CN1780909A - Nucleotide Chain Modification Methods - Google Patents

Abstract

The method for modifying a nucleotide chain is characterized in that a target modifying substance (e.g., NH having an amino group) is added to the 3 'end of a nucleotide chain (I) to be modified, the nucleotide chain having a nucleotide sequence having a specific base such as hypoxanthine (Hx) at the 3' end, by acting on a lytic enzyme specific to a nucleotide containing the specific base₂-R) a functional group (e.g. aldehyde group) having a binding ability, and the modifying substance is bound at the 3' -end of the nucleotide chain having the functional group. It is also characterized in that the 3' -terminus is an enzyme baseThe nucleotide chain of the nucleotide sequence of the specific base of the substance is a modification target, and only the nucleotide sequence portion is decomposed to form a functional group which reacts with and binds to the target modification substance. Thus, the nucleotide chain can be directly modified with the modifying substance, and labeling can be carried out easily, and the modifying substance can be stably and firmly immobilized as a linker when the purpose is to immobilize the nucleotide chain.

Description

Nucleotide Chain Modification Methods

technical field

The present invention relates to a method for modifying nucleotide chains, in particular to a method for directly modifying the 3' end of a nucleotide chain with a modifying substance, and marking, marking and immobilizing the nucleotide chain.

Background technique

In the past, in genetic analysis, radioactive isotopes have been used as modified substances for labeling and labeling nucleotide chains such as DNA, RNA, oligonucleotides, and nucleic acids. Due to problems such as space restrictions, problems of being exposed to radiation, and problems of disposal, its use has gradually decreased. In recent years, a method of modifying a nucleotide chain with a fluorescent substance such as fluorescein or biotin as a modifying substance instead of a radioactive isotope, and labeling and labeling it has been widely used. Methods for modifying nucleotide chains can be classified into three types: 5' end modification method, 3' end modification method, and internal modification method.

As a method for modifying the 5' end, a method of modifying the phosphate group at the 5' end with biotin has been proposed (see, for example, Non-Patent Document 1 below). Regarding biotin, it has been reported that using the high affinity of biotin and avidin, modifying it with enzymes such as alkaline phosphatase and horseradish peroxidase, and using the chemiluminescence of these enzymes to make gene analysis highly sensitive method (see, for example, Non-Patent Documents 2 to 3). Many other methods have been proposed by modifying the phosphate group at the 5' end (see Patent Document 1 and Non-Patent Documents 4 to 13).

However, in the method of modifying the phosphate group at the 5' end, the modification amount of the modified substance is generally 1 for the nucleotide chain, and alkaline phosphatase will dissociate the phosphate group at the 5' end of the nucleotide chain, so in order to realize For high sensitivity, the combination with alkaline phosphatase must generally be avoided. Alkaline phosphatase is also present in bacteria floating in the air, and when it is mixed into genetic analysis, the modified substance bound to the phosphate group at the 5' end is easily dissociated, and the modified state becomes less stable, which also becomes the background main cause of noise. On the other hand, the phosphoramidite method, which is widely used as a method for modifying the 5' end, has to be separated and removed by liquid chromatography to remove unreacted substances due to incomplete reaction, and the operation is complicated.

Internal modification method, developed in the reaction of nucleotide chain replication, the deoxynucleotide triphosphates used for labeling and labeling modification substances are pre-modified into the nucleotide chain, so that the nucleoside Acid chain labeling, labeled random primer method, and nick translation method (see, for example, Patent Document 4 and Non-Patent Documents 20 to 22).

This modification method can simultaneously perform amplification and Labeling and labeling (see, for example, Non-Patent Document 26), thereby bringing about high sensitivity of gene analysis, and realizing high efficiency of gene analysis represented by gene chips (see, for example, Patent Document 9 and Non-Patent Document 27- 29).

In these random primer methods, nick translation methods and PCR methods, in terms of the incorporation efficiency of nucleotide chains and the synthesis of modified nucleotide chains, it is often used to use thymidine with uracil or with fluorescent substances in advance. Labeled, labeled uracil replaces the formation of incorporated nucleotide chains. Furthermore, as an advantage of using uracil instead of uracil, when using the PCR method, uracil is incorporated into the nucleotide chain derived from exogenous contamination contained in the reaction mixture, and DNA glycosylation with uracil A method for preventing amplification of nucleotide chains from contamination by enzymatic action (see, for example, Patent Documents 11 to 13 and Non-Patent Documents 31 to 33).

However, the number of incorporation of modified substances into nucleotide chains by the PCR method is about 10 to 20 or 30 (see, for example, Non-Patent Document 30), which is random and not completely quantified. Since the labeling and labeling of deoxynucleotide triphosphates are cumbersome, arbitrary labeling and labeling products cannot be easily obtained by everyone, so the types of modified substances are limited. Furthermore, since the modifying substance is incorporated into the nucleotide chain, steric hindrance due to the modifying substance will occur during hybridization for gene analysis, that is, when the nucleotide chain forms a double strand. This method also has the disadvantage of being limited to labeling the nucleotide chain with a fluorescent substance or the like from the beginning.

The 3' end modification method has been developed to synthesize deoxynucleotide triphosphates or dideoxynucleotide triphosphates that have been labeled and labeled in advance (see, for example, Non-Patent Document 14 and Patent Document 2), and the labeled, Labeled nucleotides are added to the 3' end of the nucleotide chain by terminal deoxynucleotidyl transferase (tailing method) (see, for example, Patent Document 3 and Non-Patent Documents 15 to 18). The 3' end modification method can modify the nucleotide chain as required. Compared with the 5' end modification method, the modified substance has higher continuous stability and is more suitable as a modification site, so it is widely used.

However, the labeling and labeling of deoxynucleotide triphosphates or dideoxynucleotide triphosphates is cumbersome, and arbitrary labeling and labeling products are not easily available to everyone, so the types of modified substances are limited. . In addition, in the tailing stage, since an unnecessary nucleotide sequence is added at the 3' end of the nucleotide chain, especially in the case of using deoxynucleotide triphosphate, the number of added nucleotides is random depending on the reaction conditions (see, for example, Non-Patent Document 19), although it is possible to achieve high sensitivity in gene analysis, there is a problem with quantification. Addition of an unnecessary nucleotide sequence to the 3' end is also a major cause of mismatches during hybridization in gene analysis.

Regarding the addition of unnecessary bases, Patent Document 10 proposes to decompose the glycosidic bond of added uracil with uracil DNA glycosidase. However, in the random primer method, the nick translation method and the PCR method described above, the formation of a nucleotide chain in which thymidine is replaced with uracil is widely used. When the PCR method is used, uracil, which is used to prevent amplification from contaminating nucleotide chains, is also incorporated into the amplified nucleotide chains. Therefore, if the method described in Patent Document 10 is used for a nucleotide chain incorporating uracil, uracil DNA glycosylase acts not only on the uracil added at the 3' end but also on the nucleotide chain to be modified. of uracil, and as a result the nucleotide chain itself is decomposed.

Other 3' end modification methods also include the method of directly combining the modified substances for labeling and labeling on the 3' end nucleotides in the initial stage of chemically synthesized nucleotide chains, so as to realize the modification of nucleotide chains. The modified substances have higher sustained stability. However, the number of nucleotides in the nucleotide chain obtained by chemical synthesis is about 100 due to the relationship between synthetic by-products and yields. The modification of the 3' end of the nucleotide chain with more than 100 nucleotides amplified by extraction and PCR etc. is not applicable. That is, the chain length of the nucleotide chain to be modified is limited.

