WO2002004627A1 - Highly efficient method of constructing rna probes - Google Patents

Abstract

RNA probes specific to a number of genes can be simultaneously constructed in a short period of time without resort to the step of DNA cloning by a novel method of constructing RNA probes which comprises a series of steps, i.e., (1) the step of effecting a polymerase chain reaction or reverse transcription and a polymerase chain reaction with the use of a polynucleotide originating in an individual tissue or cells to thereby synthesize double-stranded DNAs specific to target genes; and (2) the step of synthesizing RNAs in vitro by using the DNAs synthesized in the above (1) as probes to thereby give RNA probes specific to the target genes.

Description

 Description High-efficiency RNA probe production method Technical field

 The present invention relates to a novel method for producing specific RNA probes for a large number of genes simultaneously and in a short time.

Background art

 In order to elucidate the molecular mechanisms of various life phenomena, for example, morphogenesis and disease onset, conventionally, the isolation of genes related to these by some method (differential display, subtraction, etc.) has been followed. Strategies that followed the trend of examining the detailed expression distribution of the gene and further analyzing its biological function were common. In other words, it is a method of isolating and acquiring a specific gene of interest as a target, and there was little opportunity to handle many genes at the same time.

 The identification of disease-related genes and drug target genes is expected to accelerate in the future due to the determination of genomic nucleotide sequences of various organisms by recent genome projects and the development of various genetic analysis technologies.

 DNA microarray technology, for example, can comprehensively analyze the fluctuation pattern of gene expression between normal and diseased states for thousands to tens of thousands of genes. By this method, a large number of expression-variable genes that can be associated with a disease can be identified at one time. However, among the expression-variable genes identified by the microarray technology, there are genes that cause disease, and genes that have secondary expression fluctuation due to the disease. Therefore, some means is needed to narrow down the “causal gene” of the disease from these expression-variable genes.

One of the effective means is to analyze the temporal and spatial expression patterns of expression-variable genes. For such analysis, an in situ hybridization method is often used. In order to analyze the expression of the target gene by the in situ hybridization method, it is necessary to use a pro- tein that specifically hybridizes to the mRNA of the target gene. A probe (generally, a cRNA probe) is required, and the production of the probe has conventionally been performed by obtaining the cDNA of the target gene and cloning it.

 The cloning of this cDNA includes (1) incorporation (ligation) of the obtained target gene cDNA into a plasmid having an RNA polymerase recognition sequence (promoter), (2) transformation of E. coli with the plasmid DNA, and (3) transformation of E. coli. Culture of transformed E. coli (recombinant), (4) Extraction and purification of plasmid DNA from E. coli, (5) Confirmation of plasmid DNA incorporating target cDNA (selection of clones) I have. Furthermore, by cutting the plasmid DNA obtained by the above operations (1) to (5) with a restriction enzyme, the plasmid DNA can be used for the first time for the in vitro type II for RNA probe synthesis. Preparation of such RNA probes, including cloning of cDNA, usually required more than one week.

 As described above, there is a disadvantage that it takes a long time to obtain a cRNA probe for a target gene by the conventional method. In addition, since the operation is complicated, it was difficult to obtain cRNA probes for many genes at the same time. As mentioned above, in the future genomics / genetic research, it is necessary to analyze many genes at the same time, so there is a limit to the conventional approach. Against this background, there has been a long-awaited need for a technique for efficiently preparing specific cRNA probes for a large number of genes.

Disclosure of the invention

 An object of the present invention is to provide a novel RNA probe production method capable of producing specific RNA probes for a large number of genes simultaneously and in a short time without going through the DNA cloning step. . Specifically, a DNA specific to the target gene is obtained by a polymerase chain reaction using a primer having an RNA polymerase recognition sequence (promoter sequence) attached to the 5 ′ end, and the DNA is directly synthesized as an RNA probe. An object of the present invention is to provide a novel method for preparing an RNA probe, which is used as a mirror form of the RNA probe.

As described above, before the RNA probe is synthesized by the conventional method, the DNA containing the sequence of the target gene must have an RNA polymerase recognition sequence (promoter). It was necessary to clone the plasmid DNA downstream (3 'side) of the promoter and clone it.

 As a result of intensive studies, the present inventors have come to develop a new method that is completely different from the conventional method. That is, a primer having an RNA polymerase recognition sequence (promoter) previously added to the 5 'end is prepared, and a cDNA specific to the target gene is synthesized using the primer. The synthesized cDNA is directly used for in vitro RNA synthesis. We have developed a new method to use for molds.

 Specifically, first, an oligonucleotide primer having a target sequence of about 20 bp of the target gene was prepared downstream (3 'side) of an RNA polymerase recognition sequence (promoter) such as Pacteriophage T3, Τ7, or SP6. . At that time, when preparing RNA probes for both the sense strand and the antisense strand, a promoter sequence derived from a different pacteriophage was added to each of the 5- and 3'-oligonucleotide primers. .

