JPH06329694A - Fixation of polynucleotide - Google Patents

Abstract

PURPOSE:To provide a high-sensitive detection method by improving the efficiency of fixation of the target nucleic acid or the target probe in fixation of a gene to a solid phase carrier. CONSTITUTION:A high-sensitive nucleic acid hybridization method is realized by allowing the target nucleic acid or the target probe to contain a linker arm nucleotide represented by the general formula in the sequence in a method for fixation of the target nucleic acid or the target probe to a solid phase carrier. Formula: GPQ (G shows a ribose or deoxyribose residue from which a carbon atom at 1 position of the reducing end group is removed. P shows one of adenosine, guanosine, cytosine, thymidine and uridine. Q denotes a univalent organic group containing 1 to 15 carbon atoms).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for immobilizing a polynucleotide on a carrier. More specifically, it relates to a method of binding a target nucleic acid or a gene probe (polynucleotide) for capturing a target to a solid phase carrier.

[0002]

2. Description of the Related Art Genetic information encoded by a gene (DNA) is expressed as a protein such as an enzyme via a messenger RNA, and an organism exists as an aggregate of various compounds produced by the action of these proteins. The total number of such genes in humans is said to be 50,000 to 100,000, but some abnormalities or changes (for example, deletions, duplications, etc.) may occur therein. In that case, the characteristics, type, amount, etc. of the produced protein change, and as a result, the balance of the biological system is lost, causing disease. Therefore, conversely, it becomes possible to identify or prevent the disease by detecting the gene causing the disease. With recent advances in genetic engineering, it has become possible to make diagnoses based on such genes themselves (called gene diagnosis).

Considering the mechanism of gene expression, it is presumed that most changes at the biochemical level are preceded by changes in the gene. Thus, genetic diagnosis allows for diagnosis and prediction prior to phenotypic changes in disease (ie, presymptomatic, latency of disease, or very early). In addition, since the genes of cells in the living body are all the same, the organ or tissue to be analyzed is not specified for the inherited disease. Especially for the fetus,
This is a very important diagnostic method because it can be diagnosed simply by collecting amniotic fluid from a pregnant woman and examining the cells of the fetus floating in the amniotic fluid.

Generally, genetic diagnosis is carried out as follows. First, a gene is extracted from a sample and, if necessary, cut with an appropriate restriction enzyme. The gene is then electrophoresed, followed by Southern blot. Then, after hybridizing with a gene probe corresponding to the gene of interest (usually labeled with a radioisotope, an enzyme, a fluorescent substance, etc.), the label of the probe is detected to obtain the target. Determine the presence or absence of the gene. In this method, a radioisotope has been conventionally used as a labeling agent for a gene probe.However, the radioisotope has a limited diagnostic site and requires careful handling of reagents. Development of safe labeling agents that replace elements is underway. The one utilizing a biotin-avidin bond, the one utilizing an enzyme or a fluorescent substance, etc. have already been developed, and they are about to reach a level comparable to a radioisotope in terms of ease of use and sensitivity.

By the way, in the above-mentioned method, it takes at least several days until the gene is detected, and the measuring operation is considerably complicated. Therefore, in order to simplify the measurement operation, especially the washing process, and shorten the measurement time, the so-called "sandwich hybridization method" was proposed (Ranki et.al., G.
ene. Vol.21, p77-85, 1983, JP 60-93355 A, JP 62-50107 A, and JP 6
No. 2-205800). In this method, a first gene probe having a base sequence complementary to a target gene is immobilized on a solid phase carrier, hybridized with a target nucleic acid having a specific sequence in a sample, and further, A second gene probe for detection labeled with a labeling agent, which is capable of hybridizing with another sequence portion of the target nucleic acid, is reacted.

[0006]

However, even in the above-mentioned sandwich hybridization method, there is a problem that the efficiency of immobilization of the first gene probe on the solid phase carrier is poor. In addition, there is also a problem that other genes besides the target gene are nonspecifically adsorbed on the solid-phase carrier during the hybridization. In order to prevent this non-specific adsorption, a method of previously blocking with DNA such as beef thymus or salmon sperm for a long time has been conventionally used. However, this is an obstacle to simplifying the operation and shortening the detection time. The present invention has been made to solve the above problems, and provides a method capable of immobilizing a gene, particularly a gene probe (polynucleotide), easily and efficiently on a carrier in a short time. The purpose is to do.

