JP2002253238A - Method of isolating and collecting nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of isolating and collecting nucleic acids that can simply isolate and collect a plurality of nucleic acids. SOLUTION: The method of isolating and collecting nucleic acids comprises the step in which a sample of nucleic acid solution is brought into contact with the substrate and the nucleic acid-immobilized substrate having two or more kinds of single-strand nucleic acids separately immobilized on the substrate whereby the single-strand nucleic acid is hybridized with the complementary single-strand nucleic acid and the step in which the hybridized single-strand nucleic acids are separated every site to which one single nucleic acid is immobilized and collected, the substrate to which the nucleic acids are immobilized are kept in one integral body without division.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for separating and recovering a nucleic acid used for determining the nucleotide sequence of a nucleic acid or analyzing a DNA.

[0002]

2. Description of the Related Art Conventionally, a subtraction method, a differential display method and the like have been used as a method for separating and recovering nucleic acids. Further, in Japanese Patent Application Laid-Open No. Hei 4-325092, a single-stranded nucleic acid having a different base sequence is immobilized on each of a plurality of types of separable supports, and a sample nucleic acid mixed solution is brought into contact with each of the immobilized nucleic acids. And a method for simultaneously recovering a plurality of types of nucleic acids by hybridizing with a single-stranded nucleic acid in the mixture having a complementary base sequence.

However, in the method described in JP-A-4-325092, there is a problem that the type of the support to be used is greatly limited because the nucleic acid is separated by separating the support. In addition, since it is necessary to separate a support in order to separate nucleic acids, there is also a problem that it is difficult to simultaneously separate many types of nucleic acids. Furthermore, in order to separate many types of nucleic acids, a large amount of a sample nucleic acid mixed solution is required, and there is a problem that it takes time and effort.

[0004]

SUMMARY OF THE INVENTION The present invention has been made in view of the above, and an object of the present invention is to provide a method for separating and recovering a plurality of nucleic acids in a simple manner.

[0005]

Means for Solving the Problems In order to solve the above problems, the present inventors carried out hybridization using a substrate on which a plurality of types of nucleic acids were immobilized, and separated each of the hybridized nucleic acids. The present inventors have found that the nucleic acid can be easily separated and recovered by directly recovering the nucleic acid from the substrate as it is, and the present invention has been completed.

That is, the present invention is as follows.

(1) A sample nucleic acid solution is brought into contact with a nucleic acid-immobilized substrate having a substrate and two or more single-stranded nucleic acids separated and immobilized on the substrate, respectively. A step of hybridizing the single-stranded nucleic acid complementary to the single-stranded nucleic acid, and, while keeping the nucleic acid-immobilized base material integral, the hybridized single-stranded nucleic acid is removed at each location of the fixed single-stranded nucleic acid. And recovering the nucleic acid.

(2) The method for separating and recovering nucleic acids according to (1), wherein the nucleic acid-immobilized substrate is a substrate carrying a compound having a carbodiimide group.

(3) The method for separating and recovering nucleic acids according to (1) or (2), wherein the nucleic acid-immobilized substrate is a DNA microarray.

(4) The base material is flat (1)-
The method for separating and recovering nucleic acid according to any one of (3).

[0011]

Embodiments of the present invention will be described below.

<Nucleic acid-immobilized substrate> The nucleic acid-immobilized substrate used in the nucleic acid separation / recovery method of the present invention comprises a substrate and two or more types having a known sequence, usually immobilized on the substrate. And a single-stranded nucleic acid.

The substrate used in the present invention plays a role in immobilizing nucleic acids, and is basically solvent-insoluble and in a temperature range at or around room temperature (usually 0 to 100 ° C.). ) Is not particularly limited as long as it is a solid or gel. Incidentally, that the substrate is solvent-insoluble, single-stranded nucleic acid is immobilized on the substrate as described below,
Thereafter, it is substantially insoluble in various solvents such as an aqueous solvent and an organic solvent used in each step when used in the nucleic acid separation and recovery method of the present invention.

Specific examples of the material of the substrate include plastics, inorganic polymers, metals, natural polymers, and ceramics.

Specific examples of the plastic include polyethylene, polystyrene, polycarbonate, polypropylene, polyamide, phenolic resin, epoxy resin,
Polycarbodiimide resin, polyvinyl chloride, polyvinylidene fluoride, polyfluoroethylene, polyimide, acrylic resin and the like can be mentioned. Further, specific examples of the inorganic polymer include glass, quartz, carbon, silica gel, and graphite. Specific examples of the metal include gold, platinum, silver, copper, iron, aluminum, a magnet, and a paramagnet. Examples of natural polymers include cellulose, chitin, chitosan, alginic acid and derivatives thereof. Specific examples of the ceramic include apatite, alumina, silica, silicon carbide, silicon nitride, and boron carbide.

Examples of the shape of the substrate include a film, a flat plate, and a fiber, and the size is not particularly limited.

The single-stranded nucleic acid immobilized on the nucleic acid immobilization substrate used in the present invention includes natural or synthetic DNA.
Single-stranded ones (including oligonucleotides) and RNAs (including oligonucleotides) are not particularly limited. The single-stranded nucleic acid immobilized on the nucleic acid immobilization substrate of the present invention may have a known sequence or may have an unknown sequence. Although the length of the single-stranded nucleic acid is not particularly limited, it is usually 6 to several thousand bases.

Two or more single-stranded nucleic acids are immobilized while being separated from each other. The single-stranded nucleic acid immobilized in one separated place may be composed of one kind of single-stranded nucleic acid, or may be composed of a mixture of single-stranded nucleic acids.
Therefore, "two or more" means that the base sequences of the single-stranded nucleic acids to be immobilized are different and / or the composition of the mixture constituting the single-stranded nucleic acids to be immobilized is different. . The arrangement and the like of each nucleic acid can be appropriately selected depending on the use form, application, and the like of the obtained nucleic acid-immobilized substrate.

As a method for immobilizing a single-stranded nucleic acid on the substrate, a known method can be used.
(I) a method in which a single-stranded nucleic acid is directly immobilized on a substrate by physical adsorption, and (ii) an amino acid contained in the nucleic acid when the substrate has a group capable of covalently bonding to an amino group or an imino group in advance. A method of immobilizing a nucleic acid on a substrate by covalent bonding of a group or an imino group with each group on the substrate, (iii) immobilizing the nucleic acid on the substrate via a compound capable of binding to both the substrate and the nucleic acid And the like.

The method (i) can be carried out, for example, by spotting (dropping) a solution containing a single-stranded nucleic acid on the above-mentioned substrate using a spotter or a micropipette and drying the solution.

In the method (ii), examples of the group capable of covalently bonding to an amino group or an imino group include functional groups such as a hydroxyl group, an amino group, a carboxyl group, an isocyanate group, an isothiocyanate group, and a carbodiimide group. . As the substrate having such a functional group, a substrate made of a material having the above functional group may be used in advance, or a substrate having the above functional group introduced into the substrate may be used. Also,
As a method for introducing a single-stranded nucleic acid into such a substrate, a conventionally known method can be used, and it is appropriately selected according to the functional group of the substrate.

In the method (iii), the compound capable of binding to both the substrate and the nucleic acid is, for example, a compound having a carbodiimide group or a group capable of introducing an alkyl group into another compound by a substitution reaction or an addition reaction (hereinafter referred to as a compound). , Which may be referred to as an “alkylating group”). Particularly, a compound having a carbodiimide group is preferable. According to this aspect, since the oligomer can be covalently bonded, hybridization conditions can be selected from a wide range, and thereby the separation accuracy by hybridization can be improved. For example, a condition under which a mixture of nucleic acids having one base difference can be separated can be used.

A compound having a carbodiimide group (hereinafter, referred to as a carbodiimide group)
Examples of the “carbodiimide compound” are, for example, the method disclosed in JP-A-51-61599 and the method of LM Alberino et al. (J. Appl. Po.
lym. Sci., 21, 190 (1990)) or JP-A-2-29.
Monocarbodiimide synthesized by a commonly used method for producing carbodiimide such as dehydration from polycarbodiimide or urea, desulfurization from thiourea, which can be produced by the method disclosed in No. 2316,
Low molecular weight carbodiimides such as dicarbodiimide can be exemplified. The polycarbodiimide may be partially crosslinked.

Further, other carbodiimide compounds such as those described in JP-A-63-172718 and JP-A-63-172718
A carbodiimide compound of a type imparted with hydrophilicity by adding a polyoxyethylene chain in a molecular structure as described in JP-A-264128 can also be used in the present invention. In addition, low molecular weight carbodiimide compounds such as monocarbodiimide compounds and dicarbodiimide compounds are also carbodiimide compounds that can be used in the present invention.

The carbodiimide group in the carbodiimide compound has high reactivity and reacts with almost all active hydrogen groups of alcohols, amines, thiols, phenols, carboxylic acids and the like. Therefore, the nucleic acid can be firmly fixed to the base material via the carbodiimide compound by utilizing the reactivity of the carbodiimide group.

The compound having an alkylating group is used as an alkylating reagent, and examples thereof include alkyl halides, dialkyl sulfates, aromatic sulfonate alkyl esters, and alkyl metal compounds.

As such an alkylating reagent, nitrogen iperate can be preferably used. Nitrogen ipert is disclosed in U.S. Pat.
No. 7,399,701 and US Pat. No. 5,387,707.

Specific examples of the nitrogen pipette include halogenated alkyl-N-alkylaminobenzene, dihalogenated alkyl-N-aminobenzene, halogenated alkoxy-N-alkylaminobenzene, dihalogenated alkoxy-N-aminobenzene, halogenated Alkyl-N
-Alkoxyaminobenzene, halogenated alkoxy-
N-alkoxyaminobenzene, halogenated alkylphenylaminobenzene, halogenated alkoxy-N-sulfonylalkylaminobenzene, halogenated alkyl-N-sulfonylalkoxyaminobenzene, halogenated alkyl-N-carboxyalkylaminobenzene,
Examples thereof include halogenated alkoxy-N-carboxyalkylaminobenzene and halogenated alkoxy-N-carboxyalkoxyaminobenzene.

The above-mentioned nitrogen iperate may have another functional group or atom as a substituent. As such a substituent, specifically, a hydroxyl group, a halogen atom, a halogenated alkyl group, a halogenated acyl group, a halogenated allyl group, an acyl group, an allyl group, a carboxyl group, a sulfoxyl group, a phosphonium group, a ketone group, Functional groups such as an aldehyde group, an isocyanate group, an isothiocyanate group, a carbodiimide group, a thiol group and an amino group are exemplified.

The position of the above-mentioned substituent can be any position where the alkylation reaction by nitrogen iperate is not inhibited. Specifically, it is a position other than the alkylating group involved in the alkylation reaction and the tertiary nitrogen atom. The substituent is preferably introduced into an aromatic ring.

In the present invention, as a method for immobilizing a nucleic acid on a substrate via a carbodiimide compound or an alkylating reagent, a carbodiimide compound or an alkylating reagent is supported on a substrate, and the carbodiimide group of the carbodiimide compound on the substrate is provided. Or a method in which a nucleic acid is immobilized on a substrate by utilizing the reactivity of an alkylating group of an alkylating reagent.

As a method for supporting the carbodiimide compound or the alkylating reagent on the base material, there are a method in which the carbodiimide compound or the alkylating reagent is supported simply by using physical adhesiveness, and a method in which the carbodiimide compound or the alkylating reagent is chemically supported via a covalent bond or the like. . In the nucleic acid-immobilized substrate used in the present invention, it is preferable that the carbodiimide compound or the alkylating reagent is carried on the substrate via a covalent bond.

The carbodiimide compound to be used when the carbodiimide compound is supported on a substrate by utilizing physical adhesiveness includes, without particular limitation, a high molecular compound among the carbodiimide compounds.
The range of the molecular weight is usually 1,000 or more, and preferably 100,000 or less.

When the above-mentioned alkylating reagent is supported on a substrate utilizing physical adhesiveness, a compound in which the above-mentioned alkylating reagent is bound to a polymer compound by a covalent bond (hereinafter referred to as “alkyl”) Chemical compound ”). Such a compound is not particularly limited as long as the above-mentioned alkylating reagent is bonded to the polymer compound, but the molecular weight is preferably in the range of 500 to 1,000,000.

A typical form in which the above-mentioned carbodiimide polymer compound and the above-mentioned alkylating reagent polymer compound are supported on a substrate by utilizing physical adhesiveness is a coating film. As a method of supporting the carbodiimide polymer compound or the alkylating reagent polymer compound on the base material by a coating, spraying, dipping, brushing, stamping,
Known means such as vapor deposition and coating using a film coater can be employed.

Next, a method for supporting a carbodiimide compound on a substrate by a covalent bond will be described. The carbodiimide compound supported on the substrate via a covalent bond may be any of the above carbodiimide types.

To support the compound having the carbodiimide group on the surface of the substrate via a covalent bond, for example, a carbodiimide group for immobilizing a nucleic acid when supported on the substrate and a group other than the carbodiimide group may be used. If the carbodiimide compound having a functional group for covalently bonding to the material surface is covalently bonded to the functional group of the base material having a functional group capable of covalent bonding with the functional group of the carbodiimide compound on the surface by a suitable method. Good.

More specifically, a compound having two or more carbodiimide groups or a compound having one or more carbodiimide groups and a functional group other than one or more carbodiimide groups is treated with a carbodiimide group or a compound other than the carbodiimide group on the surface. A carbodiimide compound can be supported on a substrate by leaving at least one carbodiimide group of the compound covalently bonded to a functional group of the substrate having a functional group that can be covalently bonded to the functional group.

Next, when the alkylating reagent is supported on the substrate by a covalent bond, any of the above-mentioned alkylating reagents can be used. The alkylating reagent having a covalent bond on the substrate has 3 to 3
Those having 300 alkylated groups are preferred.
When the number of the alkylating groups is 3 to 300, a better ability for immobilizing the nucleic acid is obtained, and the solution has an appropriate viscosity, which is preferable in terms of handling.

In order to carry the above-mentioned alkylating reagent on the surface of the above-mentioned substrate via a covalent bond, for example, when a nucleic acid-immobilized substrate is used, an alkylating group for immobilizing nucleic acids and another group are used. An alkylating reagent having a functional group for covalently bonding to a material surface is covalently bonded to a functional group of a substrate having a functional group capable of covalently bonding to the functional group of the alkylating reagent on the surface by a suitable method. Just do it.

More specifically, a compound having two or more alkylating groups or a compound having one or more alkylating groups and a functional group other than one or more alkylating groups is provided on the surface with an alkylating group or the above-mentioned compound. The compound is obtained by covalently bonding to a functional group of a base material having a functional group capable of covalently bonding to a functional group other than the alkylating group while leaving at least one alkylating reagent of the compound.

In addition, a carbodiimide group or a functional group other than a carbodiimide group possessed by the compound or an alkylating group or an alkylating group other than the alkylating group possessed by the reagent is used when the carbodiimide compound or the alkylating reagent is carried on a substrate. Examples of the substrate having a functional group capable of covalently bonding with a functional group on its surface include, for example, a substrate in which the functional group capable of covalent bonding is introduced on the surface of the substrate. As the functional group to be introduced, a functional group capable of covalently bonding to a carbodiimide group or a functional group other than the carbodiimide group of the compound, a functional group covalently bondable to an alkylating group, or a functional group other than the alkylating group of the reagent The functional group is not particularly limited as long as it is a functional group that can be covalently bonded to a group, and specific examples include a hydroxyl group, an imino group, an amino group, a carboxyl group, a carbodiimide group, and an aldehyde group. These functional groups are appropriately selected according to the functional groups provided for the covalent bond of the carbodiimide compound or the alkylating reagent, and can be introduced into the surface of the substrate by a conventionally known method.

As a method for immobilizing a single-stranded nucleic acid to a substrate via the above-mentioned carbodiimide compound or alkylating reagent, a portion on the substrate on which the carbodiimide compound or alkylating reagent is supported is prepared under appropriate conditions. By supplying two or more types of single-stranded nucleic acids, the carbodiimide compound or the alkylating reagent may be brought into contact with the nucleic acids to react them. The nucleic acid is covalently bonded to the carbodiimide compound or the alkylating reagent by a reaction between the carbodiimide group of the carbodiimide compound supported on the substrate or the alkylating group of the alkylating reagent and the amino group, imino group, or the like of the nucleic acid. When a thiol group has been introduced into the nucleic acid in advance, the covalent bond is also formed by the reaction between the carbodiimide group or the alkylating group and the thiol group. As a result, the nucleic acid is immobilized on the substrate.

Specifically, for example, the nucleic acid is usually supplied in a form contained in water or a buffer so that the activity of the immobilized nucleic acid is maintained in the contact reaction between the two. Further, the temperature at the time of contact is preferably about 0 to 100 ° C. so that the activity of the immobilized nucleic acid is not lost.

In the present invention, as a means for supplying a nucleic acid, usually water or a buffer containing the nucleic acid, to a substrate, there are a method using a dispenser, a method using a pin, a method using a bubble jet, and the like. However, the present invention is not limited to these.

In the nucleic acid separation / recovery method of the present invention, a sample nucleic acid solution is brought into contact with a nucleic acid-immobilized substrate, and a single-stranded nucleic acid complementary to the single-stranded nucleic acid on the nucleic acid-immobilized substrate is hybridized. To prevent non-specific binding of nucleic acids other than the immobilized nucleic acid to unreacted carbodiimide groups of the carbodiimide compound supported on the substrate or unreacted alkylated groups of the alkylating reagent, as described above. After immobilizing the nucleic acid on the base material, contacting the base material with an excess amount of bovine serum albumin (BSA), casein, salmon sperm DNA, etc., and blocking the free carbodiimide group or alkylating group Is preferred.

The nucleic acid-immobilized substrate may be used as a DNA microarray (DNA chip). In this case, two or more single-stranded nucleic acids are spotted in a size and arrangement suitable for using the nucleic acid-immobilized substrate as a DNA microarray.

<Nucleic Acid Separation / Recovery Method> In the nucleic acid separation / recovery method of the present invention, a sample nucleic acid solution is brought into contact with the above-described nucleic acid-immobilized base material to obtain one or more nucleic acid-supported nucleic acid-supported substrates. A step of hybridizing a single-stranded nucleic acid complementary to a single-stranded nucleic acid, and separating the hybridized single-stranded nucleic acid at each position of the fixed single-stranded nucleic acid while keeping the nucleic acid-immobilized base material in one body. And collecting.

The sample nucleic acid solution includes natural or synthetic DNA (including oligonucleotide) or RNA.
Solutions containing (including oligonucleotides) can be mentioned without particular limitation. The nucleic acid contained in the sample nucleic acid solution may be one kind or two or more kinds.

For the hybridization, various known methods and reaction conditions can be adopted. After the hybridization, it is preferable to remove the non-specifically adsorbed nucleic acid by performing post-hybridization washing by a known method before collecting the hybridized single-stranded nucleic acid.

As a method for recovering the hybridized single-stranded nucleic acid, the hybridized single-stranded nucleic acid is separated for each location of the fixed single-stranded nucleic acid while the nucleic acid-immobilized base material is kept in one body. Various known methods can be used as long as they can be recovered by the following method. For example, the following methods can be used.

1. Scrape only the site of the nucleic acid-immobilized substrate after hybridization where the nucleic acid has been immobilized with the tip of a micropipette tip.

2. Using a spotter in which the pin tip is deformed into a shovel shape, the site (dot) where the nucleic acid is fixed together with the substrate is scraped off.

3. Fill a spotter pin or capillary pipette with a DNA denaturant such as an alkaline solution,
The tips of these pins or capillary pipettes are brought into contact with the dots on the nucleic acid-immobilized substrate on which the nucleic acids to be recovered have been hybridized to denature the nucleic acids, and the nucleic acids are transferred into the pins or capillary pipettes. The nucleic acid may be collected by immersing the pin or the capillary pipette in another solution, or the nucleic acid in the pin or the capillary pipette may be physically transferred to another container and collected.

Alternatively, the nucleic acid is denatured by dropping the above-mentioned denaturing agent onto the dots of the nucleic acid-immobilized substrate. The nucleic acid may be sucked by a membrane or the like and fixed to the membrane as it is, or the nucleic acid may be sucked and collected by an empty capillary pipette or the like. Alternatively, the nucleic acid may be recovered using a material in which an absorbent such as a sponge is fixed at the tip of the pin.

4. Fill the pin or capillary pipette of the spotter with water or buffer, and heat the substrate while the pins or capillary pipette tips are in contact with the hybridized dots on the nucleic acid-immobilized substrate where the nucleic acids to be recovered are hybridized. Alternatively, the nucleic acid is denatured by heating the substrate after dripping the water or the buffer solution onto the dots. The denatured nucleic acid is as described in 3. above. Collect by the same method as described above.

5. The tip of a capillary pipette filled with water, buffer or DNA denaturant is fixed on a conductive material. A conductive substrate is also used for the nucleic acid-immobilized substrate, and the tip of the capillary pipette is brought into contact with the dots of the nucleic acid-immobilized substrate. The nucleic acid is recovered in the capillary pipette by applying a potential difference to the capillary pipette. At this time, when filling the capillary pipette with water or a buffer, the substrate is slightly heated.

By recovering nucleic acids using each of the above methods, even when separating and recovering two or more nucleic acids hybridized on the nucleic acid-immobilized substrate, the nucleic acid-immobilized substrate may be cleaved. In addition, individual nucleic acids can be easily recovered with high separation accuracy while being integrated.

The method for separating and recovering nucleic acid of the present invention can be used, for example, in the following applications.

1. Library preparation based on expression frequency information: a plurality of types of samples for which it is desired to detect a difference in expression frequency, for example, mRNAs extracted from cancer tissues and normal tissues are respectively hybridized, and the detected results are hybridized only from different dots. Collected samples individually. Nucleic acids recovered from dots detected only in cancer tissue (only one dot or a combination of multiple dots) can be cloned by preparing cDNA as a cancer tissue-specific expression gene or using an adapter PC
By performing R or the like, a cancer tissue-specific expressed gene can be recovered at a considerably high probability (or a library containing the gene at a high probability can be prepared). Also,
By cloning and PCR similarly for nucleic acids recovered from dots detected only in normal tissues, a gene whose expression is specifically inhibited by cancer is recovered with a high probability (or the gene can be recovered with a high probability). To make a library containing the same).

[0061] Even if a gene is expressed in both cancer and normal cells, a dot having a difference in the expression level can be cloned as a gene having a different expression frequency by collecting each dot individually (or). Library). For example, in cancer cells, 5 of normal cells
A library can be prepared as a gene (group) that is double-expressed.

[0062] 2. Preparation of library based on base substitution sequence such as SNPs: DNA (oligomer) immobilized on nucleic acid-immobilized substrate was set and hybridized to obtain information on base substitution, and hybridized DNA was recovered. In this case, a library of actually existing SNPs or the like can be prepared.

3. Preparation of a library based on database information such as a promoter sequence: DNA to be immobilized on a nucleic acid immobilization substrate was set so that information such as a partial sequence of a gene or EST extracted from a database or the like, a promoter sequence and a regulatory gene could be understood. In this case, the presence of the gene or the like can be investigated and the existing one can be collected. In the case where the nucleic acid-immobilized substrate is not used, it is practically difficult to individually examine a sequence which may or may not be present, since a large amount of sample is required.

4. Preparation of library chip: The recovered product from the nucleic acid-immobilized substrate is directly fixed to a chip (membrane or the like) without performing cloning processing or the like, and a library chip or the like is prepared. For example, a library chip of a gene specifically expressed in a normal tissue and a library chip of a gene specifically expressed in a cancer tissue are prepared from the recovered nucleic acid-immobilized substrate. The effect of the drug is detected by hybridizing mRNA or the like of the gene expressed in the administered cancer tissue. The recovered nucleic acid may or may not contain a known base sequence.

When the recovered product is a nucleic acid having a known nucleotide sequence, the library chip is prepared from the nucleic acid recovered from the nucleic acid-immobilized substrate, and a labeled probe having a nucleotide sequence different from that of the capture oligomer sequence is used. By hybridizing and detecting the presence or absence of a signal, the target substance can be more easily confirmed than by examining the nucleotide sequence.

As described above, by using the nucleic acid separation and recovery method of the present invention, a plurality of types of nucleic acids can be easily separated and recovered, and a large number of nucleic acids fixed on a nucleic acid-immobilized substrate can be immobilized on a nucleic acid. It becomes possible to separate and collect directly from the modified base material. Further, it is possible to directly examine the base sequence of the recovered nucleic acid. For this reason, it is possible to avoid the risk of being mistaken for another nucleic acid having a similar base sequence.

When the method for separating and recovering nucleic acids of the present invention is used, the recovered nucleic acids can be directly immobilized on a solid phase and investigated by hybridization. In addition, by using the nucleic acid separation and recovery method of the present invention, a nucleic acid having an unknown base sequence can be recovered and cloned very efficiently.

[0068]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

[0069]

Example 1 (1) Preparation of Oligonucleotides Using a DNA synthesizer, oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 1 to 3 (hereinafter, these may be referred to as “oligomers 1 to 3”) are prepared. did. In addition,
An oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 3 was synthesized with an amino linker attached to the 5 'end.

(2) Preparation of DNA Immobilization Substrate The solution containing oligomer 3 prepared in (1)
A spotter and a micropipette were spotted on a fixing substrate (slide glass). When using a spotter, the spot size of the DNA solution is about 7 mm in diameter.
5 μm, 250 μm and 350 μm, and the amount of DNA solution when using a micropipette is 0.5
There were two types: μl / spot and 3 μl / spot. After spotting, thoroughly dry the substrate
An immobilized substrate was used.

(3) Amplification of target by PCR A partial fragment of pUC119 DNA was amplified by PCR (polymerase chain reaction) using the above oligomer 1 and oligomer 2 as primers. Put 12.8 μl of sterile water into a PCR tube, and further oligomer 1 (100 pmol / μl),
Oligomer 2 (100 pmol / μl) and pUC1
19 DNA (10 ng / μl), 1 μl each, PCR buffer (Takara Shuzo Co., Ltd.) and dNTP (dA
TP, dGTP, dCTP, dTTP 2.5 mM each)
For 2 μl each, and 0.2 μg of Taq enzyme (Takara Shuzo Co., Ltd.)
μl was added, and a PCR reaction was performed using a PCR device.
The conditions of the PCR reaction are as follows.

[Conditions of PCR reaction] Thermal denaturation: 95 ° C., 30 seconds Annealing: 57 ° C., 25 seconds Polymerase reaction: 72 ° C., 30 seconds

After completion of the PCR reaction, 2% agarose electrophoresis was carried out using 1 μl of the amplified sample, and PCR amplification (1
24 bp). The results are shown in FIG. FIG.
In lane 1, lane 1 is a kb marker and lane 2 is a PCR marker.
The results of the amplified samples are shown respectively.

(4) Hybridization 3 μl of the target subjected to PCR amplification in the above (3) and a hybridization solution (ArrayIt ^™ (Tel
12 μl) was placed in an Eppendorf tube, heated at 100 ° C. for 5 minutes, and then cooled in ice for 5 minutes to make the target a single strand. This target solution was dropped on the DNA-immobilized substrate, covered with a cover glass, and allowed to stand at 42 ° C. for 1 hour.

(5) Washing after hybridization The DNA-immobilized substrate was mixed with 50 ml of a 2 × SSC solution having the following composition.
After immersing in the DNA for 5 minutes, the solution was discarded, and further immersed in 50 ml of a fresh 2 × SSC solution for 5 minutes to remove extra unhybridized target from the DNA-immobilized substrate.

[Composition of 2 × SSC solution] 1.753 g of NaCl 0.882 g of trisodium citrate dihydrate H ₂ O was added so that the total amount becomes 100 ml.

(6) DNA Recovery from DNA Immobilized Substrate The spotted portion of the oligomer 3 was rubbed with the tip of a pipette tip to recover the hybridized DNA. In addition, the unspotted portion of the oligomer 3 (two places, a place close to the spot and a place slightly away) was similarly rubbed with the tip of a pipette tip, and used as a negative control.

(7) Confirmation of DNA recovery DN was placed in a PCR tube containing 12.8 μl of sterile water.
The collected DNA was transferred into sterile water by inserting the pipette tip from which the collection A was performed and rubbing against the inner wall of the tube several times. Oligomer 2 (100 pmol / μl) and oligomer 2 (100 pmol / μl)
l, PCR buffer (Takara Shuzo) and d
2 μl of NTP (2.5 mM each) and 0.2 μl of Taq enzyme (Takara Shuzo Co., Ltd.) were added, and a PCR reaction was performed using a PCR device. 2 using 1 μl of amplified sample
% Agarose gel electrophoresis, and PCR amplification (124 bp) from spots of all sizes where DNA was immobilized
It was confirmed. The results are shown in FIG. FIG. 2 shows only the results of spots with a diameter of 75 μm by a spotter (lane 2) and spots of 3 μl / spot by a micropipette (lane 3).

A PCR reaction and electrophoresis were performed on the pipette tip rubbed on the portion where the oligomer 3 was not spotted by the same method as described above.
No CR amplification (124 bp) was confirmed. The results are shown in FIG. In FIG. 2, lane 4 is the result of a place close to the spot, lane 5 is the result of a place slightly away from the spot, and lane 6 is the PC obtained in the above (3).
The results for the R-amplified samples are shown.

[0080]

Example 2 (1) Preparation of Oligomers Using a DNA synthesizer, oligonucleotides having the base sequences shown in SEQ ID NOs: 1 to 6 (hereinafter, these may be referred to as “oligomers 1 to 6”) were prepared. . In addition,
Oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 3 and 6 were synthesized with an amino linker attached to the 5 'end.

(2) Preparation of DNA-immobilized substrate Oligomers 3 and 6 prepared in (1) above
Were spotted on a DNA fixing substrate (slide glass) using a spotter and a micropipette. DNA with spotter
The size of the solution spot was about 75 μm in diameter, 250 μm.
μm and 350 μm, and the amount of the DNA solution using a micropipette was 0.5 μl / spot and 3 μl / spot. After spotting, the substrate was thoroughly dried to obtain a DNA-immobilized substrate.

(3) Amplification of target by PCR A partial fragment of pUC119 DNA was amplified by PCR (polymerase chain reaction) using the above oligomer 1 and oligomer 2 as primers. Put 12.8 μl of sterile water into a PCR tube, and further oligomer 1 (100 pmol / μl),
Oligomer 2 (100 pmol / μl) and pUC1
19 DNA (10 ng / μl), 1 μl each, PCR buffer (Takara Shuzo Co., Ltd.) and dNTP (dA
TP, dGTP, dCTP, dTTP 2.5 mM each)
For 2 μl each, and 0.2 μg of Taq enzyme (Takara Shuzo Co., Ltd.)
μl was added, and a PCR reaction was performed using a PCR device.
The conditions of the PCR reaction are as follows.

[PCR Reaction Conditions] Thermal denaturation: 95 ° C., 30 seconds Annealing: 57 ° C., 25 seconds Polymerase reaction: 72 ° C., 30 seconds

After completion of the PCR reaction, 2% agarose electrophoresis was carried out using 1 μl of the amplified sample, and PCR amplification (1
24 bp). The results are shown in FIG. 3 (lane 2). In FIG. 3, lane 1 indicates a kb marker.

Further, pBl was obtained by a PCR reaction using oligomers 4 and 5 as primers.
A partial fragment of usscript II SK (manufactured by STRATAGENE) was amplified. 12.8 μl of sterile water is added to a PCR tube, and oligomer 4 (100
pmol / μl), oligomer 5 (100 pmol / μl)
l) and pBluescript II SK (10 ng)
/ Μl), 1 μl each, PCR buffer (Takara Shuzo Co., Ltd.) and dNTPs (dATP, dGTP, dCT
2 μl each of P and dTTP (2.5 mM each) and 0.2 μl of Taq enzyme (manufactured by Takara Shuzo Co., Ltd.) were added, and a PCR reaction was performed using a PCR device. The conditions of the PCR reaction are as follows.

[Conditions of PCR reaction] Thermal denaturation: 95 ° C., 30 seconds Annealing: 57 ° C., 25 seconds Polymerase reaction: 72 ° C., 30 seconds

After completion of the PCR reaction, 2% agarose electrophoresis was carried out using 1 μl of the amplified sample, and PCR amplification (1
87 bp). The results are shown in FIG. 3 (lane 3). In FIG. 3, lane 1 indicates a kb marker.

(4) Hybridization The target (124b) subjected to PCR amplification in (3) above
p) and 1.5 μl each of the target (187 bp) were mixed in an Eppendorf tube, and 12 μl of a hybridization solution (ArrayIt ^™ (manufactured by Telechem)) was added. For 5 minutes to make the target single-stranded. This target solution was dropped on the DNA-immobilized substrate, covered with a cover glass, and allowed to stand at 42 ° C. for 1 hour.

(5) Washing after hybridization The DNA-immobilized substrate was immersed in 50 ml of a 2 × SSC solution having the same composition as in Example 1 for 5 minutes, and the solution was discarded. By further immersion, an extra target that did not hybridize was removed from the DNA-immobilized substrate.

(6) DNA Recovery from DNA Immobilized Substrate Each spotted portion of the above oligomer 3 was rubbed with the tip of a pipette tip to recover hybridized DNA. In addition, each spot of the oligomer 6 was rubbed with the tip of another pipette tip, and the hybridized DNA was recovered. further,
The unspotted portion of the oligomer was similarly rubbed with the tip of a pipette tip to serve as a negative control.

(7) Confirmation of DNA Recovery The pipette tips from which DNA was recovered were placed in 11 PCR tubes containing 12.8 μl of sterile water, and the recovered DNA was rubbed several times against the inner wall of the tubes to remove the recovered DNA. Transferred to sterile water. In each tube, oligomer 1 (100 pmol / μl), oligomer 2 (100
pmol / μl), oligomer 4 (100 pmol / μl)
l) and oligomer 5 (100 pmol / μl), 2 μl each; PCR buffer (manufactured by Takara Shuzo Co., Ltd.) and dNTP (2.5 mM etch) each 2 μl, Ta
0.2 μl of q enzyme (manufactured by Takara Shuzo Co., Ltd.) was added to each, and a PCR reaction was performed using a PCR device. 2% agarose electrophoresis was performed using 1 μl of each amplified sample. Some of the results are shown in FIG. In FIG. 4, lane 1 is a kb marker, and lane 2 is pUC119 DN.
As a result of the PCR-amplified sample of the partial fragment of A (124 bp), lane 3 was a result of the PCR-amplified sample of the partial fragment of pBluescript II SK (187 bp), and lane 4 was 75 μm in diameter by the spotter of oligomer 3 of the DNA-immobilized substrate. As a result of the PCR amplification sample of the DNA recovered from the spot part, lane 5 is the result of the PCR amplification sample of the DNA recovered from the spot part having a diameter of 75 μm by the spotter of the oligomer 6 of the DNA immobilization substrate, and lane 6 is a negative control. The results of the PCR amplified samples are shown respectively.

From the spots of all sizes to which the oligomer 3 was immobilized, the target 1
PCR amplification of 24 bp DNA was confirmed. In this case, PCR amplification of 187 bp DNA was not confirmed. In addition, from the spots of all sizes to which the oligomer 6 was immobilized, 1
PCR amplification of the 87 bp DNA was confirmed. In this case, PCR amplification of 124 bp DNA was not confirmed. For any pipette tip rubbed on the spot where the oligonucleotide is not spotted, use any PC
R amplification (124 bp / 187 bp) was not confirmed. From this, it was confirmed that a plurality of types of DNAs hybridized to the DNA-immobilized substrate could be separated and recovered on the chip.

[0093]

Example 3 (1) Production of carbodiimide compound solution 4,4'-dicyclohexylmethane diisocyanate 1
17.9 g and cyclohexyl isocyanate 12.5 g
With a carbodiimidation catalyst (3-methyl-1-phenyl-
The mixture was reacted with 1.3 g of 2-phospholene-1-oxide at 180 ° C. for 4 days in a nitrogen atmosphere, and then a carbodiimide compound (polymerization degree 10, number average molecular weight 240
0) was obtained. Take 10 g of this and add 200
of the carbodiimide compound solution.

(2) Preparation of aminated slide glass To 180 ml of distilled water, 20 ml of a 10% (v / v) 3-aminopropyltriethoxysilane / ethanol solution was added and stirred well. 6N HCl was added thereto, and the pH was 3-4.
After adjusting the temperature to 15 ° C, immerse 15 slide glasses at 75 ° C.
For 2 hours. After the heat treatment, the slide glass is pulled out of the solution, washed well with distilled water, and then
It was dried by heating at 15 ° C. for 4 hours to obtain an aminated slide glass.

(3) Preparation of carbodiimidated slide glass The carbodiimide compound solution 200 obtained in the above (1) was prepared.
15 ml of the aminated slide glass obtained in the above (2) were immersed in ml, immediately pulled up, and dried by heating at 60 ° C. for 1 hour. Next, these slide glasses were washed twice with 200 ml of dichloromethane for 10 minutes each, and then dried at 40 ° C. for 2 hours to obtain carbodiimidated slide glasses.

(4) Preparation of DNA Immobilization Substrate The solution containing oligomer 3 prepared in (1) of Example 1 was applied to the carbodimidated slide glass prepared in (3) above using a spotter and a micropipette. Spotted. The spot size of the DNA solution when using a spotter is about 75 μm, 250 μm, 350 μm in diameter.
μm and D when using a micropipette
The amount of the NA solution was two types, 0.5 μl / spot and 3 μl / spot. After spotting, the substrate was thoroughly dried, and a buffer (0.2 M sodium chloride, 0.1 M Tris-HCl (p) containing 3% bovine serum albumin (SIGMA) was used.
H 7.5), 0.05% Triton X-100; hereinafter, referred to as "buffer A") solution for 5 minutes, and a buffer (T
E solution) and dried to obtain a DNA-immobilized substrate.

(Composition of TE solution) Tris (hydroxymethyl) aminomethane 121.14 g Hydrochloric acid Adjusted to pH 8 Water added to make total 1000 ml

(5) Hybridization and DN
A recovery As a DNA solidification substrate, the DN prepared in (4) above
Except for using the A-immobilized base material, (4) to (4) of Example 1 were used.
When the same operation as in (7) was performed, the same result as in Example 1 was obtained.

[0099]

Example 4 (1) Preparation of DNA Immobilization Substrate Carbodimidated slide glass prepared in Example 3 (3) was prepared by using a solution containing each of the oligomers 3 and 6 prepared in Example 1 (1). Was spotted using a spotter and a micropipette, respectively. The spot size of the DNA solution when using a spotter is three types of diameters of about 75 μm, 250 μm, and 350 μm, and the amount of the DNA solution when using a micropipette is 0.5 μl / spot and 3 μl / spot. 2
Type. After spotting, the substrate was thoroughly dried, immersed in 50 ml of buffer A solution containing 3% bovine serum albumin (manufactured by SIGMA) for 5 minutes, washed with a TE solution, dried, and immobilized with DNA. A substrate was used.

(2) Hybridization and DN
A recovery As a DNA solidification substrate, the DN prepared in (1) above
Except for using the A-immobilized base material, (4) to (4) of Example 2
When the same operation as in (7) was performed, the same result as in Example 2 was obtained.

[0101]

According to the nucleic acid separation and recovery method of the present invention, a plurality of types of nucleic acids can be easily separated and recovered, and
It is possible to directly separate and recover many types of nucleic acids fixed on the nucleic acid-immobilized substrate from the nucleic acid-immobilized substrate. Also,
It is possible to directly examine the nucleotide sequence of the recovered nucleic acid. For this reason, it is possible to avoid the risk of being mistaken for another nucleic acid having a similar base sequence.

Further, if the method for separating and recovering nucleic acid of the present invention is used, it is possible to directly fix the recovered nucleic acid on a solid phase and to conduct an investigation by hybridization. In addition, by using the nucleic acid separation and recovery method of the present invention, a nucleic acid having an unknown base sequence can be recovered and cloned very efficiently.

[0103]

[Sequence list] SEQUENCE LISTING <110> Nisshinbo Industries, Inc. <120> Nucleic acid separation and recovery method <130> P-8361 <150> JP 2000-21842 <151> 2000-01-26 <150 > JP 2000-392948 <151> 2000-12-25 <160> 6 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer for PCR <400 > 1 caggaaacag ctatgac 17 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer for PCR <400> 2 gttttcccag tcacgac 17 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 3 gtaaaacgac ggccagt 17 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer for PCR <400> 4 accctcacta aagggaa 17 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer for PCR < 400> 5 gtaaaacgac ggccagt 17 <210> 6 <211> 17 <212> DNA <213> Artificial Sequence <2 20> <223> Description of Artificial Sequence: Synthetic DNA <400> 6 tacgactcac tataggg 17

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of target amplification by PCR according to Example 1 of the present invention.

FIG. 2 is a view showing the results of confirming DNA recovery in Example 1 of the present invention.

FIG. 3 is a diagram showing the results of target amplification by PCR according to Example 2 of the present invention.

FIG. 4 is a view showing the results of confirming DNA recovery in Example 2 of the present invention.

Claims (4)

[Claims] A sample nucleic acid solution is brought into contact with a nucleic acid-immobilized substrate having a substrate and two or more types of single-stranded nucleic acids separated and immobilized on the substrate, respectively. A step of hybridizing a single-stranded nucleic acid complementary to a single-stranded nucleic acid, and leaving the nucleic acid-immobilized base material as an integral unit, the hybridized single-stranded nucleic acid is removed for each location of the fixed single-stranded nucleic acid. Separating and recovering the nucleic acid. 2. The method for separating and recovering nucleic acids according to claim 1, wherein the nucleic acid-immobilized substrate is a substrate carrying a compound having a carbodiimide group. 3. The method for separating and recovering nucleic acids according to claim 1, wherein the nucleic acid-immobilized substrate is a DNA microarray. 4. The method for separating and recovering nucleic acids according to claim 1, wherein the substrate is flat.