 Next, using the oligonucleotide primer, a polynucleotide (DNA, RNA) derived from a tissue or a cell is used as a material to perform a polymerase chain reaction (reverse transcription-polymerase chain reaction when RNA is a material), and the promoter is used. A DNA having a sequence at the 5 ′ end and containing the target sequence of the target gene was synthesized (referred to as promoter + DNA). The structure of the promoter + DNA synthesized here is the same as the promoter + DNA on the plasmid DNA produced by the conventional method, that is, the component required for in vitro RNA synthesis.

 Next, the present inventors made the promoter + DNA type II and performed in vitro RNA synthesis using a FITC-labeled nucleotide as a substrate to prepare a specific cRNA probe for the target gene. As a result of actually performing in situ hybridization using the cRNA probe, the distribution of mRNA as reported in the literature was shown for all the target genes examined, and the method for producing the RNA probe of the present invention was excellent for the purpose. It became clear that it was a technique. FIG. 1 schematically illustrates an example of such an RNA probe production method of the present invention.

The novel RNA probe preparation method of the present invention can greatly reduce the time compared to the conventional method including a DNA cloning step, and can handle a large number of genes simultaneously. is there. Specifically, it has the following merits as compared with the conventional method.

 (1) In the conventional method, it takes more than one week to prepare RNA probes for several genes, but in the method of the present invention, RNA probes for many genes can be prepared in only 1.5 to 2 days. Can be manufactured.

 (2) The sequence of the RNA probe must have high specificity to hybridize only to the mRNA of the target gene. In the method of the present invention, a homologous search is performed using a genome sequence database to predict nonspecific binding that hinders the reaction, thereby arbitrarily selecting a highly specific sequence, This can be turned into an RNA probe. In addition, if the existing cDNA clone contains a region having high homology with other genes, the conventional method may require subcloning of the cDNA omitting this region. With this method, it is possible to select only highly specific sequences as RNA probes without performing subcloning.

 (3) At present, cDNA clones (EST clones and the like) of many genes are commercially available, but in the method of the present invention, RNA probes can be produced without obtaining such clones.

 (4) In the method of the present invention, the length of cDNA synthesized by PCR (polymerase chain reaction) or RT-PCR (reverse transcription and polymerase chain reaction) is used to infiltrate the subsequently synthesized RNA probe into the thread. It can be set arbitrarily in consideration of the characteristics and sensitivity. For example, the best results can be obtained with a probe length of 500 b in Honolemount in situ hybridization, but the technique of the present invention can easily produce a probe of a required length.

 The present invention has been completed based on the above findings.

 That is, the present invention

 (1) A highly efficient method for producing an RNA probe, comprising the following steps (a) and (b):

(a) Using a polynucleotide derived from an individual tissue or cell as a starting material, a polymerase chain reaction, or reverse transcription and polymerase chain reaction, Performing double-stranded DNA synthesis;

 (b) performing in vitro RNA synthesis using the DNA synthesized in (a) as type I to produce a specific RNA probe for the target gene,

 (2) The method according to (1) above, wherein the 5′- and 3′-oligonucleotide primers used for the polymerase chain reaction or the reverse transcription and the polymerase chain reaction have a sequence specific to the target gene. High efficiency RNA probe preparation method,

 (3) The above (1) or (2), wherein an RNA polymerase recognition sequence is added to the end of 5, one oligonucleotide primer used for polymerase chain reaction or reverse transcription and polymerase chain reaction. (4) For polymerase chain reaction, or reverse transcription and polymerase chain reaction, 5,-and 3, RNA polymerase recognition sequence at the 5 'end of one oligonucleotide primer The method for producing a highly efficient RNA probe according to the above (3), wherein

(5) The method for preparing a high-efficiency RNA probe according to (4), wherein a recognition sequence for another RNA polymerase is added to the 5 'end of the 5'- and 3'-oligonucleotide primers. ,

 (6) The method according to any one of (1) to (5) above, wherein the RNA polymerase used for preparing the RNA probe is selected from T3 RNA polymerase, T7 RNA polymerase and SP6 RNA polymerase. High efficiency RNA probe preparation method,

 (7) The method for producing a high-efficiency RNA probe according to any of (1) to (6) above, wherein the polynucleotide derived from an individual thread or cell is genomic DNA, cDNA or RNA.

 (8) the method of producing a high-efficiency RNA probe according to any one of (1) to (7), wherein the target gene is a gene whose expression fluctuates between a normal state and a disease state;

 (9) The method for producing a high-efficiency RNA probe according to any one of (1) to (8), wherein a hapten or a radiolabeled nucleotide is used as a substrate in in vitro RNA synthesis.

(10) The method according to any of (1) to (9) above, further comprising the step of verifying the specificity of the RNA probe for the target gene by homology search on a genome database. Highly efficient RNA probe preparation method described in

 (11) (3) to (10) used in the polymerase chain reaction or the reverse transcription and the polymerase chain reaction in the method for producing a high-efficiency RNA probe described in any one of (5) to (5). An oligonucleotide primer or group thereof,

 (1 2) The oligonucleotide primer or the group thereof according to (11), wherein a recognition sequence for a separate RNA polymerase is added to the 5 'end of the 5' one and three, one oligonucleotide primers.

