JPH08296A - Particle carrier for nucleic acid binding - Google Patents

Abstract

PURPOSE:To obtain a granular carrier for bonding nucleic acid, capable of readily and accurately separating and extracting a protein affinitive to DNA and useful in the field of genetic engineering, etc., by fixing a specified oligonucleotide to the surface of a granule composed mainly of an organopolymer. CONSTITUTION:This granular carrier for bonding nucleic acid is composed of a double stranded oligonucleotide and fixed to a granule composed mainly of an organopolymer, containing surface carboxylic groups and having 0.1 to 15mum average particle diameter. This double stranded oligonucleotide is composed of single stranded oligonucleotides A1 and A2 respectively containing a recognition site for a specified restriction enzyme and having 5 to 80 total number of base. Both the single stranded oligonucleotides are not complementary to each other at one end side and the other side of each single stranded oligonucleotide contains a recognition site for a specified enzyme at least in the end part. The double stranded oligonucleotide is fixed to the carrier through an amide bond between one or more amino groups of a base contained in each single stranded oligonucleotide A1, A2 and the carboxyl groups on the surface of the granule.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a nucleic acid binding particle carrier, and more particularly to binding, recovery or DNA binding of a nucleic acid fragment having a specific restriction enzyme cleavage end in the fields of genetic engineering, cloning, genomic nucleic acid analysis and the like. The present invention relates to a particle carrier effective for primary screening of restriction enzyme landmarks for separating and extracting a sex protein, particularly for decoding genomic nucleic acid of living plants.

[0002]

2. Description of the Related Art A water-insoluble solid-phase carrier on which a single-stranded oligonucleotide is immobilized as a probe and a specific restriction enzyme formed by binding a complementary nucleotide sequence to a probe and a complementary base sequence Attempts have been made for a long time to combine a target nucleic acid (or a fragment thereof) with a carrier having a recognition site to extract and separate a specific base sequence. A conventional technique for immobilizing an oligonucleotide on a water-insoluble solid-phase carrier is, for example, binding to a desired functional group present on the surface of the water-insoluble solid-phase carrier via a substance such as an arm or a spacer at the terminal portion of the oligonucleotide. Method, a method of binding to the functional group on the surface of the water-insoluble solid-phase carrier by chemically modifying the terminal base of the oligonucleotide without interposing a spacer, etc. And a method of binding to a functional group on the surface of the resin (Japanese Patent Laid-Open No. 61-130305, Japanese Patent Laid-Open No. 61-246201,
JP-A-63-27000, JP-A-1-171499
No. 3, JP-A-3-147800, Cell 5,301
(1975) etc.). However, in any case, this is a technique for selectively immobilizing the ends of single-stranded oligonucleotides on the surface of a solid-phase carrier and cannot be used for immobilization of double-stranded oligonucleotides. In addition, generally known chemical reactions or biochemical reactions such as biotin / avidin reaction can also be used for immobilizing single-stranded oligonucleotides on a solid phase surface, but double-stranded oligonucleotides Can not be used for immobilization of. Therefore, the carrier on which the single-stranded oligonucleotide is immobilized by these reactions is limited to the use for capturing and detecting a specific nucleic acid, probe nucleic acid or target nucleic acid using the hybridization method.

On the other hand, when the solid-phase carrier on which the single-stranded oligonucleotide is immobilized is used for the recovery, connection or binding with a DNA-binding protein of a nucleic acid fragment treated with a specific restriction enzyme, the single-stranded oligonucleotide is used. It is necessary to anneal the complementary strand to form a recognition site for the specific restriction enzyme. Therefore, for example, the immobilized single-stranded oligonucleotide is once annealed under the condition of excess complementary strand to form the immobilized double-stranded oligonucleotide, and further a specific restriction enzyme recognition site is formed at the end thereof. After that, a carrier on which a double-stranded oligonucleotide having a specific restriction enzyme recognition site at the end, which is prepared by removing an extra complementary strand is immobilized, is separated from the DNA fragment cleaved by the restriction enzyme, The method used for extraction may be mentioned (JP-A-3-147800). However, in order to form an immobilized double-stranded oligonucleotide in the above method, an excessive complementary strand and a long time are required, and the binding of the immobilized double-stranded oligonucleotide depends on the base of several tens of bases. Since there is only a hydrogen bond, there is a drawback in that a restriction enzyme recognition site cannot be satisfactorily formed at the end due to poor annealing efficiency. further,
The resulting immobilized double-stranded oligonucleotide has poor storage stability and must be stored under the conditions at the time of double-strand formation, which cannot be said to be efficient in use and storage.

