KR20020026473A - Substrates for nucleic acid immobilization onto solid supports - Google Patents

Abstract

Substrates and methods are disclosed for immobilizing nucleic acids on solid supports. Polyanions containing activated esters are covalently attached to the solid support. In one preferred embodiment, the polyanion is polyacrylic acid.

Description

Substrate for fixing nucleic acid on a solid support {SUBSTRATES FOR NUCLEIC ACID IMMOBILIZATION ONTO SOLID SUPPORTS}

The genomic structure of some organisms has been found according to genome analysis techniques. In parallel, techniques for analyzing genomic function have been developed. In this situation, DNA chips (DNA microarrays) are an attractive technique. DNA chips are microarrays with many different genes or fragments (DNA fragments) immobilized on the surface of a solid substrate, such as a glass slide. DNA chips (DNA microarrays) are useful for the analysis of gene expression, mutations, and polymorphism.

The method of immobilizing the target nucleic acid on the substrate is a key technique for the production of DNA chips. There are two main methods used to produce DNA chips. One method is described, for example, in Science, Vol. 251, 767-773 (1991) and Nucleic Acid Research, Vol. As described in 20, 1679-1684 (1992), it involves the chemical synthesis of nucleic acids, which are linkers covalently bound to the substrate on the tip. In this method, DNA is confined to oligonucleotides and special equipment is needed to control the reaction. Therefore, this is not the usual way.

Another method is described, for example, in Science, Vol. 270, 467-470 (1995) and Nucleic Acid Research, Vol. As described in 22, 5456-5465 (1994), a method in which pre-synthesized DNA or DNA (PCR amplification products) produced by PCR (polymerase chain reaction) is covalently or non-covalently bound to a substrate. to be.

In a non-covalent fixation method, DNA (or RNA) is dissolved in a salt solution such as SSC (sodium chloride / sodium citrate) with or without denaturation. DNA is coated on a basic polycation such as polylysine, polyethyleneimine, or spotted on glass slides silanized with amine-containing silane compounds (eg 3-aminopropyltriethoxysilane) and subjected to UV-irradiation. Is fixed by. All forms of DNA (or RNA) must be able to be fixed in this way.

In practice, however, oligonucleotides and short lengths of DNA (less than 0.3 Kb) are difficult to fix using this method. Even when long length DNA (0.3 Kb or more) is non-covalently attached, the process of washing and hybridization can remove DNA from the substrate, which can reduce the detectability of the target nucleic acid.

Another limitation of the non-covalent fixation method is the increased background signal due to the nonspecific binding of the reactor nucleic acid (anionic) and the probe nucleic acid (anionic) of the residue base on the substrate surface. Residue amino groups can be blocked by acetylation (converting amines to carboxylic acids using anhydrides such as succinic anhydride). However, the transition is not always perfect.

It is desirable to develop substrates that can be applied for high sensitivity detection of target nucleic acids and are suitable for the fixation of many types of nucleic acids, including short or long length nucleic acids, single stranded nucleic acids, double stranded nucleic acids, DNA, and RNA.

Summary of the Invention

The main objects of the present invention are: (1) preparation of a novel substrate for nucleic acid immobilization capable of immobilizing nucleic acid with high efficiency; (2) a substrate immobilized with nucleic acid (ie prepared by DNA microarray); (3) a method for producing a fixed nucleic acid; And (4) detecting the nucleic acid using a substrate immobilized with the nucleic acid.

In summary, the present invention mainly comprises a nucleic acid fixing substrate; In particular it relates to the surface of the polyanion base for nucleic acid attachment. In a preferred embodiment, the polyanion is polyacrylic acid, but any polyanion can be used.

Another aspect of the invention involves immobilizing nucleic acid on a substrate via covalent bonds. Preferred methods include converting carboxylic acid functionality to activated esters. The activated ester is then reacted with the amine modified nucleic acid.

In the first and second embodiments, the substrate is a non-porous surface. Preferred form is glass.

A third aspect of the embodiment comprises a substrate immobilized with a nucleic acid; That is to say a nucleic acid immobilized substrate, wherein the nucleic acid is covalently bound to the substrate via an activated ester of polyanions. In a third embodiment, the polyanion is polyacrylic acid as the preferred form. Preferred activated esters are pentafluoro phenol esters prepared via carbodiimide coupling from polyanions and pentafluorophenols.

In a fourth embodiment, the present invention relates to a method for immobilizing nucleic acid relative to a substrate, ie, a method for immobilizing nucleic acid on a substrate coated with an activated ester derivative of polyanions. In a fourth embodiment, the polyanion is polyacrylic acid and the substrate is non-porous as the preferred form; Glass is the best non-porous substrate.

In a fourth embodiment, the nucleic acid is modified with a functional group that is reactive toward the activated ester derivative, in which the nucleic acid is modified with an amino group.

A fifth embodiment relates to a method for detecting a target nucleic acid in a sample, including but not limited to:

a) the nucleic acid fixing substrate is an activated ester derivative of polyanion. Polyanions are previously covalently attached to the surface of the glass slide.

b) a reaction process between a nucleic acid modified with a functional group reactive with the above-mentioned activated ester and ester derivative.

c) hybridization process between nucleic acid (target nucleic acid) and complementary nucleic acid (probe nucleic acid) immobilized on a substrate.

d) detection of hybridized nucleic acids.

A sixth embodiment relates to methods of detecting nucleic acids in a sample, including but not limited to:

a) a reaction between an activated derivative of a substrate and a nucleic acid modified with a functional group that is reactive to an activated ester derivative.

b) hybridization process between nucleic acid immobilized on a substrate and complementary nucleic acid.

c) detection process for hybridized nucleic acid.

The present invention relates to a nucleic acid fixing substrate used for DNA chip production. The invention also relates to a method of immobilizing nucleic acid on such a substrate and a method for detecting a target nucleic acid on the substrate.

Polyanions are polymers containing two or more negative charges.

The term nucleic acid refers to deoxyribonucleic acid (DNA) and ribonucleic acid in the form of oligonucleotide messenger RNA, anti-sense, plasmid DNA, portions of plasmid DNA or genetic material derived from viral linear DNA (viral DNA), or chromosomal DNA. RNA).

A humidity lock is any chamber that can maintain a fixed humidity range.

In the present invention, the nucleic acid fixing substrate is not limited as long as it can be applied to a DNA chip or a biosensor. Solid supports having non-porous and smooth surfaces, for example substrates composed of materials such as glass slides or silica beads, are used in the preferred form.

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

Polyanions contain acidic functional groups that have the effect of preventing non-specific binding (electrostatic) with probe nucleic acids such as carboxy groups, phosphate groups, sulfate groups and the like. Polyanions are covalently attached to the surface of the substrate; Polyacrylic acid, polyglutamic acid, polyaspartic acid, and polyphosphate are preferred forms. These polyanions are attached to the silanized surface of the substrate via carbodiimide chemistry (or other activated ester chemistry).

In order to immobilize the target nucleic acid on the polyanion coated substrate, it is necessary to activate the acid function of the polyanion (form an activated ester). For example, pentafluorophenol and imidazole esters are activated ester derivatives that can be used in the carboxy and phosphate groups, respectively.

There is no particular restriction on the nucleic acid immobilized on the substrate. All active oligonucleotides, polynucleotides, and derivatives thereof can be used. Single stranded and double stranded nucleic acids (DNA or RNA) can be used. Derivatives are not limited as long as they have a modifier that allows fixation to the substrate surface. Examples include derivatives modified at the amino acid, thiol, phosphate, and aldehyde groups at the 5 'end of DNA. In addition, crosslinkers such as alkylamines or derivatives modified with linkers at the 5 ′ end are also usable. In the present invention, modification at the 5 'end is particularly effective for fixing short nucleic acids such as oligonucleotides. Any nucleic acid covalently modified to introduce various functional or signaling groups can also be used. Mirus' Label IT reagent can be used to modify the nucleic acid.

