WO2007011016A1 - Procédé de production d’étiquettes géniques à simple brin, chacune ayant un site de lancement de la transcription - Google Patents

Procédé de production d’étiquettes géniques à simple brin, chacune ayant un site de lancement de la transcription Download PDF

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WO2007011016A1
WO2007011016A1 PCT/JP2006/314459 JP2006314459W WO2007011016A1 WO 2007011016 A1 WO2007011016 A1 WO 2007011016A1 JP 2006314459 W JP2006314459 W JP 2006314459W WO 2007011016 A1 WO2007011016 A1 WO 2007011016A1
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gene
rna
dna
stranded
primer
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PCT/JP2006/314459
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English (en)
Japanese (ja)
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Shin-Ichi Hashimoto
Kouji Matsushima
Akio Ametani
Yukie Sameshima
Kayo Shimizu
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Post Genome Institute Co., Ltd.
The University Of Tokyo
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Publication of WO2007011016A1 publication Critical patent/WO2007011016A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • the present invention relates to a method for producing a single-stranded gene tag group including a transcription initiation site.
  • the present invention also relates to a method for measuring the expression level of a gene in a eukaryotic cell, comprising a step of hybridizing the single-stranded gene tag group to a solid phase on which DNA or RNA containing a transcription initiation site is immobilized.
  • the present invention relates to a method for creating gene expression profiles by integrating the obtained gene expression information.
  • Cells can be characterized by comparison of gene expression status of various cells.
  • a cell catalog that expresses the state of a cell by a gene expression pattern.
  • cells can be identified from the gene expression state.
  • genes characteristic of each cell For example, when the expression state of a gene is compared between a normal cell and a cell subjected to artificial treatment, a gene whose expression level has changed as a result of the artificial treatment is found. This gene is a gene whose expression level has changed as a result of artificial processing.
  • genes related to diseases can be found by comparing gene expression states between patient cells and healthy subject cells.
  • a method for expression analysis is the 5 'SAGE method (Non-patent Document 1, Patent Document 1).
  • the 5 ′ SAGE method is an effective method for comprehensive analysis of gene expression at the transcription start site as sequence information.
  • SAGE method Non-patent document 2
  • MPSS method Non-patent document 3
  • a second example of a method for expression analysis is a DNA array method.
  • the expression state of a gene is determined from the signal strength of the hybrid by hybridizing the mRNA extracted from a specific cell force to tens of thousands of gene probes arranged at high density on the substrate.
  • Non-patent Document 4 This is an exhaustive determination method (Non-patent Document 4).
  • probes constituting a DNA array are designed based on known base sequence information!
  • mRNA extracted from a relatively small amount of cells By hybridizing to a DNA array using mRNA extracted from a relatively small amount of cells, it is possible to easily analyze qualitatively or semi-quantitatively the difference in the expression state of genes among many cells. is there.
  • RT-PCR method (Non-patent Documents 5-7) can be mentioned.
  • the RT-PCR method is a method for detecting DNA obtained through reverse transcription and PCR from a relatively small amount of mRNA extracted from cells by electrophoresis or the like.
  • the RT-PCR method is widely used in the study of expression analysis, and has a difficulty as a method for simultaneously processing a large number of samples that are more quantitative than microarrays.
  • the DNA array method is most advantageous in terms of completeness.
  • the position of a gene probe used in a DNA array on a sequence and a genome sequence is specified. Therefore, it should be possible to comprehensively test which genes are expressed by appropriately setting the conditions for the hybridization between the mRNA extracted from the cell force and the gene probe.
  • Non-patent Documents 8-9 How to reduce this unpredictable signal DNA array It is essential for the effective use of the law.
  • Patent Literature l WO 2005/054465
  • Non-Patent Document 1 Hashimoto et al., Nature Biotechnol. 22, 1146-1149 (2004)
  • Non-Patent Document 2 Velculescu et al., Science, 270, 484-487 (1995)
  • Non-Patent Document 3 Brenner et al., Nature Biotechnol, 18, 630-634 (2000)
  • Non-patent literature 4 Gerhold et al., Nature Genetics, 32, supplement, 547-552 (2002)
  • Non-patent literature 5 Kawasaki, ES and Wang, AM PCR Technology (Erlich, HA ed), Stockton Press 89—97 ( 1989)
  • Non-Patent Document 6 Lynas et al., J Pathol. 157, 285-289 (1989). Erratum in: J Pathol. 159, 358 (1989).
  • Non-Patent Document 7 Frohman et al., Proc. Natl. Acad. Sci. USA 85, 8998-9002 (1988)
  • Non-Patent Document 8 van Ruissen et al., BMC Genomics 6, 91 (2005)
  • Non-Patent Document 9 Pollock, Chem Phys Lipids 121, 241-256 (2002)
  • Non-Patent Document 10 Belosludtsev et al., Biotechnique 37, 654-658, 660 (2004) Disclosure of the Invention
  • the present inventors have a large number of regions unnecessary for obtaining information on mRNA and hybridization. Focusing on the fact that there are cases where this occurs, we have theoretically verified whether this is one of the causes of unpredictable signals.
  • Theoretical verification example Assume that gene A and gene B exist, and that transcription termination points A and B are at the same position.
  • the transcription start point is assumed to be A: 550 base upstream of the transcription termination point and B: 500 base upstream of the transcription termination point. That is, the A gene mRNA is 50 bases longer than the B gene mRNA.
  • the 5 'SAGE method reveals that the B gene is expressed specifically in cancer cells, and that the A gene is also expressed in normal cells / cancer cells.
  • the sample necessary for hybridization is required to be single-stranded like mRNA. Also, from a thermodynamic point of view, hybridization has a molecular weight of DNA. / J, it gets harder when it gets smaller.
  • the present invention has been made in view of such circumstances, and the production of single-stranded gene tag groups reflecting the type and quantitative ratio of the base sequence group of mRNA 5 'end extracted from eukaryotic cell force.
  • the issue is to provide a method.
  • Another object of the present invention is to provide a method for measuring the expression level of a gene in a eukaryotic cell, comprising a step of hybridizing the single-stranded gene tag group to a solid phase on which DNA or RNA containing a transcription initiation site is immobilized. To do.
  • a single-stranded gene tag group reflecting the type and quantity ratio of the base sequence group of the full-length mRNA 5 'end should be It is considered effective to use as a sample.
  • the present inventors have added a technique and an ionic modification method for converting a double-stranded DNA into a single strand in the method disclosed in the 5 'SAGE method.
  • a single-stranded gene tag group reflecting the type and quantity ratio of the 5 ′ end nucleotide sequence of mRNA was prepared.
  • the single-stranded gene tag group was found to be effective in confirming the expression of genes targeting the expression start site.
  • the present invention relates to the following method for producing a single-stranded gene tag group and uses of the tag group obtained by this method.
