridization and Measurement of Current Amount The DPV was measured in the same manner as in Example 1 except that the DNA analysis element described in (1) above was used as the DNA analysis element and “WT +” was used as the sample DNA fragment (DPV1).
2). The DPV was measured in the same manner as above except that no sample DNA fragment was present (DPV11).
As a result, the peak current value of DPV12 at 460 mV was changed by 9.7% as compared with the peak current value of DPV11 at the same potential.

[Detection of Partial Complementary DNA Fragment Using Model Compound of LPL Abnormal Gene as Sample DNA Fragment (3C)] DNA analysis was performed in the same manner as the above detection (3A) except that oligonucleotide “S447X +” was used. An element was manufactured, and its DPV was measured (DPV14). The DPV was measured in the same manner as above except that no sample DNA fragment was present (DPV13). As a result, the peak current value of DPV14 at 460 mV is
2 compared to the DPV13 peak current value at the same potential
It changed by 9.4%. The rate of this change is the above detection (3A)
Is the control value. In Example 3, the same results as in Examples 1 and 2 were observed.

Example 4 Detection of Partially Complementary DNA Fragment [Sample D fragment of LPL gene abnormal patient
Detection of partially complementary DNA fragment using NA fragment (4
A)] (1) Preparation of DNA analysis element A DNA analysis element was prepared in the same manner as in Example 1 except that oligonucleotide "WT-" (2 picomoles / μL) was used. (2) Preparation of sample DNA fragment Using chromosomal DNA purified from leukocytes (1 mL) of a patient with LPL gene abnormality, the PLP gene part was subjected to PCR amplification (40 cycles) to obtain oligonucleotide "S447".
X + / S447X + "was obtained. This sample DNA fragment
Purification was performed by dialysis and used for the following hybridization. (3) Hybridization and Measurement of Current Amount The DNA analysis device of (1) and the sample D of (2)
The DPV was measured in the same manner as in Example 1 except that the NA fragment (5 picomoles) was used (DPV16). Also,
The DPV was measured in the same manner as above except that no sample DNA fragment was present (DPV15). as a result,
The peak current value of DPV16 at 460 mV is 1.4% of the peak current value of DPV15 at the same potential.
Although reduced, in fact little change was observed.

[Detection of Partial Complementary DNA Fragment Using DNA Fragment of LPL Gene Abnormal Patient as Sample DNA Fragment (4B)] (1) Preparation of Sample DNA Fragment Purification from human leukocytes (1 mL) having a normal LPL gene Using the chromosomal DNA obtained, the LPL gene portion (“WT
The portion corresponding to "+") was subjected to PCR amplification (40 cycles) to obtain oligonucleotide "WT + / WT +". As a sample DNA fragment, this oligonucleotide “WT +
/ WT + ”and the oligonucleotide“ S447X + / S447X + ”obtained in Example 8 above were mixed at a ratio of 1: 1 (“ WT + / S447X + ”). This sample DNA fragment was purified by dialysis and used for the following hybridization. (2) Hybridization and measurement of electric current The same operation as in Example 8 was performed except that the sample DNA fragment (5 picomoles) obtained in (1) above was used, and the DPV was measured (DPV18). The DPV was measured in the same manner as above except that no sample DNA fragment was present (DPV17). As a result, DP at 460 mV
The peak current value of V18 changed by 16.4% compared to the peak current value of DPV17 at the same potential.

[Detection of partially complementary DNA fragment using normal LPL gene DNA fragment as sample DNA fragment (4C)] The oligonucleotide “WT + / WT +” obtained in Example 9 (5. The same operation as in Example 3 was performed except that 0 picomole was used, and the DPV was measured (DPV20). This sample DNA fragment was previously purified by dialysis. Also, sample DNA
In the same manner as above except that no fragment is present, the DP
V was measured (DPV19). As a result, the peak current value of DPV20 at 460 mV is
The change was 38.4% compared to the peak current value of PV19.
The rate of this change corresponds to the control value of the detection (4A) and the detection (4B).

The results of Examples 1 to 4 are summarized in Table 1 below.