 (13) DNA synthesized using the oligonucleotide primer or the group thereof according to the above (11) or (12), or the group thereof,

 (14) a reagent kit for producing a high-efficiency RNA probe, comprising the oligonucleotide primer or the group thereof according to (11) or (12), or the DNA or the group thereof according to (13); And

 (15) A reagent kit for producing a high-efficiency RNA probe, containing the following (i) and (ii):

 (i) Enzymes, reagents and buffers required for the polymerase chain reaction, or enzymes, reagents and buffers required for reverse transcription and the polymerase chain reaction;

 (ii) enzymes, reagents and buffers required for in vitro RNA synthesis,

About.

BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a diagram schematically illustrating an example of the method for producing an RNA probe of the present invention.

 FIG. 2 is a photograph showing the results of in situ hybridization (hole mount method) using cRNA probes of 13 types of genes. A: Semaphorin 3A, B: Semaphorin 6B, C: Semaphorin 6C, D: Neuropilin-1, E: Neuropilin-2, F: Plexin-Al, G: Plexin-A3, H: Netrin-1, I: DCC, J: unc-5hl,: Ephrin ^ L: Eph-Bl, M: Neurofilament-M

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides a large number of relics without going through the DNA cloning steps conventionally performed. The present invention provides, for the first time, a high-efficiency RNA probe production method capable of simultaneously producing a specific RNA probe for a gene in a short time. Specifically,

It refers to a method that includes a series of steps (a) and (b).

 (a) A step of using a polynucleotide derived from an individual tissue or cell as a starting material, and performing double-stranded DNA synthesis specific to the target gene by polymerase chain reaction or reverse transcription and polymerase chain reaction.

 (b) A step of performing in vitro RNA synthesis using the DNA synthesized in (a) above as a type II to prepare a specific RNA probe for the target gene.

 In other words, preparation of an RNA probe containing two consecutive steps of a polymerase chain reaction or a reverse transcription-polymerase chain reaction (step a) and an RNA synthesis of the DNA obtained by the reaction (step b). The method corresponds to the method for producing a high-efficiency RNA probe of the present invention.

 As the “polynucleotide derived from an individual tissue or cell” in the step (a), any polynucleotide may be used as long as it is a polynucleotide prepared from an individual tissue or cell. Specifically, a genome (chromosome) DNA, cDNA or RNA is mentioned. Here, as the RNA, the total RNA and the level of the mRNA and the deviation thereof can also be used. The genomic DNA and cDNA may be in the form of a library, that is, in a form incorporated in a plasmid, or in a form not incorporated in the plasmid.

 The positive nucleotide can be easily prepared by those skilled in the art according to, for example, Molecular Cloning, A Laboratory Manual, edited by T Maniatis et al., Second Edition (1989), Cold Spring Harbor Laboratory, and the like. In addition, a genomic DNA extraction kit (manufactured by Kuchish Inc .: DNA isolation kit, etc.), a cDNA preparation kit (manufactured by Gibco BRL: Superscript Chiyois System, etc.) And the like are commercially available, and the polynucleotide of the present invention can be easily prepared by using these. Furthermore, genomic DNA, cDNA and RNA prepared from tissues of various animal species are commercially available from, for example, Pyochain or the like, and can be purchased.

In the step (a), a polynucleotide derived from an individual tissue or cell is Specific to the target gene by either "polymerase chain reaction (hereinafter sometimes referred to as PCR reaction)" or "reverse transcription and polymerase chain reaction (hereinafter sometimes referred to as RT-PCR reaction)" Synthesize double-stranded DNA. Here, when the starting material is cDNA or genomic DNA, double-stranded DNA specific to the target gene can be synthesized by performing only the polymerase chain reaction. On the other hand, when the starting material is RNA, first, a single-stranded cDNA is synthesized by a reverse transcription reaction, and then a double-stranded DNA specific to the target gene is synthesized by a polymerase chain reaction.

 In the above, the term `` target gene '' refers to a gene to be analyzed, that is, an object to be subjected to analysis such as in situ hybridization or northern blot hybridization using an RNA probe prepared by the RNA probe preparation method of the present invention. Refers to the gene Specific examples include the following genes.

 (1) A gene whose expression fluctuates between normal and disease states.

 (2) Genes whose expression fluctuates between normal and drug administration.

 (3) Genes whose expression varies depending on the time of occurrence.

遺 伝 子 Tissue-specifically expressed genes.

 The target gene as described above can be selected in advance by, for example, DNA microarray technology, differential display, or subtraction. In particular, the DNA microarray technology can comprehensively analyze gene expression fluctuation patterns ゃ between normal and disease states, and gene expression fluctuation patterns between normal and drug administration, for thousands to tens of thousands of genes. You can do it. Those identified as “expression-variable genes” by these techniques are also included in the category of “target genes” in the present invention.

