JP2003504595A - Carrier for immobilizing nucleic acids on a solid support - Google Patents

Abstract

  (57) [Summary] Disclosed are carriers and methods for immobilizing nucleic acids on a solid support. The activated ester-containing polyanion is covalently bound to the solid support. In one preferred embodiment, the polyanion is polyacrylic acid.

Description

Detailed Description of the Invention

BACKGROUND ART 1. TECHNICAL FIELD The present invention relates to a nucleic acid-immobilized carrier used for producing a DNA chip. The present invention also relates to a method for immobilizing nucleic acid on the carrier and a method for detecting target nucleic acid on the carrier.

2. Related Technology With the progress of genome analysis technology, the structure of various organism genomes is being elucidated. In parallel with this, technological development for functional analysis of the genome is being promoted. In such a situation, the DNA chip (DNA microarray) is a technology that has been drawing attention. The DNA chip is a microarray in which a large number of different genes or fragments thereof (DNA fragments) are immobilized on the surface of a solid phase carrier such as a slide glass, and is useful for gene expression, mutation and polymorphism analysis.

The method of immobilizing a target nucleic acid on a carrier is a basic technique required for producing a DNA chip, and two methods are roughly used for producing a DNA chip. One is a method of chemically synthesizing a nucleic acid at the tip of a linker covalently bonded on a carrier,
For example, Science, Vol. 251, pp. 767-773 (1991).
And Nucleic Acids Research, 20th
Vol., Pp. 1679-1684 (1992). In this way DNA is uncommon because it is limited to oligonucleotides and requires specialized equipment to control the reaction.

Another method is pre-synthesized DNA or PCR (polymerase chain reaction).
By a method of covalently or non-covalently binding the DNA (PCR amplification product) prepared in 1. to a carrier, for example, Science, Volume 270, pp. 467-470 (1995) and Nucleic Acids Research (Nucleic Acids). Resear
ch), Vol. 22, pp. 5456-5465 (1994).

In the immobilization method by non-covalent bond, DNA (or RNA) dissolved in a salt solution such as SSC (sodium chloride / sodium citrate) is treated as it is or after denaturation, and then a basic substance such as polylysine or polyethyleneimine is used. A polycation-coated silane compound or a silane-treated silane compound (for example, 3-aminopropyltriethoxysilane) is spotted on a glass slide and fixed by UV irradiation. This method should be able to immobilize any DNA (or RNA).

However, in practice, in this method, oligonucleotides and short DNA (
(Less than 0.3 Kb) is hard to be immobilized. Further, even in the case of non-covalently binding long-chain DNA (more than 0.3 Kb), the DNA may be separated from the carrier in the washing and hybridization steps, which may be a factor that lowers the detection sensitivity of the target nucleic acid.

Further, in the immobilization method by non-covalent bond, the background signal is high due to the non-specific bond between the basic functional group (cation) remaining on the surface of the carrier and the probe nucleic acid (anion). There is a constraint that A method for blocking the remaining amino group by acetylation (conversion of amine to carboxylic acid with anhydride such as succinic anhydride) is known, but this conversion is not always complete.

As the nucleic acid, short-chain nucleic acid, long-chain nucleic acid, single-stranded nucleic acid, double-stranded nucleic acid, DNA, RNA
There is a need for development of carriers that are suitable for immobilization of each type of nucleic acid and that can be used for detection of target nucleic acid with high sensitivity.

SUMMARY OF THE INVENTION The main objects of the present invention are (1) preparation of a novel carrier for immobilizing nucleic acid capable of immobilizing nucleic acid with high efficiency; (2) carrier on which nucleic acid is immobilized (ie, prepared) DNA microarray); (3) method for producing immobilized nucleic acid; and (4) method for detecting nucleic acid using a carrier on which nucleic acid is immobilized.

When the present invention is outlined, the first invention of the present invention relates to a carrier for immobilizing a nucleic acid having a polyanion on the surface for binding a nucleic acid. In one preferred embodiment, the polyanion is polyacrylic acid, but any polyanion can be used.

The second aspect of the present invention involves immobilizing nucleic acid to a carrier via a covalent bond. A preferred method involves conversion of the carboxylic acid functionality to an activated ester. The activated ester is then reacted with the amine modified nucleic acid.

In the first and second aspects of the present invention, the carrier has a non-porous surface, and glass is preferable.