[0004]

The object of the present invention is to specify that a nucleic acid fragment having a specific restriction enzyme cleavage end can be separated and extracted or a DNA-binding protein can be separated and extracted easily and accurately. Another object of the present invention is to provide a particle carrier on which a double-stranded oligonucleotide having a recognition site for the restriction enzyme is immobilized.

[0005]

[Means for Solving the Problems] The above-mentioned problem is a particle carrier for nucleic acid binding in which a double-stranded oligonucleotide having a recognition site for a specific restriction enzyme is immobilized on the surface of a particle containing an organic polymer as a main component. (1) The particles have a carboxyl group on the surface and have an average particle diameter of 0.1 to 0.1.
15 μm, (2) the double-stranded oligonucleotide has a number of bases of 5 to 80, and one end thereof is composed of single-stranded oligonucleotides A1 and A2 having a number of bases of 1 to 30 that are not complementary to each other. And the other end is composed of a double-stranded oligonucleotide B having a recognition site for a specific enzyme at least at the end, and (3) the double-stranded oligonucleotide and the particle are the double-stranded oligonucleotide. For binding to a nucleic acid, characterized in that it is immobilized by an amide bond between an amino group present in each of the bases of the single-stranded oligonucleotides A1 and A2 constituting the above and an carboxyl group present on the surface of the particle. Particle carrier.

The present invention will be described in detail below. This will make the purpose, structure and effect of the present invention clearer. The particles used in the present invention are water-insoluble particles containing an organic polymer as a main component. Examples of the organic polymer serving as the main component here include styrene, chlorostyrene, chloromethylstyrene, α-methylstyrene, divinylbenzene, sodium styrenesulfonate, (meth) acrylic acid, methyl (meth) acrylate, (meth ) Ethyl acrylate, n-butyl (meth) acrylate, 2-hydroxyethyl (meth) acrylate, polyoxyethylene (meth) acrylate, glycidyl (meth) acrylate,
Ethylene glycol-di- (meth) acrylic acid ester, (meth) acrylic acid tribromophenyl, tribromopropyl acrylate, (meth) acrylonitrile,
Aromatic vinyl compounds such as (meth) acrolein, (meth) acrylamide, methylenebis (meth) acrylamide, butadiene, isoprene, vinyl acetate, vinylpyridine, N-vinylpyrrolidone, vinyl chloride, vinyl bromide, α, β-unsaturated Water obtained by polymerizing at least one vinyl monomer consisting of carboxylic acid esters or amides, α, β-unsaturated nitrile compounds, halogenated vinyl compounds, conjugated diene compounds, and lower fatty acid vinyl esters. Insoluble organic polymers can be mentioned. Examples of other organic polymers include crosslinked polysaccharides such as agarose, dextran, cellulose and carboxymethylcellulose, and crosslinked proteins such as methylated albumin, gelatin, collagen and casein.

These particles can be produced by emulsion polymerization, suspension polymerization, solution precipitation polymerization and the like. Further, the particles can be obtained by dispersing an organic polymer in a non-solvent and crosslinking it, or volatilizing the solvent, but the method for producing the particles is not limited thereto.

If necessary, these particles may contain or coat an organic dye, a pigment or a fluorescent dye on the inside or the surface of the particles, or a filler made of an inorganic substance,
For example, a particle having magnetism or superparamagnetism such as a metal such as magnetite, hematite, nickel or a metal compound may be impregnated to composite the particles.

The surface of the particles obtained as described above is
It is preferably non-porous. Here, that the surface of the particle is “non-porous” means that the surface of the particle does not have pores having a major axis of 0.01 μm or more. If the surface of particles is not non-porous, that is, 0.01 μm in major axis
The presence of the above pores on the surface of the particle may cause problems of sensitivity and accuracy due to the fact that the nucleic acid fragment having the restriction enzyme cleavage end used in the method for detecting nucleic acid is trapped inside the particle. . Although the surface of the particle is preferably non-porous as described above, closed cells may exist inside the particle.