  • a method for producing a single-stranded gene tag group comprising the following steps.
  • RNA linker contains a recognition sequence for a type II restriction enzyme.
  • a reagent kit for producing a single-stranded gene tag group of eukaryotic cells comprising the following elements:
  • RNA linker comprising an oligonucleotide containing a recognition sequence for an IIS type restriction enzyme
  • a method for measuring the expression level of a gene in a eukaryotic cell comprising the following steps.
  • a gene expression profile comprising a step of obtaining gene expression profiles of different types of cells by the method according to [13], comparing gene expression profiles, and selecting gene tags having different expression frequencies between cells. Analysis method.
  • the CAP structure is a structure existing at the 5 'end of eukaryotic cells or viral mRNAs that infect eukaryotic cells. Specifically, 7-methylguanosine forms a CAP structure by binding to the 5'-terminal nucleotide of mRNA via a 5'-3 phosphate bridge! /.
  • the present invention provides a method for obtaining only the 5 'end region of mRNA so that the transcription start site can be analyzed by hybridization.
  • RNA with a cap structure in the cell.
  • the RNA having this cap structure includes mature mRNA, which is a cocoon that is translated into a protein.
  • Non-coding RNA (non-coding RNA) ) Is also included.
  • RNA when RNA is obtained from a cell, some RNA may be degraded.
  • cells do not have a trapezoidal shape that is translated into protein, have a cap structure, and contain RNA!
  • the oligo-cabbing method is effective in analyzing a mature mRNA that is a trapezoid that includes a transcription initiation site and is translated into a protein in a sample containing RNA that is not subject to such analysis. is there.
  • the oligo cabbing method is a known method (Maruyama and Sugano. 1994. Gene, 138: 171-4). This method is characterized in that after treatment with two enzymes, the RNA linker is linked only to mRNA having a cap structure. After binding the RNA linker, purify only the one that has this linker and remove other RNA components. In the present invention, only the mRNA having the cap structure is taken out and processed so as to be able to bind to the oligonucleic acid as a probe for analysis of the iridescence.
  • the sample to be analyzed by hybridization was the full-length gene in the mRNA fraction obtained from the cells.
  • probes prepared for hybridization are based on a database and have a length of several tens of bases (for example, 50 bases) designed so that different mRNAs do not bind to the same probe.
  • the single-stranded portion of the sample DNA has a length of about 20 bases and corresponds to the 5 ′ end containing the transcription start site of mRNA.
  • the present invention makes it possible to analyze the transcription start site by means of hybridization.
  • the probe in the compartment on the DNA array is DNA having a sequence complementary to the transcription initiation site.
  • its length is 20 bases.
  • This sequence is preferably obtained by the sequence information obtained by the method described in the “method for obtaining a gene tag” (WO 2005/054465), which is another invention of the inventors. This makes the analysis near the transcription start site more reliable.
  • nucleotide sequence data obtained by the “Genetic Tag Acquisition Method” cannot be applied to the genome sequence (for example, 25% of the nucleotide sequence data cannot be applied in the actual experimental example, but the same sequence tag) If it appears repeatedly, it is counted as one tag, and it is included if it is not possible to apply a sequence that differs from the genome sequence by 2 or more base sequences). However, the reason why a large number of base sequence data cannot be applied to the genome sequence is not necessarily clear.
  • the analysis according to the present invention and the “method for obtaining a gene tag” can be applied to a biological species whose genome sequence is not clear. It is not only eukaryotes but also viruses that have a cap structure in mRNA. Many genome sequences of viruses have been elucidated, and viruses can also apply the method of the present invention.
  • the present invention relates to a method for producing a single-stranded gene tag group comprising the following steps.
  • the cap structure refers to a structure in which 7-methylguanosylribonucleic acid is bound via a 5'-3 phosphate bridge at the 5 'end of mRNA.
  • the CAP structure protects mRNA from degradation by 5'-3 'ethanuclease activity.
  • the CAP structure of mRNA that has finished its role is removed by decapping enzyme.
  • mRNA that has lost its CAP structure is degraded by 5 '3' ethasonuclease (LaGradeur et al, EMBO J, 17: 1487-1496, 1998).
  • the CAP structure is thought to be added to the 5 'end of RNA at an early stage of the transcription reaction by RNA polymerase II.
  • RNA derived from a eukaryotic cell can be used as the RNA.
  • polyA (+) RNA or total RNA can be used.
  • animals Cells derived from any species having a CAP structure in mRNA, such as plants, yeast, or slime molds, can be used.
  • RNA produced by eukaryotes infected with intracellular parasites such as viruses, viroids, or mycoplasmas, and RNA transcribed from the introduced genetic information are also subject of the present invention.
  • a prokaryotic cell gene originally supposed to have no CAP structure can be given a CAP structure by introducing it into a eukaryotic cell in a transcribable form.
  • the RNA thus transcribed is also included in the RNA derived from a eukaryotic cell in the present invention.
  • RNA transcribed from genetic information artificially introduced into a eukaryotic cell as a vector also has a cap structure and can be analyzed for expression by the method of the present invention.
  • RNA is first extracted from a eukaryotic cell having these RNAs.
  • RNA extraction methods are known. For example, using a commercial kit such as RNeasy (Qiagen) based on the GPTC method, high-purity RNA can be easily obtained. In the case of RNA extraction, if it is necessary to disrupt cells, they can be disrupted by methods known to those skilled in the art.
  • the method of the present invention includes a step of linking an RNA linker to the CAP structure of the extracted RNA.
  • Any method can be used to bind oligo RNA or oligo DNA to the cap structure.
  • the above oligo cabbing method can be exemplified as a preferred method for binding an RNA linker.
  • the oligo-cabbing method was developed as a method capable of protecting the 5 ′ end of mRNA in order to clone the full length of a gene (Maruyama and Sugano. 1994. Gene, 13 8: 171-4)).
  • a primer consisting of a linker sequence bound to mRNA with a cap structure at the 5 'end and an oligo dT force binding to the poly A structure at the 3' end of the mature full-length mRNA are also provided.
  • a full-length gene can be cloned by PCR amplification using primers.
  • the RNA fraction is treated with bacterial alkaline phosphatase (BAP) to hydrolyze the phosphate group at the 5 'end of RNA without a cap structure to give a hydroxyl group. .
  • BAP bacterial alkaline phosphatase
  • the 5 'end of a newly produced RNA fragment that has been cleaved for some reason, or mitochondrial RNA can serve as the substrate for this enzyme.
  • TAP tobacco acid pyrophosphatase
  • RNA linker binds to RNA with a phosphate group.
  • Ligation with T4 RNA ligase requires a phosphate group at the 5 'end. However, it does not react with RNA having a hydroxyl group at the 5 'end. In this way, the linker binds only to RNA derived from mRNA that is mature and complete at the 5 ′ end (having a cap structure).
