WO2002002814A1 - Highly sensitive method of detecting nucleic acid - Google Patents

Highly sensitive method of detecting nucleic acid

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Publication number
WO2002002814A1
WO2002002814A1 PCT/JP2001/005783 JP0105783W WO0202814A1 WO 2002002814 A1 WO2002002814 A1 WO 2002002814A1 JP 0105783 W JP0105783 W JP 0105783W WO 0202814 A1 WO0202814 A1 WO 0202814A1
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WO
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Patent type
Prior art keywords
labeled
nucleotide
nucleic acid
polynucleotide probe
de
Prior art date
Application number
PCT/JP2001/005783
Other languages
French (fr)
Japanese (ja)
Inventor
Junichi Mineno
Edy Meiyanto
Norihiro Ishida
Tatsuo Takeya
Kiyozo Asada
Ikunoshin Kato
Original Assignee
Takara Bio Inc.
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Publication date

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/158Expression markers

Abstract

A labeled polynucleotide probe characterized by being partly hybridizable with a polyadenine nucleotide moiety; a process for producing the same; and a method of detecting a nucleic acid by using the same.

Description

High sensitivity detection methods the art of Akira fine manual nucleic acid

The present invention relates to a highly sensitive detection method using the method and the probe preparation of useful labeling probes in genetic engineering. BACKGROUND

Hybrida I See Chillon method for identifying the type of mRNA A expressed in cells or tissues, or a normally well-utilized by Ru method to quantify each mR NA molecular weight. Therefore, the identification of mRNA A, hybrida I See sucrose emission techniques for quantification, is very important in the field of biology and medicine.

For example, as the High Priestess die internalization method, all R NA or purified RN A was immobilized on a porous carrier such as Nai port down membranes were labeled with a fluorescent substance or radioactive same position elements (RI), etc., detected gene (RNA) to be cane mixed with complementary probe and a method for Haiburidizu. Northern High Priestess die internalization method can be cited as a technology that belong to this category.

As another method, a mixture containing a gene (R NA) to be'll detection, Wakashi Ku is labeled R NA purified from the mixture with a fluorescent substance or radioisotope, etc., it is fixed to this on the membrane there is a method of probe hybridized. The techniques belonging to this category include reverse dot blot method. These two methods have been used in a variety of experimental studies of the genetic engineering field.

Normally a conventional High Priestess die See Chillon method, have been used nucleic acid immobilized on a membrane, recently, for hybrida See Chillon techniques described above, on the carrier of non-porous rigid such as glass to have been developed hybrida I See Chillon techniques used primarily immobilized known D NA fragment. In this technique, it can be extremely densely fixed a number of types of DN A fragment predetermined area on the carrier surface. Such high density carrier DN A fragment is fixed I arsenide is called D NA microarray (D NA chip) In general. The use of the D NA microarray, the identification of many kinds of mRNA A small amount of sample, it has become possible to perform quantitative at once.

When using the DNA microarray, a nucleic acid in a sample, for example, labeled with a fluorescent substance, detecting by Haiburidizu and immobilized DNA fragments on the support it is generally used. That is, when detecting mRNA in the sample with a DNA microarray I are, for example, Ru can be used to label the sample in the following manner.

1. How the mRNA A in a sample by 添Ka 卩 fluorescently labeled nucleotides during the c DNA synthesis was 鎵型 creating a fluorescent labeled c 'DNA or,

2. After synthesizing the c D NA from mRNA in the sample, and carry out transcription reaction with RNA polymerase to the c DNA in the presence of fluorescently labeled nucleotide as a mirror-type, a method of creating a fluorescent labeled RNA probes.

Labeled D NA or RNA had it occurred in manufacturing the above method, the base sequence derived from m RNA in the sample, or complementary thereto the nucleotide sequence has, on a microarray suitable D NA fragment was fixed in can be detected.

However, in the case of manufacturing a labeled nucleic acid having a base sequence derived from mRNA in a sample by a method as described above, since the label number of compounds to be incorporated into the labeled nucleic acid is proportional to the chain length of the nucleic acid, short chain the length of the labeled nucleic acid has a disadvantage that the number of labeled nucleotides incorporated per minute child compared to those of long chain length is reduced. Accordance connexion, labeled nucleic acid carrier of the same number molecular, for example when the fixed are I spoon DNA fragment and Haiburidizu on a microarray, the intensity of the label signal from labeling compounds are obtained for each array of the hybridizing nucleic acid strands It will be greatly affected by the length. This means that in the experiments sought a quantitative property, is a very important issue.

On the other hand, as a nucleic acid labeling method which is not affected by the chain length of the nucleic acid labeling, U.S. Patent No. 6 0 0 4 7 5 5 JP end labeling method according thereof. While with Kaka, the method is a method of using the end-labeled oligo d T primers to synthesize c DNA, are limited labeled number of compounds to be added to the primer. Therefore, if the gene-derived mRNA to be detected is a small amount, it may have signal intensities unable weakly detectable.