The average particle size of the particles is 0.1 to 15 μm, preferably 0.3 to 5 μm. If the average particle size is less than 0.1 μm, it becomes difficult to collect particles by a simple operation such as centrifugation. On the other hand, if it exceeds 15 μm, the surface area of the particles per unit volume becomes small, and the particles do not disperse uniformly in the separation and extraction of nucleic acid fragments, so that high separation rate, extraction rate, etc. may not be achieved.

Further, the particles used in the present invention are
As described below, single-stranded oligonucleotides A1 and A
In order to form an amide bond with the amino group of the base in 2, it is necessary to have a carboxyl group involved in the formation of the amide bond on its surface. Therefore, when the particles do not have a carboxyl group on the surface, it is necessary to introduce a carboxyl group on the surface in advance. It is preferable that at least one carboxyl group is present on average per 1 nm ² of the particle surface, more preferably 3 or more, and further preferably 5 or more. Examples of the particles made of the organic polymer having a carboxyl group on the surface thereof include, for example, Imtex SSM-60, SSM-58, SSM-57,
G0101, G0303, G0302, G0301, G
0201, G0202, G0501, L0101, L0
102, DRB-F1, DRB-F2, DRB-F3 and the like, which are commercially available under trade names (Nippon Synthetic Rubber Co., Ltd.). The particles having a carboxyl group on the surface can be used without any trouble in the enzymatic reaction and have heat resistance up to 100 ° C. In order to introduce a carboxyl group to the surface of particles having no carboxyl group, various conventionally known methods can be used.

Next, the double-stranded oligonucleotide used in the present invention (hereinafter referred to as "specific double-stranded oligonucleotide") will be described. The specific double-stranded oligonucleotide has 1 to 30 bases whose one ends are not complementary to each other.
Single-stranded oligonucleotides A1 and A2, and the other end of the double-stranded oligonucleotide B having a recognition site for a specific restriction enzyme at least at the end, and the total number of bases is 5 to 80 bases. (Per single strand), preferably 10 to 50 bases (per single strand). Here, if the total number of bases is less than 5 bases, it becomes difficult to form a recognition site for a restriction enzyme,
Alternatively, the enzymatic reaction becomes inefficient, and when the number of bases exceeds 80, the synthesis efficiency of the specific double-stranded oligonucleotide decreases,
Moreover, the separation of nucleic acid fragments having specific restriction enzyme cleavage ends becomes inefficient.

The double-stranded oligonucleotide B has the same or different kinds of specific restriction enzymes (eg EcoR I, Not I, Sca) at its ends and, if necessary, in its complementary strand.
I or the like) having one or more recognition sites, and the recognition site has a function of binding to a nucleic acid fragment having a specific restriction enzyme-cleaving end by the action of ligase or a function of being cleaved by a specific restriction enzyme Is. Here, the recognition site of the restriction enzyme at the end of the double-stranded oligonucleotide B may be a sticky end type having a different number of bases or a blunt end type having a uniform number of bases. When the double-stranded oligonucleotide B has two or more recognition sites for a specific restriction enzyme, the double-stranded oligonucleotide B binds to a nucleic acid fragment having a specific restriction enzyme cleavage end, It becomes possible to cut out with a specific restriction enzyme at another site and directly use for cloning and the like.

The single-stranded oligonucleotides A1 and A2 have 1 to 30 bases, preferably 5 to 15 bases. Here, the number of bases of the single-stranded oligonucleotides A1 and A2 is preferably close to the number of bases in consideration of the efficiency of the immobilization reaction with particles described below and the symmetry of the oligonucleotide, but the number of bases is not always the same. It doesn't have to be. Further, the single-stranded oligonucleotides A1 and A2 are not complementary to each other and always maintain the single-stranded state even after annealing. Therefore, the single-stranded oligonucleotides A1 and A2 contain one type of single base, for example, deoxyadenylic acid (dA), deoxytidylic acid (dC) or deoxyguanylic acid (dG), in order to avoid mutual complementarity. It is preferably composed of