The third invention of the present invention relates to a carrier on which nucleic acid is immobilized, wherein the nucleic acid is covalently bound to the carrier via the activated ester type of polyanion. In a third inventive aspect of the invention, the polyanion is preferably polyacrylic acid and the preferred activated ester is a pentafluorophenol ester prepared from the polyanion and pentafluorophenol via carbodiimide coupling.

A fourth invention of the present invention is a method for immobilizing a nucleic acid on a carrier, which comprises contacting a carrier coated with an activated ester derivative of a polyanion with an amine-modified nucleic acid. Regarding In the fourth aspect of the present invention, the polyanion is preferably polyacrylic acid, the carrier is preferably non-porous, and glass is suitable as the non-porous carrier.

In the fourth aspect of the present invention, the nucleic acid is a nucleic acid modified with a functional group reactive with an activated ester derivative, preferably the nucleic acid is modified with an amino group.

A fifth invention of the present invention relates to a method of detecting a target nucleic acid in a sample (or sample), including but not limited to the following steps: a) for immobilizing nucleic acid In the step in which the carrier becomes an activated ester derivative of a polyanion, the polyanion is previously covalently bound to the surface of the glass slide; and b) the activated ester derivative and a functional group reactive with the ester derivative. A step of reacting with a modified nucleic acid; c) a nucleic acid immobilized on a carrier (target nucleic acid) and a complementary nucleic acid (probe nucleic acid)
And a step of hybridizing; and d) a step of detecting hybridized nucleic acids.

A sixth invention of the present invention relates to, but is not limited to, a method of detecting a nucleic acid in a subject, comprising the steps of: a) an activated ester derivative of a carrier, Reacting an activated ester derivative with a nucleic acid modified with a reactive functional group; b) hybridizing a nucleic acid immobilized on a carrier with a complementary nucleic acid; c) a hybridized nucleic acid Of detecting.

[0018] Detailed description   A polyanion is a polymer containing two or more negative charges.

The term nucleic acid refers to an oligonucleotide, messenger RNA, antisense, plasmid DNA, a portion of plasmid DNA, or a virus (virus DN).
A), deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in the form of genetic material derived from linear or chromosomal DNA.

[0020]   A humidity chamber is any chamber that can maintain a fixed humidity range.

The carrier for immobilizing the nucleic acid in the present invention is not particularly limited as long as it can be used in a DNA chip or a biosensor, but it is preferably a non-porous support having a smooth surface and a slide. Materials such as glass or silica beads are preferably used.

In one embodiment, the support is treated with 3-aminopropyltriethoxysilane to provide the support surface with a functional group (in this case an amino group) capable of reacting with a polyanion. Any silane capable of introducing functional groups on the surface of the glass carrier can be used in the present invention. The polyanion can then be covalently attached to the silanized surface of the support via a carbodiimide coupling reaction.

The polyanion has an acidic functional group such as a carboxyl group, a phosphate group and a sulfate group, which has an effect of preventing non-specific binding (electrostatic) to the probe nucleic acid, and is covalently bonded to the carrier surface. Polyacrylic acid, polyglutamic acid, polyaspartic acid and polyphosphoric acid are preferably used. These polyanions are attached to the silanized surface of the support via carbodiimide (or other activated ester) chemistry.

In order to immobilize the target nucleic acid on these polyanion-coated carriers, it is necessary to activate the acidic functional group of the polyanion (form an activated ester). For example, pentafluorophenol can be used as a carboxyl group, and imidazole ester can be used as a phosphoric acid group as an activated ester derivative.

The nucleic acid immobilized on the carrier is not particularly limited, and synthetic oligonucleotides,
Any of the polynucleotides or their derivatives can be used. These nucleic acids (DNA or RNA) can be used in either single-stranded or double-stranded form. The derivative is not particularly limited as long as it has a modification that enables it to be immobilized on the surface of a carrier. For example, the 5'end of DNA is an amino group, a thiol group,
Examples thereof include derivatives modified with a phosphate group and an aldehyde group. Further, a derivative obtained by adding a cross-linking agent or a linker as a spacer, such as an alkylamine, to the modified 5 ′ end can also be used. In the present invention, modification of the 5'end is particularly effective for immobilizing short-chain nucleic acids such as oligonucleotides. Any nucleic acid covalently modified to introduce a variety of functional or signaling groups can be used, and the Mirrus LabelIT reagent can be used to modify the nucleic acid.