The modified nucleic acid mentioned above is 0.01-2.0 mg / ml, preferably 0.1 in a suitable fixative solution such as 20 mM MOPS (3-morpholino propane sulfate) buffer (pH 7.5) or 50 mM carbonate buffer (pH 9.5). Dissolve at a concentration of -1.0 mg / ml. With or without the denaturation process, the desired nucleic acid can be immobilized via contact to a substrate having an ester function activated by surface treatment. That is, using a micropipette or DNA chip making apparatus (DNA arrayer), a constant amount of the above-mentioned DNA solution is spotted on the surface of the substrate having the ester functional group activated by the surface treatment described above. Store in a humidifier for 30-60 minutes and wash with 2% SDS (sodium dodecyl sulfate) and distilled water respectively. Furthermore, if necessary, the substrate immobilized with nucleic acid is immersed in 0.3 M MaOH for 5 minutes at room temperature, or for 2 minutes in boiling water to denature the nucleic acid, washed with distilled water and absolute ethanol, and dried. By this treatment, the activated ester group does not react with the nucleic acid but is hydrolyzed back to the carboxylic acid moiety (ie negatively charged). Acid groups interfere with nonspecific electrostatic binding between the probe nucleic acid and the substrate. In general, the succinic anhydride blocking process required for polylysine slides can be omitted. These acid groups also lower background signals.

The amount of nucleic acid immobilized on the substrate can be measured using fluorescently-labeled nucleic acids. Labeled nucleic acids are covalently immobilized and residual fluorescence signals can be detected. In the present invention, the fixation method can detect high sensitivity of expression levels, mutations, polymorphisms, etc. in the gene, because this method provides high efficiency fixation. Furthermore, the immobilized nucleic acid does not fall under hybridization conditions (because the nucleic acid is covalently attached to the substrate).

Gene hybridization techniques are used to detect target nucleic acids covalently attached to a substrate (such as a DNA chip). Probe nucleic acids are labeled with a fluorophore or other signal group and denatured by alkali or heat. Labeled probe nucleic acids are added to the nucleic acid solution. 5 μl to 20 μl of hybridization solution was dropped onto the surface of the array and covered with cover glass, taking care not to allow air bubbles to form under the cover glass. It was stored in a humidifier at the appropriate temperature and time. The cover glass is removed, the slide is thoroughly washed, the moisture is removed and finally the fluorescence signal is detected by a fluorescence scanner (fluorescence reader).

Specific polyanion coated glass slides in the present invention can efficiently fix nucleic acids. The three-dimensional matrix formed on the surface of the substrate by covalently attached polyanions allows the increased number of nucleic acids to be immobilized.

Substrates and immobilized nucleic acids in the present invention provide for high sensitivity detection signals and low background signal detection of nucleic acids. Activated ester derivatives of polyanions that do not react with the target nucleic acid revert to polyanions when the activated ester is hydrolyzed, thereby inhibiting non-specific binding of the negatively charged probe nucleic acid to the substrate.

Using the present invention, an improved sensitivity of nucleic acid detection is obtained as a result of: (1) the target nucleic acid (including oligonucleotide) is attached to the substrate with high sensitivity, and (2) the blocking process can be omitted after fixation. (3) nonspecific binding between the target nucleic acid and the substrate is kept to a minimum, and (4) the three-dimensional effect of the polyanion treatment allows for high efficiency hybridization.

The invention is illustrated in detail by the following examples. However, the present invention is not limited to these detailed examples.

Example # 1

(1) Two kinds of phage DNA fragments (1.0 Kb and 0.3 Kb, respectively, the latter are part of the former) shown in SEQ ID NOs # 1 and # 2 of the sequence listing, respectively, were prepared by PCR as standard DNA.

PCR was performed with two combinations of the following primers using phage DNA as a template; In the Sequence Listing, the combination of Primer S and Primer A1000 shown in SEQ ID NOs: # 3 and # 4, respectively, and the combination of Primer A300 as Primer S and SEQ ID NO: 5. After PCR, each amplified fragment was purified using Qiaquick PCR Purification Kit 96 (Qiagen) according to the instructions. To elute DNA from the column, distilled water was used. DNA was concentrated by lyophilization.

Each DNA fragment was dissolved in 20 mM MOPS buffer (pH 7.5) and the array was made by GMS 417 Arrayer (Genetic MicroSystems) on commercial glass slides with amino groups on the surface. The glass slides used were poly-L-lysine coated POLY-PREP-SLIDES (abbreviated as Sigma: PLL coated slide glass) and MAS, amino alkyl silanes, coated slides (Matsunami Glass Ind .: MAS coated slides). Abbreviated glass), and these slides are widely used for the manufacture of DNA chips. Spots consisted of sizes and intervals of 150 μm and 375 μm, respectively. Five spots of the same DNA were arrayed.

After DNA spotting, each slide was preserved for 1 hour in an incubator controlled at 37 ° C. and 90% humidity and then UV crosslinked by 60 mJ / cm ² . The slides were washed once with 0.2% SDS and twice with distilled water, dehydrated by centrifugation at low speed (about 1,000 rpm) and then air dried. The previously mentioned slides were washed thoroughly with distilled water, boiled in a water bath for 3 minutes and exposed in ice cooled ethanol to denature DNA. Ethanol was removed by low speed centrifugation. The slides were then air dried and stored in the desiccator.

Some of the slides were treated with absolute succinic anhydride to block free amino acids on the substrate surface by succinization. The blocking solution was prepared by mixing digested 1.5 g succinic anhydride in 89.5 ml of N-methyl-2-pyrrolidone with 9 ml of 1M borate buffer (pH 8.0) and the slide was added to the blocking solution at room temperature at 20 Soaked for a minute. The treated slides were washed thoroughly with distilled water, boiled in a water bath for 3 minutes and exposed to ice cold ethanol to denature DNA. Ethanol was removed by low speed centrifugation. The slide was then air dried and stored in the desiccator.

(2) A commercially available amino alkyl silane glass slide (In situ PCR glass slide, PE Biosystems; abbreviated as AAS slide glass) was immersed in 2.5% glutaraldehyde solution for 1 hour, washed three times with distilled water, and then air dried. To obtain a slide coated with an aldehyde.

A 24 mer oligonucleotide (abbreviated to NH ₂ -F Oligo), shown as SEQ ID NO: 6 in the Sequence Listing, bound to an alkyl amino linker (PE Biosystems) of chain length 6 at the 5 'end was prepared on a DNA synthesizer . Thereafter, the final concentration was adjusted to 10 mM by dissolving in 20 mM MOPS buffer (pH 7.5). This synthesized DNA was spotted on a glutaraldehyde treated slide using a GMS 417 arrayer in the same manner as mentioned in Example Example # 1- (1). The slide glass was then washed with 0.2% SDS for 2 minutes and washed successively with distilled water. To reduce the Schiff-base formed by the covalent bond between the aldehyde on the substrate and the amino group on the DNA to convert it into a stable amine, the slides were 0.25% sodium borohydride (Wako Pure Chemical Industries) containing PBS-100% ethanol ( Soak for 5 minutes in volume ratio 3: 1) and air dry.

(3) covalently attaching polyanions to a solid support

Glass microscope slides were cleaned by ultrasonic for 10 minutes each in 2M nitric acid, 2M sodium hydroxide, pure water, and acetone. The washed slides were aminosilaned for 2 minutes in 4% 3-aminopropyltriethoxysilane dissolved in 95% ethanol, and then incubated at 100 ° C. for 10 minutes to aminoaminosilylate the surface. Polyacrylic acid was dissolved in dimethylformamide to a final concentration of 5 mg / ml, and 1-[(3-dimethylamino) propyl-3-ethylcarbodiamide hydrochloric acid (abbreviated to EDC) to a final concentration of 4.0 g / 100ml Added at. The aminosilaned slides were soaked in this solution overnight and rinsed with dimethylformamide, methanol, pure water, and acetone. Slides covered with polyacrylic acid were dried under vacuum.

(4) Activation of the Polyanion Coated Solid Support

The polyacrylic acid coated slides were converted to activated ester coated slides in the following manner. 1.8 g pentafluorophenol, 2.0 g EDC, and 13 mg dimethylaminopyridine were dissolved in 50 ml dimethylformamide. EXAMPLES Polyacrylic acid coated glass slides prepared in Example # 1- (3) were soaked in this solution for 5 hours, rinsed with dimethylformamide and methylene chloride, dried under vacuum and finally stored in a desiccator.

Example (example) # 2

(1) Two sizes of lambda phage DNA fragments (1.0 Kb and 0.3 Kb) were prepared by the same method as in Example # 1- (1). In this synthesis, DNA fragments having amino groups at the ends were amplified using an amine containing Primer S bound to an alkyl amino linker (PE Biosystems) of chain length 6 at the 5 'end.

After purification, each amine-containing DNA fragment was dissolved in 20 mM MOPS buffer (pH 7.5) to a final concentration of 0.5 mg / ml, then activated ester activated by the method of Example # 1- (4) Spotted three times on a glass slide coated with. As a control, the same array was made on PLL coated slide glass.

Activated ester coated glass slides, spotted with DNA, were preserved in 37 ° C. and 90% humidity controlled incubator (Tokyo Rikakikai: Eyela Model KC-1000) for 30 minutes and then incubated at room temperature in 0.2% SDS. Excess salt was removed and washed with distilled water. The slides were then denatured in 0.3 N NaOH for 5 minutes to denature the DNA, washed thoroughly with distilled water, dried by centrifugation at about 1,000 rpm and stored in a desiccator.

(2) hybridization

Nucleic acid arrays prepared on each glass slide were hybridized and observed with a nucleic acid probe labeled with a fluorescent dye. Each DNA array was pre-hybridized at 42 ° C. for 1 hour in a prehybrid buffer consisting of 50% formamide, 0.2% SDS, 5 × Denhardt 'solution, 0.1 mg / ml salmon sperm DNA, and 6 × SSC.

The 1.0 Kb lambda phage DNA fragment prepared in Example # 1- (1) was labeled with Cy5 ™ using the Label IT ™ Cy5 ™ Labeling Kit (Mirus), according to the instructions. This Cy5 ™ labeled nucleic acid probe was diluted with pre-hybridization buffer to final concentrations of 20, 2 and 0.2 ng / ml, heated at 95 ° C. for 2 minutes and cooled to room temperature. Insoluble material was removed by centrifugation. About 5 ul of hybridization solution was dropped onto the nucleic acid array. Overlaid with glass coverslips, with care not to form air bubbles, and incubated for 20 hours in a 42 ° C., humidifier. The cover glass was removed at room temperature in a 2 × SSC solution and washed twice with 65 × C for 5 minutes and three times for 55 minutes with 2 × SSC containing 0.2% SDS. It was then washed with 0.05 × SSC for 10 minutes at room temperature, dehydrated by centrifugation at about 1,000 rpm, and air-dried.

After hybridization, the nucleic acid array was read using a GMS 418 array scanner (GMS) at an emission wavelength of 635 nm and an excitation wavelength of 660 nm. The detected signal intensity was analyzed by image analysis software ImaGene (Biodiscovery). The results obtained are summarized in Table 1.

Table 1

Probe Fixed DNA Fragments DNA fixing substrate Concentration (mg / ml) Size (bp) Activated ester coated slide glass PLL Coated Slide Glass 0.2 1000 1484 889 300 988 856 2 1000 6640 2702 300 2635 1077 20 1000 27383 14310 300 7099 3824

As shown in Table 1, at all probe concentrations, the signal from each size of immobilized DNA on the activated ester coated glass slide was higher than that on the PLL coated glass slide. That is, in the detection of target PCR amplified nucleic acid fragments, DNA chips prepared using activated ester coated glass slides provide a higher signal than those prepared on conventional (PLL) coated glass slides.