  • the RNA linker linked to the CAP structure also has an oligonucleotide ability including at least a recognition sequence for an IIS type restriction enzyme.
  • the oligonucleotide used as the RNA linker may be DNA or RNA.
  • a preferred RNA linker is RNA.
  • the base sequence constituting the RNA linker may be any base sequence including the recognition sequence of the IIS type restriction enzyme.
  • the IIS type restriction enzyme cuts an arbitrary sequence having a specific recognition site force separated by a certain length.
  • An object of the present invention is to obtain the 5 ′ end of mRNA as a tag. Therefore, in order to prepare DNA containing the transcription initiation site used in the present invention, it is desirable to place an IIS type enzyme recognition sequence close to the 5 ′ end of the mRNA, that is, at the 3 ′ end of the RNA linker.
  • the recognition sequence is positioned so that cleavage occurs at a site downstream of the transcription start site of mRNA.
  • Various types of IIS type restriction enzymes are known. The distance between the recognition sequence and the cleavage position is almost constant by the enzyme.
  • Bsm FI or Fokl cleaves DNA at positions 9 to 10 bases from the recognition sequence, leaving a sticky end.
  • the following enzymes are known as IIS-type restriction enzymes having the same action (Szybalski, Gene 40: 169, 1985).
  • an IIS type restriction enzyme called Mme I cleaves at a position 20 bases away from the recognition sequence (5'-TCCRAC-3 '(R is G or A)) (Tucholski et al, Gene Vol. 157, pp. 87-92, 19 95).
  • An expression analysis method capable of obtaining a 20-base long tag using Mmel as a tagging enzyme is also known (US Patent 6498013). SAGE using Mmel is also called long SAGE.
  • IIS restriction enzymes have different recognition sequences depending on the enzyme used. Depending on the element, it is desirable to place the restriction enzyme recognition sequence on the RNA linker.
  • the recognition sequence is 5'_TCCRAC-3 '(where R is G or A), so UCCRAC (R is G or A) is placed at the 3' end of the RNA linker. It is desirable to do.
  • the base sequence constituting the RNA linker can also be used as a region for the primer for tag amplification to call.
  • a region for annealing the primer a length of 10 bases or more, usually 10 to 40 bases, particularly 15 to 30 bases is preferable.
  • the base constituting the primer can be designed so that the melting temperature (Tm) of the primer is 60 to 80 ° C, especially 65 to 75 ° C. It is not desirable that the primers make a dimer, or that the primer binding portion has a higher order structure. In addition, those that do not contain much GC are preferable. If the above conditions are satisfied, the base sequence of the portion where the primer anneals may be arbitrary.
  • the region constituting the recognition sequences of various restriction enzymes and the region for annealing the primer can be overlapped in the RNA linker.
  • G may be placed at the 3 'end of the RNA linker.
  • RNA linker other than the oligo-cabbing method As a method for binding an RNA linker other than the oligo-cabbing method, a method of binding a force RNA linker by purification using a solid-phased cap-binding protein (Edery, L. et al , Mol. Cell Biol. 15: 3363-3371, 1995), a cap trapper method in which biotin is bound to a diol group of the cap structure to form cDNA and then oligo DNA (Carnici, P., Genomics 37: 327-336, 1996) and the like can be used arbitrarily.
  • RNA having a cap structure bound to a solid phase if a TAP treatment is applied to mRNA having a cap structure bound to a solid phase, mRNA having a phosphate group at the 5 'end is released. Bind the RNA linker. Furthermore, reverse transcription reaction is performed on this RNA.
  • piotin is bound to the cap structure, and mRNA having the piotin bound thereto is recovered by solid-phased avidin and subjected to a reverse transcription reaction.
  • the oligo DNA adapter linker is bound to this cDNA.
  • cDNA synthesis consists of two steps: first strand synthesis and second strand synthesis.
  • the synthesis of the first strand is a reverse transcription reaction using RNA as a template.
  • the second strand is the first strand synthesized earlier.
  • the first strand of cDNA can be synthesized with a primer that anneals to an arbitrary region of RNA.
  • a primer that anneals to an arbitrary region of RNA.
  • cDNA having a sequence complementary to RNA is synthesized by an extension reaction toward the 3 'end of the oligo DNA.
  • RT reverse transcriptase
  • RNA template As a mutant of reverse transcriptase, a mutant (Superscript I I, Invitrogen) in which the RNaseH activity of reverse transcriptase is lost is commercially available.
  • enzymes such as Tth DNA polymerase that catalyze complementary strand synthesis reactions that are RNA-like but are DNA synthesis enzymes are also known. If such an enzyme is used, the first strand (RNA template) and the second strand (DNA template) can be synthesized with a single enzyme.
  • oligo dT As the primer, so-called oligo dT can be used, and oligo dT is also commercially available (Invitrogen, etc.). Since oligo dT binds to the polyA moiety at the 3 'end of mRNA, reverse transcription reaction using this will give the full length cDNA. The resulting cDNA also contains a portion complementary to the RNA linker at the 3 'end.
  • an oligo DNA having a sequence complementary to a sequence specific to a certain region can also be used as a primer.
  • an extension reaction of a gene having a specific sequence proceeds by reverse transcription, and a cDNA including a transcription initiation site and an oligo RNA portion is synthesized. Therefore, only the transcription start site of a gene having a special sequence is analyzed. This can be applied to determine the transcription start site of a known gene.
  • the expression level of each transcript can also be compared.
  • the genes that give the multiple transcripts that encode the same amino acid sequence but differ in the transcription start site should be identified. Can do.
  • each can also be used as an adapter primer. That is, an arbitrary sequence capable of binding to the oligo DNA can be connected to the 5 ′ upstream portion of each primer with a sequence different from the sequence used for the adapter portion of the RNA linker.
  • the full length of RNA is not always necessary! /.
  • tags are obtained from a small region including the 5 ′ end of RNA. Therefore, if the region containing the 5 ′ end of RNA can be synthesized as cDNA, the cDNA necessary for the present invention can be obtained. Therefore, it is also possible to use a known random primer instead of oligo dT and an adapter connected to the 5 ′ end upstream of this random primer.
  • Adapter One random primer is a mixture of multiple sequences, so it binds to various parts of RNA and the extension reaction proceeds toward the 3 'side from where it was bound.
  • the complementary sequence at the 5 'end of RNA is always included at the 3' end. Whether or not it contains a complementary sequence at the 5 ′ end of RNA can be determined by whether or not it contains an RNA linker sequence.
  • the cDNA thus obtained can be used for the analysis of the transcription initiation site. By using random primers, there is an advantage that reverse transcription reaction can be performed even if the obtained RNA is a fragment that is incomplete at the 3 'end and does not contain a polyA region. There is also an advantage that non-coding RNA can be analyzed.
  • the RNA used as the reverse transcription reaction type may be subjected to alkaline decomposition.