[0007]

As a result of intensive studies to solve these problems, the present inventors have found that linker arm nucleotides are used to increase the efficiency of immobilization of polynucleotides on a solid-phase carrier. It has been found that it is effective to bind the polynucleotide containing γ to the solid phase carrier via the linker part.

That is, the present invention is a method for immobilizing a polynucleotide, which comprises binding a polynucleotide containing at least one linker arm nucleotide represented by the following general formula (I) to a solid phase carrier.

General formula (I): GPQ (wherein G represents a residue in which the carbon atom at the 1-position at the reducing end is removed from ribose or deoxyribose. P is adenosine, guanosine, cytidine or thymidine. , And uridine, and Q represents a monovalent organic group containing 1 to 15 carbon atoms.)

In the present invention, a polynucleotide containing at least one linker arm nucleotide represented by the following general formula (I) is immobilized on a solid phase carrier, a sample containing a target nucleic acid is hybridized, and a labeled probe is further added. A method of quantifying a target nucleic acid in a sample, which comprises hybridizing and detecting the label.

General formula (I): GPQ (wherein G represents a residue in which the carbon atom at the 1-position at the reducing end is removed from ribose or deoxyribose. P is adenosine, guanosine, cytidine, thymidine. , And uridine. Q represents a monovalent organic group containing 1 to 15 carbon atoms.)

Furthermore, the present invention introduces at least one linker arm nucleotide represented by the following general formula (I) into a polynucleotide in a sample, immobilizes it on a solid phase carrier, and then hybridizes with a labeled probe, A method for quantifying a target nucleic acid in a sample, which comprises detecting the label.

General formula (I): GPQ (wherein G represents a residue in which the carbon atom at the 1-position at the reducing end is removed from ribose or deoxyribose. P is adenosine, guanosine, cytidine, thymidine. , And uridine. Q represents a monovalent organic group containing 1 to 15 carbon atoms.)

The linker arm nucleotide represented by the general formula (I) in the present invention comprises a ribose residue or a deoxyribose residue (G) and any one residue of adenosine, guanosine, cytosine, thymidine and uridine (P). ) And a monovalent organic group (Q) containing 1 to 15 carbon atoms. G is specifically a ribose residue or a deoxyribose residue represented by the following formula.

[0015]

[Chemical 1]

P is adenosine, guanosine, cytosine,
Any one residue of thymidine and uridine is shown. Thymine is exemplified below.

[Chemical 2]

Q is a monovalent organic group having 1 to 15 carbon atoms, for example

[Chemical 3]

[0018]

[Chemical 4]

[0019]

[Chemical 5]

[0020]

[Chemical 6]

(Wherein R ₁ is a hydrogen atom or a lower alkyl group having 1 to 6 carbon atoms, Y is one or more amino groups (-NH ₂ ), substituted amino groups (-NHR), amide (-CONH) ₂ ), a substituted amide (-CONHR), an aminoalkylphenyl or a substituted aminoalkylphenyl group (R represents an alkyl group having 1 to 12 carbon atoms), Y is one or more acetylimide groups or trihalogen Acetyl imide group). Specifically, the following organic groups are exemplified.

[0022]

[Chemical 7]

[0023]

[Chemical 8]

At least 1 represented by the above general formula (I)
The means for binding the polynucleotide containing the individual linker arm nucleotides to the solid phase carrier is preferably a physical adsorption method.

The linker arm nucleotide used in the present invention is one in which an amino group is introduced into an adenosine, guanosine, cytidine, thymidine, or uridine (P) which is a constitutional unit of a nucleic acid, through an organic group (Q). As described in Reference Example 1 and Reference Example 2 in Examples described later, it was prepared for the purpose of introducing a label such as at least one enzyme into a nucleotide sequence.