The specific double-stranded oligonucleotide used in the present invention comprises a base sequence which can be a single-stranded oligonucleotide A1 or A2 having 1 to 30 bases, and a double-stranded oligonucleotide which forms a recognition site for a specific restriction enzyme after annealing. It can be easily formed by annealing a single-stranded oligonucleotide having a total number of bases of 5 to 80 having a base sequence capable of becoming the double-stranded oligonucleotide B under normal annealing conditions. The specific double-stranded oligonucleotide after annealing has an amino group present in the base sequence of the double-stranded oligonucleotide B bound by a hydrogen bond by annealing, and the amino group present in the bases of the single-stranded oligonucleotides A1 and A2. Only the single-stranded oligonucleotides A1 and A2 are specifically immobilized on the particle surface because only the single-stranded oligonucleotides are in a free state. Therefore, if the specific double-stranded oligonucleotide is used, the specific sites of the single-stranded oligonucleotides A1 and A2 can be specifically and highly efficiently immobilized on the particle surface without any special chemical modification. it can. The specific double-stranded oligonucleotide obtained by the above-mentioned annealing can be purified by passing through a commercially available hydroxyapatite column to remove the unreacted specific single-stranded oligonucleotide.

In order to immobilize the specific double-stranded oligonucleotide on the particles, the carboxyl group on the particle surface and the single-stranded oligonucleotides A1 and A2 are taken into consideration in consideration of the ease of operation, the high immobilization efficiency and the accuracy. It is necessary to fix the amino group of the base inside with an amide bond.

In the reaction for immobilization, for example, after a particle having a carboxyl group on the surface and the specific double-stranded oligonucleotide are charged in a reaction vessel of an appropriate size, a dehydration condensation agent is added. By. Here, as the dehydration condensing agent, for example, 1-ethyl-3- (N, N'-
Dimethylamino) propylcarbodiimide, N-ethyl-5-phenylisoxazolium-3'-sulfonate, N-ethoxycarbonyl-2-ethoxy-1,2-
Water-soluble dehydration condensation agents such as dihydroquinoline are preferred, but oil-soluble dehydration condensation agents can also be used. These dehydration condensing agents are generally used in an amount of 1 to 20 mol, preferably 2 to 10 mol, based on 1 gram equivalent of the carboxyl group on the surface of the particles used.
The amount of the particles used is usually 0.5 to 500 g, preferably 5 to 5 g per 1 mmol of the specific double-stranded oligonucleotide.
It is 50 g. The above reaction may be carried out usually in a water-soluble medium having a pH of about 3 to 11, for example, in water at 4 to 70 ° C. for 5 minutes to overnight. When the carrier of the present invention obtained as described above is used for binding reaction with a nucleic acid fragment having a recognition site for a specific restriction enzyme of interest, the particle carrier is usually used in a state of being dispersed in an aqueous medium. The concentration of the dispersion liquid at this time is usually 0.05 to 25% by weight, and more preferably 0.1 to 15% by weight. Particle concentration is 0.0
If the amount is less than 5% by weight, the nucleic acid fragment having a specific restriction enzyme recognition site cannot be efficiently bound, and if the amount exceeds 25% by weight, the restriction enzyme adheres to the particle surface and does not work effectively.

[0018]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. Example (1) Synthesis and purification of single-stranded oligonucleotide Single-stranded oligonucleotide X having a base sequence shown below by β-cyanoethylphosphoamide method using a DNA synthesizer (Applied Biosystems model ABI381A type) (+) And X (-) were synthesized. In addition,
The following base sequence is a specific double-stranded oligonucleotide obtained by annealing: a double-stranded oligonucleotide B having a NotI recognition site at one end and a single-stranded oligonucleotide A1 having 10 bases; A2
It was designed to consist of and. Sequence X (+); 5'GGC CGC CTT TAG TGA GGG TTA ATG TCG ACA CCC CCC CCCC 3'Sequence X (-); 3'CG GAA ATC ACT CCC AAT TAC AGC TGT CCC CCC CCCC 5'where (+ ) And (-) represent + and-strands of DNA, respectively, and 5'and 3'represent 5'and 3'ends, respectively. Synthesized single-stranded oligonucleotides X (+) and X (-) were each added to concentrated ammonium hydroxide 10 ml.
Then, it was cut out from the column of the above apparatus, left at 55 ° C. overnight, and dried in vacuum. It was then purified by high performance liquid chromatography using a C18 reverse phase column with a 15 wt% acetonitrile solution as the eluent. Purified single-stranded oligonucleotides X (+) and X (-) were electrophoresed in an 18% by weight polyacrylamide gel, and the single-stranded oligonucleotides X (+) and X (-) were purified. It was confirmed. The single-stranded oligonucleotide X (+) and X (-) were eluted from the eluate, and the single-stranded oligonucleotide X was precipitated by ethanol.
(+) And X (-) were further concentrated and purified. Next, concentrated and purified single-stranded oligonucleotide X (+)
Each of X and X (−) was dissolved in 500 μl of sterilized water, and then the absorbance at 260 nm was measured and quantified. As a result, about 5 mg of single-stranded oligonucleotides X (+) and X (−) were obtained.