In cells are very many types of gene expression, polypeptide structure thereof encoded by the respective genes, because the different functions, respectively, also the chain length of mR NA corresponding to these Poribe peptide It is distributed in a wide range. Thus, detection of these mRNA as accurately as possible, also with high reproducibility, mRNA that gives excellent signal quantitative characteristic for quantifying or detecting method of nucleic acids have a nucleotide sequence derived from mRNA A is obtained which was. The purpose of the invention

An object of the present invention, the not affected by the chain length of the nucleic acid to be detected labeled nucleic acid is prepared and is to provide a highly sensitive detection method using the labeled nucleic acid. Summary of the Invention

This 宪明 we Detection of the nucleic acid according hybrida I See Chillon method performs sharpness meaning study Determination, the immobilized probe and Haiburidizu to mR NA, 3 of the mR NA, are present in the terminated poly We found a method capable of detecting excellent mRNA quantitative characteristic and detection sensitivity by detecting using the a sequence. In addition, artificially it is also found a method capable of detecting target DNA in a similar manner as above in D NA was added a polynucleotide, and completed the present invention.

If it outlined present invention, a first aspect of the present invention relates to labeled polynucleotide probe, which comprises hybridizing to the poly adenine nucleotide portion of the target nucleic acid. In the first invention of the present invention, poly adenine nucleotide moiety which is labeled polynucleotide probe for soybean High Priestess may be Poririboadeni down nucleotide tail mRNA A. Moreover, labeled Porinukureo Chidopurobu In the present invention, the chain length of the probe may be suitably used those which are 5 0 nucleotides or more. Further, the probe is preferably a polynucleotide consisting of de O carboxymethyl © La sill nucleotide 及 Pi Z or de-O carboxymethyl thymine nucleotide, Rukoto contain labeled Dokishi © La sill nucleotide 及 Pi Z or labeled de O carboxymethyl thymine nucleotides It is preferred. Further, the labeled nucleotides are radioactive isotopes, chemical Tsutomuhikari substances, fluorescent substances, those which are-labeled with a substance selected from the group consisting of ligand and receptor can be suitably used. Is not particularly limited, for example, Furuo Resein, cascade blanking / Leh, Oregon Green, BODIPY, Rodamingu lean, A lexa F 1 uor, Texas Red, with C y 3 and C y 5 Tona Ru material selected from the group labeled polynucleotide probe which is labeled can be preferably used, particularly preferably, d UT P or d TTP with the label is shown an example.

The second aspect of the present invention, a polyribonucleotide adenine nucleotide or Poridokishi adenine nucleotide and 鎳型, the first invention of the present invention which is characterized in that it comprises the step of performing a nucleotide extension reaction using oligo d T and primer methods for the preparation of labeled polynucleotide probe. In the second aspect of the present invention, nucleotide extension reaction reverse transcription reaction der connexion may that the 铸型 the polyribonucleotide adenine nucleotides. Further, the chain length of Poriripoadenin'nukureochidoa Rui as a 铸型 in the present invention is poly de O carboxymethyl adenine nucleotide, 5 0 nucleotides or der shall can be suitably used. Further, nucleotide extension reactions in the present invention may be a de-O alkoxy nucleotide polymerization reaction. Further, in the present invention, labeled chain length of the polynucleotide probe, 5 0 or more nucleotides at it is rather preferable, the nucleotide extension reaction or de O carboxymethyl nucleotide polymerization reaction labeled de O carboxymethyl © La sill triphosphates and / or a reaction performed by using the labeled de O carboxymethyl thymine nucleotidyl de triphosphate. In this case, the molar ratio of the labeled Dokishiura sill nucleotide triphosphates and Z or labeled de O carboxymethyl thymine nucleotide triphosphates and unlabeled de O carboxymethyl © La sill nucleotide triphosphates and / or unlabeled Dokishichimi down nucleotide triphosphate is 1: 1 to 1: is preferably implemented in three probes preparative reaction solution in which, also labeled substances used may group consisting radioisotopes elemental, chemiluminescent substances, fluorescent substances, ligands and receptor substance can be preferably used which are more selective. Is not particularly limited, for example Furuoresein, Cascade Blue -, Oregon Green, B OD IPY, Rhodamine Green, A 1 e X a F luor, Tekisasuretsudo, from a material that is selected the group consisting of C y 3 and C y 5 Les Shi preferred that are labeled.