Furthermore, these three modification methods are difficult to selectively modify one of the nucleotide chains when the nucleotide chain exists in a double-stranded state. That is, when labeling and labeling a nucleotide chain by the conventional method, both strands of the nucleotide chain that usually exist in a double-stranded state are modified at the same time, and it is difficult to label one of the nucleotide chains. Selectively modified while the other strand of nucleotides is isolated and removed. Therefore, when the genetic information contained in one nucleotide chain in the double strand is analyzed, the other nucleotide chain without the required information is also used for analysis, which becomes the main cause of suspicious results.

On the other hand, the modification of the nucleotide chain is not only performed for labeling or tagging, but also for fixing the nucleotide chain to the substrate. As the substrate for this application, one or both sides of hydrophobic and positively charged substrates made of nitrocellulose, nylon, or polyvinyl fluoride are used, and the immobilized nucleotide chains may dissociate. In recent years, it has gradually been used to modify the 5' end or 3' end of the nucleotide chain with functional groups such as amino groups and aldehyde groups. Using substrates such as glass and silicon, the surface is equipped with aldehyde groups, epoxy, etc. A method of immobilizing nucleotide chains on substrates by pretreatment of bases or amino groups. By using such a method to immobilize a plurality of nucleotide chains with different sequences per unit area of the substrate, a gene analysis chip can be realized. However, in the immobilization of such a nucleotide chain, there is a problem of modification of the nucleotide chain as described above.

(patent documents)

1 Japanese Patent No. 1706289

2 Japanese Patent Laid-Open No. 61-115094

3 Japanese Patent Publication No. 7-31194

4 Japanese Patent Application No. 2000-508709

5 Japanese Patent No. 1814713

6 Japanese Patent No. 2093730

7 Japanese Patent No. 2093731

8 Japanese Patent No. 2613877

9 Japanese Patent Application No. 10-503841

10 Japanese Patent Application No. 2002-049055

11 Japanese Patent No. 2103155

12 Japanese Patent Application No. 2000-502251

13 Japanese Patent Application Laid-Open No. 11-113599

(non-patent literature)

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The present invention was made in view of the above conventional problems, and an object of the present invention is to provide a nucleotide chain modification that can directly modify the 3' end with a modifying substance regardless of the base structure of the nucleotide chain and without disassembly of the nucleotide chain. method.

Another object of the present invention is to provide a nucleotide chain modification capable of selectively modifying not only the 3' end of a single-stranded nucleotide chain but also the 3' end of one of the nucleotide chains forming a double strand method.

disclosure of invention

In order to achieve the above object, the method for modifying a nucleotide chain of the present invention is characterized in that, for a nucleotide chain to be modified in which a nucleotide sequence having a specific base exists at the 3' end, a nucleotide chain containing the specific base is used. The nucleotides and specific decomposing enzymes act on the 3' end of the nucleotide chain as the modification object, adding a functional group that has the ability to bind to the target modification substance, and the nucleotide with the functional group The 3' end of the chain binds the modifying substance. By making a nucleotide chain having a nucleotide sequence of a specific base that is an enzyme substrate at the 3' end as the object of modification, only the part of the nucleotide sequence is decomposed to form a functional group that reacts with and binds to the target modification substance . By doing so, the nucleotide chain can be directly modified with a modifying substance, and while labeling and labeling can be easily performed, the modifying substance can be used as a linking substance when the nucleotide chain is immobilized For a stable, firm fixation.

A nucleotide sequence having a specific base is located at the 3' end of the nucleotide chain serving as the main chain, and the specific base is preferably a base that does not exist in the main chain. This prevents the main chain from being decomposed even when decomposing enzymes act on it, and keeps the sequence information of the main chain part intact. The sequence information of the so-called nucleotide chain preferably has a nucleotide sequence of at least about 10 bases.

A nucleotide sequence having a specific base can be added to the 3' end of the nucleotide chain as the main chain. The method is a method of purposefully adding nucleotides having a base as an enzyme substrate. Regardless of whether the nucleotide chain to be modified is single-stranded or double-stranded, the 3' end of the nucleotide chain can be restricted to the 3' end of the nucleotide chain and modified with a modifying substance easily. By appropriately selecting the added nucleotide sequence, incorporation of unnecessary base sequences is also excluded.

Specifically, annealing a nucleotide chain having a sequence complementary to the 3' end region of the nucleotide chain at the 3' end region to the single-stranded nucleotide chain as the modification object can be performed in the modification object A complementary nucleotide sequence containing a specific base is added to the 3' end of the nucleotide chain.

In addition, at least one strand of the double-stranded nucleotide chain to be modified is cut with a restriction enzyme that can specifically recognize the base sequence of the 3' end region of the nucleotide chain to be modified. A complementary nucleotide sequence containing a specific base is added to the 3' end of the cleaved nucleotide chain to be modified.

In addition, at the 3' end of at least one of the double-stranded nucleotide chains to be modified, the double-stranded nucleotide chain having a nucleotide sequence containing a specific base in the 5' end region is used DNA ligase ligation, by cleaving the base sequence from the specific base to the 3' end of the linked nucleotide chain with a restriction enzyme that specifically recognizes, the nucleotide that is the object of modification can be A nucleotide sequence containing specific bases is added to the 3' end of the strand.

Addition of nucleotide sequences can be performed by incorporation of nucleotides using DNA polymerase.

A nucleotide chain in which a nucleotide sequence having a specific base exists may be a chemically synthesized nucleotide chain.

It is preferable to add an oligonucleotide having a base sequence complementary to a specific base before acting with a decomposing enzyme. The substrate specificity of the enzyme can be improved and the reaction efficiency can be improved by causing complementary binding between bases of double-stranded nucleotides.

The 3' end of the nucleotide chain to be modified may have, for example, an aldehyde group. Specifically, by using DNA glycosidase as a decomposing enzyme, an aldehyde group can be attached to the 3' end of the nucleotide chain to be modified. More specifically, when the specific base is hypoxanthine, an aldehyde group can be attached to the 3' end of the nucleotide chain to be modified by using 3-methyladenine DNA glycosidase as a decomposing enzyme.

The specific base and the decomposing enzyme may be in various combinations, but sometimes it is not desirable to use it as a specific base, for example, because incorporation of uracil into a nucleotide chain is widely used in various methods of gene analysis. By using low-frequency hypoxanthine and its decomposing enzyme, the user's convenience for modifying the 3' end of the nucleotide chain can be greatly improved. In addition, the 3' end of the nucleotide chain can be directly and easily modified quantitatively and stably with any modifying substance regardless of the base structure and chain length of the nucleotide chain. This method is a method in which only the 3' end of the nucleotide chain can be modified with a modifying substance by excluding unnecessary base sequences and the incorporation of modified bases into the nucleotide chain.

In addition to the above-mentioned combination of hypoxanthine and 3-methyladenine DNA glycosidase, for example, combinations shown in the following Table 1 can be used to make the 3' end of the nucleotide chain to be modified with an aldehyde group .