(2) Terminal phosphorylation treatment of single-stranded oligonucleotide 88 μl (3 mg) of single-stranded oligonucleotide X (+) synthesized in (1)
(-) Was taken in an amount of 91 µl (3 mg), and each was mixed with 500 units of polynucleotide kinase (manufactured by Toyobo Co., Ltd.) and 2 nmol of adenosine-5 'triphosphate, and a 10-fold protrading endokinase buffer solution (manufactured by Toyobo Co., Ltd.)
After adding 50 μl, the volume was adjusted to 500 μl with sterile distilled water. These were reacted in a water bath at 37 ° C. for 2 hours, left at 95 ° C. for 3 minutes, and precipitated and purified using ethanol. Subsequently, 1 mg each of the purified single-stranded oligonucleotides X (+) and X (−) subjected to terminal phosphorylation treatment was treated with TE buffer (10 mM Tris hydrochloride buffer (p
H8) and 1 mM ethylenediaminetetraacetic acid buffer solution) (500 μl).

(3) Formation of specific double-stranded oligonucleotide The terminal phosphorylated single-stranded oligonucleotides X (+) and X (-) obtained in (2) are each 6 nmo.
1 l, put in a 2 ml Eppendorf tube, 1
0-fold annealing solution (100 mM Tris hydrochloride buffer (pH 8), 60 mM magnesium chloride, 60 mM β-
10 μl of a buffer solution consisting of mercaptoethanol and 500 mM sodium chloride) was added to 100 μl with sterile distilled water.
Adjusted to ml. 1 in a 80 ° C water bath
The mixture was heated for 0 minutes and then left to stand at room temperature (required time 4 hours). By this reaction, two single-stranded oligonucleotides A1 and A2 each having 10 bases at one end and the restriction enzyme N at the other end at the other end
Double-stranded oligonucleotide B having a recognition site for otI
A specific double-stranded oligonucleotide composed of and was obtained. The obtained specific double-stranded oligonucleotide was applied to a hydroxyapatite column (Biogel HPHT column,
Purification was performed using BioRad Co., Ltd.) with 0.6 mol phosphate buffer as an eluent. When the specific double-stranded oligonucleotide after purification was recovered, concentrated with butanol and desalted, the yield was 91% before purification.

(4) Immobilization of specific double-stranded oligonucleotide on particles Average particle size 1.0 μm, standard deviation of average particle size 5
%, And polystyrene particles Immunotex L01 having an average of 5 carboxyl groups per particle surface area 1 nm ²
01 (manufactured by Nippon Synthetic Rubber Co., Ltd.) 10 (W / V)% 0.
1000 μl of 1 mN hydrochloric acid suspension, 4 nmol of the specific double-stranded oligonucleotide obtained in (3) and 0.5 (W / V)% of 1-ethyl-3- (N, N′-dimethylamino) propylcarbodiimide 0.1mN hydrochloric acid solution 50
0 μl was placed in an Eppendorf centrifuge tube and mixed overnight in a thermostat set at 10 ° C. to mix the amino groups and particles in the dc base sequence of the single-stranded oligonucleotides A1 and A2 in the specific double-stranded oligonucleotide. An amide bond was formed with the carboxyl group on the surface. Then 10,000
After centrifugation at rpm for 2 minutes, particles having the specific double-stranded oligonucleotide immobilized thereon were collected as a precipitate. Next, binding buffer (10 mM Tris hydrochloride buffer, pH 8, 5
1 ml of a buffer solution consisting of 00 mM sodium chloride, 0.1 (W / v)% sodium dodecylsulfate and 1 mM ethylenediaminetetraacetic acid) was added to the oligonucleotide-immobilized particles and redispersed, and then 3 times at 10,000 rpm.
After centrifugation for minutes, the particles having the specific double-stranded oligonucleotide immobilized thereon were collected. This operation was repeated 3 times, and finally the particles having the specific double-stranded oligonucleotide immobilized thereon were dispersed in 1 ml of TE buffer (solid content 10 (W / V)%) (hereinafter referred to as “dispersion liquid a”). .).