In the present invention, the polynucleotide containing the linker arm nucleotide in the sequence is about 2
It is a single-stranded nucleotide of defined sequence, which is shorter than 00 base units in length. Substituents capable of functioning as one or more detectable reporter groups, or alternatively bound to one or more detectable reporter groups, are sterically tolerable sites on the base. (For example, pyrimidine C-5, purine C-
8) A single-stranded oligonucleotide containing at least one nucleotide unit which is bound to 8).

More specifically,

[Chemical 9] [In the formula, R'is a hydrogen atom or a hydroxy group, P is a pyrimidine base or a purine base, and Q is a linker arm. ] A substantially single-stranded oligonucleotide consisting of a preselected sequence which basically has no more than 200 nucleotides and has at least one nucleotide residue represented by the above formula] at a preselected position.

The non-enzymatic synthetic method comprises coupling an activated nucleotide monomer with a terminal unit having a free hydroxyl group of an elongating nucleotide chain, at least one of which monomer and terminal unit A sterically tolerable site on the base is bound by a substituent capable of functioning as one or more detectable reporter groups or capable of binding to one or more detectable reporter groups. Is qualified by.

The orthonucleotide may be a riboxynucleotide or a deoxyribonucleotide. Prior to its synthesis, the reporter group is preselected as well as the specific nucleotide units to which the reporter group is attached.

As a method for incorporating the linker arm nucleotide used in the present invention, a random primer method (Feinberg et.al., Analytical Biochemistr) using a primer having a linker arm nucleotide in the sequence is used.
y.Vol.132, P6-13,1983, Vol.137, P266-267,1984)
And a PCR method using a primer having a linker arm nucleotide in the sequence (Saiki et.al., Science. Vo.
L.230, P1350-1354, 1985), etc., and can be detected by using a labeled probe that binds to a solid phase carrier via a linker portion introduced by these methods.

The carrier used in the method of immobilizing the polynucleotide of the present invention may be a membrane carrier such as nitrocellulose or nylon, latex particles, microtiter plate, or the like which has been conventionally used for hybridization and is considered to be usable. There is no particular limitation as long as it can be used.

In the method for immobilizing a polynucleotide of the present invention, the gene is immobilized on the carrier by physically adsorbing or covalently bonding the linker part (Q) of the linker arm nucleotide in the polynucleotide to the carrier. In the case of physical adsorption, a gene probe (polynucleotide) having a linker arm nucleotide is used as a reaction solution having an appropriate concentration of 1 to 1000 nM, preferably 10 to 100 nM, as in the case of being usually used in the immunization method. It can be realized by dipping the carrier, or adding it to the carrier, and allowing it to stand or suspend for a suitable time.

In the case of a covalent bond, the linker part (Q) of the linker arm nucleotide in the gene probe (polynucleotide) and the functional group present on the surface of the carrier react to bond. At this time, the reaction for forming a covalent bond depends on the functional group existing on the surface of the carrier.
Therefore, in forming a covalent bond between the functional group existing on the surface of the carrier and the linker part (Q) of the linker arm nucleotide in the gene, the reaction method and conditions are not particularly limited. Examples of the reaction for forming the covalent bond include an amide bond with the amino group of the linker portion when the carrier has a carboxyl group.

The polynucleotide used in the present invention brings about an increase in the efficiency of immobilization on a carrier, as compared with a polynucleotide not containing the linker arm nucleotide in the sequence.

The carrier immobilized by the immobilization method of the present invention can be preferably used for detection of a gene using sandwich hybridization. In addition, in the conventional method of extracting a gene from a sample and appropriately treating it, and then immobilizing it on a carrier, the linker arm nucleotide used in the present invention may be incorporated during the treatment such as the random primer method or the PCR method described above. The same effect can be obtained.

As a specific method for quantifying a target nucleic acid, a polynucleotide containing at least one linker arm nucleotide represented by the general formula (I) is immobilized on a solid phase carrier, and a sample containing the target nucleic acid is prepared. There is a method of quantifying a target nucleic acid in a sample, which comprises hybridizing and then hybridizing a labeled probe to detect the label.

Further, after introducing at least one linker arm nucleotide represented by the above general formula (I) into the polynucleotide in the sample and immobilizing it on a solid phase carrier, a labeled probe is hybridized and the label is labeled. There is a method of quantifying a target nucleic acid in a sample, which is characterized by detecting.

As a sample, a sample that can be collected from a living body can be widely used, and specifically, feces, urine, tissue, blood or the like can be used directly or after an appropriate treatment. As the labeled probe, an oligonucleotide having a length of 10 to several thousand bases in which a label is bound to a nucleic acid that hybridizes with a target nucleic acid that may exist in a sample is used. Desirably, the length is about 10 to 100 bases, but it may be longer when the linker arm nucleotide in the polynucleotide in the sample is incorporated. As the label, radioactive elements, enzymes, fluorescent substances, antigens, antibodies, haptens, and the like can be used so long as they are commonly used for labeling nucleic acids. As a method for detecting the label, any of the detection methods usually used for detecting the above can be used.

[0039]

INDUSTRIAL APPLICABILITY In the present invention, the efficiency of immobilization of a gene probe (polynucleotide) on a solid phase carrier in the hybridization method is improved by binding the gene probe (polynucleotide) having the above-mentioned linker arm nucleotide to the carrier. Be improved. By this,
The signal value during the hybridization reaction increases,
As a result, the minimum detection sensitivity can be improved.

[0040]

EXAMPLES The effects of the present invention will be further clarified by giving examples and reference examples of the present invention below. In the examples, “n × SSC” means a (20 / n) -fold diluted solution of a mixed solution of 0.3M sodium citrate (pH 7.0) and 3.0M NaCl.

Reference Example 1 Synthesis of Capture Probe and Label Probe for Detection of Vibrio parahaemolyticus The capture probe and the label probe were DNA synthesizer 392.
Type (Applied Biosystems) was used to synthesize by the phosphoramidite method. The base sequence of the capture probe is 5'-CGGTCATTCTGCTGXGTTCGTAAAAT-3 '. The base sequence of the labeled probe is 5'-CCCCGGTTCTGAXGAGATATTGTT-3 '. In the sequence, X represents the following linker arm nucleotide.

[0042]

[Chemical 10]

Reference Example 2 Labeling of Labeled Probe with Enzyme / Alkaline Phosphatase The binding of the labeled probe synthesized in Reference Example 1 with alkaline phosphatase via the linker arm was described in the literature (Nucleus
ic Acids Research, vol.14, P.6155, 1986). Linker arm nucleotide (labeled probe) 1.
0 A ₂₆₀ was dissolved in 60 μl of 0.2 M sodium bicarbonate, 1.25 mg of disuccinimidyl suberate (DSS) was added thereto, and the mixture was reacted at room temperature for 2 minutes. The reaction solution was added with 1 mM sodium acetate (p
Equilibrated with H5.0).

Next, Sephadex G-25 (Pharmacia)
Excess DSS was removed by gel filtration through a column (1 cm φ x 30 cm). A linker arm nucleotide in which the terminal amino group was activated was further added with alkaline phosphatase (Boehringer Mannheim) at a molar ratio of 2 times and at room temperature.
An alkaline phosphatase-labeled nucleic acid probe was obtained by reacting for a time. The obtained labeled probe was purified using negative ion exchange high performance liquid chromatography MONO-QFPLC (Pharmacia). Fractions containing the labeled probe were collected and concentrated by an ultrafiltration method using Centricon 30K (Amicon).

Reference Example 3 Preparation of Vibrio parahaemolyticus Gene A strain of Vibrio parahaemolyticus was inoculated into Brain Heart Infusion Medium and cultured at 37 ° C. overnight. The grown colonies were scraped into 1.5 ml Eppendorf tubes and suspended in 300 μl of the dilution buffer. Further to this, proteinase K (Nacalai Tesque) 0.6 mg, lysate (8 M urea, 0.25% sodium dodecyl sulfate, 0.25% sodium lauryl sarcosine, 50 mM EDTA, pH 7.6) 600
μl was added, stirred, and incubated at 60 ° C. for 30 minutes. The resulting solution was extracted twice with phenol and once with chloroform, followed by ethanol precipitation to obtain a nucleic acid.

Example 1 (1) Method for binding capture probe to solid phase carrier As a solid phase carrier, a polystyrene microtiter plate (Microlite 2, Dynatech) was used. The capture probe obtained in Reference Example 1 was diluted to 10 pmole / ml with 50 mM borate buffer. 100 μl of the solution was dispensed into wells of a microtiter plate and incubated at room temperature overnight. Further, as Comparative Example 1, a capture probe having no linker arm nucleotide was prepared and similarly bound to a solid phase carrier.

(2) Blocking method of capture probe binding plate The capture probe solution was removed from the plate obtained by the above method with an aspirator, and the block buffer was added to each well.
Dispense 150 μl each and block at room temperature for 2 hours.

(3) Detection of Vibrio parahaemolyticus Gene by Sandwich Hybridization Method Using the reagents and samples prepared by the methods of Reference Examples 2 and 3,
The detection of the Vibrio parahaemolyticus gene as a positive sample was performed by the method described below. Add an equal volume of 0.6N NaOH to the sample at room temperature.
It was denatured for 15 minutes. As a control (negative sample), a human placenta-derived DNA (Sigma) denatured by the same method was used.

After binding the capture probe, the blocking solution was removed from the blocked plate and the denatured sample was washed with 10
μl was added. Then add hybridization buffer 1
00 μl was added, and hybridization was carried out at 50 ° C. for 60 minutes. Remove the solution from the wells and wash solution I (2 x SSC, 1.
200 μl of 0% sodium lauryl sulfate) was added, and the mixture was washed at 50 ° C. for 5 minutes. Subsequently, the cleaning solution I is removed and 1 × SSC 2
The well was rinsed with 00 μl, 100 μl of alkaline phosphatase-labeled probe solution was added, and hybridization was carried out at 50 ° C. for 60 minutes. Remove the probe solution from the well, add 200 μl of Wash Solution I and wash at 50 ° C for 10 minutes.
Next, wash solution II (1 x SSC, 0.5% Triton X-100)
00 μl was added and the mixture was washed at room temperature for 10 minutes. Finally 1 x SSC
After rinsing the well by adding 200 μl of Lumiphos480, the chemiluminescent substrate for alkaline phosphatase, Lumiphos480
(Wako Pure Chemical Industries, Ltd.) (100 μl) was added, and a luminescence reaction was carried out at 37 ° C. in the dark for 15 minutes. The amount of light emission was measured with Microlight 1000 (Dynatech).

As is clear from Table 1 below, when the capture probe using the linker arm nucleotide of the present invention is used, the value of the positive sample measured is higher than that of Comparative Example 1, and the negative sample is Due to the reduced measured values, excellent results with regard to sensitivity and specificity were obtained.

[0051]

[Table 1]

Example 2 (1) Introduction of linker arm nucleotide into Vibrio parahaemolyticus The Vibrio parahaemolyticus gene prepared in Reference Example 3 was mixed with random primer, dNTP and linker arm nucleotide, and heated at 95 ° C. for 3 minutes. Then, it was rapidly cooled in ice and left for 5 minutes. Add 2 units of Klenow Fragment to this, 37 ℃
After reacting for 3 hours at 95 ° C., it was heated at 95 ° C. for 3 minutes. This was rapidly cooled in ice to prepare a Vibrio parahaemolyticus gene fragment having a linker arm nucleotide in the sequence.

(2) Method for binding Vibrio parahaemolyticus gene to solid phase A nylon membrane (Hybond N +; Amersham) was used as the solid phase. Membranes were lightly rinsed with 5 × SSC solution before immobilizing nucleic acids. The Vibrio parahaemolyticus nucleic acid obtained in (1) above was diluted with sterile water to a concentration of 10 μl / 50 μl. An equal volume of 0.3 N NaOH was added to the nucleic acid aqueous solution and treated at room temperature for 15 minutes for denaturation. After denaturation, 100 μl of each nucleic acid was added onto the nylon membrane using a dot blotter (BRL) and slowly sucked. After all the solution has been aspirated, 100 μl of 5 × SSC
Add each one and suck, remove the membrane from the blotter and remove 5 × S
Rinse lightly with SC, and remove water from the membrane with filter paper to dry.

(3) Blocking of Vibrio parahaemolyticus Gene-Immobilized Membrane and Gene Detection Method The nucleic acid-immobilized membrane was placed in a hybrid bag (BRL), and a block buffer was added to seal with a policyr. Block at 15 ° C for 15 minutes. The block buffer was removed from the hybrid bag, 100 μl of a hybridization buffer solution containing an alkaline phosphatase-labeled probe was added, and hybridization was carried out at 50 ° C. for 60 minutes while shaking. Remove the membrane from the hybrid bag, transfer it to a vat containing Wash Solution I (2 x SSC, 1.0% sodium lauryl sulfate), and shake it while shaking.
It was washed at 0 ° C for 10 minutes. Next, wash solution II (1 x SSC, 0.1
It was transferred to a vat of 5% Triton X-100) and shaken and washed at room temperature for 10 minutes. Put the washed membrane into a hybrid bag, add a substrate solution of nitroblue tetrazolium and alkaline phosphatase containing 5-bromo-4-chloro-3-indolyl phosphate, seal and leave in the dark at 37 ° C for 1 hour. I made it react. After the color development, the film was washed with distilled water and dried, and the color development was measured using a color difference meter CR-221 (Minolta Camera Co., Ltd.). ΔL * a * b * mode was used for the measurement.

As is clear from Table 2 below, when the gene fragment using the linker arm nucleotide of the present invention is bound to a solid phase and used, the value of the positive sample measured for Comparative Example 2 increases. In addition, since the measured value of the negative sample was decreased, excellent results were obtained regarding sensitivity and specificity.

[0056]

[Table 2]

[0057]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 26 Sequence type: Nucleic acid Topology: Single-stranded Sequence type: Synthetic DNA Sequence features Location: 1..26 Method of determining features: S Other features : Vibrio parahaemolyticus TDH (Thermostable Direct
Haemolysin) has a sequence homologous to the 339th to 364th nucleotide sequences. Sequence CGGTCATTCT GCTGTGTTCG TAAAAT 26

SEQ ID NO: 2 Sequence length: 24 Sequence type: Nucleic acid Topology: Single-stranded Sequence type: Synthetic DNA Sequence features Location: 1..24 Method of determining features: S Other features: Vibrio parahaemolyticus TDH (Thermostable Direct
Haemolysin) has a sequence homologous to the 102nd to 125th nucleotide sequences. Sequence CCCCGGTTCT GATGAGATAT TGTT 24

Claims (3)

[Claims] 1. A method for immobilizing a polynucleotide, which comprises binding a polynucleotide containing at least one linker arm nucleotide represented by the following general formula (I) to a solid phase carrier. General formula (I): GPQ (In the formula, G represents a residue in which the carbon atom at the 1-position at the reducing end is removed from ribose or deoxyribose. P is adenosine, guanosine, cytidine, thymidine, or uridine. Represents any one residue, and Q represents a monovalent organic group containing 1 to 15 carbon atoms.) 2. A polynucleotide containing at least one linker arm nucleotide represented by the following general formula (I) is immobilized on a solid phase carrier, a sample containing a target nucleic acid is hybridized, and a labeled probe is hybridized. A method for detecting a target nucleic acid in a sample, which comprises detecting the label. General formula (I): GPQ (In the formula, G represents a residue in which the carbon atom at the 1-position at the reducing end is removed from ribose or deoxyribose. P is adenosine, guanosine, cytidine, thymidine, or uridine. Represents any one residue, and Q represents a monovalent organic group containing 1 to 15 carbon atoms.) 3. At least one linker arm nucleotide represented by the following general formula (I) is introduced into a polynucleotide in a sample and immobilized on a solid-phase carrier, and then a labeled probe is hybridized with the labeled probe. A method for detecting a target nucleic acid in a sample, which comprises detecting General formula (I): GPQ (In the formula, G represents a residue in which the carbon atom at the 1-position at the reducing end is removed from ribose or deoxyribose. P is adenosine, guanosine, cytidine, thymidine, or uridine. Represents any one residue, and Q represents a monovalent organic group containing 1 to 15 carbon atoms.)