[0087]

Table 1 Table 1 DNA fragment Sample DNA fragment Current value Example 1 dA ₂₀ dT ₉ dAdT ₁₀ 22.2 dA ₂₀ dT ₈ dA ₄ dT ₈ 15.1 dA ₂₀ dT ₂₀ 37.3 ─────────────────────────── {Example 2 WT-S447X + 6.3 WT- WT + 65.4} {Example 3 S447X- WT + 9.7 S447X- S447X + 29.4} ─────────── Example 4 WT- 447X + / S447X + -1.4 WT- WT + / S447X + 16.4 WT- WT + / WT + 38.4 ─────────

From the results of Examples 1 to 4, the double-stranded DNA fragment having a special DNA structure such as a mismatch structure or a bulge structure was identified as an example of a sample DNA fragment by LP
It was confirmed that sufficient detection was possible even when a model compound of the L abnormal gene was used. Also, as a sample DNA fragment,
It can be seen that the above detection is possible even using actual DNA of a patient with LPL gene abnormality. That is, in the normal LPL gene, the current value increased by about 38%,
LPL gene abnormalities (heterozygotes: those in which two LPL genes are present on the chromosome and one of them has a mutation) increase by about 16%, and LPL gene abnormalities (homozygotes: two (In which two LPL genes are present and in which both are mutated), no increase was observed. This indicates that a genetic diagnosis of a genetic disease relating to the LPL gene is possible by examining the polymorphism of the LPL gene.

Example 5 Confirmation that the Rate of Change in Current Value Depends on the Amount of Intercalator Bound to Double-Stranded DNA Fragment Hybridization and measurement of current value were performed at the same temperature (20 ° C.). The temperature was 15, 25,
DPVs were measured in the same manner as in Example 1 except that the temperature was changed to 35 and 45 ° C., respectively, and each peak current value at 450 mV was obtained (− △ − in FIG. 6). 20 ° C
The peak current value (Example 1) in the case of (1) is also shown.

Each peak current value was determined in the same manner as described above except that the sample DNA fragment was changed to dT ₈ dA ₄ dT ₈ (-−- in FIG. 6). However, the current value at 30 ° C. was also measured.

FIG. 6 shows that when dT ₂₀ was used as the sample DNA fragment, the peak current decreased when the temperature was higher than 30 ° C., and when dT ₈ dA ₄ dT ₈ was used, the peak current decreased. A decrease in the peak current value was observed from around ℃. It is known that the melting temperature of dA ₂₀ and dT _{20 in} a solution of a double-stranded DNA fragment is 42 ° C., but the former result shows good agreement with this fact. In the former, since the formed double-stranded DNA fragment has a full-match structure, the double-stranded structure is stable. Up to about 30 ° C., ferrocene-naphthalenediimide is stably bonded to the double-stranded DNA fragment. On the other hand, in the latter, the double-stranded DNA fragment has a bulge structure, and the stability is lower than that of the full-match structure. It is thought that until. Therefore, it can be seen that the peak current value corresponds to the amount of the naphthalenediimide bound to the double-stranded DNA fragment, and does not correspond to the amount of the double-stranded DNA fragment formed on the electrode. The same relationship as described above was also observed in an experiment in which the state of change in absorbance with respect to change in temperature was measured (data not shown). Therefore, as long as the hybridization and the electrochemical measurement are performed at a low temperature (around 20 ° C.), a change in the current value depending on the amount of the formed double strand is not observed, so that the double-stranded DNA fragment can be accurately detected. be able to.

<Example system 2> [Example 6] [DNA- (A) ₄ GTAGAG as sample DNA fragment]
(1) Synthesis of PNA fragment: PNA fragment represented by the following formula: PNA-CTCT (C)
₂ (T) ₄ in the literature (PENielsen et al., Journal of A
American Chemical Society, 114, 1895-1897 (1992) and
114, 9677-9678 (1992)). T represents thymine and C represents cytosine. In order to distinguish PNA fragments from DNA fragments, PNA-, DNA
-Is shown.