The modified nucleic acid is 0.01 to 2.0 mg / ml, preferably 0.1 to 1.0 m
A solution suitable for immobilization, such as 20 mM MOPS (3
-Dissolve in morpholino propane sulfonic acid) buffer (pH 7.5) or 50 mM carbonate buffer (pH 9.5) and contact with the above-mentioned carrier which has been surface-treated to undergo ester surface functionalization as it is or after denaturing treatment. By so doing, the desired nucleic acid can be immobilized on the carrier. That is, the DNA solution is spotted on a carrier having an ester functional group activated by the surface treatment by using a micropipette or a DNA chip manufacturing apparatus (DNA arrayer) by a predetermined amount. 3 in the humidity chamber
After holding for 0 to 60 minutes, 2% SDS (sodium dodecyl sulfate) and distilled water are washed in this order. Further, if necessary, the carrier on which the nucleic acid is immobilized can be treated with 0.3 N Na at room temperature.
The nucleic acid is denatured by immersing in OH for 5 minutes or in boiling water for 2 minutes, washed with distilled water and absolute ethanol in this order, and dried. By this treatment, the activated ester group that has not reacted with the nucleic acid is hydrolyzed (that is, negatively charged) to the carboxylic acid moiety.
Since these acidic groups prevent nonspecific electrostatic binding between the probe nucleic acid and the carrier,
Generally, the succinic anhydride blocking operation required for polylysine slides can be omitted. These acidic groups also reduce the background signal.

The amount of nucleic acid immobilized on the carrier in this manner can be measured using a fluorescently labeled nucleic acid. The remaining fluorescent signal can be detected by performing an immobilization operation by covalent bonding of the labeled target nucleic acid. The immobilization method of the present invention has high immobilization efficiency, and the immobilized nucleic acid does not peel off under the hybridization conditions (since the nucleic acid is covalently bound to the carrier), the gene expression level, It can be used for highly sensitive detection of mutations and polymorphisms.

A general hybridization method is used for detecting the target nucleic acid covalently bound to a carrier (for example, a DNA chip). The probe nucleic acid is labeled with a fluorescent dye (or other signal group), denatured with alkali or heat, and then added to the hybridization solution, and 5 to 20 μl thereof is dropped on the array surface so that air is not put on it. Place a cover glass, put it in a humidity chamber, hold it at a suitable temperature for a suitable time, remove the cover glass, wash the slide glass thoroughly, remove water droplets, and detect the fluorescence signal with a fluorescence scanner (fluorescence reader) To do.

The polyanion-coated slide glass of the present invention can efficiently immobilize nucleic acid. The three-dimensional matrix formed on the surface of the carrier by the covalently bound polyanion increases the amount of nucleic acid immobilized.

The nucleic acid-immobilized carrier of the present invention enables detection of nucleic acid signals with high sensitivity and low background. The activated ester derivative of the polyanion unreacted with the target nucleic acid is hydrolyzed to the polyanion, and the nonspecific binding of the negatively charged probe nucleic acid to the carrier is blocked.

According to the present invention, (1) the target nucleic acid (including the oligonucleotide) is immobilized on the carrier with high efficiency, (2) the blocking operation after immobilization can be omitted, and (3) the target nucleic acid and the carrier. As a result that non-specific binding with and is minimized and (4) the efficiency of hybridization is increased by the three-dimensional effect of the polyanion treatment, the target nucleic acid can be detected with high sensitivity.

EXAMPLES The present invention will be described in more detail by the following examples, but the present invention is not limited to the scope of the examples.

Example 1 (1) As standard DNA, PCR was carried out using two types of phage DNA fragments (1.0 Kb and 0.3 Kb, respectively, the latter part of the former) shown in SEQ ID NOS: 1 and 2 of the sequence listing. Was prepared by.

That is, a combination of the primer S shown in SEQ ID NO: 3 of the sequence listing and the primer A1000 shown in SEQ ID NO: 4 of the sequence listing and the combination of primer S and primer A300 shown in SEQ ID NO: 5 of the sequence listing using phage DNA as a template. And P respectively
CR was performed. Then, each amplified fragment was subjected to Qiaquick PCR purify.
The product was purified using a catation kit 96 (manufactured by Qiagen) according to the attached instruction. Elution of DNA from the column was performed with distilled water and concentrated by freeze-drying.