Table 1 The base of a nucleotide incorporated into the 3' end of a nucleotide chain Applicable DNA glycosidases, DNA repair enzymes · Hypoxanthine · Hypoxanthine DNA Glycosidase 3-Methyladenine 3-Ethyladenine 3-Methyladenine DNA glycosidase type I 3-methyladenine Hypoxanthine Xanthine 3-methylguanine 7-methylguanine 1,N ⁶ -acetyl adenine 8-oxoguanine 7-ethyl Purine·3-ethylpurine·7,3-diethylpurine·1-carboxyethyladenine·7-carboxyethylguanine·O ² -methylpyrimidine·7(2-ethoxyethyl) Guanine·7(2-hydroxyethyl)guanine·7(2-chloroethyl)guanine·1,2-bis(7-guaninyl)ethane·3-ethylthioethylpurine·N ^2,3 -Ethylideneguanine N ^2,3 -vinylideneguanine 5-hydroxymethyluracil 5-formyluracil 1,N ⁴ -vinylidenecytosine 1, N ² -vinylideneguanine · 3,N ² -vinylideneguanine 3-Methyladenine DNA glycosidase type II

Table 1 (continued) The base of a nucleotide incorporated into the 3' end of a nucleotide chain Applicable DNA glycosidases, DNA repair enzymes · Uracil · 5-hydroxyuracil · 5,6-dihydroxyuracil · 5-fluorouracil · Uracil DNA Glycosidase Thymine glycol 5,6-dihydrothymine 5,6-dihydroxydihydrothymine hydroxycytosine urea 5-hydroxy-5-methylhydantoin 6-hydroxy- 5,6-dihydroxypyrimidine · 5-hydroxyuracil · 5-hydroxy-6-hydrothymine · 5,6-dihydrouracil · uracil glycol · 5-hydroxy-6-hydrouracil · Endonuclease III 8-oxoguanine 7,8-dihydro-8-oxoguanine 8-oxoadenine formamidoguanine methylformamidoguanine 8-Oxoguanine DNA Glycosidase 7-methylguanine 1,6-diamino-5-carboxamidopyrimidine carboxamidoguanine methylformamidoguanine carboxamidoadenine 8-oxoadenine 8 -Oxoguanine · 7,8-dihydro-8-oxoguanine · Hydroxycytosine · 5-Hydroxyuracil · Aflatoxin combined with imidazole-opened guanine · Imidazole-opened N-2-aminofluorene -8-guanine · Formamidopyrimidine DNA Glycosidase 8-Oxoguanine Adenine/guanine mismatch ·MutY-DNA Glycosidase 1, N ⁴ -vinylcytosine Uracil/guanine mismatch Mismatch uracil DNA glycosylase · Pyrimidine dimer · Pyrimidine dimer DNA glycosylase Thymine/guanine mismatch Guanine/guanine mismatch · Thymine DNA Glycosidase

As the modified substance, a substance having an amino group that forms a chemical bond with a functional group attached to the 3' end of the nucleotide chain can be used.

A modifying substance may be a substance that labels or marks a nucleotide chain. Thus, the modified nucleotide chain can be used as a detection probe or target in gene analysis.

Such modified substances can be applied to fluorescent substances, vitamins, lipids, amino acids, oligopeptides, proteins or exogenous substances. Since amino acids, oligopeptides, and proteins have amino groups, by making other substances have amino groups, they can be fused with the aldehyde group at the 3' end of the nucleotide chain. Thus, functions of fluorescent substances, vitamins, lipids, amino acids, oligopeptides, proteins, or exogenous substances can be added to the nucleotide chain.

The modifying substance may be a substance having a binding ability to a substrate for gene analysis. Thereby, the modified nucleotide chain can be stably immobilized on the substrate as a detection probe or a target.

As such modified substances, aminoalkanethiol or aminosilane coupling compounds can be used. These compounds can be stably bonded to noble metal, glass or resin substrates.

A brief description of the drawings

FIG. 1 is an explanatory diagram showing the principle of a nucleotide chain modification method according to Embodiment 1 of the present invention;

Fig. 2 has represented the partial process of the nucleotide chain modification method shown in Fig. 1, the chemical reaction formula of the process that the aldehyde derivative that will generate is modified with fluorescein;

Fig. 3 has represented the partial process of the nucleotide chain modification method shown in Fig. 1, the chemical reaction formula of the process of modifying the aldehyde derivative generated with aminoalkane thiol and the process of immobilizing it on the noble metal substrate;

Fig. 4 is an explanatory diagram showing the principle of a nucleotide chain modification method according to Embodiment 2 of the present invention;

Fig. 5 has represented the chemical reaction formula of the reaction center part of the nucleotide chain of Fig. 4;

Fig. 6 is an explanatory diagram showing the principle of a nucleotide chain modification method according to Embodiment 3 of the present invention;

7 is an explanatory diagram showing the principle of the nucleotide chain modification method according to Embodiment 4 of the present invention;

Fig. 8 is an explanatory diagram showing the principle of a nucleotide chain modification method according to Embodiment 5 of the present invention;

Figure 9 shows the results of polyacrylamide electrophoresis of nucleotide chains in each reaction process in the nucleotide chain modification method of Example 1 of the present invention;

Fig. 10 has represented the result of the film exposure of the nucleotide chain of each reaction process in the nucleotide chain modification method of the embodiment 2 of the present invention;

Fig. 11 shows the results of polyacrylamide electrophoresis and film exposure of nucleotide chains in each reaction process in the method for modifying nucleotide chains in Example 3 of the present invention.

The best way to practice the invention

Hereinafter, embodiments of the present invention will be described with reference to the drawings.

(Embodiment 1)

Embodiment 1 of the present invention will be described based on FIG. 1 . In the figure, 1, m and n represent 0 or any natural number, base represents any base such as adenine, guanine, cytosine, thymine, uracil, etc., Hx represents hypoxanthine, and Cyt represents cytosine.

By combining a nucleotide chain (I) of an arbitrary base sequence and 2'-deoxyinosine-5'-triphosphate (A) (constructed with inosine Hx as shown below) with a terminal deoxynucleotide The transferase acts to add a nucleotide sequence containing hypoxanthine to the 3' end of the nucleotide chain (I) to obtain the nucleotide chain (II).

Nucleotide chain (III) is obtained by annealing oligonucleotide chain (B) of several tens of nucleotides in length having cytosine to obtained nucleotide chain (II).

By acting on the partially double-stranded nucleotide chain (III) with 3-methyladenine DNA glycosylase type II and performing alkaline heat treatment, the glycosidic bond of the nucleotide having hypoxanthine is cut off, The remaining deoxyribose-phosphate bond is cleaved to obtain a nucleotide chain (IV) having an aldehyde group at the 3' end.

By reacting a modifying substance having an amino group (H ₂ NR (discussed later on the modifying substance)) with the nucleotide chain (IV), a Schiff base obtained by dehydration condensation of the nucleotide chain (IV) and the modifying substance is obtained, That is, the nucleotide chain (V) whose 3' end is directly modified by the modifying substance.

If the target nucleotide chain is a nucleotide chain of several tens of nucleotides long which can be chemically synthesized, the first-stage reaction can be omitted by pre-synthesizing a nucleotide chain having a specific base sequence.

As shown in Figure 2, if fluorescein cadaverine (C), known as a fluorescent substance, is reacted with a nucleotide chain (IV) having an aldehyde group at the 3' end, a Modified nucleotide chain (VI) that directly binds fluorescein cadaverine.

Such modification with fluorescein enables the nucleotide chain to be fluorescently labeled (labeled), and the nucleotide can be used as a probe or target for genetic analysis such as hybridization.

As shown in Figure 3, if the thiol with amino group, 8-amino-1-octylthiol (D) is used here to react with the nucleotide chain (IV) having an aldehyde group at the 3' end, the obtained Modified nucleotide chain (VII) in which 8-amino-1-octanethiol residue is directly bonded to the 3' end of the nucleotide chain.

By reacting the modified nucleotide chain (VII) with the noble metal substrate (E), the thiol group and the substrate (E) form a strong covalent bond, and the nucleotide chain (VII') can be immobilized on the substrate (E) on.

By immobilizing on the substrate (E) in this way, gene analysis can be performed by electrochemical measurement methods using noble metals on substrates, crystal oscillator microbalance methods, surface plasmon resonance methods, and the like.

The modification substance used to label and label the nucleotide chain should only have the ability to bind to the functional group attached to the 3' end of the nucleotide chain. For example, fluorescent substances such as fluorescein, Texas red (Texasred), rhodamine, cyanine compounds represented by Cy3·Cy5, or non-fluorescent substances such as biotin, digoxigenin, etc., through An amino group is formed at the terminal and can be used as a modification substance. Nucleotide chains labeled (labeled) with these modifying substances can be used as probes or targets for hybridization isogenic analysis.

Amino acids, peptides, proteins can also be used as modifying substances. Among amino acids, phenylalanine, tryptophan, and tyrosine are fluorescent and can be used for labeling and marking. Non-fluorescent amino acids and peptides can also be used for labeling and labeling by binding fluorescein or the like to their side chains (hereinafter referred to as labeling compounds).

Since the peptide formed by linking amino acids has side chains corresponding to the number of amino acids, multiple labeling compounds can be linked. For example, there are 3 amino groups on the side chain in trimeric lysine, and a labeling compound having a carboxyl group, a succinimide group, etc. can be arbitrarily combined up to a total of 3.

Not limited to peptides, substances obtained by modifying hydrocarbons such as alkyl groups, allyl groups, cycloalkyl groups, aromatics, sugars, etc. Modification, to achieve labeling and labeling of nucleotide chains.

Proteins such as alkaline phosphatase, peroxidase, colored or fluorescent transferrin, hemoglobin, green fluorescent protein, blue fluorescent protein, aequorin (aeguorin) and the like can also be used as modifying substances. It is very useful for gene analysis, especially in situ hybridization, to label and mark the nucleotide chain.

In addition, noble metal colloids such as gold colloids and silver colloids with amino groups formed on the surface, magnetic particles, polymer particles such as polystyrene beads, etc. can also be used as the modification substance of the nucleotide chain.

When immobilizing a nucleotide chain on a noble metal, a compound having an amino group among thiol compounds collectively called alkanethiols can be used as a modifying substance. Since nucleotide chains can be immobilized on noble metals by modifying substances, genetic analysis can be performed by electrochemical measurement methods using noble metals as substrates, crystal oscillator microbalance methods, and surface plasmon resonance methods.

The alkanethiol can be used without any particular limitation on the structure and the like as long as it has a thiol group and an amino group bonded to a noble metal. The hydrocarbon residue between the amino group and the thiol group can be in various forms such as straight chain, branched chain, ring, allyl chain, aromatic ring, etc. The number of carbon atoms and the position of the amino group also vary. possible.

In substrate crystal resonator microbalance method, surface plasmon resonance method, etc., gold substrates are widely used, but depending on measurement methods, etc., platinum, silver, copper, palladium, indium, nickel, iron, aluminum, and their derivatives can also be used. alloy etc. as the substrate.

Modified substances can use silane coupling compounds with amino groups, thus, nucleotide chains can be immobilized on glass, silicon, silicon dioxide, alumina, mica, and polymer resins such as polystyrene, nylon, and epoxy resin. It can also be applied to various gene analysis methods in the past.

As long as the silane coupling compound is a compound having an amino group and an alkoxy group, it can be used without any particular limitation on the structure or the like, like the alkanethiol. Silane coupling compounds that can be used are, for example, γ-aminopropyltriethoxysilane, N-β(aminoethyl)-γ-aminopropylmethyldimethoxysilane, N-β(aminoethyl) - γ-aminopropyltrimethoxysilane, N-β(aminoethyl)-γ-aminopropyltriethoxysilane, γ-aminopropyltrimethoxysilane and the like.

Substances that do not originally have an amino group can also be modified in the nucleotide chain by using an appropriate spacer group (spacer) having an amino group. For example, a substance having a carboxyl group can modify a nucleotide chain by using a substance having a diamino structure such as 1,2-diaminoethane or 1,6-diaminohexane as a spacer.

Furthermore, a substance having a hydrazine group, an aminooxy group, or a cyano group, or a substance having a magnesium halide similar to a Grignard reagent can also be used as a modifying substance.

(Embodiment 2)

FIG. 4 shows the principle of the nucleotide chain modification method according to Embodiment 2 of the present invention, and FIG. 5 shows the chemical reaction formula of the reaction center part of the nucleotide chain in FIG. 4 .

In Fig. 4 and Fig. 5, N ₀ represents any base in adenine, guanine, thymine, cytosine, and N ₁ represents adenine, guanine, thymine, which forms a complementary base pair with N ₀ Any base in cytosine. N ₂ represents any base among adenine, guanine, thymine, and cytosine that does not form _a complementary base pair with N 0, and N ₃ represents adenine and hypoxanthine that form a complementary base pair with N ₂ , thymine, cytosine any base. C represents cytosine, Hx represents hypoxanthine, and these N ₀ , N ₁ , N ₂ , N ₃ , C, and Hx are all contained in 2'-deoxynucleotides (hereinafter referred to as nucleotides). N ₀ , N ₁ , N ₂ , and N ₃ only represent part, and the number is not limited.

dITP, dATP, dCTP, and dTTP represent 2'-deoxyinosine-5'-triphosphate, 2'-deoxyadenosine-5'-triphosphate, 2'-deoxycytidine-5 '-triphosphate, 2'-deoxythymidine-5'-triphosphate. NH ₂ -R represents any modified substance having an amino group as described above.

The nucleotide chain to be modified (2-1: indicates the number in FIG. 4 ; the same applies hereinafter) has a sequence of base _N0 in the 3' end region. The additionally obtained nucleotide strand (2-2) is annealed to this nucleotide strand (2-1) to form a double-stranded nucleotide. The nucleotide chain (2-2) has a sequence of any base N ₁ complementary to the base N ₀ of the 3' end region of the nucleotide chain (2-1) at the 3' end, and has a sequence near the 5' end at it. Cytosine C, followed by a sequence of any base _N2 that is not complementary to base _N0 , the 5' end region protrudes after annealing.

For the partially double-stranded nucleotides, DNA polymerase is used as a catalyst to incorporate dITP, dATP, dCTP, and dTTP to form complete double-stranded nucleotides. The nucleotide chain (2-3) that forms at this moment is that the 3' end of nucleotide chain (2-1) adds the nucleotide sequence that is complementary to nucleotide chain (2-2) single strand region Nucleotide chain with hypoxanthine Hx corresponding to cytosine C of nucleotide chain (2-2).

By acting on the formed complete double-stranded nucleotides with 3-methyladenine DNA glycosidase type II and performing alkaline heat treatment, the carbon atom at the 1' position of the nucleotide (deoxyribose) containing hypoxanthine Hx The glycosidic bond between the glycoside and the oxygen atom is specifically decomposed, and the 3' end of the nucleotide chain (2-4) thus formed has an aldehyde group.

Then, a modifying substance (NH ₂ -R) having an amino group is added to react each aldehyde group with the amino group to form a Schiff base. The nucleotide chain (2-5) formed at this time is the target product, and is a nucleotide chain in which the modifying substance is directly bonded to the 3' end of the starting material nucleotide chain (2-1).

After the modification, the unnecessary nucleotide chain (2-2) is separated and removed. The removal of the nucleotide chain (2-2) can be carried out by heat-treating the double-stranded nucleotide bound to the modified substance, or making the 5' end of the nucleotide chain (2-2) a phosphate group, so that its specificity Decomposition is easily performed by the action of lambda exonuclease. By substituting in advance the guanine portion of the bases N ₁ and N ₂ of the nucleotide chain (2-2) with hypoxanthine, it can be fragmented into small fragments by 3-methyladenine DNA glycosidase type II.

(Embodiment 3)

Fig. 6 shows the principle of the nucleotide chain modification method according to Embodiment 3 of the present invention. In this method, a nucleotide sequence containing hypoxanthine as a substrate of the above-mentioned 3-methyladenine DNA glycosidase type II enzyme is added to one of the nucleotide strands to be modified among double-stranded nucleotides .

In the figure, the meanings of N ₀ , N ₁ , N ₂ , C, Hx, 5' and 3' are the same as above, and N ₄ represents adenine, guanine, thymine, and cytosine that form a complementary base pair with N ₂ Any base in A, G, and T represent adenine, guanine, and thymine, respectively.

The double-stranded nucleotide is composed of a first nucleotide strand (2-11) and a second nucleotide strand (2-12), and the nucleotide strand (2-11) is the object of modification. The nucleotide chain (2-11) has a sequence of base N ₀ in the 3' end region, and the nucleotide chain (2-12) has a sequence of base N ₁ in the 5' end region.

The double-stranded nucleotides are dissociated into single strands by heat treatment or alkaline treatment, and the third nucleotide strand (2-13) is annealed to the dissociated nucleotide strand (2-11), forming a partial double-stranded nucleotides. The second nucleotide strand (2-13) has a sequence of any base N _{1 complementary to the base N 0 of} the 3' end region of the nucleotide chain (2-11) at the 3' end, and the sequence near the 5 The 'end has a sequence of cytosine C, followed by an arbitrary base _N2 not complementary to base _N0 , and after annealing, the 5' end from cytosine C becomes a single strand.

For the partially double-stranded nucleotides, DNA polymerase is used as a catalyst to incorporate dITP, dATP, dCTP, and dTTP to form complete double-stranded nucleotides. The nucleotide chain (2-14) that forms at this moment is that the 3' end of nucleotide chain (2-11) adds the complementary nucleotide sequence with nucleotide chain (2-2) single strand region The nucleotide chain has hypoxanthine Hx at the position corresponding to the cytosine C of the nucleotide chain (2-2).

The complete double-stranded nucleotide can be carried out by performing the same operation as Embodiment 2, so that the 3' end of the nucleotide chain (2-11) has an aldehyde group, which can be combined with a modification substance to remove unnecessary nuclei. nucleotide chain.

Here, the modification method is exemplified in the case where only one of the nucleotide chains is the object of modification, but it is understood that modification is also possible when both nucleotide chains are the object of modification.

(Embodiment 4)

Fig. 7 shows the principle of the nucleotide chain modification method according to Embodiment 4 of the present invention. This method is a nucleotide chain modification method under the condition that an appropriate restriction enzyme cleavage site exists on the double-stranded nucleotide chain.

In the figure, the meanings of N ₀ , N ₁ , A, G, T, C, and Hx are the same as those mentioned above, and N ₂ and N ₄ represent any one selected from adenine, guanine, thymine, cytosine, hypoxanthine, urine Bases that are complementary to bases such as pyrimidine.

The double-stranded nucleotide is composed of a first nucleotide strand (2-21) and a second nucleotide strand (2-22), and the nucleotide strand (2-21) is the object of modification. In the 3' end region of the nucleotide chain (2-21) (the 5' end region of the nucleotide chain (2-22)), there is a base sequence (GGATCC/CCTAGG) that can be recognized and cut by the restriction enzyme Bam HI ).

First, the double-stranded nucleotides are cut with Bam HI to form nucleotides with only G at the 3' end of N ₀ (2-23) and CCTAG nucleotides at the 5' end of N ₁ (2- 24) A partially double-stranded nucleotide.

For the partially double-stranded nucleotides, DNA polymerase is used as a catalyst to incorporate dITP, dATP, dCTP, and dTTP to form complete double-stranded nucleotides. The nucleotide chain (2-25) that forms at this moment is that the 3' end of nucleotide chain (2-23) adds the nucleotide sequence that is complementary to nucleotide chain (2-24) single-stranded region The nucleotide chain has hypoxanthine Hx at the position corresponding to the cytosine C of the nucleotide chain (2-24).

The complete double-stranded nucleotide can be carried out by carrying out the same operation as Embodiment 2, only adding 1 nucleotide to the 3' end of the nucleotide chain (2-21) and making it have an aldehyde group, making it Combine modifying substances to remove unwanted nucleotide strands.

(Embodiment 5)

Fig. 8 shows the principle of the nucleotide chain modification method according to Embodiment 5 of the present invention. In this method, double-stranded nucleotides in which hypoxanthine has previously been incorporated in the base sequence are ligated.

In the figure, the meanings of N ₀ , N ₁ , C, and Hx are the same as those described above, and N ₂ and N ₄ represent the interaction of any bases selected from adenine, guanine, thymine, cytosine, hypoxanthine, uracil, etc. complementary bases.

Double-stranded nucleotides are composed of a first nucleotide strand (2-31) and a second nucleotide strand (2-32), and the nucleotide strand (2-31) is the object of modification. The nucleotide chain (2-31) has a sequence of base N ₀ in the 3' end region, and the nucleotide chain (2-32) has a 3' sequence with the nucleotide chain (2-31) in the 5' end region. The terminal region forms a sequence of bases _N1 of complementary base pairs.

On this double-stranded nucleotide, another double-stranded nucleotide is ligated using DNA ligase as a catalyst. This additional double-stranded nucleotide uses a nucleotide chain (2-33) consisting of a sequence with base _N4 on both sides of hypoxanthine Hx and a sequence complementary to base _N4 on both sides of cytosine C A nucleotide chain composed of a nucleotide chain (2-34) of base _N2 , which is connected so that the 3' end of the nucleotide chain (2-31) to be modified is a nucleotide chain (2-33) . Thereby, form by nucleotide chain (2-35) (nucleotide chain (2-31) and nucleotide chain (2-33)) and nucleotide chain (2-36) (nucleotide chain (2-31) and nucleotide chain (2-33)) composed of double-stranded nucleotides.

The complete double-stranded nucleotide can be carried out by performing the same operation as Embodiment 2, so that the 3' end next to the _N0 sequence of the nucleotide chain (2-31) has an aldehyde group, so that it can be combined with a modified substance, Remove unwanted nucleotide strands.

In this Embodiment 5, the 5' end from the position of hypoxanthine Hx (and the _{3 '} end from the position of cytosine C) are made to have Nucleotide sequence, this part of nucleotide sequence can also be omitted.

However, if a sequence recognized by a restriction enzyme is formed at the junction, the efficiency of the ligation reaction by DNA ligase increases, so the 5' end starting at the position of hypoxanthine Hx (and the 3' end starting at the position of cytosine C end) so that it has a nucleotide sequence corresponding to 3 to 4 base pairs.

For example, the sequence of the base N ₀ at the 3' end of the nucleotide chain (2-31) and the sequence of the base N ₁ at the 5' end of the nucleotide chain (2-32) are in the case of the following sequence (I), if The 5' end starting from the position of hypoxanthine Hx in the nucleotide chain (2-33) and the 3' end starting from the position of cytosine C in the nucleotide chain (2-34) have the following sequence (II), Sequence (III) is formed at the junction, and since this sequence (III) is a sequence recognized and cut by the restriction enzyme Hinc II, the ligation reaction proceeds efficiently.

··GTC ^{3-5 -} GAC····GTCGAC··

··CAG 5 ^- 3 ^- CTG····CAGCTG··

(I) (II) (III)

The ends of the two sets of double-stranded nucleotides connected can be the blunt ends of the above-mentioned nucleotide chains, or sticky ends protruding from a nucleotide chain at the cutting site of Bam HI.

Such cleavage by restriction enzymes and ligation by DNA ligase have been conventionally used for insertion into gene vectors and the like. By using the method of the present invention at the same time, insertion into a gene (nucleotide chain) carrier, etc., and modification by a modifying substance can be realized under the same reaction conditions, and it is possible to avoid wasting time amplified from a small amount of sample by PCR method, etc. Precious nucleotide chain samples are used efficiently. For example, a pET-like vector (Studier, F. et al., Methods Enzymol, Vol. 185, p. 60) as a gene expression vector has a Bam HI site at its gene insertion site, so it can be The method realizes the modification of the nucleotide chain and the insertion into the vector at the same time.

The length of the nucleotide chain to be modified is at least 3 or more, but the sequence of the nucleotide chain must be limited for gene analysis, so the chain length of the nucleotide chain is preferably at least 10 or more.

The nucleotide sequence provided at the 3' end of hypoxanthine Hx may correspond to a chain length of at least 1 base pair when using the methods of Embodiment 2 and Embodiment 3, but when using Embodiment 4 and Embodiment In the case of the method of Embodiment 5, the chain length is preferably at least 2 to 4 base pairs. This is because a chain length slightly longer than the sequence region recognized by each enzyme is required in order for the restriction enzyme and the ligase to function sufficiently.

In the above-mentioned various embodiments, the method of adding a nucleotide sequence containing hypoxanthine Hx and using the nucleotide sequence containing hypoxanthine Hx as an enzyme substrate with 3-methyladenine DNA glycosidase type II is exemplified. , but not limited to this. Combinations of nucleotides containing bases in Table 1 (shown above) at the 3' end and DNA glycosidases can similarly modify the 3' end of the nucleotide chain with a target modifying substance.

Hereinafter, the present invention will be further described in detail by citing specific examples.

(Example 1)

As the nucleotide chain to be modified, a single-stranded chemically synthesized nucleotide chain having 30 nucleotides (synthesized by commissioning Sigma-Genosya) was used. The nucleotide chain encodes the 8th to 37th positions of the Escherichia coli 16S ribosomal nucleic acid gene, and has the following base sequence from the 5' end. A, T, G, and C represent the bases adenine, thymine, guanine, and cytosine in deoxynucleotides, respectively.

AGAGTTTGATCATGGCTCAGATTGAACGCT (Sequence 1)

First, 2 units (hereinafter abbreviated as u)/μl terminal deoxynucleotidyl transferase (manufactured by Invitrogen), 10 mM 2'-deoxyinosine-5' -in 50 μl of reaction solution of triphosphoric acid, 2mM cobalt chloride, 1mM dithiothreitol, and 0.1M potassium cacodylate (pH 7.2), reacted at 37°C for 4 hours, and the hypoxanthine base The nucleotide sequence is tailed to the 3' end of the nucleotide chain.

The nucleotide chain was recovered by ethanol precipitation, and 100 μM oligodeoxycytosine (nucleotide number 18, manufactured by New England BioLabs) was added to anneal to the nucleotide tailed to the 3' end of the nucleotide chain on the hypoxanthine base of the sequence.

Next, the nucleotide chain was mixed with 0.3u/μl 3-methyladenine DNA glycosylase type II (manufactured by Trevigen), 1mM ethylenediaminetetraacetic acid, 1mM glycol ether diaminetetraacetic acid, 1mM In the reaction solution of thiothreitol, 10mm 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid-potassium hydroxide (pH7.4), react at 37°C for a day and night, and the Glycosidic bond cleavage of a nucleotide sequence tailed at the 3' end of a nucleotide chain.

Add 0.1M sodium hydroxide to the reaction solution and heat at 100°C for 5 minutes to dissociate the remaining phosphate group at the 3' end of the nucleotide chain and form an aldehyde group at the 3' end.

The nucleotide chain was recovered by ethanol precipitation, mixed with 5 mM fluorescein cadaverine (manufactured by Molecular Probes) under the condition of 0.1 M sodium carbonate (pH 9.5) to make the reaction solution volume 50 μl, and reacted at room temperature for 2 hours , to form a Schiff base between the aldehyde group at the 3' end of the nucleotide chain and the amino group present in fluorescein cadaverine. Then, sodium borohydride was added at 1 mg/ml so that the volume of the reaction solution was 100 μl, and the reaction was carried out at 4° C. for a whole day and night to reduce the Schiff base and stabilize the binding.

Figure 9 shows the results of tracking this modification process by 7-molar urea-polyacrylamide gel electrophoresis (Molecular Cloning, 2nd edition, p. 12.23, 1989). Numbers and arrows on the left side of the figure indicate electrophoretic positions of nucleotide chains having respective base numbers. The detection of the nucleotide chain was carried out according to the silver staining method (Electrophoresis, Vol. 4, p. 92, 1983).

Lanes A, B and C represent molecular weight markers, oligocytosine and untreated nucleotide chains, respectively. Swimming lane D shows the nucleotide chain with the nucleotide sequence of hypoxanthine added to the 3' end, and the number of nucleotides is increased to more than 100. Lane E represents the nucleotide chain to which oligodeoxycytosine annealed. Lane F represents the chain of nucleotides forming an aldehyde group at the 3' end, with the number of nucleotides reduced to 30. Lane G represents the 3' end modified and stabilized nucleotide chain with fluorescein cadaverine.

In lane D, no tailing of nucleotides with hypoxanthine was found, but some unreacted nucleotide chains remained, which may be due to the prolongation of tailing reaction time, terminal deoxynucleotidyl transferase As the amount of addition increases, the nucleotide chain becomes easily digested.

Adenine, guanine, and cytosine, which are the nucleotide chains to be modified, also have amino groups and may react inside the nucleotide chain to self-polymerize, but as shown in lane F, self-polymerization did not occur. However, if necessary, the amino group of the nucleotide chain can be protected with an appropriate protecting group such as benzoyl group and isobutyryl group used in the chemical synthesis of the nucleotide chain.

(Example 2)

The nucleotide chains in each process of electrophoresis in Example 1 were blotted onto a nylon membrane (manufactured by Schleicher & Schuell) according to Southern's method (J.Mol.Biol. Vol. 98, p. 503, 1975) .

The anti-fluorescein antibody (manufactured by Amersham Biosciences) labeled with horseradish peroxidase was added dropwise to the nucleotide chain on the nylon membrane, and the negative film (manufactured by Amersham Biosciences) was made by chemiluminescence (manufactured by Amersham Biosciences, ECL detection reagent). Manufactured by the company, Hyperfilm ECL) was exposed to detect the presence or absence of fluorescein. The results are shown in Figure 10. The numbers and arrows on the left side of the figure are the same as those in Fig. 9, and indicate the positions of nucleotide chains having respective base numbers.