 The “double-stranded DNA specific to the target gene” synthesized in the step (a) is a double-stranded DNA corresponding to all or a part of the target gene, and a base specific to the target gene. It means a double-stranded DNA consisting of a sequence. The preferred length of the double-stranded DNA is about 100 bp to 5000 bp, more preferably about 500 bp to 2000 bp. The fact that it is “specific” for the target gene will be described later.

5'-oligonucleotide primers and 3'-oligonucleotides used in the polymerase chain reaction or the reverse transcription and polymerase chain reaction of the step (a). All of the tide primers contain a nucleotide sequence portion specific to the target gene. That is, in order to prevent the primer from annealing to a gene other than the target gene to synthesize an untargeted DNA, the oligonucleotide primer must contain a nucleotide sequence portion specific to the target gene. . Here, the length of the base sequence portion specific to the target gene in the oligonucleotide primer is preferably about 10 bases to 30 bases, and more preferably about 20 bases. These oligonucleotide primers can be easily prepared using a DNA synthesizer.

 In the above description, the nucleotide sequence “specific” for the target gene means that the nucleotide sequence is not found in the nucleotide sequence of another gene with high homology. Specifically, a nucleotide sequence specific to a target gene is defined as a sequence of homology between the target gene and another gene, and a homology of at least 80% (preferably at least 95%). It means such a base sequence portion for which no sequence is found.

 In recent years, with the rapid progress of genomic analysis, a vast database of human genomic or cDNA sequences has been constructed. Examples of accessible databases include the GenBank database and the EMBL database. In particular, the GenBank genome database is effectively used. By performing a homology search using these databases, a nucleotide sequence portion specific to the target gene can be specified. Furthermore, the use of a genome sequence database has the advantage that the specificity of a probe can be predicted completely, providing information that is qualitatively different from the past. Therefore, it is preferable to perform a homology search using a registered database of complete genome sequences.

 Specifically, NCBI (National Center for

Access Biotechnology Information) and open the BLAST program. Furthermore, select Human Genome BLAST and enter the sequence of the target gene or the accession number in the database of the target gene and perform homology search. When a similar sequence is found in another gene by homology search, the sequence of the oligonucleotide primer is selected from a sequence portion other than the similar sequence. Verification of specificity by homology search on the database as described above is based on oligonucleotide nucleotide priming. It is preferable to carry out the selection at the time of selection of the primer, but it is also effectively used when verifying that the oligonucleotide primer selected by another means has specificity.

 As mentioned above, the 5'- and 3'-oligonucleotide primers used in the polymerase chain reaction or the reverse transcription and the polymerase chain reaction need to contain a nucleotide sequence specific to the target gene. Stated.

 In addition, the addition of an RNA polymerase recognition sequence (hereinafter sometimes referred to as a promoter) to the 5 'end of the oligo nucleotide primer enables the simultaneous and short-time preparation of a large number of genes, which is the object of the present invention. It is possible to prepare a specific RNA probe for Hereinafter, this point will be described in detail.

 5. Two strands having the RNA polymerase recognition sequence at the 5 'end by performing polymerase chain reaction or reverse transcription and polymerase chain reaction using an oligonucleotide primer having an RNA polymerase recognition sequence added to the end. Strand DNA is synthesized (hereinafter, the synthesized double-stranded DNA is sometimes referred to as promoter + DNA). The structure of the promoter + DNA synthesized here is equal to the promoter + DNA on the plasmid DNA produced by the conventional method, that is, the component necessary for in vitro RNA synthesis. That is, by performing the above step (a) using an oligonucleotide primer having an RNA polymerase recognition sequence added to the 5 ′ end, the obtained promoter + DNA is converted into the in vitro RNA in the step (b) It has the great advantage that it can be used directly for synthesis.

 Here, the RNA polymerase recognition sequence to be added to the 5 'end of the oligonucleotide primer may be any RNA polymerase recognition sequence available to those skilled in the art. Recognition sequences for phage-derived RNA polymerase are included. Specific examples include a recognition sequence for T3 RNA polymerase, T7 RNA polymerase, or SP6 RNA polymerase. Here, as a specific example of the T3 RNA polymerase recognition sequence, GenBank Acc.

No. U13871, base sequences described in base numbers 172 to 191 of the pT7T3D vector, and the like. As an example, the base sequence described in SEQ ID NO: 1 can be mentioned. As specific examples of the T7 RNA polymerase recognition _{sequence, GenBank Acc. No. U13871, P} T7T3D vector The base sequence described in one base number 275 to 294 (complementary strand) and the like are exemplified. As an example, the base sequence described in SEQ ID NO: 2 is exemplified. Specific examples of the SP6 RNA polymerase recognition sequence include GenBank Acc. No. X65328, the base sequence described in base numbers 3014 to 3 of the pSP64 vector, and the like. Oligonucleotide primers in which a nucleotide sequence specific to the target gene is linked to the third side of these RNA polymerase recognition sequences can be easily synthesized using a DNA synthesizer.