Each DNA fragment was dissolved in 20 mM MOPS buffer (pH 7.5), and GMS4 was added.
An array was prepared using 17 arrayer (manufactured by Genetic Micro Systems) on a commercially available slide glass having an amino group on the surface. The slide glass is poly-L-lysine-coated POLY-PREP, which is widely used for DNA chip production.
-SLIDES (manufactured by Sigma, hereinafter abbreviated as PLL coated slide glass) and aminoalkylsilane-based MAS coated slide glass (manufactured by Matsunami Glass Industry Co., Ltd., hereinafter abbreviated as MAS coated slide glass) were used. The spot size is 150μ
m, the interval was set to 375 μm, and the same DNA was aligned in 5 spots.

After spotting the DNA, each slide glass was kept in a thermo-hygrostat at 37 ° C. and a humidity of 90% for 1 hour, and then UV cross-linked at 60 mJ / cm ² . This slide glass was washed once with 0.2% SDS and twice with distilled water, and after removing water by low speed centrifugation at about 1,000 rpm, it was air dried. The slide glass pretreated in this way was thoroughly washed with distilled water, boiled in a boiling water bath for 3 minutes, denatured the DNA by exposing it to ice-cold ethanol, removed ethanol by low-speed centrifugation, and then air-dried. , Stored in a dessicator.

A part of the slide glass was treated with succinic anhydride, and free amino groups on the surface of the carrier were blocked by succinylation. 1.5 g of succinic anhydride was added to 89.5 ml of N 2.
-After dissolving in methyl-2-pyrrolidone, 9 ml of 1 M borate buffer (pH 8.0)
Were mixed to prepare a blocking solution. The slide glass was immersed in the blocking solution for 20 minutes at room temperature. The slide glass treated in this way was thoroughly washed with distilled water, boiled in a boiling water bath for 3 minutes, and then exposed to ice-cold ethanol for DN treatment.
A was denatured, ethanol was removed by low-speed centrifugation, air-dried, and stored in a desiccator.

(2) Aminoalkylsilane-treated commercial slide glass (In situ
PCR glass slides, manufactured by PE Biosystems, abbreviated as AAS slide glass hereinafter) is immersed in a 2.5% glutaraldehyde solution for 1 hour, washed with distilled water 3 times, air-dried, and a slide glass coated with an aldehyde group. Got

An oligonucleotide having a length of 24 bases (hereinafter, referred to as NH ₂ -F oligo) was synthesized using a DNA synthesizer, and
It was dissolved in 20 mM MOPS buffer (pH 7.5) so as to be mM. This synthetic DNA was spotted on a slide glass treated with glutaraldehyde using GMS417 arrayer in the same manner as in Example 1- (1). The slide glass after spotting was washed with 0.2% SDS for 2 minutes and then continuously washed with distilled water. PBS-containing 0.25% sodium borohydride (manufactured by Wako Pure Chemical Industries, Ltd.) on a slide glass in order to reduce a Schiff base by a covalent bond between an aldehyde group of a carrier and an amino group of DNA to a stable amine. It was immersed in 100% ethanol (volume ratio 3: 1) for 5 minutes and then dried.

(3) Covalent Bonding of Polyanion to Solid Support A microscope slide glass was washed by ultrasonic treatment in 2M nitric acid, 2M sodium hydroxide, pure water, and acetone for 10 minutes each. 4%
3-aminopropyltriethoxysilane is dissolved in 95% ethanol so that the above becomes, and the slide glass washed previously is aminosilanized in this solution for 2 minutes,
The surface of the glass slide was aminopropylsilanated by holding it at 100 ° C. for 10 minutes. Next, polyacrylic acid was dissolved in dimethylformamide so as to have a concentration of 5 mg / ml, and 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride (at a concentration of 4.0 g / 100 ml). (Hereinafter abbreviated as EDC) was added. Immerse the aminosilanized slide glass in this solution overnight,
Rinse with dimethylformamide, methanol, pure water, and acetone. The polyacrylic acid-coated slide glass was dried under reduced pressure.

(4) Activation of Polyanion-Coated Solid Support A polyacrylic acid-coated slide glass was made into an activated ester-coated slide glass as follows. 1.8 g pentafluorophenol, 2.0 g E
DC and 13 mg dimethylaminopyridine were dissolved in 50 ml dimethylformamide. The polyacrylic acid-coated slide glass prepared in Example 1- (3) was immersed in this solution for 5 hours, rinsed with dimethylformamide and methylene chloride, dried under reduced pressure, and stored in a desiccator.

Example 2 (1) Two lambda phage DNA fragments (1.0 Kb and 0.3 Kb) were prepared in the same manner as in Example 1- (1). In this synthesis, a DNA fragment having an amino group at the end was amplified by using an amine-containing primer S in which an alkylamino linker having a chain length of 6 (manufactured by PE Biosystems) was bound to the 5'end. Each amine-containing DNA fragment after purification was dissolved in 20 mM MOPS buffer (pH 7.5) so that the final concentration was 0.5 mg / ml, and Example 1- (4) was used.
Three spots were spotted on each of the activated ester-coated glass slides activated by the method described in 1. As a control, the same array was prepared on a PLL-coated slide glass.

The activated ester-coated slide glass spotted with DNA was kept in a thermo-hygrostat (Tokyo Rika Kikai: Isla KCL1000 type) at 37 ° C. and a humidity of 90% for 30 minutes, and then 0.2% SDS at room temperature. Keep in to wash away excess salt and then with distilled water. Then, immerse in 0.3N NaOH for 5 minutes to denature the DNA,
After thoroughly washing with distilled water, the product was dried by centrifugation at about 1,000 rpm and stored in a desiccator.

(2) Hybridization Each of the nucleic acid arrays prepared on each slide glass was hybridized with a nucleic acid probe labeled with a fluorescent dye and observed. Each DNA array was loaded with 50% formamide, 0.2% SDS, 5 × Denhardt's solution, 0.1 mg / ml salmon sperm DNA.
, And 6 × SSC in prehybridization buffer at 42 ° C. for 1 hour.

The 1.0 Kb λ phage DNA fragment prepared in Example 1- (1) above was replaced with Lab.
It was labeled with Cy5 ^™ according to the attached instruction manual using el IT ^R Cy5 ^™ Labeling Kit (manufactured by Miras), and then purified by ethanol precipitation. This Cy5 ^™ -labeled nucleic acid probe was diluted with prehybridization buffer to a final concentration of 20, 2 or 0.2 ng / ml, and 9
After heating at 5 ° C for 2 minutes and cooling to room temperature, insoluble matter was removed by centrifugation. About 5 μl of the hybridization solution was dropped on the nucleic acid array, covered with a cover glass so that bubbles did not enter, and then placed in a humidity chamber and kept at 42 ° C. for 20 hours. Room temperature 2
Remove the cover glass in SSC solution, and in 2 SSC containing 0.2% SDS.
It was washed twice at 65 ° C. for 5 minutes and 55 ° C. for 30 minutes. Then at room temperature 0.05 ×
It was washed with SSC for 10 minutes, and after removing water by centrifugation at about 1,000 rpm, it was air-dried.

The nucleic acid array after hybridization is analyzed by a GMS418 array scanner (G
(Manufactured by MS Co., Ltd.) was used for measurement wavelength: 635 nm and excitation wavelength: 660 nm. The intensity of the detected signal was analyzed using image analysis software ImaGene (manufactured by Biodiscovery). The results are summarized in Table 1.

[0047]

[Table 1]

As is clear from Table 1, at all probe concentrations, the signal of immobilized DNA of any size was PL on activated ester-coated glass slides.
It was stronger than the L-coated slide glass. That is, in the detection of a target PCR amplified nucleic acid fragment, DN prepared using activated ester-coated glass slides
The A-chip was shown to give a stronger signal than that produced on conventional (PLL) coated glass slides.

Example 3 (1) Preparation of DNA Chip Having Oligonucleotide DNA Array An alkylamino linker having the sequence shown in SEQ ID NO: 6 of the sequence listing and having a chain length of 6 at the 5 ′ end (manufactured by PE Biosystems) ) Modified oligonucleotide of 24 bases (hereinafter referred to as NH ₂ -F oligo) and an oligonucleotide modified at the 5 ′ end to include a hydroxyl group (hereinafter referred to as OH-F oligo). , And each was prepared using a DNA synthesizer. Furthermore, an oligonucleotide having a length of 24 bases, which has a sequence shown in SEQ ID NO: 7 complementary to the oligonucleotide and has Cy3 ^TM bound to the 5'end thereof (hereinafter, referred to as Cy3-R oligo), and these An oligonucleotide having a length of 24 bases, which has a sequence unrelated to the oligonucleotide and has a sequence represented by SEQ ID NO: 14 and having an alkylamino linker bonded to the 5'end (hereinafter, referred to as N
Two H ₂ referred to as -N oligo) were synthesized.