(5) Preparation of Nucleic Acid Fragment Having NotI Terminus Plasmid BluescriptII SK (+) (pBSII Toyobo Co., Ltd.) 75 μg (about 38 pM), NotI (10 units / μl) 200 units, 10 times HB buffer 40 μm
l (both manufactured by Takara) and 294 μl of sterile distilled water
Were mixed and reacted at 37 ° C. for 3 hours. After the reaction,
An equal volume of P. C. I (phenol: chloroform: isoamino alcohol = 25: 24: 1 (volume ratio)) solution was added and mixed well, 15,000 rpm
After centrifuging at 10 minutes, the upper aqueous layer was collected.
100 μl of 8M ammonium acetate in the recovered water layer
Was added, mixed well, and then centrifuged at 15,000 rpm for 5 minutes, and the obtained NotI-digested plasmid Bluescript II SK (+) was added to 20 μl of TE buffer.
Dispersed.

(6) Labeling with ³² P NotI digested plasmid Bluesc prepared in (5)
About 2 μl of alkaline phosphatase (manufactured by Takara) in TE buffer of riptII SK (+) (40 units in terms of enzyme activity), 10 times buffer solution for alkaline phosphatase 2
μl and 16 μl of sterilized distilled water were added and reacted at 37 ° C. for 2 hours, and then heated at 65 ° C. for 30 minutes. 50 μl of ethanol was added to this solution, the mixture was allowed to stand at −80 ° C. for 10 minutes and then centrifuged at 15,000 rpm for 5 minutes, and the obtained precipitate was dissolved in 20 μl of TE buffer. To this solution, 1 μl of T4 polynucleotide kinase (manufactured by Boehringer Heim) (8 units in terms of enzyme activity), 5 μl of γ- ³² P adenosine triphosphate (50 μC in radiation dose)
i) 5 µl of 5'-phosphorylation buffer having a 10-fold concentration and 19 µl of sterilized water were added and mixed, and the mixture was incubated at 37 ° C for 30 minutes. Next, T4 polynucleotide kinase was extracted from the reaction solution with phenol (“Molecular Clonin”).
g ", Cold Spring Harbor Laboratories, 1982, 458), and then the reaction solution was swollen with TE buffer Sephadex G-50 (Pharmacia) column (1
ml) was used for gel filtration. The 10-fold concentrated 5'-phosphorylation buffer solution used above had the following composition. Composition: 0.5M Tris-hydrochloride buffer, pH 7.6 0.1M magnesium chloride 50mM dithiothreitol 1mM spermidine 1mM ethylenediaminetetraacetic acid 100μl of the eluate by gel filtration is dispensed in order, and the radiation dose of each fraction is Cherenkov. The flow-through fraction was determined by the counting method and collected. As a result, No. of the plasmid BluescriptII SK (+) labeled with ³² P
20 μl of a TE buffer solution of the tI digest (hereinafter referred to as “TE buffer solution I”) was obtained.

(7) Digestion with ScaI ScaI (100 / μ was added to the TE buffer I obtained in (6).
1, manufactured by Takara Co., Ltd.) and 10 times HB buffer (40 μl) were added to 400 μl of sterilized water, and the reaction was carried out at 37 ° C. for 3 hours. After completion of the reaction, an equal amount of P. C. Solution I was added and mixed well, then centrifuged at 15,000 rpm for 10 minutes to recover the upper aqueous layer. 8 in the collected water layer
M ammonia acetate 100 μl and ethanol 8
After adding 00 μl and mixing well, 15,000 rp
Plasmid digested with ScaI after centrifugation for 5 minutes at m.
The fragment of BluescriptII SK (+) was dissolved in 100 μl of TE buffer. Cherenkov-in the same manner as (6)
When the radiation dose was measured by the counting method, 1 μl
The solution had a radiation dose of 40,000 rpm per count. In addition, the plasmid BluescriptII SK
The concentration of the (+) fragment is 0.30 pmol / μl, Not
The I-terminal site was 0.6 pmole / μl.