  • the obtained cDNA has a portion complementary to the sequence of the RNA linker at the 3 'end in common.
  • RNA linker As a cage.
  • Methods for synthesizing double-stranded cDNA are well known.
  • the oligo DNA used here contains a recognition sequence for the IIS restriction enzyme! /. Except for the recognition sequence of IIS type enzyme, the sequence of the part should be sufficient It is usual to design an RNA linker sequence.
  • DNA polymerase is used as an enzyme in addition to oligo DNA as a primer.
  • An example of the DNA polymerase is T4 DNA polymerase.
  • the second strand may be synthesized using the modified single-stranded oligo DNA as a primer.
  • the double-stranded by single-stranded oligo DNA in DNA polymerases that have undergone modifications do not adequately be obtained, it is possible to use a primer duplexes with modifications.
  • the double-stranded primer is the annealed two of the modified oligo DNA and the unmodified oligo DNA.
  • the modified strand should be an oligo DNA that has two regions: the modified 5 'end and the 3' region that has the same sequence as the RNA linker.
  • the 5 ′ half of this modified oligo DNA is annealed with another oligo DNA having a sequence complementary to this portion, and the remaining 3 ′ half of the modified oligo DNA having a sequence in common with the RNA linker is Leave as single strand.
  • the unmodified oligo DNA has a complementary sequence in the 5 ′ half region of the modified oligo DNA.
  • the 5 'half and the 3' half do not necessarily have the same length.
  • the single-stranded portion of the double-stranded primer is annealed to the cDNA.
  • the second strand of the cDNA can be efficiently modified (Shiraki et al., 2003. Proc. Natl. Acad. Sci. USA. 100: 157 76-81) o
  • a known DNA ligase is used to connect the first strand of the cDNA with the primers used here.
  • known methods such as pyotinization and DIG binding can be used.
  • a known Taq polymerase can also be used to make the cDNA double-stranded.
  • two types of primers may be used.
  • the other oligo DNA differs depending on the primer used in the reverse transcription reaction.
  • an oligo DNA having an adapter is used in the reverse transcription reaction, an oligo DNA having a sequence complementary to a part of the adapter can be used.
  • oligo dT is used as a primer during reverse transcription
  • oligo dT is also used as a primer for double-stranded reaction.
  • a specific sequence is used as a primer during reverse transcription reaction
  • a primer having a specific sequence can also be used for the double-stranded reaction.
  • Double-stranded DNA can be obtained by reacting with Taq polymerase using the above two primers.
  • Taq polymerase performs a gene amplification reaction, but it does not need to be excessively amplified here.
  • the present invention includes a step of converting cDNA into a double-stranded gene tag group using an IIS type restriction enzyme.
  • the double-stranded cDNA synthesized by the above method is treated with an IIS-type restriction enzyme, and the restriction enzyme recognition site force is cleaved at an arbitrary length in the direction corresponding to the mRNA.
  • the length of the fragment generated by cleavage varies depending on the IIS type restriction enzyme used, but the restriction enzyme recognition site power is preferably 12 to 26 bases, more preferably 20 bases.
  • a fragment having a sequence of about 20 bases from the 5 ′ end of the gene (19 bases in the case of the RNA linker of SEQ ID NO: 2) can be mentioned. If this fragment has a modification capable of binding to the solid phase, the double-stranded gene tag can be purified by retaining this fragment on the solid phase and recovering it from the solid phase.
  • the recovered double-stranded DNA tag can be made into a single strand by denaturation. Denaturation can be carried out by a known method, for example, heating (95 ° C, 3 minutes) followed by rapid cooling (on ice). If there is a modification capable of binding to the solid phase, it can be immediately rebound to the solid phase and the strand corresponding to the complementary sequence of mRNA released by denaturation (first strand) can be removed. After this, an oligo DNA having a sequence complementary to the sequence of the RNA linker is excessively covered and hybridized to the portion corresponding to the RNA linker, so that only the portion corresponding to the 5 'end of the mRNA is 1 It can be left as it is. After washing the solid phase, recover the bound DNA from the solid phase. By the above method, the single-stranded gene tag group of the present invention can be obtained.
  • the modification portion used for binding of the DNA to the solid phase is chemically modified to produce It can be used for analysis.
  • the chemical modification include techniques known to those skilled in the art, such as radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.
  • Examples of the modifying substance used in the chemical modification of the present invention include the following.
  • Preferred labeling enzymes include, for example, peroxidase, alkaline phosphatase, ⁇ -D-galactosidase, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, ⁇ -glycerol phosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase Examples thereof include xidase, asparaginase, glucose oxidase, ribonuclease, urease, catalase, glucose monophosphate dehydrogenase, darcoamylase, and acetylcholinesterase.
  • Preferred fluorescent substances include, for example, fluorescein isothiocyanate, phycobiliprotein, rhodamine, phycoerythrin, phycocyanin, and aloficocyanine.
  • luminescent substances include isorminol, lucigenin, luminol, aromatic ataridum ester, imidazole, ataridium salt and its modified ester, luciferin, luciferase, and equorin.
  • Preferred radioactive materials include 125 I, I, 131 1, 14 C, 3 H, 32 P, or 35 S.
  • Suitable techniques for binding the modifying substance to the single-stranded gene tag of the present invention are known. Specifically, direct labeling and indirect labeling can be used.
  • a direct labeling method a method in which a single-stranded gene tag and a label are covalently covalently bound by a crosslinking agent is generally used.
  • Cross-linking agents include N, ⁇ '-orthophenol dimaleimide, 4- ( ⁇ -maleimidomethyl) cyclohexanoic acid ⁇ ⁇ -succinimide ester, 6-maleimidohexanoic acid ⁇ ⁇ -succinimide ester, 4,4 '-Dithiopyridine and other known crosslinking agents can be used.
  • the reaction between these cross-linking agents and single-stranded gene tags may be performed according to a known method depending on the properties of the respective cross-linking agents.
  • a low molecular hapten such as piotin, dinitrophenol, pyridoxal, or fluoresamine is bound to a single-stranded gene tag and indirectly labeled with a binding component that recognizes it.
  • piotin avidin and streptavidin are used as recognition ligands.
  • antibodies that recognize these haptens are labeled with dinitrophenyl, pyridoxal, or fluoresamine.
  • horseradish peroxidase can be used as a labeling enzyme.
  • This enzyme is advantageous because it can react with many substrates and can be easily bound to antibodies by the periodate method.
  • an antibody is used as an antibody, for example, Fab ′, Fab, F (ab ′).
  • Polyclonal antibodies and monoclonal antibodies are used as an antibody, for example, Fab ′, Fab, F (ab ′).
  • an enzyme label can be obtained by the same treatment. If the enzyme label obtained using the above-mentioned crosslinking agent is purified by a known method such as affinity chromatography, a more sensitive immunoassay system can be obtained.
  • the purified enzyme-labeled antibody is preserved by adding thimerosal or the like as a preservative and glycerin or the like as a stabilizer.
  • the labeled antibody can be stored for a longer period of time by lyophilization and storage in a cool and dark place.
  • examples of the modifying substance of the present invention include chemical modification using piotin, avidin beads, avidin Cy3 and the like.
  • the present invention relates to a method for measuring the expression level of a gene in a eukaryotic cell, comprising the following steps.
  • the single-stranded DNA tag group reflecting the type and quantity ratio of the 5 ′ end nucleotide sequence of the mRNA of the present invention can be used as a sample for the DNA array method.
  • sample DNA or RNA
  • RNA RNA
  • oligo DNA is bound to a small section on a chip such as a slide glass, and hybridization is performed with this oligo DNA to probe.
  • the power to detect DNA binding to ⁇ is well known.
  • the synthesized oligo DNA is fixed on a glass chip by various methods.
  • a non-porous tip such as a slide glass (Biostrand) or a porous membrane such as a trocellulose membrane is used. You can also
  • the probe for performing hybridization is not particularly limited as long as it has a complementary sequence of DNA expected to be contained in the sample.
  • the design of the probe used here is very important. It is a key to the National Center for Biotechnology Information (NCBI) such as ReSEQ and Unigene. It is a probe of the transcription start site by using databases such as European Bioinformatics Institute ⁇ Ens ble ble, GoldenPath, etc. It is possible to design the base sequence of DNA.
  • NCBI National Center for Biotechnology Information
  • a probe specific for analysis of the 5 'end of mRNA can also be prepared in advance.
  • the genetic data in the public database does not always contain the transcription start point.
  • the sequence of the start site could not be obtained.
  • WO 2005/054465 which the inventors previously applied for a patent, many transcription initiation sites that were not found in databases such as ReSEQ and Unigene were revealed. (Hashimoto et al., 2004. Nat. Biotechnol. 22: 1146-9).
  • a single-stranded gene tag group prepared by the above method is added to the above DNA array.
  • the hybridization conditions are, for example, “2 X SSC, 0.1% SDS, 50.C”, “2 X SSC, 0.1% SDS, 42.C”, “1 X SSC. , 0 • 1% SDS, 37 ° C, and more stringent conditions: 2 X SSC, 0.1% SDS, 65 ° C, 0.5 X SSC, 0.1% SDS, 42 ° C, and 0.2 X SSC , 0.1% SDS, 65 ° C. ”.
  • the probe is added for 1 hour or more. Hybridize at ° C, then wash 3 times for 20 minutes in 2 X SSC, 0.1% SDS at room temperature, followed by 20 minutes at 37 ° C in 1 X SSC, 0.1% SDS It can be done 3 times, and finally it can be washed twice in 1 X SSC, 0.1% SDS at 50 ° C for 20 minutes.
  • Prehybridization Solution (CLONTECH)
  • prehybridization at 55 ° C for 30 minutes or more add the labeled probe and incubate at 37-55 ° C for 1 hour or more. It is possible to wash 3 times for 20 minutes at room temperature in 2 X SSC, 0.1% SDS, and once for 20 minutes at 37 ° C in 1 X SSC, 0.1% SDS.
  • the temperature of the prehybridization and the hybridization can be set to 60 ° C
  • the stringent condition can be set to 68 ° C.
  • the conditions such as the salt concentration and temperature of the buffer in addition to the other conditions such as the probe concentration, the probe length, the probe base sequence composition, and the reaction time. Can be set.
  • a preferred example of a method for measuring the expression level is a method for quantifying a single-stranded gene tag that has been probed or hybridized by the DNA array method.
  • the single-stranded gene tag group used as a sample can be fluorescently modified by the above method.
  • the position of the probe on the chip fluoresces, and the position of the probe to which nothing is bound does not fluoresce .
  • the fluorescence intensity changes depending on the amount of DNA that hybridizes to a probe.
  • the single-stranded gene tag that hybridizes to a probe can be quantified, and the expression level of a gene having a sequence complementary to the probe can be clarified. Fluorescence modification should be performed prior to hybridization, and single-stranded gene It can be performed on all of the tag groups, or can be performed only on the single-stranded gene group that has been hybridized to the probe on the DNA chip after hybridization. Alternatively, hybridization can be detected without using fluorescence. In any case, since the complementary sequence portion of the sample DNA is derived from the 5 ′ end of the mRNA, the expression of a gene having a transcription initiation site complementary to the probe can be detected.
  • two types of samples can be compared using a known method.
  • fluorescence such as a DNA chip
  • it can be performed as follows.
  • different fluorescent materials are used for the two types of samples.
  • Cy3 can be used for one sample
  • Cy5 can be used for the other samples. If these two types of samples are premixed and then placed on the chip, and the fluorescence observation on the chip is examined to determine whether the fluorescence is derived from Cy3 or Cy5, either It turns out that it is DNA from the sample. This reveals which gene expression level was higher in which sample.
  • the advantage of this method is that, as already well known, two samples can be compared by analysis on one chip ⁇ ;
  • RNA linker (1 or 2) of the same sequence may be used. May be used.
  • Comparison of gene expression of two types of samples is useful in the following cases.
  • a certain cancer cell is prepared, one is cultured in a normal medium, and the other is cultured by adding an anticancer agent to this medium.
  • a gene that was expressed only in the case where an anticancer drug was added is considered to be induced directly or indirectly by the anticancer drug.
  • Genes that were found in those who did not have expression after adding an anticancer drug are considered to have been suppressed by the anticancer drug.
  • tissue-specific gene expression can be examined by comparing cells from different tissues, such as the liver and kidney. Even if the same tissue is compared with normal tissue and diseased tissue, disease-specific genes can be expressed, which is useful for disease diagnosis. Or, if you compare gene expression between Japanese cattle and F1 cattle made by crossing Japanese cattle and non-Japanese cattle, you will be able to distinguish Japanese cattle from other cattle if you find a characteristic gene for each. .
  • the single-stranded gene tag group of the present invention can be used as a sample for a DNA array method.
  • a single-stranded DNA tag group that reflects the type and quantity ratio of the 5′-end base sequence of the mRNA of the present invention as a sample for the DNA array method, the transcription start site and the gene targeting the transcription start site Can be easily and accurately examined.
  • the method of the present invention it is considered possible to eliminate the occurrence of an unpredictable signal that occurred when full-length mRNA was used for gene expression analysis.
  • the present invention relates to a reagent kit for producing a gene tag including the following elements.
  • kits examples include the following. Several types of kits can be created by combining the primers. In addition, the power to analyze one cell and the power to make two fluorescent substances can be selected depending on whether two types of cells are compared.
  • Elements for performing RNA ligation including an RNA linker having a recognition site for a type IIS restriction enzyme,
  • a cDNA second comprising an oligo DNA having a sequence complementary to a part of the RNA linker and having a label capable of binding to a solid phase and an oligo DNA having a sequence complementary to the oligo DNA of (2).
  • RNA ligation elements including an RNA linker with a recognition site for type IIS restriction enzyme Bacterial alkaline phosphatase
  • primers for cDNA first strand synthesis are examples.
  • Primer that has an adapter sequence and is complementary to a specific mRNA.
  • V A random primer that has an adapter sequence.
  • a cDNA second comprising an oligo DNA having a sequence complementary to a part of the RNA linker and having a label capable of binding to a solid phase and an oligo DNA having a sequence complementary to the oligo DNA of (2).
  • one primer for cDNA second strand synthesis is
  • RNA linker When analyzing the expression of fluorescently labeled avidin in one cell, only one kind of fluorescent substance such as Cy3 labeled avidin is used. When comparing expression in two cells, two types of fluorescent dyes such as Cy3-labeled avidin and Cy5-labeled avidin are used. Apply these two types of fluorescent labels to single-stranded gene tags from each cell! ⁇ Hybridize to the probe competitively on the DNA chip as described above.
  • the adapter part of the RNA linker can be arranged differently for each label to be bound, and the IIS type enzyme recognition site can be made the same sequence.
  • a specific example of a reagent kit for producing a single-stranded gene tag group includes a kit in which the following elements are combined.
  • RNA linker comprising an oligonucleotide containing a recognition sequence for an IIS type restriction enzyme
  • kits which is any primer for which a group force of 0-iiO force below the primer for cDNA first strand synthesis is selected.
  • gene expression information (expression level) at the 5 'end of mRNA, which is a transcription product can be obtained.
  • the expression information at the 5 'end is particularly important in gene analysis.
  • gene expression information at the 5 ′ end that can be obtained by the present invention can be used for the following uses.
  • the present invention can be used to obtain a gene expression profile. That is, the present invention relates to a method for obtaining a gene expression profile in a eukaryotic cell, comprising the following steps.
  • an expression profile refers to a list of gene information accompanied by expression information.
  • Expression information is a quantitative parameter that indicates the level of expression.
  • Gene information usually refers to information for specifying a gene. Specifically, gene base sequence, gene name, gene ID number, etc. constitute gene information. The number of genes that make up the list is arbitrary. Moreover, the object is not limited. Depending on the purpose of the analysis, information on necessary genes is accumulated to construct an expression profile.
  • gene expression information at the 5 'end can be obtained from RNA having a CAP structure.
  • the base sequence information and the appearance frequency are associated with each other.
  • an expression profile can be obtained.
  • RNAs are targeted as RNA
  • an expression profile for all genes can be obtained.
  • a gene tag can also be generated for a specific gene or a group of genes having structural commonality. In such a case An expression profile of a specific gene or group of genes is generated.
  • the expression profile obtainable by the present invention more accurately expresses the expression status of the gene in the cell. It can be said that it is reflected in.
  • the expression profile obtained by the present invention can be used as a database.
  • a database is a set of electronic data in which information constituting an expression profile is stored as machine-readable data.
  • the database of the present invention includes gene expression information at the 5 ′ end of mRNA. Furthermore, the database of the present invention can record the ID number of each base sequence information and the origin of the RNA from which the expression information was obtained. Furthermore, it is possible to add information such as the relationship with the expression information of known genes and the result of mapping onto the genome.
  • a comparison target of the expression level information of the tag information stored in a database accumulated in advance can be used.
  • gene tag information is accumulated in advance based on the method of the present invention. This information can be shared on a computer network. Alternatively, it can be distributed commercially by attaching to the reagent kit. It is also possible to compare the gene tag information obtained in this way with the gene tag information obtained through experiments.
  • a database indicating which gene transcription start site probe is present in each section of the chip is prepared. It is good to keep. There can be a chip for each eukaryote, including humans, which is not a single type of chip. In addition, one species of probe is not always on one chip. A single species may make a chip that covers a variety of genes, or it may specialize in a certain type of product. For example, it is possible to make a chip specially designed for site force-in, receptors, cancer suppressor genes, molecules with CD numbers (ie, CD antigens).
  • the result can be displayed by software that has the function of displaying which promoter worked and which response element or transcription factor was involved.
  • the software captures this table data file of the chip used when the fluorescence intensity is observed.
  • the software should have functions such as sorting and searching for each section data.
  • a display arranged in order of fluorescence intensity and a display arranged for each gene will be possible.
  • the fluorescence intensity of one transcript is set to 1, and the fluorescence intensity of other transcripts can be expressed as a ratio. Kutoyo ⁇ .
  • the gene is labeled with fluorescence of a different color for each cell.
  • fluorescence intensity When fluorescence intensity is observed, it is better to write two colors in the table: fluorescence color and intensity. If it is shown separately for each color when displaying, it shows which gene expression is most likely in which cell.
  • This software may be included in the kit as described above.
  • the expression profile database of the present invention can be stored in an electronic medium.
  • Examples of electronic media include various disk devices, tape media, and flash memory. These electronic media can be shared on a network.
  • the database of the present invention can be shared on the Internet.
  • a function for referring to the database information of the present invention can be added to the software for analyzing the expression level of the tag via the Internet.
  • new expression profile information generated based on the present invention can be added to the database via the Internet.
  • An expression profile analysis can be performed using the expression profile of the present invention. That is, the present invention comprises gene expression profiles comprising the steps of obtaining gene expression profiles of different types of cells based on the present invention, comparing gene expression profiles, and selecting gene tags having different expression frequencies between cells.
  • the present invention relates to a profile analysis method.
  • An analysis method for obtaining genes having different expression levels between different cells is called expression profile praying. By such analysis, for example, many genes related to diseases have been obtained.
  • the expression profile of the present invention can also be used for such expression profile praying.
  • different cells to be analyzed refer to all cells having different origins. Even cells derived from the same yarn and weave are cells that have different origins if there are some differences in conditions such as the presence or absence of disease, race, age, and sex. Depending on the purpose of the analysis, if the conditions to be considered are different, the cell has a different origin. On the other hand, if only negligible differences can be found for the purpose of the analysis, they are considered the same cell. For example, different organs, different tissues, or origins and cultures By comparing expression profiles between cells with different conditions and the like, genes with high (or low) expression levels can be selected between organs, tissues, or cells. Examples of combinations of analysis objects to which the present invention can be applied are shown below.
  • expression information of gene tags characteristic of cancer can be obtained by comparing expression profiles between cancer tissue and normal tissue.
  • genetic tags related to malignancy can be identified by comparing particularly high-grade cancer and low-grade cancer.
  • the above software is the first analysis performed on the data obtained by the chip, and the data has significance by performing this analysis.
  • the data obtained with this chip can be used for more meaningful analysis.
  • the expression of such genes is subject to transcriptional regulation in the promoter sequence upstream from the transcription start site on the genome.
  • the promoter sequences that have been clarified so far are not necessarily sufficient. For example, only a few hundred promoter sequences are shown in humans. However, if the gene whose transcription amount has been changed is able to show what kind of promoter it has undergone, it becomes important information.
  • the software first associates each probe sequence on the chip with a promoter sequence located upstream in the genome, and when the fluorescence is detected by hybridizing to the probe, that promoter sequence. It is possible to have a function of displaying as a product.
  • This promoter sequence information can be an extension of the data in the table file used in the above software! /.
  • the response element may be found at a position distant from the promoter sequence on the genome sequence.
  • force in this response element.
  • a transcription factor binds here, and when several factors bind to the transcription factor, it binds to the promoter sequence, and further, RNA polymerase binds to the complex and the promoter. The downstream force transfer of the array is started. Therefore, the response elements and transcription factors related to each probe are stored in the table file as extended data, and when the fluorescence is observed, it is possible to display which response element or transcription factor is activated. it can.
  • intracellular signal transduction pathways, receptors, ligands that bind to receptors, etc. can be stored in a table file as extended data for the probes on each chip. .
  • the corresponding promoter, response element, transcription factor, intracellular signal transduction pathway, receptor, and ligand can be displayed. Such a display is more effective when comparing two cells.
  • the set of genes to be transcribed is clear for activation of one receptor or intracellular signal transduction pathway, it can be included as extended data.
  • the gene set transcribed for a cell by the method according to the present invention is clarified, and this result is associated with the gene set in the extended data, so that what pathway is activated in the cell and under what circumstances. It can be estimated.
  • a single-stranded gene tag sample was prepared by the following method.
  • HT-29 Cultured cells HT-29 (Fogh, J., and G. Trempe, 1975, Human Tumor Cells in Vitro, J. Fogh, editor, Plenum Publishing Corp., New York, 115-141)
  • the culture was performed using McCoy's 5A medium containing infant serum (McCoy, TA, Maxwell, M. and Kruse, PF (1959) Proc. Soc. Exper. Biol. Med., 100: 115).
  • the oligo cap method was performed by modifying the method of Maruyama and Sugano (Gen e. 1994 138: 171-174.). 10 7 cells were solubilized using RNA-Bee (Tel Test, Friendswood, TX, USA), and total RNA was obtained according to the protocol.
  • RNAeasy kit Qiagen, Hilden, Germany. Next, it was treated with 5 U of bacterial alkaline phosphatase at 37 ° C. for 60 minutes in the presence of 200 U of RNase inhibitor. Components other than RNA were removed by phenol chloroform extraction and ethanol precipitation, followed by treatment with 20 U tobacco acid pyrophosphatase in the presence of RNase inhibitor at 37 ° C for 60 minutes. After extraction and precipitation again, an RNA linker having one of the following sequences was ligated at 250C for 3 hours using 250 U of T4 RNA ligase.
  • RNA with poly A was converted to mRNA Isolation Kit for Total RNA (Miltenyi Biotech, Bergisch
  • the resulting single-stranded cDNA was synthesized with 10 U Taq polymerase using a piotinylated linker primer and a 3 ′ primer.
  • the reaction was performed by repeating 13 steps 13 times: 94 ° C for 1 minute, 58 ° C for 1 minute, 72 ° C for 10 minutes.
  • Primer sequences are as follows.
  • DNA was collected by phenol Z chloroform extraction and ethanol precipitation, and subjected to electrophoresis using a 1% agarose gel to obtain a fraction having a length of 500 bp or more.
  • a QIAEX II Gel Extraction kit Qiagen
  • Double-stranded cDNA was cleaved using 8 U of Mmel (New England Biolabs, Beverly, MA, USA), which is an IIS type restriction enzyme. Heat at 96 ° C for 5 minutes and then cool on ice to bind the pyotinylated fragment containing the transcription start site to avidinized magnetic beads (Dynabeads M-280 strept avidin, Dynal, Oslo, Norway).
  • a DNA chip to be hybridized with the single-stranded gene tag sample was prepared by the following method. Based on the tag sequence information obtained from HT-29 cells according to the “method for obtaining gene tags” (WO 2005/054465) already filed by the inventors, the oligo DNA to be placed on the DNA chip Selected. The tag sequence was compared with the genome and EST database, and the gene whose transcription start position was identified at one location was selected as the oligo DNA to be placed on the chip. In addition, among the tags identified by the “Genetic Tag Acquisition Method”, the number of identified tags is large (the expression level is large, the one) and the small number (the expression level is small). Arbitrarily selected).
  • One base was added to the 3 'end of the selected tag sequence so as to be the same as the identified genomic sequence, resulting in 20 bases.
  • oligo DNA having a complementary sequence with the above 20-base tag sequence and an amino group introduced at the C-6 position at the 3 'end were synthesized, and each oligo DNA was placed in the chip compartment at a concentration of 50 ⁇ . Combined.
  • the sequences of oligo DNAs arranged on the chip are shown in Table 1 below.
  • SEQ ID NOs: 1, 2, 7-14, and 16 are obtained by adding 1 base to the 3 ′ end and adding 20 bases to the tag sequence obtained according to “Genetic tag acquisition method”. The added base was determined by identifying the gene as genomic data or EST data and comparing it with the data. Also, distribution Column numbers 3-6 and 15 are gene tags that appeared when HT-29 cells were cultured with 5-aza-2'-deoxycitidine.
  • the collected DNA was dried with Speed Vac for 20 minutes. Dissolved in 15 ⁇ l of 0.5% SDS, 5 X SSC solution. This solution was obtained by diluting 10% SDS and 20 ⁇ SSC with water. 20 X SSC is obtained by autoclaving 3 M NaCl, 0.3 M trisodium citrate dihydrate, pH 7.0. The DNA solution was heated at 99 ° C for 3 minutes, allowed to stand at room temperature for 15 minutes, and then incubated on the chip at 42 ° C for 16 hours.
  • LoTE is a mixture of 300 1 1 M Tris-HCl, pH 7.5 and 40 ⁇ 1 500 mM EDTA, pH 8.0, and made up to 100 ml with water. Next, ethanol precipitation was performed, and the precipitate was dried with Speed Vac. The precipitated DNA was dissolved in 15 ⁇ l of 0.5% SDS, 5 ⁇ SSC solution. The DNA solution was heated at 99 ° C for 3 minutes, allowed to stand at room temperature for 15 minutes, and then incubated on the chip at 42 ° C for 16 hours.
  • the recovered DNA was dried with Speed Vac for 5 minutes. In 15 ⁇ l of 0.5% SDS, 5 X SSC solution Dissolved. The DNA solution was heated at 99 ° C for 3 minutes and allowed to stand at room temperature for 15 minutes and then incubated on the chip at 42 ° C for 16 hours.