A third aspect of the present invention, Poriade - down the High Priestess soybeans to labeled polynucleotide probes to nucleotide moiety with a sample potentially containing the target nucleic acid the target nucleic acid to include the step of immobilized carrier and High Priestess soybean a method for detecting a target nucleic acid shall be the said. In a third aspect of the present invention, Poriadenin'nu Kureochido portion may be a polyribonucleotide adenine nucleotide tail mR NA, both are preferably used those artificial poly adenine nucleotide portion later allowed to Tsukeka 卩it can. That is, the labeled polynucleotide probe of the first aspect of the present invention efficiently, as long as it specifically Haiburidizu is not particularly limited. In a third aspect of the present invention, labeled polynucleotide probes, labeled de O carboxymethyl © La sill nucleotide 及 Pi / or labeled de O carboxymethyl thymine nucleotides Dressings containing can be suitably used. The labeled nucleotides, radioisotopes, chemiluminescent substances, fluorescent substances, preferably those which are-labeled with a substance selected from the group consisting of ligand and receptor is not particularly limited example, Furuoresein, cascading pull one, Oregon Green, BODIPY, rhodamine green, A

1 e X a F luor, Tekisasuretsudo, the labeled polynucleotide probe that is labeled include a material that is selected from the group consisting of C y 3 and C y 5. Step A fourth invention of the present invention, a) the first sample that may contain the target nucleic acid, thereby Porinu Kureochi de probe Aniru labeled with a first label that hybridizes to the poly adenine nucleotide moiety; b ) that may contain a target nucleic acid, the second sample is different from the first sample, Poriade - to High Priestess soybean to down nucleotide moiety, a first polynucleotide labeled with different second label and labeling step of probe and Aniru; c) step a and step resulting labeled Porinukureo Chidopurobu is in b to support the Haiburidizu possible samples ized immobilize the target nucleic acid containing the target nucleic acid was Aniru; d) on a support step for detecting the first and second of label Haiburidizu; and e) comparing the presence or extent of the first and second label detected Step, characterized in that it comprises a method for the analysis of target gene expression. In a fourth aspect of the present invention, if example particularly but not limited to examples, Furuoresein, cascading pull one, Oregon Green, BODIPY, rhodamine green, A lexa F 1 uor, the group consisting of Texas Red, C y 3 and C y 5 labeled polynucleotide probe that is labeled with a substance that is more selectively can be suitably used.

A fifth invention of the present invention, Poriade - characterized by High Priestess soybean Poriade portion other than the two N'nu Kureochido with a labeled polynucleotide probe, wherein the hybridizing expressed genes down nucleotide moiety labeled Porinukureo Chidopurobu to include the step of sample and Haipuridai's that may contain an expression gene by combining about analysis method expressed genes characterized by. In the onset Ming fifth invention, but it is not for example, full Oresein, cascade blue, Oregon green, BODIPY, rhodamine green, A lea F 1 uor, Texas Red, C y 3 and C y 5 particularly limited labeled polynucleotide probe that is labeled with a substance selected from the group can be suitably used. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: is a diagram showing a contact Keru specificity for hybridization of the labeled polynucleotide probes of the present invention.

Figure 2: is a diagram showing the detection sensitivity of labeled polynucleotide probes of the present invention. Figure 3: is a view to view the detection of expressed genes using a labeled polynucleotide probes of the present invention.

Figure 4: shows a contact Keru specificity to High Priestess die internalization of labeled polynucleotide probes of the present invention.

Figure 5: is a diagram illustrating a detection using labeled polynucleotide probes of the present invention in D NA chip.

Figure 6: shows the expression analysis of genes using two kinds of labels Porinu Kureochi de probes with different labels of the present invention in DNA chips. Detailed Description of the Invention

The present invention will be described in detail.

The nucleotide used herein, refers to a de-O alkoxy ribonucleotides or ribonucleotide, for example, in the case of de O carboxymethyl lipoic nucleotides refers to a nucleotide sugar moiety is composed of D-2-Dokishiriposu, for example, adenine salt moiety, cytosine, Guanin, thymine, include those having a Urashiru. Also, in the case of lipoic nucleotides refers to a nucleotide sugar moiety is composed of D- ribose, adenine base moiety, cytosine, Guanin include those having a Urashiru. Further, modifications to the nucleotide Dokishiriponu Kureochido or modified ribonucleotide les, displacement also is included, for example, modified ribonucleotides replaced with oxygen atom of position of a phosphate group to a sulfur atom [(shed one S) ribonucleotide ( alpha-S) also referred to as New] or other derivatives are also included. In the present specification, the poly adenine nucleotide, refers to a polymer of lipoic adenine Nucleotidyl de or de O carboxymethyl adenine nucleotides. The polymer also include any of those present in those or natural artificially preparation. It is not particularly limited, for example, poly Α tail eukaryotic origin mRNA (Poririboa De Nin nucleotide tail) and the like.

The labeled nucleotide as used herein refers to a nucleotide labeled with 檫識 material. The labeling substance, directly to the de O carboxymethyl nucleotides or ribonucleoprotein tides, or any of those are linked via a linker, First include. The labeled polynucleotide herein, labeled, refers to the port Rimmer nucleotides, the polymer, the polymer is preferably composed of a labeled nucleotide and / or unlabeled Nukure Ochido.