Example # 3

(1) Construction of DNA Chips with Oligonucleotide-DNA Arrays

Two oligonucleotides of 24 nucleotides in length were prepared on a DNA maker; Oligonucleotides represented by SEQ ID NO: 6 in the Sequence Listing, one oligonucleotide is modified at the 5 'end with an alkyl amino linker (PE Biosystems) of chain length 6 (abbreviated to NH ₂ -F Oligo) and the other oligonucleotide is 5 Modified to contain hydroxy groups at the ends (abbreviated to OH-F Oligo). Two other oligonucleotides of 24 nucleotides in length were also synthesized; One oligonucleotide, with Cy3 ™ at the 5 'end, has a sequence complementary to the first oligonucleotide shown in SEQ ID NO: # 7 (Cy3-R Oligo); The other oligonucleotide has an unrelated sequence shown in SEQ ID NO: 14 with an alkyl amino linker (NH ₂ -N Oligo) at the 5 'end (NH ₂ -N Oligo).

OH-F Oligo, NH ₂ -F Oligo, and NH ₂ -N Oligo were dissolved in 20 mM MOPS buffer (pH 7.5) at final concentrations of 5, 50, and 500 μm, and then in Examples # 1- (4). Arrayed on glass slides coated with esters activated by the method. At this time, seven spots of each DNA were arrayed. After spotting, the slides were dried as described in Example # 2 and finally treated as oligonucleotide chips. As a control, the same oligonucleotide array was prepared on glutaraldehyde as described in Example # 1- (2).

(2) Hybridization of Oligonucleotide Chips

The oligonucleotide chip prepared on each slide was hybridized with Cy3-R Oligo prepared separately as a probe, and the resulting fluorescence signal was observed. Briefly, about 5 ul of hybridization solution consisting of 1 mM Cy3-R Oligo, 0.2% SDS, 5 × Denhardt 'solution, 0.1 mg / ml salmon sperm DNA, and 6 × SSC was added to the oligonucleotide array. This overlaps with the glass coverslips, taking care not to form air bubbles, and incubated for 20 hours in a 42 ° C., humidifier. The cover glass was removed at room temperature in 2x SSC solution and washed twice at room temperature in 2x SSC. The water was then removed by centrifugation at about 1,000 rpm and air-dried. After hybridization, the total fluorescence for the oligo-DNA chip was measured using a GMS 418 array scanner (GMS) with an emission wavelength of 635 nm and an excitation wavelength of 660 nm. The detected signal intensity was analyzed by image analysis software ImaGene. The results obtained are summarized in Table 2.

TABLE 2

Concentration of Oligonucleotide Spotting Solution (μM) Oligo-DNA Activated ester coated slide glass Glutaraldehyde-treated slide glass 5 OH-F Oligo 19.8 0.0 NH ₂ -F Oligo 5516.7 0.9 NH ₂ -N Oligo 0.0 0.0 50 OH-F Oligo 2067.0 0.0 NH ₂ -F Oligo 12104.1 0.0 NH ₂ -N Oligo 0.0 0.0 500 OH-F Oligo 4385.8 1113.5 NH ₂ -F Oligo 14828.7 201.9 NH ₂ -N Oligo 0.0 8.5

As shown in Table 2, on the slide glass coated with the activated ester, the spot of the oligonucleotide having an amino group at the 5 'end showed a stronger signal than without the amino group. This indicates that the activated ester groups on the slide glass react with the amino groups of the oligonucleotides. For all oligonucleotides, the signal from the spot on the glass slide coated with the activated ester was higher than that on the glutaraldehyde treated glass slide.