 As described above, when an RNA polymerase recognition sequence is added to the 5 and 5 ends of an oligonucleotide primer, the RNA polymerase recognition sequence should be added only to the 5 and 5 ends of an oligonucleotide primer according to the purpose. If possible, the RNA polymerase recognition sequence can be added to the 5 'end of both the 5'- and 3'-oligonucleotide primers.

 That is, in the in vitro RNA synthesis in the step (b), when only the RNA probe of the antisense strand for the target gene is synthesized, the RNA polymerase is used only for the 3′-oligonucleotide primer used in the step (a). You can add a recognition sequence. The RNA probe of the antisense strand is used to detect the expression of the mRNA of the target gene in in situ hybridization or Northern plot hybridization.

 On the other hand, in the in vitro RNA synthesis in the step (b), when synthesizing both the antisense strand and the sense strand RNA probes for the target gene, the 5′- and 3,-used in the step (a) are used. It is necessary to add an RNA polymerase recognition sequence to both oligonucleotide primers. The RNA probe of the antisense strand is used for detecting the expression of the mRNA of the target gene in in situ hybridization and Northern blot hybridization as described above, while the RNA probe of the sense strand is It is used as a control for detecting the expression of knock background.

 The “antisense strand” means having a sequence complementary to raRNA that defines the amino acid sequence of the protein, and the “sense strand” means having the same sequence as the mRNA.

5,-and 3,-oligonucleotide primers used in step (a) as described above When an RNA polymerase recognition sequence is added to both 5′-ends, it is preferable that each primer be provided with a recognition sequence for RNA polymerase derived from a different species. That is, since the recognition sequences for different RA polymerases are added to the 5′- and 3, -oligonucleotide primers, the sense strand RNA and the antisense strand RNA are separated in step (b). It has the great advantage that it can be synthesized (independently). Examples of combinations of the RNA polymerase recognition sequence (promoter) added to the 5′- and 3′-oligonucleotide primers are as follows. However, the present invention is not limited to this example, and it is sufficient if the promoters on both the 5 ′ and 3 ′ primers can be recognized by different RNA polymerases.

 5, one side of oligo nucleotide primer Z3, one side of oligonucleotide primer

 T3 promoter / T7 promoter

T7 promoter / T3 promoter

T3 promoter / SP6 promoter SP6 promoter / T3 promoter

T7 promoter Z SP6 promoter

SP6 promoter / T7 promoter Techniques for performing the polymerase chain reaction or the reverse transcription and polymerase chain reactions using the 5,-and 3, -oligonucleotide primers as described above are presently known to those skilled in the art. For example, the method can be performed with reference to Molecular Cloning, A Laboratory Manual, edited by T Maniatis, 2nd edition (1989), Cold Spring Harbor Laboratory, PCR A Practical Approach, IRL Press (1991). In addition, kits and the like are commercially available for these reactions. Specifically, a PCR kit (Gibco BRL: PCR supermix, etc.) is used for the polymerase chain reaction, and an RT is used for the reverse transcription and the polymerase chain reaction. -PCR kits (Gibco BRL, Superscript One-Step RT-PCR System, etc.) can be used. The reaction conditions for the reverse transcription reaction and the polymerase chain reaction can be appropriately set based on the protocol attached to the above-mentioned basic manual kit. In the case of the RT-PCR reaction, for example, reverse rotation at 50 ° C for 30 minutes is performed. After the transcription reaction, the mRNA and single-stranded cDNA were heated at 94 ° C for 2 minutes. Separate, and then perform the PCR reaction at 94 ° C, 15 seconds → 60 ° C, 30 seconds → 72 ° C, 1 minute 40 times, and then perform the reaction at 72 ° C, 1 minute.

 Next, the step (b) will be described.

 The step (b) is a step of performing in vitro RNA synthesis using the DNA synthesized in the step (a) as a type III to prepare a specific RNA probe for the target gene. As described above, when a DNA having an RNA polymerase recognition sequence added to the 5 'end is synthesized in step (a), step (b) can be performed directly using the synthetic DNA.

 The technique of in vitro RNA synthesis itself is currently a common means for those skilled in the art, and can be performed with reference to the aforementioned Molecular Cloning and the like. Specifically, RNA (cRNA) is synthesized by converting the DNA synthesized in step (a) into type I, and reacting the substrate (NTPs) and RNA polymerase at 37 ° C for about 2 to 5 hours. Can be. At that time, it is preferable to carry out the above reaction in the presence of a ribonuclease inhibitor in order to avoid RNA degradation.

It is preferable to use a hapten or a radiolabeled nucleotide as the substrate. In that case, it is not necessary that all four nucleotides are labeled, and it is sufficient that at least one nucleotide is labeled. Here, the hapten includes fluorescein (FITC) digoxigenin and the like, and the radiolabel includes ³² P and the like. Thus, by using hapten or radiolabeled nucleotide as a substrate, the synthesized RNA is hapten or radiolabeled, so that the RNA is in situ hybridization, Northern plot hybridization. It is used effectively for mRNA detection (gene expression detection) of the method.