The nucleotide extension reactions herein is not particularly limited as long as it is a reaction to extend the nucleotide reverse transcription reaction using polyribonucleotide adenine nucleotide and 鎳型, D NA polymerase reaction or, terminal de O carboxymethyl nucleotides transfer reaction any of by de O carboxymethyl nucleotide polymerization reaction can be preferably used.

The target nucleic acid herein, a nucleic acid sequence to be'll detection, for example, refers to a nucleic acid sequence of any gene. The nucleic acid sequence, it may also be either a D NA or RNA Rere.

(1) labeled polynucleotide probes of the present invention

Labeled polynucleotide probes of the present invention may be a polynucleotide probe capable of hybridizing to Poriade two down nucleotide moiety is not particularly limited, de O carboxymethyl © lysine nucleotides (dUTP) polymer, de Okishi polymers of thymine nucleotides (dTTP), or any of the mixed polymer dUTP and dTT P can be suitably used. In this specification, the Poriade Nin nucleotide moiety, refers to a labeled polynucleotide probe a portion of the target nucleic acid to soybean High Priestess in part of poly adenine nucleotide of the present invention. Chain length of labeled polynucleotide probes of the present invention is not particularly limited, preferably 50 nucleotides or more, more preferably can be suitably used those of 100 nucleotides or more. Labeled nucleotide probes of the present invention, as long as it contains a substantially constant amount of a labeled compound to the probe per molecule is not particularly limited example, Due to the fact that contain nucleotides labeled compound is added (labeled nucleotides) it can be there. The labeled nucleotides, especially not name limitation for example, a suitable label de O carboxymethyl © La sills nucleotides or labeled de O carboxymethyl thymine Nutare Ochido, may be used by mixing them. Furthermore, the label Nukure Ochido a radioactive isotope, a chemical light material, a fluorescent material, any of the labeled nucleotides in the ligand or receptions terpolymer can be suitably used. Is not particularly limited, for example, radioisotopes such as 32 P, 33 P, chemiluminescent substances, such as AM PPD, Furuoresein, Cascade Blue, Oregon Green, BOD I PY, Rhodamine Green, Al exa F l uo r, Tekisasuretsudo, Cy 3 is Rere is C y fluorescent substance such as 5, Biochin, Abijin, Rere deviation of nucleotides labeled with ligand or receptor, such as digoxigenin may also be suitably used. In the present invention, preferably a fluorescent substance, particularly preferably A 1 e X a F 1 uor, dUTP labeled with Cy 3 or Cy 5 can be suitably used.

Further, the label in another embodiment, using a modified Nukure Ochido having a functional group such as an amino group Ya thiol groups after preparing the polymer of the modified nucleotides were then take advantage of the functional groups by adding said labeling substance the polynucleotide contained in a labeled Porinuku octreotide probes of the present invention.

Labeled polynucleotide probes of the present invention is not particularly limited preferably those that hybridize to the poly adenine nucleotide moiety in Sutorinji Ento conditions. Examples of the stringent conditions, for example, Molecular click opening one Jung, § Laboratory Manual (Molecular cloning, A laboratory manual), Second Edition (1989) according the condition can be suitably used.

Signal intensity obtained in the detection of mRNA using labeled polynucleotide probes of the present invention, regardless of the size of the mRNA A, it depends on the copy number of only each mRNA. Further, there is no need to prepare a probe for each mRNA to be'll detection, probes can be used in common in different samples.

Furthermore, the probe of the present invention provides that in the nucleotide Ri Contact is a plurality of locations labels, the strong signaling Le than Endorabe polynucleotide or cDNA A probe. Therefore, if a continuous binding of the labeled dUTP does not affect the nucleotide extension reactions, or Slight extension product, Te does not cause steric hindrance, labeled polynucleotide probes of the present invention, for example, labeled dUTP can be obtained about 4 times stronger signal than c DNA probe obtained were end labeled in the same length by one nucleotide labeled by placing four.

(2) process for the preparation of labeled polynucleotide probes of the present invention