  • the cover glass was removed by immersing the chip in 2X SSC solution containing 0.2% SDS.
  • the chip was then left for 12 minutes in a 2X SSC solution containing 0.2% SDS. Furthermore, it was left in 2 X SSC for 12 minutes. Thereafter, the chip was taken out, and water droplets on the chip were removed by air spray.
  • PCR was performed twice to synthesize the double-stranded DNA in Example 1.
  • the solution once PCR was diluted 100 times with water, and the primer, enzyme, buffer, etc. were collected and the reaction was performed again.
  • the reaction was performed by repeating the steps described in Example 1 21 times. This sample was treated in the same manner as condition 3.
  • Fluorescence intensity is the value obtained by subtracting the fluorescence intensity where no oligo DNA is spotted from the fluorescence intensity of each SEQ ID NO.
  • condition 1 fluorescence staining was performed after hybridization with oligo DNA on the chip, while in condition 2, fluorescence staining was performed before hybridization. Comparing the two results, it was found that fluorescence staining was necessary after the hybridization because condition 2 did not show any fluorescence.
  • Condition 3 the number of washings after the hybridization was increased. Although there was no change in the intensity of the fluorescence intensity compared to condition 1, the fluorescence intensity (background) of the V and part of the oligo DNA spot could be kept low.
  • condition 4 the fluorescence was observed only in SEQ ID NO: 7 in condition 1 and condition 3, Due to the low fluorescence intensity, PCR was repeated twice to increase the number of cycles in order to increase the amount of DNA in the sample. In conditions 1 to 3, 1/5 of the total amount of the sample after PCR was used, while in condition 4, the second PCR was performed with 1/5 of the total amount after the first PCR and the second PCR. After that, the entire amount was used in the next step.
  • the experimental method is the same as Condition 3. As a result, fluorescence was observed in SEQ ID NOs: 2, 5, 7, 8, 10, and 13.
  • SEQ ID NO: 7 is a sequence starting from the transcription initiation point of a product having 534 tags and a particularly large transcription amount in HT-29 cells. Strong fluorescence was also observed under condition 3, and the large number of tags corresponded well with the fluorescence intensity.
  • SEQ ID NO: 13 is derived from the same gene as SEQ ID NO: 7, and SEQ ID NO: 13 is 5 bases downstream of SEQ ID NO: 7 and has an overlapping portion. There are not many transcripts starting from the 5 'end of SEQ ID NO: 13 with the number of tags starting from the 5' end of SEQ ID NO: 13, starting from the 5 'end of SEQ ID NO: 13. Strong fluorescence was observed against SEQ ID NO: 13. This is reasonable if it is assumed that the transcript starting with the 5 'end force of SEQ ID NO: 7 also binds to SEQ ID NO: 13 on the chip and that strong fluorescence was observed due to the large amount of this product.
  • the present invention provides a method for producing a single-stranded gene tag group reflecting the type and quantity ratio of the nucleotide sequence group at the 5 'end of mRNA extracted from eukaryotic cells. Sarakuko succeeded in confirming the expression of the desired gene by using the gene tag group as a sample of DNA and hybridization for the DNA chip.
  • the present invention has an advantage that the transcription start site and expression level of a gene expressed in a target cell can be examined simply, accurately and comprehensively, and can be used in various industrial fields. .
  • the transcription start site reflects the state of cells. It is possible to investigate the difference between the transcription start site of normal state and disease state in cells of the same tissue site, and to perform gene cloning based on the difference. Furthermore, genes that are specifically expressed in disease cells, or genes in the form of alternative splicing specific to diseases can be clarified. Therefore, it is possible to develop PCR primers and DNA probes for disease diagnosis.
  • Nucleic acid drugs are designed to specifically destroy the mRNA of a disease-inducing gene or to specifically inhibit transcription of such a gene.
  • the elucidation of the structure is important.
  • the overall pharmacological action can be clarified, and side effects and toxicity can be clarified at the same time. If the side effects and toxicity can be clarified in the laboratory, their usefulness is high. In other words, if testing with cultured cells is possible, animal experiments will be reduced, and if side effects and toxicity can be estimated before clinical trials, accidents in clinical trials will be prevented.
  • the method of the present invention is a technique for comprehensively clarifying the expression of a target gene targeting a transcription initiation site immediately before development of a diagnostic method, drug development, and clinical trial. It can be used as
  • the present invention provides a sample preparation method suitable for analysis of the transcription start site on a DNA chip, and at the same time a method for designing a probe for a DNA chip based on the data obtained in the “method for producing a gene tag”. provide.

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Abstract

La présente invention a trait à un groupe d’étiquettes géniques à simple brin qui peut rendre compte des types et du rapport des montants de séquences nucléotidiques, située chacune à la terminaison 5' du mRNA ; ledit groupe peut être produit en employant une procédure pour convertir les ADN à double brin en ADN à simple brin combinée au procédé énoncé dans le 5'-SAGE. Lorsque l’hybridation est réalisée sur une puce à ADN en utilisant comme échantillon le groupe d'étiquettes géniques à simple brin, on a découvert que le groupe d’étiquettes géniques à simple brin est utile pour la confirmation de l’expression d’un gène dans lequel le site de lancement de l’expression est employé en tant que cible. En utilisant le groupe d’étiquettes géniques à simple brin, il devient possible de réaliser une analyse exhaustive de l’expression de gènes dans laquelle divers sites de lancement de transcription sont employés comme cibles.
PCT/JP2006/314459 2005-07-22 2006-07-21 Procédé de production d’étiquettes géniques à simple brin, chacune ayant un site de lancement de la transcription WO2007011016A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005054465A1 (fr) * 2003-12-01 2005-06-16 Post Genome Institute Co., Ltd. Procede permettant d'obtenir une etiquette de gene

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005054465A1 (fr) * 2003-12-01 2005-06-16 Post Genome Institute Co., Ltd. Procede permettant d'obtenir une etiquette de gene

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HARBERS M. ET AL.: "Tag-based approaches for transcriptome research and genome annotation", NAT METHODS, vol. 2, no. 7, July 2005 (2005-07-01), pages 495 - 502, XP009058036 *
HASHIMOTO S.: "5' SAGE-ho (5' End Idenshi Hatsugen Kaiseki). (SAGE for 5'-ends transciptome)", EXPERIMENTAL MEDICINE, vol. 23, no. 10, 1 June 2005 (2005-06-01), pages 1569 - 1575, XP003006075 *
SUZUKI Y. ET AL.: "mRNA Tensha Kaishiten no Dotei to Idenshi Hatsugen no Kaiseki", PROTEIN, NUCLEIC ACID AND ENZYME, vol. 42, no. 17, 25 December 1997 (1997-12-25), pages 2836 - 2843, XP003006076 *

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