(8) Plasmid Blue digested with NotI
Evaluation of capture and separation of scriptII SK (+) fragments 10 μl each of dispersion a obtained in (4) A-1 to A-
Placed in 4 centrifuge tubes up to 4. Next, plasmid Bluesc digested with NotI and ScaI prepared in (7)
The riptII SK (+) fragments were 1, 2, 4, and 6p, respectively.
A-1 to A-4 were added to four centrifuge tubes so as to have a mol. Each of these four centrifuge tubes was supplied with a 10-fold ligation buffer (660 mM Tris-HCl buffer (pH
8), 66 mM magnesium chloride and 100 mM D
Buffer consisting of TT) 5 μl, 50 mM 5′-adenosine triphosphate 5 μl, 50% polyethylene glycol 5
μl and 5 μl of 3M sodium chloride were added. Then, 5 μl of T4 DNA ligase (350 units / μl, manufactured by Takara) was added to each centrifuge tube, and sterile distilled water was added to adjust the volume to 50 μl, and the reaction was allowed to proceed at 17 ° C. for 12 hours. After the reaction was completed, the Cherenkov counts of the four centrifuge tubes were measured, and each was used as the total count of each centrifuge tube. In addition, after the above reaction is completed, MB is added to four centrifuge tubes.
Buffer (50 mM Tris-HCl buffer (pH 7.5),
A buffer consisting of 0.01% gelatin, 100 mM sodium chloride and 10 mM magnesium chloride) 1000 μ
The reaction was stopped by adding 1 each. Then, the four centrifuge tubes were centrifuged at 10,000 rpm for 3 minutes, the supernatant was removed, and then dispersed in 50 μl of TE buffer to measure the Cherenkov count, and each was designated as count 2 of each centrifuge tube. Next, 40 units of NotI (100 units / μl) and 10 μl of 10 times HB buffer (Takara) were added to the four centrifuge tubes, adjusted to 100 μl with sterile distilled water, and reacted at 37 ° C. for 3 hours. Then 10,000 r
Centrifuge and wash for 5 minutes at pm and remove the precipitate with TE buffer / 0.2.
After centrifugal washing with a 5 M sodium chloride solution three times, the solution was redispersed in 50 μl with TE buffer, and the Cherenkov count was measured. The capture rate and recovery rate of NotI-digested plasmid Bluescript II SK (+) were calculated by the following formulas and shown in Table 1.

[0026]

[Equation 1]

[0027]

[Equation 2]

[0028]

[Table 1]

[0029]

INDUSTRIAL APPLICABILITY According to the present invention, a specific restriction enzyme capable of easily and accurately ligating and recovering a nucleic acid fragment having a specific restriction enzyme cleavage end or separating and extracting a DNA-binding protein is provided. A particle carrier having a double-stranded oligonucleotide having a recognition site immobilized thereon can be obtained.

Claims (1)

[Claims] 1. A surface of particles having an organic polymer as a main component,
A nucleic acid binding particle carrier having a double-stranded oligonucleotide having a recognition site for a specific restriction enzyme immobilized thereon,
(1) The particles have a carboxyl group on the surface,
And the average particle size is 0.1 to 15 μm, (2) the double-stranded oligonucleotide has a total number of bases of 5 to 80, and one end has a number of bases of 1 to 30 that are not complementary to each other.
Which is composed of single-stranded oligonucleotides A1 and A2, and the other end of which is composed of a double-stranded oligonucleotide B having a recognition site for a specific enzyme at least at its end, (3) The double-stranded oligonucleotide The particle is immobilized by an amide bond between an amino group present in at least one of the bases of the single-stranded oligonucleotides A1 and A2 constituting the double-stranded oligonucleotide and a carboxyl group present on the surface of the particle. ing,
A particle carrier for nucleic acid binding, comprising: