JPH1014572A - Probe library, its production and purification of probe hybridized from the same with another dna or rna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a probe library capable of corresponding to mutation of a virus to be detected by polymerizing one of deoxyribonucleotides, including deoxyribonucleotide analogs, in the presence of a protein with no template nor primer. SOLUTION: This probe library having diversity sufficiently capable of corresponding to mutation of a virus to be detected is obtained by polymerizing one of deoxyribonucleotides, including deoxyribonucleotide analogs, such as 1,N<6> -esenodeoxy adenosine-5'-triphosphate, fluorescein-modified deoxyuridine-5'- triphosphate, biotin-modified deoxyuridine-5'-triphosphate, biotin-modified dioxyadenosine-5'-triphosphate, digoxigenin-modified deoxyuridine-5'-triphosphate, and dinitrophenyl-modified deoxyuridine-5'-triphosphate, in the presence of a protein composed of thermostable DNA polymerase originating from cells which belong to Cellumococcus or Cellumus bacteria, with no template nor primers.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing a DNA (polydeoxyribonucleotide; hereinafter the same) probe library containing a deoxyribonucleotide analog, a DNA probe library obtained by the method, and the DNA probe library. For purifying a DNA probe that forms a hybrid with another DNA or RNA from DNA, a method for producing a DNA probe obtained by the method, a method for producing an RNA probe library containing a ribonucleotide analog, and an RNA probe library obtained by the method , Other R from the RNA probe library
The present invention relates to a method for purifying an RNA probe that forms a hybrid with NA or DNA, and an RNA probe obtained by the method. Specifically, in the presence of a protein, a deoxyribonucleotide and an analog thereof can be used independently of a template and / or a primer. The present invention relates to a method for synthesizing a DNA containing the analog by polymerization.

DNA or RN synthesized according to the present invention
A (In the following description, "DNA or RNA"
For convenience, it is abbreviated as “polynucleotide”. ) Makes it possible to provide non-radioactive probes useful in fields such as medicine, pharmacy, fermentation engineering, agriculture, agricultural chemistry, biochemistry and the like.

[0003]

BACKGROUND OF THE INVENTION In recent years, genetic engineering has made remarkable progress. Medicine, agriculture,
In the field of fermentation engineering and the like, many techniques using this genetic engineering have been developed. For example, gene diagnosis and gene therapy in the medical field are typical examples.

[0004] Here, genetic diagnosis will be described. The following methods are currently used. That is, (1) a "probe" which is a short polynucleotide having a base sequence complementary to a part of the base sequence of a gene to be detected is chemically modified with biotin or the like or fluorescently labeled with fluorescein or the like. For example, a fluorescence emission in situ hybridization method (FIS)
H method, Hori et al., Laboratory Manual Human Genome Mapping, Maruzen, 1991), Southern Blot method (Ichino,
Cell Engineering Experiment Protocol, Shujunsha, 1992)
And Northern Blot method (Ichino, Cell Engineering Experiment Protocol, Shujunsha, published in 1992) and the like. (2) Polymerase chain reaction method using primers for the upstream and downstream nucleotide sequences of the gene to be detected [PCR method, Siki et al., Science
e), 239, 487-491 (1988
Year)] and amplifying the base sequence of the gene and performing electrophoresis for detection.

[0005] Genetic diagnosis using these techniques can be used only for those whose base sequence has already been determined. In other words, it is necessary to determine the nucleotide sequence of the gene to be detected and to recognize it before performing the genetic diagnosis.

[0006] For this reason, genetic diagnosis of infectious diseases in which the pathogen gene mutation is small is not so problematic. For example, human immunodeficiency virus (hereinafter referred to as "HI
V) (abbreviation), there is a problem that once a mutation occurs in the virus, the probe or primer before the mutation can no longer be used. Since determination of the base sequence generally requires a lot of time and a large cost, the inability to use the developed probe or primer causes a great loss.

[0007] Therefore, although it is desired to develop probes and primers having a variety of hybridization ability to sufficiently cope with gene mutation, unfortunately, they are synthesized by conventional techniques. Since the nucleotide sequence of a polynucleotide is uniquely determined by a coexisting template, it is necessary to simultaneously produce molecular groups of polynucleotides having various nucleotide sequences (that is, a DNA probe library having diversity, RNA Producing a probe library) was not possible in principle.

Hereinafter, further explanation will be given. That is, D
Not to mention NA, the sugar component of D-deoxyribose and adenine (A), guanine (G), cytosine (C),
A nucleotide having a base component of tisine (T) is a polynucleotide in which a single linear polymer is formed by a phosphodiester bond, and the biosynthesis of this DNA is performed by dATP, dG
DNA polymerase and reverse transcriptase (in the same order) in a template-dependent (template-complementary) manner for DNA and RNA using four types of nucleotide triphosphates, TP, dCTP and dTTP, as substrates.
It was done by. In addition, these reactions always required a "primer" which was an oligonucleotide that was complementary to a part of the template in advance. As described above, the base sequence of a DNA synthesized by a conventional method is mechanically determined by a template to be used.
A probe library could never be obtained at one time.

The same applies to RNA. That is,
In RNA, nucleotides having a sugar component of D-ribose and a base component such as adenine (A), guanine (G), cytosine (C) and uracil (U) form a single linear polymer by a phosphodiester bond. The formed polynucleotide, the biosynthesis of this RNA, ATP, GTP, CTP,
It is carried out by RNA polymerase in a DNA or RNA-dependent manner using a nucleotide triphosphate such as UTP as a substrate.
The reaction products are messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA
(RRNA) etc., all of which are DNA or RNA
The template was required in advance, and the reaction product was only RNA complementary to the template. In other words, it was impossible to synthesize RNA having sequence diversity depending on information of only the protein. Therefore, it was impossible to obtain a diverse RNA probe library at a time by conventional techniques.

[Object of the Invention] The present invention has been made in view of the above-mentioned circumstances, and its object is to provide, for example, a variety of polymorphs that can sufficiently cope with a case where a virus to be detected is mutated. In providing a nucleotide probe library, and a method for producing the same,
It is intended to provide a method for purifying a useful polynucleotide probe from the library, and a purified probe.

[0011]

According to a first aspect of the present invention, there is provided a method for producing a probe library, comprising polymerizing a deoxyribonucleotide containing a deoxyribonucleotide analog in the absence of a template and / or a primer and in the presence of a protein. It is a method characterized by the following.

[0012] The method for producing a probe library according to claim 2 is a method for producing a probe library in the absence of a template and / or a primer.
A method characterized by polymerizing a ribonucleotide containing a ribonucleotide analog in the presence of a protein.

[0013] A method for producing a probe library according to claim 3 is the method according to claim 1 or 2, wherein the protein is a thermostable DNA polymerase.

According to a fourth aspect of the present invention, in the method for producing a probe library according to the third aspect, the heat-resistant D
The method is characterized in that the NA polymerase is at least one selected from the group consisting of bacterial DNA polymerases belonging to the genus Sermococcus and the genus Serumus.

[0015] A method for producing a probe library according to a fifth aspect is characterized in that, in the method according to any one of the first to fourth aspects, the polymerization is carried out at a temperature of about 20 to 90 ° C. Is the way.

According to a sixth aspect of the present invention, there is provided a method for producing a probe library according to any one of the first to fifth aspects, wherein the polymerization is carried out under neutral to weak alkaline conditions (pH of about 7 to about 5).
A method characterized in that the method is performed under the condition of 10).

The method for producing a probe library according to claim 7 is the method according to claim 1, wherein the deoxyribonucleotide analog is an analog having at least a different base.

The method for producing a probe library according to claim 8 is the method according to claim 7, wherein the deoxyribonucleotide analog is 1, N ⁶ -ethenodeoxyadenosine-5'-triphosphate, fluorescein-modified deoxyribonucleotide. Uridine-5'-triphosphate, biotin-modified deoxyuridine-5'-triphosphate, biotin-modified deoxyadenosine-5'-triphosphate, digoxigenin-modified deoxyuridine-5'-triphosphate, and dinitrophenyl-modified deoxyuridine-5 ' -At least one selected from the group consisting of triphosphoric acid.

A method for producing a probe library according to claim 9 is the method according to claim 2, wherein the ribonucleotide analog is an analog having at least a different base.

[0020] The method for producing a probe library according to claim 10 is the method according to claim 9, wherein the ribonucleotide analog is 1, N ⁶ -ethenoadenosine-5 '.
Triphosphate, fluorescein-modified uridine-5'-triphosphate, biotin-modified uridine-5'-triphosphate, biotin-modified adenosine-5'-triphosphate, digoxigenin-modified uridine-5'-triphosphate, and dinitrophenyl-modified uridine At least one selected from the group consisting of 5'-triphosphoric acid
A method characterized by being a seed.

[0021] The probe library according to the eleventh aspect is obtained by the production method according to any one of the first to tenth aspects.

[0022] The method for purifying a probe according to claim 12 is as follows:
Other DNA from the probe library according to claim 11.
Alternatively, a method for purifying a probe capable of forming a hybrid with RNA, which is performed by using affinity chromatography in which another DNA or RNA is covalently bound as a ligand.

[0023] The method for purifying a probe according to claim 13 is as follows:
13. The method according to claim 12, wherein the other DNA or RN is used.
A is a method characterized in that A is derived from a virus.

[0024] The method for purifying a probe according to claim 14 is as follows:
14. The method of claim 13, wherein the virus is a human immunodeficiency virus.

The probe according to claim 15 is the probe according to claim 12
15. It has the ability to form a hybrid with another DNA or RNA, which is purified by the method according to any one of the above items 14 to 14.

[0026]

DETAILED DESCRIPTION OF THE INVENTION The present inventors have made various discoveries in the study of the synthesis of polynucleotides containing nucleobase analogs. That is, in a reaction system in which a template and a primer are not present, a protein, for example, a thermostable DNA polymerase, specifically, Sermococcus litoralis ( The The
romococus litralis ) DNA polymerase (trade name: Vent (Vent) DNA polymerase, New England Bio-Raves, hereinafter)
" Tli DNA polymerase") at pH 7
Or, in the case of DNA synthesis under weak alkaline conditions of 10 or less, deoxyribonucleotide triphosphate (dATP,
dCTP, dGTP, dTTP) and nucleic acid analogs as substrates, or in the case of RNA synthesis, ribonucleotide triphosphates (ATP, CTP, GTP, UTP) and nucleic acid analogs as substrates and the polymerase is not inactivated. By reacting at an elevated temperature of 20 ° C. or more for 1 hour or more, DNA (hereinafter abbreviated as “NTPA-DNA”) or RNA (hereinafter “NTPA-DNA”) containing a nucleic acid analog even in a reaction system in which a template and a primer are not present. RNA "(abbreviated as" RNA ").

The “NTPA-DN” synthesized according to the present invention
"A or NTPA-RNA", that is, "NTPA-polynucleotide" includes a large number of polynucleotides having various base sequences. Thereby, the NTP
It can be said that A-polynucleotide is rich in its hybridization forming ability. In other words, it can be said that hybridization has diversity. By using such an NTPA-polynucleotide, for example, when detecting a certain kind of virus, the operation can be simplified and the required time can be drastically shortened. That is,
When a suspected virus is detected from a virus-infected cell, the DNA or RNA base sequence of the virus is first determined. Determining the nucleotide sequence requires a complicated operation and a lot of cost, but once the nucleotide sequence has been determined, a DNA probe that hybridizes to the nucleotide sequence is prepared.
Virus detection was performed using the probe.

However, by using the NTPA-polynucleotide of the present invention, the RNA of the suspected virus
Need not be determined in advance. For example, when using affinity chromatography,
An affinity chromatography carrier to which the virus RNA is covalently bound as a ligand is used as a carrier, and an NTPA-polynucleotide is flowed on the carrier.
In the NTPA-polynucleotide, the viral RNA
If a polynucleotide probe that hybridizes with is contained, both bind at this point (hybridize). Thereafter, by passing a polynucleotide elution buffer solution secondarily, the polynucleotide probe which has hybridized up to that time can be eluted and collected. The recovered polynucleotide probe should, of course, hybridize with the virus RNA or DNA and the DNA derived from the virus introduced into the chromosome of the cell infected with the virus. It can be used as

As described above, according to the present invention, there is no need to previously determine the base sequence of the virus RNA or DNA to be detected and to recognize the base sequence by the operator, as in the prior art. Detection is simplified and the processing time is drastically reduced.

The reduction in the time required for virus detection is significantly different from the reduction in the processing time for the detection of other biochemicals (for example, enzymes and hormones).
The effect is enormous. Because in the case of a virus,
There are many "mutations", that is, changes in the base sequence, indicating that the detection of the virus is 1
Because it is better to be quick in minutes and it is not permissible to spend time unnecessarily.

The nucleic acid analogs (specific D
Detection or quantification of NA or RNA, or various DNA
To provide DNA probes and RNA probes used to determine the affinity between various RNAs, etc.
The analog of the nucleic acid used for imparting the labeling property to DNA or RNA is not particularly limited, and in the case of producing a DNA probe library, for example, 1, N ⁶
- Essey Bruno deoxyadenosine-5'-triphosphate, fluorescent-labeled deoxyribonucleotide triphosphates, chemically modified deoxyribonucleotide triphosphates such (more specifically, 1, N ⁶ -
Ethenodeoxyadenosine-5'-triphosphate, fluorescein-modified deoxyuridine-5'-triphosphate, biotin-modified deoxyuridine-5'-triphosphate, biotin-modified deoxyadenosine-5'-triphosphate, digoxigenin-modified deoxyuridine-5 '-Triphosphate, and dinitrophenyl-modified deoxyuridine-5'-triphosphate and the like. In the case of producing an RNA probe library, for example, 1, N ⁶ -ethenoadenosine-5′-triphosphate, fluorescently labeled ribonucleotide triphosphate, chemically modified ribonucleotide triphosphate, and the like (more specifically, 1, ^{N 6} - Essey Bruno adenosine-5'-triphosphate, fluorescein modified uridine-5'-triphosphate, biotin-modified uridine-5'-triphosphate, biotin-modified 5'-triphosphate, digoxigenin-modified uridine -5 '-Triphosphate, and dinitrophenyl-modified uridine-5'-triphosphate and the like.
It is sufficient for these nucleic acid analogs only to acquire the labeling property by the binding of the indicator substance to the base portion (although it functions sufficiently as a probe). And an analog having a different pentose (for example, as shown in [Formula 1] below, ribose (a
Nucleotide analogs containing arabinose (see b) and xylose (see c) in place of S (sulfur) (see FIG. 4d), in which O (oxygen) constituting the 5-membered ring is replaced by S (sulfur). Analogs) and / or analogs that also have different phosphate groups (eg, as shown in
Use of an analog in which O (oxygen) bonded to P (phosphorus) is replaced by S (sulfur) or another atom does not disturb the present invention (in addition, adenosine is exemplified as a base in [Chemical Formula 2]). However, the present invention is not limited to this, and is effective for guanine and cytosine).

[0032]

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One of these analogs may be used alone, or two or more thereof may be used in combination. The mixing ratio of the analog is not particularly limited, but is preferably 1 or more (molar ratio) based on 100 of the total amount of deoxyribonucleotides or ribonucleotides. The mixing ratio of the analog to the total amount of (deoxy) ribonucleotide 100 is 1
If the ratio is less than the above, there is a possibility that a problem that the labeling efficiency of the NTPA-polynucleotide is lowered and the probe cannot be used as a probe may occur.

In the polymerization of deoxyribonucleotides containing a deoxyribonucleotide analog, 1-
Formulation of (2'-deoxyribofuranosyl) -3-nitropyrrole, 1- (2'-deoxyribofuranosyl) -nitroindole, and polymerization of ribonucleotides containing ribonucleotide analogs resulted in 1- (2 '-
Ribofuranosyl) -3-nitropyrrole, 1- (2'-
The formulation of (ribofuranosyl) -nitroindole is also within the scope of the present invention. At the time of probe synthesis, the above compound (hereinafter referred to as “diversifying agent”) is replaced with (deoxy)
The diversification agent is introduced into the probe molecule to be synthesized by being incorporated into the reaction system together with the ribonucleotide analog and the (deoxy) ribonucleotide (that is, the diversification within the NTPA-polynucleotide molecule). Agent is incorporated). Once the diversification agent is incorporated, NTP
A-polynucleotides have reduced sequence specificity, which increases the diversity of the probe more than usual. Even if a probe screened for a certain gene is attached to a mutant form of the gene, it can be used as it is (for example, a certain H
Probes screened for IV-1 can also be used directly for mutated HIV-1). Accordingly, in addition to providing a probe library having more diversity than usual, it is possible to provide a probe that can be used for a mutant other than a specific gene.

When the above-mentioned diversifying agent is used, the addition amount thereof is preferably 5 to 80 (molar ratio) based on 100 of the total amount of deoxyribonucleotides or ribonucleotides. When the addition amount of the diversifying agent exceeds 80, the specificity of the probe itself is excessively reduced and noise increases, and the possibility of false positive increases, and when it is less than 5, the sequence specificity decreases so much. Without the above, the above-mentioned effects are hardly obtained.

It is preferable that the DNA polymerase does not inactivate even at a temperature of about 20 ° C. or more, preferably about 60 ° C. or more, and more preferably about 70 ° C. or more. For example,
Sermococcus litoralis listed above ( Thermoc
occus litoralis ) and other species of the genus Sermococcus, such as Cellus aquaticus ( Thermus)
aquaticus ), the same as Sermophilus ( T. th .
and DNA polymerases of bacteria belonging to the genus Cellmas, such as E. thermophilus .

One of the above DNA polymerases may be used alone, or a mixture of two or more (two, three,...) May be used. When two or more DNA polymerases are used in combination to polymerize deoxyribonucleotides or ribonucleotides, the polynucleotide synthesized at one time is more diversified (that is, the base sequence is more variable) than when one type is used alone. As a result, the content of the polynucleotide which can be a polynucleotide probe is increased. In this case, DNA polymerases of different bacteria belonging to the same genus may be used in combination, or DNA polymerases of bacteria belonging to different genus may be used.

The reaction temperature and time in the reaction of deoxyribonucleotide or ribonucleotide (both containing analogs) with DNA polymerase can be selected within a range that does not inactivate the DNA polymerase. By reacting under the temperature conditions in the range shown, the reaction can proceed promptly. For example, the reaction may be carried out at 74 ° C. for several hours.
A cycle of holding for 2 minutes, (2) holding for 2 minutes at 45 ° C., and (3) holding for 3 minutes at 74 ° C. may be repeated. The reaction is initiated after a short lag time initially and eventually reaches a maximum rate. It is considered that the polynucleotide synthesized by the method of the present invention does not depend on DNA or RNA to be used as a template or primer, but is synthesized depending on information of DNA polymerase present in the reaction system.

In the polymerization of (deoxy) ribonucleotide using DNA polymerase, the reaction temperature was 2
If the temperature is lower than 0 ° C., the reaction rate is reduced, and if it is higher than 90 ° C., the stability of the enzyme is lowered, which is not preferable. In addition, the reaction rate is lowered even under conditions deviating from weak alkaline conditions having a pH of about 7 or 10 or less, that is, under acidic conditions and strong alkaline conditions. The above reaction temperature and reaction system of a liquid with respect to (pH) will depend on the reaction characteristics of the protein (Kong, H. E
t . al . , J. Biol . Chem . 268,196
5-1975 (1993)).

As described above, the synthesized NTPA-polynucleotide provides a probe library for genetic diagnosis. As an example of genetic diagnosis, a probe for detecting HIV infection is screened from the probe library, and HIV infection is diagnosed using the probe. As a screening technique, affinity chromatography is used. First, HIV
The RNA is transferred to a suitable chromatographic carrier, for example, CN.
It is covalently bonded to a carrier such as Br-activated Sepharose 4B (Pharmacia) to prepare an HIV RNA affinity column, and liquid chromatography is performed. Chromatography conditions are neutral to slightly alkaline p.
The column is equilibrated with a buffer having a high salt concentration from about 6 to about 9 H, and the NTPA-polynucleotide is provided thereto.
Hybridize with V RNA. Next, HIV
To elute and recover the NTPA-polynucleotide hybridized to the RNA, the buffer is made neutral to slightly alkaline (high pH from about 6 to about 9) with high formamide concentration. At this time, in order to detect the eluted polynucleotide, ultraviolet absorption having a wavelength of about 260 nm, which is generally used for detecting the polynucleotide, is measured. Thus, HIV can be better than NTPA-polynucleotide.
A probe capable of detecting infection can be separated, and RNA or D used for the affinity chromatography can be separated.
By changing the NA, various probes can be screened. For example, influenza virus, hepatitis C virus (hereinafter abbreviated as "HCV"), D
Viral RNAs and cDNAs such as hepatitis hepatitis virus (hereinafter abbreviated as “HDV”), or mRNAs and cDNAs of oncogenes and growth factors that are deeply involved in tumorigenesis of cells, repetitive sequences present in centromeres, etc. DNA with
If an affinity column such as that described above is used, the corresponding probe can be screened.

Upon completion of the probe screening,
Next, as an example of actual genetic diagnosis, HIV infection is detected using the FISH method. First, CD4 ⁺ expressing HeL
A chromosome specimen (slide specimen) is prepared by a known method from a cell (hereinafter abbreviated as “HeLa CD4 ⁺ cell”) infected with HIV-1 and a cell not infected with HIV-1.
The above-mentioned fluorescently-labeled probe or chemically-labeled probe is added to this slide sample as an HIV-1 detection probe, and a hybrid is formed with the DNA of the sample by a known method. The specimen hybridized with the fluorescently labeled probe is directly observed with a fluorescence microscope. Also,
Specimens hybridized with chemically labeled probes include, for example, biotin-labeled avidin-FITC and avidin-rhodamine or other substances capable of binding to biotin and a fluorescent dye previously covalently bound, and fluorescein-labeled anti-fluorescein- Fluorescein, such as FITC or anti-fluorescein-rhodamine, which can bind to fluorescein, and a fluorescent dye covalently bonded in advance, or digoxigenin-labeled, anti-digoxigenin-FITC, anti-digoxigenin, rhodamine, etc. If a dinitrophenyl label is used, a substance capable of binding to dinitrophenyl such as anti-dinitrophenyl-FITC or anti-dinitrophenyl-rhodamine is preliminarily covalently bound with a fluorescent dye. It viewed in a fluorescence microscope using of covalently linked advance fluorescent dye antibodies specific for compounds. As a result, a fluorescent signal cannot be observed in a sample not infected with HIV-1, and a fluorescent signal can be observed in a sample from HIV-infected cells. In addition, the method used here can be used not only for various cultured cells but also for terminal bleeding of HIV-infected patients.

From these results, it is confirmed that NTPA-polynucleotide can provide a probe for genetic diagnosis. In addition, since this genetic diagnosis can be used for detection of viral infections such as influenza virus, HCV, HDV, etc. in addition to HIV, and for detection of oncogenes that are deeply involved in tumorigenesis of cells, the fluorescence synthesized in this study was used. NTP of the invention comprising nucleic acid analogs and chemically modified nucleic acid analogs
A-polynucleotide functions as a probe library for genetic diagnosis.

As a method of gene diagnosis, FIS
In addition to the H method, a known method such as a Southern blot method or a Northern blot method can be used.

The method for producing the thermostable DNA polymerase used in the present invention or the method for producing the gene encoding the same are known today and can be easily carried out by those skilled in the art. In addition, the related gazettes are listed below (these can also be obtained). JP-A-2-6058
No. 5, JP-B-8-24570 (Japanese Unexamined Patent Publication No. 2-4)
No. 34), Japanese Patent Application Laid-Open No. 5-6847, Japanese Patent Publication No.
-59195 (JP-A-5-130871), JP-A-5-328969, JP-A-7-51
061, JP-A-6-339373, JP-A-6-7160

[0045]

The present invention will be further described in the following examples, which by no means limit the invention.

Example 1 Synthesis of NTPA-DNA (Production of DNA Probe Library) Buffer A [10 mM KCl, 10 mM
(NH ₄ ) ₂ SO ₄ , 20 mM Tris / HCl (pH
8.8), 6 mM MgCl ₂ , 0.1% octoxynol (Triton X-100) (all final concentrations)],
And deoxyribonucleotide triphosphate [dATP, dC
TP, dGTP, dTTP (final concentration 200μ
M)] and the following deoxyribonucleotide triphosphate analogs (manufactured by Sigma and: Boehringer
Mannheim, Gibco, Funakoshi)
One each selected from each (final concentration 200
μM) and 100 μl of a reaction solution composed of 20 units / ml Tli DNA polymerase were reacted at 74 ° C. for 3 hours.

1, N ⁶ -ethenodeoxyadenosine-
5'-Triphosphoric acid (see [Chemical Formula 3]; hereinafter, "ε-dATP"
Fluorescein-modified deoxyuridine-5'-triphosphate (see [Chem. 4]; hereinafter, abbreviated as "Flu-dUTP") Biotin-modified deoxyuridine-5'-triphosphate (see [Chem. 5]; hereinafter referred to as "Bio") -DUTP) Biotin-modified deoxyadenosine-5'-triphosphate (hereinafter abbreviated as "Bio-dATP") Digoxigenin-modified deoxyuridine-5'-triphosphate (see [Chem. 6]; hereinafter, "Dig-dUTP") Abbreviation) Dinitrophenyl-modified deoxyuridine-5'-triphosphate (hereinafter abbreviated as "Dnp-dUTP")

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The reaction solution containing ε-dATP (described above) was electrophoresed on a 0.8% agarose gel and then observed as it was under ultraviolet irradiation. As a result, a band emitting light was observed at 50 to 10,000 base pairs (hereinafter, abbreviated as “bp”).

Next, with respect to the reaction solution containing other deoxyribonucleotide triphosphates (above), 0.8
% On an agarose gel, and then put on a nitrocellulose filter (Schleicher & Sewell)
NA was transferred. The filter was dried at 80 ° C. for 2 hours and the prehybridization buffer [50%
Formamide, 4 × SSC (600 mM NaCl, 6
0 mM trisodium citrate dihydrate), 50 mM
HEPES (Nakarai-Tex Inc.) / NaO
H (pH 7.0), 0.5% polyvinylpyrrolidone,
0.5% Ficoll 400 (manufactured by Sigma), 0.5%
Calf serum albumin, 10 mg / ml denatured salmon sperm D
NA (manufactured by Sigma)], and prehybridized at 42 ° C for 2 hours. Thereafter, the filter was buffered with buffer T [50 mM Tris / HCl (pH 7.5),
[150 mM NaCl], and left to stand at room temperature for 5 minutes. Then, the filter was transferred to 10 ml of buffer solution T and immersed. Among them, the following (1) is used for the reaction solution containing the analog, the following (2) is used for the reaction solution containing the analog, and the following (3) is used for the reaction solution containing the analog. To the reaction solution containing the body, 50 μg of each of the following (4) was added, and reacted at room temperature for 1 hour.

(1) Anti-fluorescein-alkaline phosphatase-labeled antibody (anti-Flu-AP antibody, manufactured by Funakoshi) (2) Alkaline phosphatase-labeled streptavidin (hereinafter abbreviated as “SA-AP”, manufactured by Funakoshi) (3) Anti- Digoxigenin-alkaline phosphatase-labeled antibody (anti-Dig-AP antibody, Boehringer Mannheim).

(4) Anti-dinitrophenol alkaline phosphatase-labeled antibody (anti-Dnp-AP antibody, manufactured by Funakoshi).

The filters were washed three times with 50 ml of buffer solution T, and these filters were washed with a coloring solution [100 mM Tris / HCl (pH 8.0), 50 mM MgCl ₂ , 1
00 mM NaCl, 250 μg / ml 2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-tetrazolium chloride (manufactured by Sigma, hereinafter abbreviated as “INT”), 250 μg / ml 5-bromo-
10 ml of 4-chloro-3-indolyl phosphate (manufactured by Sigma, hereinafter abbreviated as "X-phosphate")], allowed to react at room temperature for 30 minutes, washed with water after completion of color development, and dried. As a result, 50-10,00
A blue band distributed at 0 bp was confirmed.

From these results, all nucleic acid analogs were NT
It was confirmed that it was incorporated into PA-DNA. That is, it was confirmed that template-primer-independent DNA can be chemically modified and fluorescently labeled.

Example 2 (Purification of HIV-RNA Specific Probe in NTPA-DNA Using HIV-1 RNA Affinity Chromatography) An HIV-1 RNA affinity chromatography column was prepared.

First, CNBr-activated Sepharose 4B
(Pharmacia) was washed with 1 mM HCl to swell the gel. The gel was then coupled with a coupling buffer [0.1 M NaHCO ₃ (pH 8.3), 0.5 M
NaCl]. Dissolve 10 μg of HIV-1 RNA in this coupling buffer, mix with the gel suspension,
Mix for 2 hours at room temperature. The gel was blocked with blocking reagent (0.2 M glycine, p
H8.0) and mixed at room temperature for 2 hours. After washing once with a coupling buffer, the plate was washed with a washing solution [0.1 M acetate buffer (pH 4.0), 0.5 M NaCl], and then again with a coupling buffer.

Next, an HIV-RNA-specific probe in NTPA-DNA was purified using the HIV-1 RNA affinity chromatography carrier.

First, a starting buffer [0.7 M NaCl,
The column was equilibrated with 50 mM Tris / HCl (pH 7.5), 25% formamide, 20 mM EDTA], and digoxigenin-modified NTPA-DNA was added to the column.
(Hereinafter abbreviated as “Dig-DNA”) 10 μg was provided. After washing the column with the starting buffer, the DNA elution buffer [10 mM potassium phosphate buffer (pH 7.5), 1
0 mM EDTA, 0.2% lauroyl-sarcosine,
90% formamide], and Dig-DNA specific to HIV-1 RNA was eluted. Dig-DNA was detected and recovered by monitoring the absorption at 260 nm.

Example 3 (Detection of HIV-Infected Cells Using Dig-DNA Probe) First, HIV-1 infected cells were prepared. He
La CD4 ⁺ cells were added to a culture supplement [10% fetal calf serum,
10 μg / ml hexadimethrine bromide (trade name: Po
lybrene)] at 37 ° C. in a modified Dulbecco's modified Eagle medium (hereinafter abbreviated as “DMEM”, manufactured by Gibco).
And cultured in a 12-well culture plate.

Thereafter, the cells were subjected to DMEM containing a culture auxiliary solution.
, And suspended in 200 μl of the same medium.
1 was added, and the virus was adsorbed for 2 hours at 37 ° C. Thereafter, the cells were washed twice with DMEM and then 10%
The cells were suspended in 1 ml of DMEM containing fetal calf serum and cultured at 37 ° C. for 5 days. Thereafter, 0.1 μM methotrexate (Methotrexate, manufactured by Nippon Redley Co., Ltd.) was added to stop the DNA de novo synthesis pathway, block the progress to the S phase, further culture for 1 day, and wash twice with DMEM.

The cells were suspended in 1 ml of DMEM containing 10% fetal bovine serum and 10 μM thymidine, and cultured for 6 hours. 30 μm before the start of sampling, 5 μg / ml actinomycin D was added to suppress chromosome condensation in the G2 phase, and 20 μg / ml colcemide was further added 10 minutes before. The cells were collected by centrifugation, suspended in 7 ml of 75 mM KCl, and 15 minutes later, an equal volume of a fixative (methanol: acetic acid = 3: 1) was added thereto, followed by pipetting to centrifuge the cells. Fix cells 14m with fixative
The operation of washing with 1 was repeated three times and dropped on a slide glass. Dry for 1 day and dehydrate through 70, 80, 100% alcohol series. The sample is baked at 62 ° C. for 3 hours, placed in a denaturing solution (70% formamide, 2 × SSC) heated to 72 ° C., and left for 2 minutes. Cold 70
The denaturation was stopped with% ethanol, dehydrated and dried.

Next, a probe solution was prepared. First, 25
0 ng of Dig-DNA and 2 μg of Cot-1 DNA
(Gibco) and 2 μg of salmon sperm DNA were added, and a total of 50 μl of the solution was boiled for 10 minutes, and then allowed to stand on ice for 5 minutes. Hybridization solution (50% formamide,
10% dextran sulfate (2 × SSC)
And pre-annealed at 37 ° C. for 30 minutes to suppress repetitive sequences. Place this solution on the specimen,
Cover with a cover glass and seal with liquid glue, then 37
Cultured overnight at ℃. The sample was transferred to 2 × SSC, shaken for 10 minutes, the cover glass was peeled off, and 50% formamide, 1%
Shake twice at 42 ° C. for 15 minutes in × SSC, shake at room temperature for 15 minutes with 1 × SSC, and shake at room temperature for 10 minutes with 2 × SSC.
It was left still for 5 minutes in SC. Place 40 μl of staining solution A (200 μg / ml anti-digoxigenin-rhodamine, 4 × SSC, 1% bovine serum albumin), react at 37 ° C. for 30 minutes, 4 × SSC for 10 minutes, 4 × SSC + 0.01%
Triton X-100 (supra) 10 minutes, 4 × SSC
Washing was performed for 10 minutes. Subsequently, the chromosomes in the specimen were stained with staining solution B (0.5 μg / ml propidium iodide,
(1 μg / ml 4 ′, 6′-diamidino-2-phenylindole) for 20 minutes. Thereafter, observation was performed with a fluorescence microscope at an excitation wavelength of 596 nm.

As a result, a portion exhibiting a deep red color was observed only in a chromosome specimen from cells infected with HIV-1.
Thereby, the probe for HIV-1 RNA is:
In fact, it was confirmed that HIV-derived DNA integrated into the chromosome could be detected. Example 4 (Synthesis of NTPA-RNA (Production of RNA Probe Library)) Buffer R [10 mM KCl, 10 mM
(NH ₄ ) ₂ SO ₄ , 20 mM Tris / HCl (pH
8.8), 15 mM MnCl ₂ , 0.1% Triton X-100 (all final concentrations)] and ribonucleotide triphosphate [ATP, CTP, GTP, UTP (all final concentrations 500 mM)] and the following (A) To (D) ribonucleotide triphosphate analogues (A: manufactured by Sigma, BD: manufactured by Boehringer Mannheim), each one selected (final concentration: 200 μM), 20 units / ml
Reaction solution 10 composed of Tli DNA polymerase
0 μl was reacted at 74 ° C. for 3 hours.

(A) 1, N ⁶ -ethenoadenosine-5 '
-Triphosphoric acid (ε-ATP) (B) Fluorescein-modified uridine-5'-triphosphate (F
lu-UTP) (C) Biotin-modified uridine-5'-triphosphate (Bio-
UTP) (D) digoxigenin-modified uridine-5'-triphosphate (D
ig-UTP).

Thereafter, the synthesized RNA was detected in the same manner as in Example 1. As a result, it was found that all the synthesized RNAs contained the ribonucleotide analog described above.

Example 5 (Purification of HIV-RNA specific probe in NTPA-RNA using HIV-1 RNA affinity chromatography, and HI by purified probe
Detection of V-infected cells) An HIV-1 RNA affinity chromatography column was prepared. First, CNBr-activated Sepharose 4B (Pharmacia) was added to 1 mM
Washed with HCl to swell the gel.

Next, the gel was mixed with a coupling buffer [0.1
M NaHCO ₃ (pH 8.3), 0.5 M NaC
l]. 10 μg of HIV-1 RNA was dissolved in this coupling buffer, mixed with the gel suspension, and mixed at room temperature for 2 hours. To block the remaining active groups, the gel was run with a blocking reagent (0.2 M glycine, p
H8.0) and mixed at room temperature for 2 hours. After washing once with a coupling buffer, the plate was washed with a washing solution [0.1 M acetate buffer (pH 4.0), 0.5 M NaCl], and then washed again with a coupling buffer.

Next, using this HIV-1 RNA affinity chromatography carrier, NTPA-RN
Purification of the HIV-1 RNA-specific probe in A was performed. First, a starting buffer [0.7 M NaCl 50 mM
The column was equilibrated with Tris / HCl (pH 7.5), 25% formamide, 20 mM EDTA], and digoxigenin-modified NTPA-RNA (hereinafter, referred to as “column”).
10 g of “Dig-RNA”). After washing the column with the starting buffer, the RNA elution buffer [10 mM
Potassium phosphate buffer (pH 7.5), 10 mM ED
TA, 0.2% lauroyl-sarcosine, 90% formamide] with HIV-1 RNA-specific Dig-
RNA was eluted, and Dig-RNA was detected and recovered by monitoring the absorption at 260 nm.

Using the recovered Dig-RNA probe, detection of HIV-1-infected cells was attempted in the same manner as in Example 3, and it was confirmed that HIV-derived DNA integrated into the chromosome could actually be detected. Was done.

[0069]

According to the present invention, a probe library can be easily obtained. This probe library was synthesized in a reaction system in which no template and no primer were present, and contained DNA or RNA having various base sequences. Thus, the polynucleotide probe library of the present invention has diversity in its hybridization ability, and even if the virus to be detected is mutated, if it falls within the aforementioned diversity, the probe is sufficient as a probe. Take the role of.

Continuing from the front page (54) [Title of the Invention] A method for producing a probe library, a probe library obtained by the production method, a method for purifying a probe that hybridizes with another DNA or RNA from the probe library, and Probe purified by the method

Claims (15)

[Claims] 1. A method for producing a probe library, comprising polymerizing a deoxyribonucleotide containing a deoxyribonucleotide analog in the absence of a template and / or a primer in the presence of a protein. 2. A method for producing a probe library, comprising polymerizing a ribonucleotide containing a ribonucleotide analog in the absence of a template and / or a primer and in the presence of a protein. 3. The method according to claim 1, wherein the protein is a thermostable DNA polymerase. 4. The production method according to claim 3, wherein the thermostable DNA polymerase is at least one selected from the group consisting of bacterial DNA polymerases belonging to the genus Sermococcus and the genus Serumus. 5. The method according to claim 1, wherein the polymerization is carried out at a temperature of about 20 to 90 ° C. 6. The process according to claim 1, wherein the polymerization is carried out under neutral to weakly alkaline conditions. 7. The method of claim 7, wherein the deoxyribonucleotide analog is
The production method according to claim 1, wherein at least the base is a different analog. 8. The method of claim 8, wherein the deoxyribonucleotide analog is
1, ^{N 6} - Essey Roh deoxy-adenosine-5'-triphosphate,
Fluorescein-modified deoxyuridine-5'-triphosphate,
Selected from the group consisting of biotin-modified deoxyuridine-5'-triphosphate, biotin-modified deoxyadenosine-5'-triphosphate, digoxigenin-modified deoxyuridine-5'-triphosphate, and dinitrophenyl-modified deoxyuridine-5'-triphosphate. 8. The production method according to claim 7, wherein the production method is at least one selected from the group consisting of: 9. The method according to claim 2, wherein the ribonucleotide analog is an analog having at least a different base. 10. The method of claim 10, wherein the ribonucleotide analog is 1, N ⁶
-Esenoadenosine-5'-triphosphate, fluorescein-modified uridine-5'-triphosphate, biotin-modified uridine
At least one selected from the group consisting of 5'-triphosphate, biotin-modified adenosine-5'-triphosphate, digoxigenin-modified uridine-5'-triphosphate, and dinitrophenyl-modified uridine-5'-triphosphate. Item 10. The production method according to Item 9. 11. A probe library obtained by the production method according to any one of claims 1 to 10. 12. A method for purifying a probe capable of forming a hybrid with another DNA or RNA from the probe library according to claim 11, wherein the affinity is obtained by covalently binding the other DNA or RNA as a ligand. A method for purifying a probe, which is performed by using chromatography. 13. The purification method according to claim 12, wherein the other DNA or RNA is derived from a virus. 14. The method according to claim 13, wherein the virus is a human immunodeficiency virus. 15. A probe purified by the method according to any one of claims 12 to 14 and capable of forming a hybrid with another DNA or RNA.