Process for the preparation of labeled polynucleotide probes of the present invention is particularly limited to not but for example, poly-lipoic adenine nucleotide or polypeptide de O carboxymethyl adenine nucleocapsid tides and 铸型, the Seisuru adjusted by nucleotide extension reaction and primer oligo dT can. In the nucleotide extension reaction, the enzyme used is not particularly limited as long as the present onset Ming labeled polynucleotide probe can be efficiently prepared, mesophilic, thermophilic, be either refractory enzymes good. Further, 該Nu Kureochido extension reaction, a reverse transcription reaction using polyribonucleotide adenine nucleotide and 铸型 can be preferably used. In this case, it is not particularly limited but for example, avian myeloblastosis virus-derived reverse transcriptase (AMV RT ase), Morro - A murine leukemia virus-derived reverse transcriptase (M- MLV RTa se), Rous associated virus 2 from reverse transcription enzyme (RAV- 2), Bacillus Kano TOLEDO Tenax (B ca: B acilluscardotenax) derived DNA polymerase, Thermus thermophilus (T th: th er mu sthe rmo philus) derived DNA polymerase peptidase and the like. Furthermore, the nucleotide extension reaction may be complementary strand synthesis by DNA polymerase. In this case, although not particularly limited, for example Tarenou fragment, B ca DN A polymerase, Bacillus steer Rosa Moff Ira scan (B st: B acillusstearothe rmo philus) derived DNA polymerase, Thermus aquaticus (T aq: Th er mu saquaticus) derived DNA polymerase, Pyrococcus furiosus (P fur Py rococcusfuriosus) derived from DNA polymerase ^ "Ze, support Mokokkasu kodakaraensis. (KOD: T her mo coccuskodakaraensis) derived from DNA polymerase, and the like in addition, by using the DN a synthesizer chemically synthesized can be suitably used. Further, the chain length of short-chain nucleotide ligation reaction by can be suitably used those polynucleotide. above 铸型 become poly lipoic adenine nucleotides, preferably 50 nucleotides or more, more preferably 100 nucleotides or more. When using the 鍚型 range of labeled polynucleotide prepared it is 50 nucleotides or more. Further, as an aspect of another said nucleotide extension reactions may be Dokishinuku Reochido polymerization reaction. In this case, end de O carboxymethyl nucleotide transition enzyme. (T d T: Terminal deoxynucleotidyl transferase) commercial modifying enzyme, or the like may be suitably used in this case, setting the reaction conditions as the chain length of the probe is on the 50 nucleotides or more it is preferable to.

Prepared in the case of any of the above nucleotide extension reactions, the labeled de O carboxymethyl © brush Runu Kureochido triphosphate and / or labeled de O carboxymethyl thymine nucleotides 3 easily labeled polynucleotide by carrying out the reaction in the presence of phosphoric acid can do. In the above nucleotide extension reactions, labeled Dokishiurashi Le nucleotide triphosphate in the reaction solution and / or labeled de O carboxymethyl thymine nucleotide triphosphates and unlabeled de O carboxymethyl © La sill triphosphates and Z or unlabeled Dokishichimin nucleotide molar ratio of 3-phosphate is preferably 1: 1 to 1: 3 range, particularly preferably 1: 2. Moreover, labeled nucleotide triphosphates used in the preparation process of the present invention can be selected radioisotope, chemiluminescent substance, a fluorescent substance, a nucleotide triphosphate labeled with ligand or receptacle Puta, the Nukure Ochido It does not inhibit the extension reaction labeled nucleotide triphosphate or said labeled nucleotide 3 may be an analog of the phosphoric acid.

Further, a process of preparing the labeled polynucleotide probes of the present invention, a method of incorporating labeled nucleotides the-labeled compound is added during the nucleotide extension reactions are exemplified, unlabeled nucleotides another aspect use nucleotide extension reaction, the reaction was completed after further prepare Kosoi 匕学 manner or I 匕学 to be a method of introducing a labeled compound, eventually labeled Porinukureo Chidopurobu of the present invention both can of indicator introduction method that is Ru can be preferably used.

In this 突明, to prepare the indicated polyribonucleotide adenine nucleotide 及 Pi oligo de O carboxymethyl poly de reverse transcription reaction thus fluorescently labeled in the presence of thymine nucleotides O carboxymethyl © La sill nucleotides as 铸型 and primers It is Ru can. Prepared by the method of the present invention, labeled polynucleotide probe of the present invention containing a labeled de O carboxymethyl © La sills nucleotide and unlabeled de O carboxymethyl thymine nucleotides were spotted onto the membrane or glass surface (DNA chips) it can be used to detect DNA sequence and High Priestess to mRNA.

(3) The method of analyzing detection method and expressed genes of the target nucleic acid of the invention

It can be detected by a labeled polynucleotide probe that is hybridized to the poly adenine nucleotide unit amount of the target nucleic acid in the method for detecting a target nucleic acid of the present TsutomuAkira. Poly adenine nucleotide portion of the target nucleic acid may be those artificially Caro with the rear Karapo re-lipoic adenine nucleotide or polypeptide de O carboxymethyl adenine nucleotides may be poly-lipoic adenine nucleotide tail natural mR NA . That is, the labeled polynucleotide probe efficiently the present invention, not specifically particularly limited as long as it hybridizes, said labeled nucleotide is labeled with a radioisotope, chemiluminescent substance, a fluorescent substance, a ligand or receptor preferably those selected from any of the nucleotides, e.g., target identification is Furuoresein, cascade Blue, Oregon Green, B OD IPY, mouth over da Min Green, a lexa F luor, Tekisasuretsudo, either C y 3 or C y 5 It includes labeled nucleotides selected from. Contact V This method Te, for example, the target nucleic acid in the sample hybridized to the immobilized nucleic acid on the carrier, be detected using the optimum method for labeling a polynucleotide probe away with.