Example # 4

3-D effect of polyacrylic acid

Fluorescently labeled nucleic acids of different lengths are spotted and the hybridization signal from the nucleic acid array is calculated as a fixed nucleic acid length per signal. CDNA fragments of the human transferrin receptor (abbreviated to TFR) were prepared by the following method. MRNA extraction from human cell line K562 (Dainippon Pharmaceutical) and preparation of cDNA of TFR were performed according to conventional methods. Along with the cDNA as a template, a 4,738 bp PCR product was prepared using Primer S7 and Primer AS7, SEQ ID NOs: # 8 and # 9 of the Sequence Listing, TFR (GeneBank No. X01060) specific primers. Recombinant plasmid pT7TFR was obtained by ligation of the PCR fragment with pT7Blue vector (Novagen). Fluorescently labeled PCR fragments of different lengths were prepared by PCR using one primer and pT7TFR in pairs labeled Rhodamine X (PE Biosystems; abbreviated as ROX) at the 5 'end, respectively, as primers and templates; That is, PCR was performed using S7 (SEQ ID NO: 8) and AS4 (SEQ ID NO: 10), AS3 (SEQ ID NO: 11), AS2 (SEQ ID NO: 12), or AS1 (SEQ ID NO: 13) labeled with ROX. As a result, 1.0, 0.5, 0.2 and 0.1 kb fragments were produced, respectively.

After purification, each DNA fragment was dissolved in 20 mM MOPS buffer (pH 7.5) and aminated using Amino Label IT ™ reagent; That is, Amino Label IT ™ reagent was dissolved in dimethyl sulfoxide and added as a ratio of 1 / 5O (W / W) into the purified DNA solution and incubated for 1 hour at 37 ° C. Each aminated DNA was purified by ethanol precipitation method.

Amino Label IT reagents were prepared by substituting fluoro groups with amino groups, as described in patent application WO98 / 52961.

The aminated TRF fragments were dissolved in 20 mM MOPS buffer (pH7.5) at a final concentration of 0.5 mg / ml, and then each sequence was scored seven points by the method of Example # 1- (1). Spotted onto glass slides coated with activated ester activated as-(4). After spotting, it was washed and dried as described in Example # 3. As a control, the same array was prepared on MAS coated slide glass as Example # 1- (1) and treated with or without succinic anhydride blocking the free amino group after fixation.

DNA chips prepared on each slide glass were hybridized with Cy5 labeled DNA probes having different fluorescence wavelengths from ROX and the signal was observed. That is, the 1.0 Kb TFR fragment prepared above was labeled with Cy5 ™ using the Cy5 Label IT ™ Kit (Mirus) according to the manufacturer's recommendations.

About 5 ul of hybridization solution consisting of Cy5 labeled 20 ng / ml DNA probe, 0.2% SDS, 5x Denhardt 'solution, 0.1 mg / ml salmon sperm DNA, and 6x SSC was added to the DNA region immobilized on the chip. . This was overlaid with cover glass and incubated for 20 hours at 65 ° C. in a humidifier. The cover glass was removed at room temperature in 2 × SSC solution and washed twice at 65 ° C. for 5 minutes and at 55 ° C. for 30 minutes with 0.2% SDS containing 2 × SSC. It was then washed with 0.05 × SSC for 10 minutes at room temperature, dehydrated by centrifugation at about 1,000 rpm, and air-dried.

After hybridization, the ROX labeled fluorescence signal at the ends of the array DNA and the Cy5 labeled fluorescence signal at the hybridization probe on the DNA chip were measured using a GMS 418 array scanner (GMS). The obtained image was analyzed by image analysis software ImaGene and the fluorescence signal was converted into numerical data. The signal strength of Cy5 at each DNA size divided by the signal strength of ROX was considered as the signal strength per DNA. The results are summarized in Table 3. Because of the high background signal, it was impossible to measure the signal from the DNA chip prepared on the MAS coated slide glass without blocking the free amino groups.

TABLE 3

DNA size (bp) Signal strength per DNA (A) / (B) Slide glass coated with activated ester (A) MAS Coated Slide Glass (B) 1000 26.77 9.09 2.9 500 9.61 4.17 2.3 200 0.46 0.43 1.1 100 0.30 0.18 1.7

As shown in Table 3, at all DNA fragment lengths, the signal intensity of the spots per DNA on the slide glass coated with the activated ester was higher than that on the MAS coated slide glass. This result indicates that slide glass coated with activated esters allows hybridization with higher efficiency. Three-dimensional lattice made by covalently attached polyanions can allow greater access to the attached target nucleic acid.