[0093]

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(2) Preparation of PNA Analytical Element A phosphoric acid buffer of 1,2-bis (vinylsulfonylacetamide) ethane is dropped on a gold electrode having a mercapto group on the surface and having an area of 2.25 mm ^2. An aqueous solution (2 μL) of 100 pmol / 1 μL of PNA-CTCT (C) ₂ (T) ₄ was dropped onto a gold electrode having a vinylsulfonyl group at one end where the vinyl sulfonyl group was free, and left at room temperature for 1 hour to leave PNA.
An analytical element was produced. (3) Preparation of sample DNA fragment DNA- (A) ₄ GTAGAG was used as the sample DNA fragment.
Was prepared according to the method described in JP-A-10-288080. (4) Measurement of Hybridization and Current Amount 4-1 Measurement of Background Value Ferrocene-modified electrochemically active embroidery intercalator synthesized in Example 1 (0.1 M potassium chloride-0.1 M acetate buffer containing 50 μM) (PH 5.6) solution to 3
At 8 ° C., the PNA analytical element produced in the above (2) was immersed, and a voltage in the range of 100 to 700 mV was applied to measure differential pulse voltammetry (DPV). Next, when a response current value (background value) at an applied voltage of 460 mV was measured, it was -1.3 μA.
Met. The measurement was performed with a pulse amplitude of 50 mV and a pulse width of 50
The measurement was performed at an mS and a scan speed of 100 mV / sec.

4-2 Detection of Complementary DNA Fragment A 10 mM Tris buffer containing the DNA- (A) ₄ GTAGAG (70 pmol) obtained in (3) above the PNA analytical element prepared in (2). (PH 7.5) 2 μL of the solution was added dropwise and incubated at 25 ° C. for 30 minutes. Next, the surface of the PNA-modified electrode after the incubation was washed with a 0.1 M sodium dihydrogen phosphate-disodium hydrogen phosphate aqueous solution (pH 7.0), and unreacted DN was added.
A- (A) ₄ GTAGAG was removed. Then, the same operation as the above-described measurement of the background value is performed to obtain a response current value (current value of the complex of PNA and the sample DNA fragment).
Was -1.7 [mu] A. The ratio of the change in the response current value to the background value is 31
%Met.

In the same manner as described above except that the DNA analysis element prepared by dropping an aqueous solution containing DNA-CTCT (C) ₂ (T) ₄ was used instead of using the PNA analysis element prepared in the above (2). Then, when the background value and the response current value were measured, the values were −2.5 μA and −2.5 μA, respectively.
3.0 μA. The ratio of the change in the response current value to the background value was 20%.

[DNA- (A) ₄ as a sample DNA fragment]
(G) on the PNA chip prepared in ₂ AGAG the use detection 2-] wherein (2), the sample DNA fragments: DNA
- (A) ₄ instead of GTAGAG, JP-10-28
Sample DN prepared according to the method described in No. 8080
A fragment: DNA- (A) ₄ (G) ₂ Hybridization and current amount were measured in the same manner as in (4) except that AGAG was used. The background value was -1.3 μA, the response current was The value was −2.7 μA, and the rate of change of the response current value relative to the background value was 10 μA.
8%.

In the same manner as described above, except that the DNA analysis element prepared by dropping the aqueous solution containing DNA-CTCT (C) ₂ (T) ₄ was used instead of using the PNA analysis element prepared in the above (2). Then, when the background value and the response current value were measured, the values were −2.5 μA and −2.5 μA, respectively.
It was 3.5 μA. The ratio of the change in the response current value to the background value was 40%.

From the detection-1 of Example 6, PNA-CT
When a double-stranded fragment having a mismatch structure formed by CT (C) ₂ (T) ₄ and DNA- (A) ₄ GTAGAG can be confirmed, and a DNA analysis element is used even if a PNA analysis element is used. As in (Detection-2-), it can be seen that a sample DNA fragment having partial complementarity can be detected. Further, it can be seen that the background sensitivity can be further reduced when the PNA analysis element is used, and the detection sensitivity is higher than when the DNA analysis element is used.

[0100]

According to the detection method of the present invention, the binding amount of the electrochemically active embroidery intercalator to the hybrid DNA depends on the structure of the hybrid DNA formed by hybridization of the DNA fragment and the sample nucleic acid fragment. Thus, it can be detected that the sample nucleic acid fragment is a nucleic acid fragment having partial complementarity with the DNA fragment. Even if the DNA fragment is replaced with a PNA fragment, a nucleic acid fragment having partial complementarity with the PNA fragment can be detected. Since the PNA fragment has a stronger binding force to the sample nucleic acid fragment than the DNA fragment, the complementarity with the sample nucleic acid fragment can be more strictly recognized. In addition, the detection method of the present invention is a quick and simple method performed at a low temperature, and can be used for detecting any gene mutation of a corresponding abnormal gene fragment if a conductive substrate on which a normal gene fragment is immobilized is used. can do. In the conventional SSCP method, it has been difficult to detect the non-base pair-forming portion particularly at the end of the hybrid DNA, but the method of the present invention is sufficiently possible. Therefore, the present invention can provide a powerful technique for analyzing gene mutations in medicine, biology, and the like, and is expected to be used for risk diagnosis of genetic diseases.