Another aspect of the detection method of the present invention include analysis method expressed genes. In this method, by using at least two kinds of labeled polynucleotide Dopurobu with different labels, it is possible to compare the expression of many types of genes in two different samples. Specifically, a sample possibly containing the target nucleic acid, poly adenine nucleotide moiety is Aniru and High Priestess soybean labeled polynucleotide probe, another sample possibly containing the target nucleic acid, poly-adenine nucleotide moiety is another labeled with labeled polynucleotide probes and Aniru hybridizing, samples obtained labeled polynucleotide probe that may contain a target nucleic acid that § two Lumpur, for example, the target nucleic acid fixed to the is responsible body and the High Priestess soybean ized, and detecting each of label Haiburidizu on the carrier, it is possible to analyze the expression of a target gene by comparing the presence or extent of each label detected. Although labeled polynucleotide probe is not particularly limited, for example, the polynucleotide probe labeled with C y 3 and C y 5 each can be suitably used.

Still another aspect of the analysis method of the expressed gene, Poriade two emissions nucleotide moiety High Priestess soybean labeling polynucleotides Puropu the expressed gene poly Ryo de Yun hybridizing to a portion other than a nucleotide labeled polynucleotide probe (c can be combined DNA labeled polynucleotide probe). In this case, the label can be used different. Puropu which hybridizes to a portion other than poly adenine nucleotide of the expressed genes, known methods can a child prepared by, for example, c D NA synthesis reaction the mR NA in the sample was a mirror type. The method of the present invention is not particularly limited, for example, 3 'end of the polyadenylation or expression of specific genes that are controlled by deadenylated can be used, it is possible to detect the control described above .

Moreover, labeled polynucleotide probe in the detection method of the present invention, it is possible to use in common, it is not necessary to prepare c DNA probe in accordance with the prior art target nucleic acid, the detection in a short time at low cost It can be carried out. Example

More specifically explained with reference to inventive examples, but the present invention is not intended to result as being limited to these examples.

Example 1

(1) Preparation of labeled polynucleotide probe

The labeled polynucleotide probe was prepared using reverse transcription Z labeling reaction. The reaction was carried out under the following reaction solution composition. That, l O Opmo l synthesis template - primer P oly (r A) - p (dT) 12 _ 18 a (Amersham 'Fuarumashi §' manufactured by Biotech) and錶型, dATP each 0. 5 mM, dCTP, dGT P, 40 / M of d TTP and 20 μΜ of a 1 exa- dUTP (manufactured by Morekyurapu robe, Inc.), (produced by formic Buko BRL, Inc.) 1 OmM DTT, 25U lipoic nucleotide inhibitors, super script reversal of 1. 5 U Continuous enzyme (Gibco BRL) was added and the reaction volume to 50 IX 1. The reaction mixture was held 60 minutes at 30 ° C, then heated for 3 minutes at 94 ° C to inactivate the remaining enzyme, cooled on an ice bath. The reflected 応溶 solution was purified using micro-spin S- 300HR column (manufactured by Amersham Pharmacia BAS Ioteku Inc.). Then the purified product 37 ° C, 20 minutes, treated with N a OH of 0. 3N, after decomposing 鍀型 poly (r A), with hydrochloric acid 及 Pi 1 M Tris monohydrochloride buffer solution (pH7. 0) neutralized. This solution was used as a fluorescent-labeled poly dU professional ^ "Breakfast solution.

(2) cell culture, RNA extraction, preparation of cDNA probes

Human H e L a cell and WI 38 cells, Hiyuman Science Research Resources Bank: obtained from (Hum an S cie nc e Re search Re s ou rces B ank HSRRB), D ulbecco's mo dified Ea containing 10% fetalcalf serum They were cultured in glemedium. Total RNA guaiacolsulfonate from the culture cells - issued extracted with Jiu Mu phenol-chloroform method. Genomic DNA of contaminating possibility, RNa se free DNa se (RQ 1 DNa se, professional glasses:. H $ removed using a ¾ mRNA, if necessary, mRNA Pu rificati on Ki t (Amersham file Pharmacia Bio Techne ± M) was purified using. cDNA probe, the above-described 6 00 ng purified RNA of a錶型, with O 1 igo (d T) 12 18 of 100 pmo 1 as primers, cy 3- dUTP. (Amasha lambda · Pharmacia Bio Techne: hB) and RNA F luoresc en ce La beling Co re K it (Takara Shuzo) was prepared by reverse transcription reaction using the reaction conditions,

After Inkyubeto 37 ° C, 60 minutes, 10/1 1 N hydroxide Natoriumu dissolved solution were added 10 minutes, and incubated at 37 ° C, to decompose the mirror type RNA. After neutralizing by adding 25.mu. 1 of 1M Tris-HCl buffer (ρΗ8. 0) in the reaction was precipitated with Etanoru. The precipitate was redissolved in 10 1 of High Priestess buffer scratch.

(3) Preparation of DNA-immobilized membrane and DN Alpha microarray

Human Bok GAP DH (glycera丄dehyde 3- phosphate dehydrogenase), P CN A (proliferating cell nuclear antigen) p 53, R b (retinoblastoma) Λ R as oncogenes, pl 30, pl 4, pl 6, pl 8, pl 9 the gene for p 21, p 27 as a target, primer 及Pi of SEQ ID NO: 1 to 40, wherein the sequence listing T a K a R a RNA PCR Ki t (AMV) V er. 2. 1 (Takara Shuzo Co., Ltd.) used in RT- PCR was carried out. Incidentally, p 53, RB, for genes of pi 30, pi 6, pl 9, p 27, pl 8, pi 4 after the reverse transcription reaction was carried out Ne sted PCR.錶型 used the RNA obtained in Example 1 (2). The resulting amplified fragment after purification sub claw and Jung to pT7B 1 u eT cloning vector (Novagen) to confirm the DNA sequence analysis. After confirming the insertion base sequence, Hybond N + using the above-described insert the B io Do t (BioRad); and fixed on (Amersham Pharmacia Bio Techne fc ^). Moreover, DNA microarray, using the commercially available Te STARRAY (Takara Shuzo Co., Ltd.).

(4) hybrida I See Chillon and detection

Membrane filters one for holding the target gene, hybrida I See Chillon buffer membrane 1 cm 2 per lm l (5XSSC, 0. 5% BS A, 0. lmg / m 1 salmon sperm DNA, 0. 1% use SDS), 3 7 ° C, 1 hour, was subjected to pre-High Priestess die See Chillon. The above Example 1 labeled poly dU probe solution 10 ^ 1 prepared in Example to any 10 mu 1 of total RNA or purified RNA obtained in (2) 1 (1) was added initially, final volume 200 mu to become as hybridization buffer was added 1, after heating between 70 ° C, 10 minutes, the membrane filter and 6-10 hours, and hybridized with 30 ° C. Men-plane filters, 15 minutes at room temperature, wash buffer I (1 XSSC, 0. 1% SDS) shaken gently put in, then 10 minutes, wash buffer II (0. 1 XS SC, 0 . shaken placed in 1% SDS), then washed. After washing, Men-plane filter scratch, was detected by fluorescence image analyzer FMB IOII (Takara Shuzo Co., Ltd.). On the other hand, hybrida I See Chillon of DN A microarray was carried out in accordance with the instructions of the manual of Attach. That is, the pre-High Priestess die See Chillon is prehybridized da I See Chillon solution of 14μ 1 (6 XSSC, 0. 1% SDS, 5 XD enhard t's solution, 0. 1 μ g / μ 1 salmon sperm DNA) using 1 hour, KoTsuta at 65 ° C. For hybridization with poly d U probe, the Example 1 (2) obtained in the 600 ng; mRNA in adding 1〃 1 poly dU, and the capacitance 14 mu 1 and. The solution was heated 70 ° C, 10 min. Further, with the same amount of mRNA, it was prepared in c DNA probe. The reaction solution above was added to the microarray, and then 65 ° C the array, and 18-20 hours incubator Pies and incubated for an additional 30 ° C, 3 hours. After hybrida I See Chillon, the microarray 60 ° C, 15 min, washed with 2xS SC solution further 60 ° C, 15 min, with a washing solution (2 XSSC, 0. 1% SDS), 2 finally at room temperature times, and washed with 2XSSC. Signal on the microarray were detected with the array scanner one 418 (manufactured by Afime Tricks Corporation).

As an example of a labeled polynucleotide probes of the present invention in Men-plane filter, for c Ivry specificity † raw labeled polynucleotide probe labeled with A le X a-dUTP shown in Figure 1_ A. Figure 1-B shows a spot Tsu bets position of each polynucleotide. The labeled polynucleotide probe as shown in FIG. 1-A is to Haiburidizu specifically poly lipoic adenine nucleotide (po 1 yA), the other nucleotides was confirmed that not hybridize. Furthermore, we examined the sensitivity by using a membrane spotted with a concentration range of the polyribonucleotide adenine nucleotide 1 ng (3 f mo l) ~lg (3 pmo 1). The results are shown in Figure 2. It was confirmed that clear fluorescence signal is obtained in the case of fixing I spoon 1 ng to (3 f mo 1) to 1 2 mm 2 as shown in FIG. Further, using a filter spotting GAPDH DNA fragments 及 Pi pUC 1 8 plasmid DNA of l ju g on the same Menpuren, the He L a from cells 10 to 40 mu above examples total RNA g 1 (1) the in what was prepared labeled poly dU Aniringu the probe solution 1 0 ^ 1 was High Priestess soybeans. The results are shown in Figure 3. Instead pUC 1 8 DNA as shown in FIG. 3, signal was detected only those spotted GAPDH DN A.