OH-F oligo, NH ₂ -F oligo, and NH ₂ -N oligo at 20 mM M
Dissolved in OPS buffer (pH 7.5) at concentrations of 5, 50, and 500 μM, respectively, and aligned on activated ester-coated slide glass activated by the same method as described in Example 1- (4). Let At this time, each DNA sequence was aligned in 7 spots. After spotting, wash the slides as described in Example 2,
It was dried to obtain an oligonucleotide chip. As a control, an array of the same oligonucleotides was prepared on a slide glass treated with glutaraldehyde by the method described in Example 1- (2).

(2) Hybridization of Oligonucleotide Chips The oligonucleotide chips prepared from each slide glass were hybridized with the Cy3-R oligo prepared above as a probe, and the fluorescence signal was observed. That is, about 5 μl of a hybridization solution consisting of 1 mM Cy3-R oligo, 0.2% SDS, 5 × Denhardt's solution, 0.1 mg / ml salmon sperm DNA, and 6 × SSC was added to each oligonucleotide array, and foamed. It was covered with a cover glass so that it would not enter, and then placed in a humidity chamber and kept at 42 ° C. for 20 hours. The cover glass was removed in a 2 × SSC solution at room temperature, washing was carried out twice for 10 minutes in a 2 × SSC at room temperature, the water was removed by low speed centrifugation at about 1,000 rpm, and then air-dried. Total fluorescence of the oligo DNA chip after hybridization was measured using GMS418 array scanner, measurement wavelength: 635 nm, excitation wavelength:
The intensity of the detected signal measured at 660 nm was measured by the image analysis software Ima.
It analyzed using Gene. The results are summarized in Table 2.

[0052]

[Table 2]

As is clear from Table 2, in the activated ester-coated slide glass, the spot of the oligonucleotide having an amino group at the 5 ′ end showed a stronger signal than the spot of the oligonucleotide having no amino group. It was confirmed that the activated ester group on the glass was reacting with the amino group of the oligonucleotide. In each of the oligonucleotides, the signal intensity of the spots formed on the activated ester-coated slide glass was stronger than that of the glutaraldehyde-treated slide glass.

Example 4 Three-Dimensional Effect of Polyacrylic Acid Hybridization signals of nucleic acid arrays spotted with fluorescently labeled nucleic acids of different lengths were calculated as signals per immobilized unit nucleic acid length. A human transferrin receptor (hereinafter abbreviated as TFR) cDNA fragment was prepared by the following method. MRNA was extracted from a human K562 cell line (manufactured by Dainippon Pharmaceutical Co., Ltd.) by a conventional method to prepare TFR cDNA. Using the obtained cDNA as a template,
A PCR product of 4,738 bp was obtained using primers S7 and AS7 shown in SEQ ID NOS: 8 and 9 of the sequence listing, which are specific to TFR (GenBank No. X01060). This was connected to the pT7Blue vector (Novagen) to obtain a recombinant plasmid pT7TFR. Using this pT7TFR as a template, one primer is Rhodamine X (manufactured by PE Biosystems: hereinafter abbreviated as ROX).
PCR is performed using a primer labeled at the 5 ′ end with
A CR fragment was prepared. That is, ROX label S7 (SEQ ID NO: 8) and AS4 (SEQ ID NO: 10), AS3 (SEQ ID NO: 11), AS2 (SEQ ID NO: 12), or AS
PCR was performed using the primer pair of 1 (SEQ ID NO: 13), 1.0, 0.5,
0.2 and 0.1 kb fragments were prepared.

Each purified DNA fragment was dissolved in 20 mM MOPS buffer (pH 7.5) and then aminated using Amino Label IT ^R reagent. That is, the purified DNA solution was dissolved in dimethyl sulfoxide to prepare Amino Label I.
The T ^R reagent was added 1 volume of 5 minutes in a weight ratio, and incubated 1 hour at 37 ° C.. Each aminated DNA was purified by the ethanol precipitation method.

The Amino Label IT reagent was prepared by changing the fluorescent group to an amino group according to the method described in International Publication WO98 / 52961.