Example # 5

Two kinds of 1.0 Kb and 0.3 Kb lambda phage DNA fragments having amino groups at the ends were prepared by the same method as in Example # 2- (1). A portion of DNA was aminated using Amino Label IT ™ reagent as described in Example # 4. Each DNA fragment was dissolved in 20 mM MOPS buffer (pH 7.5) at a final concentration of 0.5 mg / ml and spotted at seven different sites on slide glass coated with activated ester and MAS coated slide.

Nucleic acid arrays prepared on each slide glass were hybridized with fluorescent Cy3-labeled DNA probes and the resulting signal was analyzed. That is, the 1.0 Kb lambda phage fragment prepared above was labeled with Cy3 according to the manufacturer's recommendation using the Label IT ™ Cy3 Labeling Kit (Mirus). Using this Cy3 labeled DNA probe, hybridization and washing were performed as described in Example # 4.

After hybridization, DNA chip fluorescence (ie, signal intensity) was measured using a GMS 148 array scanner at an emission wavelength as 532 nm and an excitation wavelength as 570 nm. The obtained image was analyzed and the signal intensity values were converted to numerical data using image analysis software ImaGene. Relative fluorescence intensity was calculated with the fluorescence intensity of 100 on the spot on the MAS coated slide glass. The results obtained are summarized in Table 4.

Table 4

Target DNA Relative strength of signal Size (bp) Amino Modification by Label IT MAS Coated Slide Glass Slide glass coated with activated ester 1000 - 100 183 1000 + 100 276 300 - 100 945 300 + 100 2492

As shown in Table 4, at all spots of the DNA fragment, the signal intensity from the slide glass coated with the activated ester was higher than that from the MAS coated slide glass. In particular, the signal for the 300 bp short DNA fragment was much higher than that on the MAS slide.

When spotted on slide glass coated with activated esters, the amine modified target produced a stronger signal than without amine modification. This demonstrates that amino modification with Amino Label IT ™ provides increased adhesion of the target to glass slides coated with activated esters.

The preceding embodiments are considered to merely illustrate the principles of the invention. Moreover, since numerical modifications and changes are readily made to those skilled in the art, it is not intended to limit the present invention to the precise construction and manipulation shown or described. Accordingly, all suitable modifications and equivalents are within the scope of the present invention.

Claims (19)

Solid support having a surface coated with a polyanion covalently attached to detect sample nucleic acid: Nucleic acid fixing substrate comprising a. The substrate of claim 1, wherein the polyanion is comprised of polyacrylic acid. The substrate of claim 1, wherein the polyanion contains an activated ester. The substrate of claim 3, wherein the activated ester is selected from the group consisting of pentafluorophenol and N-hydroxysuccinimide. The substrate of claim 1, wherein the solid support is non-porous. The substrate of claim 5, wherein the solid support consists of glass. Solid supports having a covalently attached polyanion coated surface attachable to a nucleic acid: A nucleic acid detection substrate comprising a. 8. The substrate of claim 7, wherein the polyanion is comprised of polyacrylic acid. 8. The substrate of claim 7, wherein the polyanion comprises an ester activated. 8. The substrate of claim 7, wherein the solid support is non-porous. The substrate of claim 10, wherein the solid support consists of glass. a) coating the solid support with a covalently attachable polyanion containing an activated ester; b) contacting nucleic acid with said polyanion for detection: A method of immobilizing a nucleic acid on a substrate comprising a. 13. The method of claim 12, wherein the polyanion consists of polyacrylic acid. The method of claim 12, wherein the solid support is non-porous. The method of claim 14, wherein the solid support consists of glass. 13. The method of claim 12, further comprising reacting the nucleic acid with an activated ester. The method of claim 16 wherein the nucleic acid contains an amino group for reacting with the activated ester. 13. The method of claim 12 further comprising a blocking process. a) fixing a polyanion containing the activated ester of claim 4; b) reacting the polyanion with an immobilizable nucleic acid containing a functional group that reacts with an ester to immobilize the nucleic acid; c) hybridizing the immobilized nucleic acid with a sample nucleic acid complementary to the immobilized nucleic acid; d) detecting the hybridized sample nucleic acid: Comprising, a sample nucleic acid in a sample.