[Brief description of the drawings]

FIG. 1 is a diagram schematically showing a conventional SSCP method.

FIG. 2 is a typical schematic diagram of the method for detecting a partially complementary DNA fragment of the present invention.

FIG. 3 shows differential pulse voltammography when HS-dA ₂₀ and dT ₉ dAdT ₁₀ are hybridized and differential pulse voltammography of HS-dA ₂₀ .

FIG. 4 shows differential pulse voltammography when HS-dA ₂₀ and dT ₈ dA ₄ dT ₈ are hybridized and differential pulse voltammography of HS-dA ₂₀ .

FIG. 5 shows differential pulse voltammography when HS-dA ₂₀ and dT ₂₀ are hybridized and differential pulse voltammography of HS-dA ₂₀ .

FIG. 6 is a graph showing a state of a change in a peak current value with respect to a change in temperature in hybridization between HS-dA ₂₀ and dT ₂₀ and measurement of an amount of electricity;
FIG. 9 is a graph showing a state of a change in a peak current value with respect to a change in temperature in hybridization between dA ₂₀ and dT _{8 and} dA ₄ dT ₈ and in measurement of the amount of electricity.

[Explanation of symbols]

11a normal gene fragment 11b abnormal gene fragment 21a single-stranded normal gene fragment 21b single-stranded abnormal gene fragment 31 denaturation treatment 41 conductive substrate 51 DNA fragment 61 DNA analysis element 71a sample nucleic acid fragment 71b complementary to DNA fragment 71b , 71c, 71d A sample nucleic acid fragment that forms one or more non-base pairing portions with a DNA fragment 81 Hybridization 91a Hybrid DNA with a full match structure 91b Hybrid DNA with a mismatch structure 91c, 91d Hybrid DNA with a bulge structure 100 -Type intercalator 101a, 101b, 101c, 101d Complex formed by hybrid DNA and electrochemically active embroidery intercalator

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/566 C12M 1/34 Z 33/58 G01N 27/46 336M // C12M 1/00 C12N 15/00 ZNAA 1/34 G01N 27/30 351 (72) Inventor Masaji Ogawa 2-26-30 Nishiazabu, Minato-ku, Tokyo Inside Fujisha Shin Film Co., Ltd. (72) Inventor Makoto Takagi 3 Shonan-cho, Hakata-ku, Fukuoka City, Fukuoka Prefecture -4-29 (72) Inventor Shigeori Takenaka 4-23-21 Mai no Sato, Koga City, Fukuoka Prefecture (72) Inventor Kenichi Yamashita 17-104 Tsutsumi Danchi, Jonan-ku, Fukuoka City, Fukuoka F-term (reference) 2G045 AA35 AA40 DA12 DA13 FB02 FB05 GC20 4B024 AA11 AA20 CA01 HA11 4B029 AA07 AA08 AA23 BB20 CC01 CC03 CC08 FA03 GA03 4B063 QA01 QA12 QA17 QQ42 QR32 QR55 QR84 QS20 QS34 QX05

Claims (14)

[Claims] (1) A sample solution obtained by dissolving or dispersing a sample nucleic acid fragment is brought into contact with a DNA analysis element in which a DNA fragment is fixed at one end to the surface of a conductive substrate, and the electrochemically active sewing is performed. In the presence of a type intercalator,
A step including an operation of measuring a current value flowing between the intercalator and the analysis element by applying a potential to the analysis element; and Using a nucleic acid fragment having complementarity to the DNA fragment fixed to the DNA analysis element, and collating with a standard current value of the complementary nucleic acid fragment obtained by the same operation as the above (1). A method for detecting whether or not a sample nucleic acid fragment used in step (1) is partially identical to the complementary nucleic acid fragment with respect to a base sequence. (1) A sample solution obtained by dissolving or dispersing a sample nucleic acid fragment is brought into contact with a DNA analysis element in which a DNA fragment is fixed at one end to the surface of a conductive substrate, and the electrochemically active sewing is performed. In the presence of a type intercalator,
A step of measuring the value of a current flowing between the intercalator and the analysis element by applying a potential to the analysis element; (2) a sample nucleic acid fragment is applied to the DNA analysis element having the same configuration as described above; By contacting an aqueous liquid containing no, and applying a potential to the analytical element in the presence of the electrochemically active sewn intercalator, the background current value flowing between the intercalator and the analytical element And (3) using the nucleic acid fragment having complementarity to the DNA fragment fixed to the DNA analysis element with the current value measured in the above step (1), Step (1), which comprises a step of checking with a standard current value of a complementary nucleic acid fragment obtained by the same operation as 1) and comparing with a current value measured in Step (2). There sample nucleic acid fragment, a method for detecting whether or not partially identical with respect to the complementary nucleic acid fragment and sequencing. 3. A DNA analysis element having a surface other than the DNA fragment fixing site, which has been subjected to a mask treatment for preventing the electrochemically active sewn intercalator from contacting the surface of the conductive substrate. The detection method according to claim 1 or 2, wherein: 4. The method according to claim 1, wherein the masking is performed by applying DN to the surface of the conductive substrate.
The method according to claim 3, wherein the fragment A is fixed at one end thereof, and then subjected to a surface treatment with 2-mercaptoethanol. 5. The DNA analysis device according to claim 1, wherein a DNA analysis device having a DNA fragment having a known base sequence bonded at one end thereof is used as the DNA analysis device. Detection method. 6. The method according to claim 1, wherein a ferrocene-modified electrochemically active stitched intercalator having a redox activity is used as the electrochemically active stitched intercalator. Detection method as described. 7. The detection method according to claim 1, wherein the measurement of the current value in each step is performed by differential pulse voltammography. 8. (1) A sample solution obtained by dissolving or dispersing a sample nucleic acid fragment is brought into contact with a PNA analysis element in which a PNA fragment is fixed to one end of the surface of a conductive substrate, and the electrochemically active sewing is performed. In the presence of a type intercalator,
A step including an operation of measuring a current value flowing between the intercalator and the analysis element by applying a potential to the analysis element; and Using a nucleic acid fragment having complementarity to the PNA fragment immobilized on the PNA analysis element, and comparing it with a standard current value of the complementary nucleic acid fragment obtained by the same operation as the above (1). A method for detecting whether or not a sample nucleic acid fragment used in step (1) is partially identical to the complementary nucleic acid fragment with respect to a base sequence. 9. A sample solution obtained by dissolving or dispersing a sample nucleic acid fragment is brought into contact with a PNA analysis element in which a PNA fragment is fixed to one end of the surface of a conductive substrate, and sewn electrochemically. In the presence of a type intercalator,
A step including an operation of measuring a current value flowing between the intercalator and the analysis element by applying a potential to the analysis element, (2) a sample nucleic acid fragment is applied to the PNA analysis element having the same configuration as above. By contacting an aqueous liquid containing no, and applying a potential to the analytical element in the presence of the electrochemically active sewn intercalator, the background current value flowing between the intercalator and the analytical element (3) using the nucleic acid fragment having complementarity with the PNA fragment fixed to the PNA analysis element using the nucleic acid fragment, Step (1), which comprises a step of checking with a standard current value of a complementary nucleic acid fragment obtained by the same operation as 1) and comparing with a current value measured in Step (2). There sample nucleic acid fragment, a method for detecting whether or not partially identical with respect to the complementary nucleic acid fragment and sequencing. 10. A PNA analysis element in which a surface other than a PNA fragment fixing site is subjected to a mask treatment for preventing the electrochemically active sewn intercalator from contacting the surface of the conductive substrate is used. The detection method according to claim 8 or 9, wherein: 11. A mask process comprising: forming a P on the surface of the conductive substrate;
The method according to claim 10, wherein the NA fragment is immobilized at one end thereof and then subjected to a surface treatment with 2-mercaptoethanol. 12. A PNA to which a PNA fragment having a known base sequence is bound at one end as a PNA analysis element.
The detection method according to any one of claims 8 to 10, wherein an analysis element is used. 13. The method according to claim 8, wherein a ferrocene-modified electrochemically active stitching intercalator having a redox activity is used as the electrochemically active stitching intercalator. Detection method as described. 14. The detection method according to claim 8, wherein the measurement of the current value in each step is performed by differential pulse voltammography.