As the RNA polymerase, it is necessary to use an RNA polymerase that recognizes a promoter sequence present at the 5 ′ end of the DNA synthesized in the step (a). When synthesizing both sense strand and antisense strand RNA, the DNA (promoter + DNA) having a promoter sequence in both strands synthesized in step (a) is placed in two separate tubes. The reaction is performed by adding RNA polymerase for the synthesis of sense strand RNA to one side and RNA polymerase for the synthesis of antisense strand to the other side. RNA polymerase used is T7 RNA polymerase, T3 RNA polymerase And SP6 RNA polymerase, all of which can be purchased from Roche.

 The RNA probe thus prepared is used for in situ hybridization, Northern plot hybridization, etc. after degrading type I DNA by adding deoxyribonuclease I. be able to.

 FIG. 1 schematically shows a specific example of a method for producing a high-efficiency RNA probe through a series of steps (a) and (b). Each setting in Fig. 1 is as follows.

Step (a): cDNA synthesis by reverse transcription and polymerase chain reaction (RT-PCR)

 5'-oligonucleotide primer: 5'-oligonucleotide primer with T3 RNA recognition sequence (T3 promoter-1) added to 5 'end

 3'-oligonucleotide primer: 5, 3'-oligonucleotide primer with a T7 RNA recognition sequence (T7 promoter-1) at the end

Target gene-specific sequence length: about 500 bp

Step (b): cRNA probe synthesis by in vitro RNA synthesis

Substrate: Fluorosein (FITC) labeled nucleotide

Sense strand cRNA: synthesized by T3 RNA polymerase

Antisense strand cRNA: synthesized by T7 RNA polymerase

The present invention further encompasses an oligonucleotide primer having an RNA polymerase recognition sequence added to its 5 'end and a DNA synthesized using the oligonucleotide primer used in the step (a). Things. These primers and DNAs have never existed in the past because they are primers and synthetic DNAs with an RNA polymerase recognition sequence added to the end.These are the highly efficient RNA probes of the present invention. It is effectively used as a reagent for the production method. Furthermore, when producing a specific RNA probe for a gene whose expression fluctuates between a normal state and a disease state and a gene whose expression fluctuates between a normal state and a drug administration, a large number of expression It is necessary to prepare the oligonucleotide primer and the DNA for the variable gene. In the present invention, such a 5′-terminal RNA polymer It also includes a group of oligonucleotide primers to which a protease recognition sequence has been added, or a group of DNAs synthesized using the group of oligonucleotide primers.

 In particular, a set or group of oligonucleotide primers with a separate RNA polymerase recognition sequence at the 5 'end of the 5'- and 3,1 oligonucleotide primers, or a set of these oligonucleotide primers Alternatively, DNA synthesized using the group or a group thereof is preferable.

 These oligonucleotide primers or groups thereof, or DNAs or groups synthesized using the oligonucleotide primers or groups thereof can also be used in the form of a kit for producing a highly efficient RNA probe. For example, by associating the Accession number on the database with the oligonucleotide primer, the primer can be designed and synthesized, and DNA synthesis (PCR or RT-PCR) using the primer can be performed. It is possible to prepare an RNA probe for the gene or genes to be analyzed. The kit is used very effectively when creating an in situ database.

 The present invention also provides a reagent kit for producing a high-efficiency RNA probe, capable of performing the series of steps (a) and (b). The kit contains the following reagents (i) and (ii).

 (i) Enzymes, reagents and buffers required for the polymerase chain reaction, or enzymes, reagents and buffers required for reverse transcription and the polymerase chain reaction.

 (ii) Enzymes, reagents and buffers required for in vitro RNA synthesis.

 When using the kit, it is necessary to prepare in advance a polynucleotide derived from an individual tissue or cell and a specific primer for the target gene (having an RNA polymerase recognition sequence at the 5 'end). However, as long as these polynucleotides and primers are prepared, RNA probes for a large number of target genes can be prepared simultaneously and in a short time by using the kit.

In the above (i), the term "enzyme necessary for the polymerase chain reaction" refers to a thermostable DNA polymerase, and the term "reagent necessary for the polymerase chain reaction" refers to a buffer solution and a substrate solution that are optimal for expressing the activity of the enzyme. Point to. "The relaxation required for the polymerase chain reaction The “impact” includes, specifically, a solution of 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl ₂ , 50 mM KC1, 1 mM dNTP at the time of the reaction.

In the above (i), “enzymes required for reverse transcription and polymerase chain reaction” refer to reverse transcriptase and thermostable DNA polymerase, and “reagents required for reverse transcription and polymerase chain reaction”. It refers to a buffer solution and a substrate solution that are optimal for expressing the activity of the enzyme. The “buffer necessary for reverse transcription and polymerase chain reaction” is, specifically, 50 mM Tris-HCl (pH 8.3), 5 mM MgCl ₂ , 50 mM KC1, 1 mM DTT, 20 mM g / ml BSA, 1 mM dNTP solution and the like.