The aminated TFR fragment was treated with 20 mM MO so that the final concentration was 0.5 mg / ml.
On the activated ester-coated glass slide dissolved in PS buffer (pH 7.5) and activated as in Example 1- (4), 7 points for each sequence were prepared by the method of Example 1- (1). Spotted. After spotting, it was washed and dried as described in Example 3.
As a control, the same array was subjected to M by the method described in Example 1- (1).
It was prepared using an AS-coated slide glass, and after immobilization treatment, one with succinic anhydride blocking of free amino groups and one without blocking treatment were prepared.

The DNA chip prepared with each slide glass was hybridized with a DNA probe labeled with Cy5 having a fluorescence wavelength different from that of ROX, and the signal was observed. That is, the 1.0 Kb TFR fragment prepared as described above was replaced with C
Using y5 Label IT ^R Kit (manufactured by Miras), labeling was carried out with Cy5 ^™ according to the attached instruction manual.

About 5 μl of a hybridization solution consisting of 20 ng / ml Cy5-labeled DNA probe, 0.2% SDS, 5 × Denhardt's solution, 0.1 mg / ml salmon sperm DNA, and 6 × SSC was immobilized on the chip. Each of these was added to each region, covered with a cover glass, and then placed in a humidity chamber and kept at 65 ° C. for 20 hours. Remove cover glass in 2xSSC solution at room temperature and add 2xSSC containing 0.2% SDS.
In the above, it was washed twice at 65 ° C. for 5 minutes and 55 ° C. for 30 minutes. Then, at room temperature, 0.
It was washed with 05 × SSC for 10 minutes, and after removing water by low speed centrifugation at about 1,000 rpm, it was air dried.

For the DNA chip after hybridization, the fluorescent signal of the terminal ROX label of the array DNA and the fluorescent signal of the Cy5 label of the hybridization probe were measured using a GMS418 array scanner, and the obtained image was analyzed with image analysis software. Fluorescence signal was quantified by analysis using ware ImaGene. The value obtained by dividing the Cy5 signal intensity by the ROX signal intensity for each size of DNA was defined as the signal intensity per unit DNA. The results are shown in Table 3.
It was shown to. Among the DNA chips manufactured with MAS-coated slide glass,
The DNA chip that had not been subjected to the blocking treatment of free amino groups had a high background, and the signal could not be quantified.

[0061]

[Table 3]

As is clear from Table 3, the signal intensity per unit DNA of the DNA fragments of any length was larger in the spots formed on the activated ester-coated slide glass than in the spots formed on the MAS-coated slide glass. I got stronger. The results show that activated ester-coated glass slides make the hybridization more efficient. The three-dimensional lattice created by the covalently bound polyanions allows for greater access of the bound target nucleic acid.

Example 5 In the same manner as in Example 2- (1), an amino group was added to the end of the DNA fragment.
Two lambda phage DNA fragments of 0 Kb and 0.3 Kb were prepared. Obtained D
A portion of NA was further aminated using the Amino Label IT ^R reagent as described in Example 4. Each DNA fragment was dissolved in 20 mM MOPS buffer (pH 7.5) so as to have a final concentration of 0.5 mg / ml, and 7 of each DNA fragment was prepared on activated ester-coated glass slides and MAS-coated glass slides. Spotted spot by spot.

The nucleic acid array prepared on each slide glass was hybridized with a fluorescent Cy3-labeled DNA probe, and its signal was analyzed. That is, the 1.0 Kb lambda phage fragment prepared as described above was labeled with Label IT ^R Cy3 Lab.
Cy using an eling Kit (manufactured by Miras) according to the attached instructions.
Labeled with 3. Using this Cy3 labeled DNA probe, hybridization and washing were performed by the method described in Example 4.

Fluorescence of DNA chip after hybridization (ie signal intensity)
Using a GMS418 array scanner, measurement wavelength: 532 nm, excitation wavelength:
The image intensity was measured at 570 nm, and the image intensity was quantified using the image analysis software ImaGene. The relative fluorescence intensity was determined with the signal of the spot on the MAS-coated slide glass as 100. The results are shown in Table 4.

[0066]

[Table 4]

As is clear from Table 4, the signal intensity of the activated ester-coated glass slide was stronger than that of the MAS-coated glass slide at any spot of the DNA fragment, and particularly, in the 300 bp short-chain DNA fragment, the signal intensity was higher than that of the MAS glass slide. The signal became noticeably stronger.