Lane A represents molecular weight markers. For this labeling, fluorescein-labeled 2',3'-dideoxyuridine-5'-triphosphate (manufactured by EnzoDiagnostics) tailed with terminal deoxynucleotidyl transferase was used.

No luminescence was detected in lanes B to F, and it was found that the nucleotide chain was not modified with fluorescein.

In lane G, the luminescence of the nucleotide chain having 30 nucleotides was detected, and it can be known that the modified nucleotide chain was indeed modified by fluorescein cadaverine.

In lane G, oligodeoxycytosine with nucleotide number 18 was also modified with fluorescein cadaverine. This is because the nucleotide chain of the tailed hypoxanthine is partially annealed with oligodeoxycytosine at random, there is no double-strand formation locally, and there is a place where 3-methyladenine DNA glycosidase type II does not act. Moreover, oligodeoxyhypoxanthine with a small number of nucleotides is thus generated, and its 3' end forms an aldehyde group, and fluorescein cadaverine modifies its aldehyde group, and in these reactions oligodeoxyhypoxanthine and oligo Polydeoxycytosine is then annealed. However, the unwanted oligodeoxycytosine is mixed with oligodeoxyhypoxanthine, oligodeoxyhypoxanthine, oligodeoxycytosine, etc. Guanine or annealed oligonucleotides obtained by mixing them can be easily separated and removed.

Above, as shown in Example 1 and Example 2, according to the method of the present invention, the 3' end of the nucleotide chain can be directly modified with a modifying substance.

The structures and chain lengths of nucleotide chains, modifying substances, reaction conditions, and reaction compositions shown in the above-mentioned embodiments are just examples, not limited thereto, and can be changed arbitrarily.

(Example 3)

As the nucleotide chain to be modified, the cDNA sequence corresponding to human skeletal muscle myosin heavy chain 1 at positions 5831-5850 (the GenBank website of the National Center for Biotechnology Information (http://www.ncbi.nlm. nih.gov/Genbank/index.html), accession number: NM_005963, as of February 2004) single-stranded deoxynucleotide chain with 20 nucleotides (synthesized by Sigma-Genosys). As the nucleotide chain complementary to this nucleotide chain, a single-stranded deoxynucleotide chain having 29 nucleotides (synthesized by commissioning Sigma-Genosys) was used. Aminooxymethylcarbonylhydrazone-Cy5 was used as the modifying substance. Each nucleotide chain has the following base sequence.

modified nucleotide chain

TGCTG AAAGG TGACC AAAGA (Sequence 2)

complementary nucleotide chain

ATCGGATCCTCTTTGGTCACCTTTCAGCA (Sequence 3)

step 1

Suspend the modified nucleotide chain (hereinafter referred to as MYH1) and complementary nucleotide chain (hereinafter referred to as MYH1-Bam HI) in 50 mM sodium chloride, 10 mM magnesium chloride, 1mM dithiothreitol, 10mM Tris-HCl (pH 7.5) buffer solution (100μl total solution), heated at 65°C for 10 minutes, then cooled to room temperature for more than 30 minutes to form 5 of cMYH1-Bam HI ' region (CTTTCAGCA) protruding double strand.

step 2

The formed double-stranded nucleotides were recovered by ethanol precipitation, and were added to a solution of 10mM dITP, 10mM dATP, 10mM dTTP, 10mM dCTP, 50mM sodium chloride, 20mM magnesium chloride, 40mM Tris-HCl (pH 7.5), according to the final Add T7 DNA polymerase modified enzyme シ-ケネ-ス ver.2.0 (USB company) (total solution 100 μl) at a concentration of 0.5u/μl, react at 37°C for 4 hours, add MYH1- The nucleotide sequence of hypoxanthine complementary to Bam HI forms a complete double-stranded nucleotide sequence of 29 base pairs. The MYH1 formed at this time is represented by (Sequence 1)-HxHxATCCHxAT.

step 3

The formed double-stranded nucleotides were recovered by ethanol precipitation by adding 1mM ethylenediaminetetraacetic acid, 1mM ethylene glycol-bis(2-aminoethyl ether)tetraacetic acid (=ethylene glycol ether diaminetetraacetic acid), 1mM In the solution of dithiothreitol and 10mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid-potassium hydroxide (pH 7.4), add 3-methyladenine according to the final concentration of 0.2u/μl DNA glycosidase II (Trevigen Company) (100 μl total solution), reacted at 37 ° C for a day and night, hydrolyzed the glycosidic bond of the deoxyribose with hypoxanthine, and made the 3' end of MYH1 bear an aldehyde group. This MYH1 is represented by (SEQ ID NO: 1)-CHO.

step 4

Double-stranded nucleotides with an aldehyde group at the end of MYH1 were added to a solution of 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid-potassium hydroxide (pH 7.2) at a final concentration of 240 μM Aminooxymethylcarbonylhydrazone-Cy5 (100 μl total solution) was added as a modification substance, and reacted at room temperature for a day and night, so that a Schiff base was formed between the aldehyde group at the 3' end of MYH1 and the amino group of Cy5. This MYH1 is represented by (SEQ ID NO: 1)-CHHNOCHNHNH-Cy5.

The results of tracking the nucleotide chains in each step with 16% polyacrylamide gel electrophoresis (90mM Tris-boric acid·2mM EDTA buffer) are shown in Figure 11. Nucleotide chains were stained with SYBR Gold (Molecular Probes). M in the figure represents a molecular weight marker, and 20bp, 30bp, 50bp, and 100bp represent the electrophoretic positions of double-stranded nucleotides of 20 base pairs, 30 base pairs, 50 base pairs, and 100 base pairs, respectively.

The band in lane 1 represents the modified nucleotide chain (MYH1); the band in lane 2 represents the complementary nucleotide chain (MYH1-Bam HI); the band in lane 3 appears around 30 base pairs Indicates the partially double-stranded nucleotides formed in step 1; the band moving slightly slower in lane 4 compared with lane 3 indicates the complete double-stranded nucleotides of 29 base pairs formed in step 2; The band moving slightly faster in Lane 5 than in Lane 4 indicates double-stranded nucleotides with aldehyde groups on MYH1; the band in Lane 6 indicates double-stranded nucleotides in MYH1 modified by modifying substances.

No significant difference in moving speed was found between lane 5 and lane 6, and the double-stranded nucleotides in each lane after electrophoresis were analyzed according to Southern's method (J.Mol.Biol. Vol. 98, p. 503, 1975) Blot onto a nylon membrane (Schleicher & Schuell Company), use horseradish peroxidase-labeled anti-cyanine antibody (Rockland Company), and use a negative film (trade name Hyperfilm ECL, Amersham Biosciences Company) to investigate the chemiluminescence caused by the luminol reaction With or without, only the double-stranded nucleotides in lane 6 expose the film (lane 6'). As a result, the horseradish peroxidase-labeled anti-cyanine antibody was bound to the double-stranded nucleotides in lane 6, indicating that the aminooxymethylcarbonylhydrazone-Cy5 to which the antibody can bind was bound to MYH1.

Aminooxymethylcarbonylhydrazone-Cy5 (cyanine pigment) as a modified substance, with Cy5-hydrazine (Amersham Biosciences Company) as an initial substance, according to Ide et al. (Ide et al., Biochemistry, volume 32, page 8276 , 1993) and the methods of Gruber et al. (Gruber et al., Bioconjugate Chemistry, Vol. 11, p. 161, 2000), synthesize and purify as follows.

1 mM Cy5-hydrazine and 3 mM tert-butoxycarbonylaminooxyacetic acid (t-Boc-NH-O-CH ₂ COOH) (Fluka) in 3 mM N, N'-dicyclohexylcarbodiimide (DCC) In the presence of 3mM 1-hydroxyl-1H-benzothiazole (HOBt) (solution total amount 300μl), react at 4°C for one day and night to make condensation, after concentrating the reaction mixture, add 50% trifluoroacetic acid (TFA ) (2000 μl), reacted at room temperature for 1 hour, the tert-butoxycarbonyl (t-Boc) of the condensate was cut off, and finally the reactant mixture was treated with 5% N, N'-diisopropylethylamine (DIPEA ) and purified by a C ₁₈ silica gel column to obtain the target compound. The yield was 13.4%.

As mentioned above, according to the present invention, the 3' end of one or two nucleotide chains selected from single-stranded nucleotides and double-stranded nucleotides can be directly used with modifying substances. Modifications for irrelevant cases. Decomposition of the main chain part can be avoided by selecting a base that does not exist in the main chain part, such as a base that does not originally exist as a gene and is not incorporated into the nucleotide chain during PCR amplification, etc., as a specific base . The method of the present invention can directly and easily quantify the 3' end of the nucleotide chain with any modification substance without considering the base structure and chain length of the nucleotide chain (main chain part). Modified steadily and stably.

Therefore, it is possible to mark and mark only the nucleotide chains having the target gene information, and at the same time, when the immobilization of the nucleotide chains is the purpose, the modification substance can be used as a linking substance for stable and firm immobilization. Immobilization also prevents suspicious results from genetic analyses.

Possibility of industrial use

The nucleotide chain modification method of the present invention can quantitatively and stably modify the 3' end of the nucleotide chain as the object of modification, regardless of whether the nucleotide chain is single-stranded or double-stranded, with any modifying substance. For gene analysis that requires labeling, labeling, immobilization, etc. of nucleotide chains.

sequence listing

<110> Matsushita Electric Industrial Co., Ltd. (MATSUSHITA ELECTRIC INDUSTRIAL CO., LTD.)

<120> Nucleotide chain modification method

<130>pct4020

<150> JP 2004-187133

<151>2004-06-25

<150> JP 2003-186151

<151>2003-06-30

<160>3

<170>PatentIn version 3.1

<210>1

<211>30

<212>DNA

<213> Artificial

<220>

<223> Bases 8-37 of the 16S rRNA gene encoding Escherichia coli

<400>1

agagtttgat catggctcag attgaacgct 30

<210>2

<211>20

<212>DNA

<213> Artificial

<220>

<223> Oligodeoxynucleotide encoding human mature myosin heavy chain 1 position 5831-5850

<400>2

tgctgaaagg tgaccaaaga 20

<210>3

<211>29

<212>DNA

<213> Artificial

<220>

<223>Sequence complementary to position 5850-5831 of human mature myosin heavy chain 1, plus 9 nucleotides recognized by Bam HI restriction endonuclease at the 5' end

<400>3

atcggatcct ctttggtcac ctttcagca 29

Claims (18)

1. method of modifying nucleotide chain, wherein, exist the conduct of nucleotide sequence to modify the nucleotide chain of object to 3 ' end with particular bases, with the lytic enzyme effect special to the Nucleotide that contains described particular bases, add the functional group that target modification material is had binding ability at described 3 ' end, and hold in conjunction with described modification material at 3 ' of the nucleotide chain with described functional group as the nucleotide chain of modifying object.

2. method of modifying nucleotide chain as claimed in claim 1, its feature also be, the nucleotide sequence with particular bases is positioned at 3 ' end as the nucleotide chain of main chain, and described particular bases is the base that is not present in the described main chain.

3. method of modifying nucleotide chain as claimed in claim 1 or 2, its feature also be, adds the nucleotide sequence with particular bases to as the nucleotide chain of main chain 3 ' end.

4. method of modifying nucleotide chain as claimed in claim 3, its feature also are, the nucleotide sequence with particular bases is added by the tailing method.

5. method of modifying nucleotide chain as claimed in claim 3, its feature also is, to have with the nucleotide chain annealed combination of this nucleotide chain 3 ' end regions complementary sequence at 3 ' end regions and modify on the strand nucleotide chain of object to conduct, thereby at the described complementary nucleotide sequence that contains particular bases as 3 ' end interpolation of the nucleotide chain of modifying object.

6. method of modifying nucleotide chain as claimed in claim 3, its feature also is, with the double chain nucleotide of at least one nucleotide chain as the modification object, with Restriction Enzyme that can specific recognition as the base sequence of 3 ' end regions of the nucleotide chain of modifying object is sheared, thereby add the complementary nucleotide sequence that contains particular bases at the 3 ' end that the nucleotide chain of object is modified in the conduct of having sheared.

7. method of modifying nucleotide chain as claimed in claim 3, its feature also is, 3 ' end of the nucleotide chain of object is modified at least one conduct in double chain nucleotide, to connect with dna ligase at the double chain nucleotide that 5 ' end regions has a nucleotide sequence that contains particular bases, by described particular bases to 3 to the nucleotide chain of this connection ' base sequence of end shears with the Restriction Enzyme of specific recognition, thereby adds the nucleotide sequence that contains particular bases at described 3 ' end as the nucleotide chain of modifying object.

8. as claim 5 or 6 described method of modifying nucleotide chain, its feature is that also the interpolation of nucleotide sequence is undertaken by using archaeal dna polymerase to mix Nucleotide.

9. method of modifying nucleotide chain as claimed in claim 1, its feature are that also having the nucleotide chain of the nucleotide sequence with particular bases is the nucleotide chain of chemosynthesis.

10. method of modifying nucleotide chain as claimed in claim 1, its feature also are, before the lytic enzyme effect, add the oligonucleotide that has with particular bases complementary base sequence.

11. method of modifying nucleotide chain as claimed in claim 1, its feature are that also making it is aldehyde radical in the functional group that 3 ' end as the nucleotide chain of modifying object has.

12. method of modifying nucleotide chain as claimed in claim 1, its feature also are, make 3 ' end as the nucleotide chain of modifying object have aldehyde radical as lytic enzyme the DNA Glycosylase.

13. method of modifying nucleotide chain as claimed in claim 1, its feature are that also particular bases is an xanthoglobulin, as lytic enzyme, make 3 ' end as the nucleotide chain of modifying object have aldehyde radical 3-methyladenine DNA Glycosylase.

14. method of modifying nucleotide chain as claimed in claim 1, its feature also are, modify material and have the amino that the functional group that has with nucleotide chain 3 ' end forms chemical bond.

15. as claim 1 or 14 described method of modifying nucleotide chain, its feature is that also modifying material is the material that makes nucleotide chain marking, signization.

16. method of modifying nucleotide chain as claimed in claim 15, its feature are that also modifying material is fluorescent substance, VITAMIN, lipid, amino acid, oligopeptides, protein or living body exogenous material.

17. as claim 1 or 14 described method of modifying nucleotide chain, its feature is that also modifying material is to have and the material that is used for the substrate bonded ability of genetic analysis.

18. method of modifying nucleotide chain as claimed in claim 17, its feature are that also modifying material is amino alkane mercaptan or aminosilane coupling compound.

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