In the above (ii), “enzymes required for in vitro RNA synthesis” refers to RNA polymerase (T7, T3, SP6) and deoxyribonuclease I, and “reagents” are buffers optimal for the activity of the enzyme. Liquid, substrate solution (including hapten label). "Buffer" refers to a solution of 40 raM Tris-HCl (ρΗ8.0), 20 mM MgCl ₂ , 50 mM NaCl, 5 mM DTT, 1 mM NTP (including hapten label) at the concentration at the time of the reaction. And the like.

 Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited to these examples, and it goes without saying that ordinary changes in the technical field of the present invention can be made. Unless otherwise specified, each operation in the examples was performed according to the method described in “Molecular Cloning, A Laboratory Manual, edited by T Maniatis et al., 2nd edition (1989), Cold Spring Harbor Laboratory].

Example 1

Preparation of RNA probe

To investigate that the target gene-specific RNA probe is prepared by the method of the present invention, the specific cRNA probes for 13 genes described below were prepared, i _n situ High Priestess die See Chillon (hole using these Puropu Mounting method).

 Semaphorin 3A (Genbank Acc.No.X95289), Semaphorin 6B (Genbank Acc.No.AB000776), Semaphorin 6C (Genbank Acc.No.AB000817), Neuropilin-1 (Genbank Acc. AF016296), Neuropilin-2 (Genbank Acc.No.AF016297), Plexin-Al (Genbank Acc.No.D86948), Pleki

A3 (Genbank Acc.No.D86950), Netrin-1 (Genbank Acc.No.AF128865), DCC (Genbank Acc.No.U68725), unc-5hl (Genbank Acc.No.U87305), Ephrin-Bl (Genbank Acc.No.U07560), Eph-Bl (Genbank Acc.No.X13411), and We chose Neurol Firament -M (Genbank Acc. No. Z12152).

 First, oligonucleotide primers (PCR primers) in which the base sequence of the 5'-end 20 base of each of the sense strand and the antisense strand of the 500 bp region specific to the target gene was attached downstream of the promoter sequence derived from pacterio phage Was synthesized using a DNA synthesizer. Here, the sequences at the 5 bases and the 20 bases were determined by conducting a homology search using a database of genome sequences. As the promoter, a T3 promoter (SEQ ID NO: 1) on the sense strand side and a T7 promoter (SEQ ID NO: 2) on the antisense strand side were used.

 The 5′-PCR primer and 3′-PCR primer of semaphorin 3A are shown in SEQ ID NOs: 3 and 4, and the 5, -PCR primer and 3′-PCR primer of semaphorin 6B are shown in SEQ ID NOs: 5 and 6. The 5'-PCR primer and 3'-PCR primer of semaphorin 6C are shown in SEQ ID NOs: 7 and 8, and the 5'-PCR primer and 3, -PCR primer of neuropilin-1 are shown in SEQ ID NOs: 9 and 10. In addition, the 5'-PCR primer of neuropilin-2 and the-3 -PCR primer are shown in SEQ ID NOS: 11 and 12, and the 5'-PCR primer of Plexin-A1 and the 3'-PCR primer are shown in SEQ ID NO: Plexin-A3 5- and -primer 3'-PCR primers are shown in SEQ ID NOs: 15 and 16, Netrin-1 5- and -PCR primers and 3'-PCR primers 13 and 14. And 5'-PCR primer and 3'-PCR primer of DCC to SEQ ID NO: 17 and 18, respectively. 9 and 20, 5'-PCR primer and 3'-PCR primer of unc-5hl to SEQ ID NO: 21 and 22, and 5'-PCR primer and 3'_PCR primer of Ephrin-B1 to SEQ ID NO: 23 and 24, the 5'-PCR primer and 3'-PCR primer of Eph-B1 are shown in SEQ ID NOS: 25 and 26, and the 5'-PCR primer and 3'-PCR primer of Neurofilament-M are shown in SEQ ID NO: 27. See page 28 for details.

The 5 '及Pi 3, - PCR primers as one set, using Aisojen the newborn rat brain-derived total RNA 1 mu _§ prepared by (Nibbonji down) in the starting material, Superscript One-Step RT-PCR System ( GIBCO BRL ) Was used to synthesize cDNA for each gene (reaction conditions: 50 ° C, 30 minutes → 94 ° C, 2 minutes → [94 ° C, 15 seconds → 60 ° C, 30 seconds → 72 ° C, 1 minute) ] x 40 times → 72 ° C, 1 minute). T3 promoter at the end of sense strand of synthesized cDNA And a T7 promoter is added to the 5 'end of the antisense strand (hereinafter, the cDNA with the promoter added is referred to as promoter + cDNA). The synthesized promoter I ~ "hcDNA was recovered by ethanol precipitation after phenol: chlorophonolem treatment, and used for the following in vitro transcription reaction (cRNA probe synthesis) type II.