In the case of spotting on activated ester-coated glass slides, a stronger signal was obtained when the target was further modified with amine, as compared with when the target was not modified with amine.
Amination modification with Label IT ^R was shown to be effective in increasing target binding to activated ester-coated glass slides.

The above examples are illustrative of the principles of the invention. Further, since many modifications and changes can be easily made by those skilled in the art, the present invention is not limited to the above-described specific configuration and operation. Accordingly, all suitable variations and equivalents are within the scope of the invention.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AU, BA, BB, BG, BR, BZ, CA, CN, CR, CU, C Z, DM, DZ, EE, GD, GE, HR, HU, ID , IL, IN, IS, JP, KP, KR, LC, LK, LR, LT, LV, MA, MG, MK, MN, MX, N O, NZ, PL, RO, SG, SI, SK, TR, TT , UA, UZ, VN, YU, ZA (72) Inventor James E. Hagstrom             United States 53562 Mi, Wisconsin             Dalton, Bergamot Way No. 4700 (72) Inventor Paul M. Sluttam             53711 Ma, Wisconsin, United States             Dison, Keting Terrace No. 4306 (72) Inventor Vladimir Gee Budkar             United States 53562 Mi, Wisconsin             Dalton, Sunrise Ridge Terrace             No. 5141 (72) Inventor Mary-Anne Watt             53719 Ma, Wisconsin, United States             Dison, Radcliffe Drive 7617,             Apartment Sea (72) Inventor Masanori Takayama             Kyotanabe City, Kyoto Prefecture 4-14-2-             321 (72) Inventor Kiyoshi Asada             3-20-9 Kibogaoka, Konan Town, Koga District, Shiga Prefecture (72) Inventor Ikunoshin Kato             1-1-15 Minamiryocho, Uji City, Kyoto Prefecture F-term (reference) 4B024 AA11 AA20 CA01 CA09 CA12                       HA12                 4B029 AA07 BB20 CC03 CC07 CC11                       FA15 HA09                 4B063 QA08 QA13 QA18 QA19 QQ01                       QQ42 QQ53 QQ62 QR08 QR14                       QR20 QR32 QR36 QR55 QR56                       QR58 QR82 QS25 QS34 QX02                       QX04

Claims (19)

[Claims] 1. A carrier for immobilizing a nucleic acid, which comprises a solid support surface-coated with a covalently bondable polyanion for detecting a sample nucleic acid. 2. The carrier according to claim 1, wherein the polyanion comprises polyacrylic acid. 3. The carrier according to claim 1, wherein the polyanion comprises an activated ester. 4. The carrier according to claim 3, wherein the activated ester is selected from the group consisting of pentafluorophenol and N-hydroxysuccinimide. 5. The carrier according to claim 1, wherein the solid support is non-porous. 6. The carrier according to claim 5, wherein the solid support is made of glass. 7. A carrier for nucleic acid detection, comprising a solid support surface-coated with a covalently bound polyanion capable of binding to a nucleic acid. 8. The carrier according to claim 7, wherein the polyanion comprises polyacrylic acid. 9. The carrier according to claim 7, wherein the polyanion comprises an activated ester. 10. The carrier according to claim 7, wherein the solid support is non-porous. 11. The carrier according to claim 10, wherein the solid support is made of glass. 12. A method for immobilizing nucleic acid on a carrier, which comprises the steps of: a) coating a solid support with a covalently bondable polyanion containing an activated ester; b) polyanion for detection. And the step of contacting the nucleic acid. 13. The method according to claim 12, wherein the polyanion comprises polyacrylic acid. 14. The method of claim 12, wherein the solid support is non-porous. 15. The method of claim 14 wherein the solid support comprises glass. 16. The method of claim 12, further comprising reacting the nucleic acid with an activated ester. 17. The method of claim 16, wherein the nucleic acid contains an amino group for reaction with the activated ester. 18. The method of claim 12, further comprising a blocking step. 19. A method for detecting a sample nucleic acid in a sample, which comprises the following steps: a) a step of immobilizing the activated ester-containing polyanion according to claim 4; and b) a polyanion and an ester. Immobilizing the nucleic acid by reacting with an immobilizable nucleic acid containing a reactive functional group; c) hybridizing the immobilized nucleic acid with a sample nucleic acid complementary to the immobilized nucleic acid; d) hybridizing Step of detecting soybean sample nucleic acid.