Promoter + 0.2 to 1.0 ^ _§ for each gene, lx transcription buffer (Roche), lx fluorescein RNA labeling mix (Roche), 10-unit liponuclease inhibitor (Nippon Gene) An in vitro transcription reaction was performed at 37 ° C for 2 to 5 hours in a 20 μ μ solution containing 20 units of RNA polymerase (Roche) to synthesize a fluorescein-labeled cRNA probe. Each gene was reacted independently with T3 RNA polymerase and T7 RNA polymerase to synthesize a sense cRNA probe in the former and an antisense cRNA probe in the latter. After the reaction, 1 μl of DNase I (Roche) was added to decompose type I cDNA, and the cRNA probe synthesized by ethanol precipitation was recovered and re-dissolved in TE buffer. It was used for the dimensioning (Honoremount method).

Example 2

in situ hybridization

 Honole mount in situ hybridization was performed according to the method described in Riddle et al. Cell 75, 1401-1416 (1993).

 The pretreated embryonic rat embryos were immersed in a hybridization solution containing 100 ng / ml of the synthesized cRNA probe at a temperature of 70 ° C overnight. After washing the excess cRNA probe, the localization of the cRNA probe (mRNA) was visualized using the detection kit for FITC (fluorescein) labeled probe (Dako). The result is shown in figure 2. As a result, all the specific signals of the mRNAs of the 13 targeted genes could be detected. These were consistent with the distributions already reported in the literature, indicating that the RNA probe production method of the present invention functions effectively.

Industrial applicability

 According to the present invention, unlike the conventional method, specific RNA probes for a large number of genes can be prepared simultaneously and in a short time.

Claims

 Scope of the claim 1. Highly efficient RNA probe production including a series of the following steps (a) and (b) (a) Starting from a polynucleotide derived from an individual tissue or cell, using the polymerase chain reaction, or Performing double-stranded DNA synthesis specific for the gene of interest by reverse transcription and polymerase chain reaction;  (b) a step of performing in vitro RNA synthesis using the DNA synthesized in the above (a) as type I to prepare a specific RNA probe for the target gene.  2. The high-efficiency RNA according to claim 1, wherein the 5,-and 3'-oligonucleotide primers used in the polymerase chain reaction or the reverse transcription and the polymerase chain reaction have a sequence specific to the gene of interest. Probe preparation method.  3. The high-efficiency RN according to claim 1 or 2, wherein an RNA polymerase recognition sequence is added to the 5, terminal of the 3, one oligonucleotide primer used for the polymerase chain reaction or the reverse transcription and the polymerase chain reaction. A probe preparation method.  4. The high efficiency according to claim 3, wherein an RNA polymerase recognition sequence is added to the 5, terminal of the 5, 1 and 3'-oligonucleotide primer used for the polymerase chain reaction or the reverse transcription and the polymerase chain reaction. RNA probe preparation method.  5. The method for producing a highly efficient RNA probe according to claim 4, wherein a recognition sequence for another RNA polymerase is added to the 5, terminal of the 5,-and 3'-oligonucleotide primers.  6. The high efficiency according to any one of claims 1 to 5, wherein the RNA polymerase used for preparing the RNA probe is selected from T3 RNA polymerase, T7 RNA polymerase, and SP6 RNA polymerase. Method for preparing RNA probe.  7. The method for producing a highly efficient RNA probe according to any one of claims 1 to 6, wherein the polynucleotide derived from an individual tissue or cell is genomic DNA, cDNA or RNA. 8. The method for producing a highly efficient RNA probe according to any one of claims 1 to 7, wherein the target gene is a gene whose expression fluctuates between a normal state and a disease state. 9. The method for producing a high-efficiency RNA probe according to any one of claims 1 to 8, wherein a hapten or a radiolabeled nucleotide is used as a substrate in in vitro RNA synthesis.  10. The method for producing a highly efficient RNA probe according to any one of claims 1 to 9, further comprising the step of verifying the specificity of the RNA probe for the target gene by homology search on a genome database.  1 1. Used in the polymerase chain reaction or reverse transcription and the polymerase chain reaction in the method for producing a high-efficiency RNA probe according to any one of claims 3 to 10. An oligonucleotide primer or a group thereof.  12. The oligonucleotide primer according to claim 11, wherein a recognition sequence for a separate RNA polymerase is added to the 5, terminal of the 5, 1 and 3'-oligonucleotide primer.  13. A DNA synthesized using the oligonucleotide primer or the group thereof according to claim 11 or 12, or a group thereof.  14. A reagent kit for producing a highly efficient RNA probe, comprising the oligonucleotide primer or the group thereof according to claim 11 or 12, or the DNA primer or the group thereof according to claim 13.  1 5. Reagent kit for producing high-efficiency RNA probes, containing the following (i) and (ii):  (i) enzymes, reagents and buffers required for the polymerase chain reaction, or enzymes, reagents and buffers required for reverse transcription and the polymerase chain reaction;  (ii) Enzymes, reagents and buffers required for in vitro RNA synthesis.