Then, the use of membrane spotting DNA fragments prepared in (4), poly dU prepared in the He L a cell and WI 1 38 total RNA and the examples of 20 mu g from the cell 1 (1) a material obtained by annealing the probe solution 1 0 1 was High Priestess soybean. The results are shown in FIG. Figure 4 one 1 is derived from the He L a cell, FIG. 4-2 shows the results using samples from WI 1 38. Figure 4 one 3 shows the position of each gene was spotted. From FIG. 4 one 1 and 4 one 2, it was confirmed that a signal depending on the gene expression is detected. Furthermore, it was confirmed to be detected by the same expression pattern was the result and compare the Northern blot of the prior art.

It was investigated using the DNA microarray and WI 38 cell-derived mRNA. The results are shown in FIG 5. In FIG. 5, DNA microarrays used is obtained by two pairs spotted 96 DNA to see the reproducibility 1 raw data (replication (duplicate) and was spot to). Arrows indicate the top and bottom of each la. 5 those labeled polynucleotide probes of the present invention to mRNA A of WI 38-derived cells, as shown in A were annealed, was shown to hive Ridizu specific target from mammalian cells. On the other hand, it did not detect any thing of Shianopakuteria gene. That is, Xia Roh bacteria are prokaryotes, for quite DNA sequence from mammals such eukaryotic differ, In such a result in order to High Priestess die internalization does not occur. From this, labeled polynucleotide professional one blanking of the present invention, it was confirmed that can be used in DNA microarrays. Also shows the same switch-up, the results have been conducted under study from the same mRNA preparations to c DNA probe prepared in a conventional manner in Fig 5 B. To show a detection method 1S conventional method basically the same results of the present invention from a comparison of A and B in FIG. 5 was confirmed.

Example 2

(1) Preparation of labeled polynucleotide probe

Example 1 (1) 1 6 3 - (11;?. Ding 3- (11; Ding, Cy 5-DUT in place of P (Amersham 'Pharmacia Biotech), labeled poly by the method of Example 1 the nucleotide probes were prepared and C y 3 labeled poly d U probe respectively, and C y 5 labeled poly d U probe.

(2) cell culture, RNA extraction

Mouse RAW247 cells Human Science Research Resources punk: was obtained from (Hum an S cience R esearcn R esources B ank HS RR B). Example 1 i (2) the same method as in cell culture was carried out extractions of mRNA. RAW24 7 min I spoon cells into osteoclasts, H su H. et al. Method (P roc Na tl Ac d S ci USA 9 6:..... P 3 540- 3 54 5,

1 9 9 9 years) I Koju Rere, using O steoclast D ifferentiation F actor (ODF).

(3) Bruno, Eve Lida I See Chillon and detection

The extracted mRNA and C y 3 labeled poly dU professional one blanking from RAW24 7 cells undifferentiated were annealed. That is, a mixture of 6 00 ng of mRNA and 1 0 1 C y 3 labeled poly dU probes were treated 1 0 minute 7 0 ° C. Also, an mRNA C y 5 labeled poly dU probes extracted from RAW24 7 cells induction osteoclast was Aniringu as above.

I ntelli G ene Mo use CH IPS et I ver. 1. with 0 (Takara Shuzo Co., Ltd.) was subjected to High Priestess die See Deployment as instructed accompanying manual. That is, the pre-High Priestess die internalization is 14 ^ 1 pre hybrida I See Chillon solution (6 XSSC, 0. 1% SDS, 5 XD enhard t's solution, 0. 1 mu g / mu 1 salmon sperm DNA) using 1 hour, was performed in 6 5 ° C. By mixing the Cy 3, C y 5 labeled poly dU probe mRNA was allowed to anneal solution and 1 4 1 As Puropu, was added to the microarray, then array 6 5 ° C, 1 8~2 0 hour and further 3 0 ° (:, after 3 hours between incubated hybrida I See Chillon, the microarray 6 0 ° C, 1 5 min, 2 XSSC solution further 6 0 ° C, 1 5 min.? washed with SakiKiyoshieki (2 XSSC, 0. 1% SDS), finally twice at room temperature, 0. washed. signal on the microarray in 2 XSSC, a 4 1 8 array scanner (manufactured by Abuime Tricks Co.) was detected using. the results are shown in FIG.

As shown in FIG. 6, even when a combination of two kinds of labeled Porinukureo Chidopuropu labeled with different fluorescent substances, conventional C y 3 or C y 5 2-color with labeled c D NA probe it was confirmed that it is possible to perform gene expression analysis similar to the law. Industrial Applicability

The present invention, useful labeled nucleic acid probe in hybrida I See Chillon, sensitive method for detecting a target nucleic acid using the method of preparation 及 Pi said probe of said probe is provided. Sequence Listing Free Text

SEQ ID N0: 1

Designed oligonucleotide primer to amplify a portion of human p53 gene

SEQ ID NO: 2

Designed oligonucleotide primer to amplify a portion of human p53 gene SEQ ID NO: 3

Designed oligonucleotide primer to amplify a portion of human p53 gene SEQ ID NO: 4

Designed oligonucleotide primer to amplify a portion of human p53 gene SEQ ID NO: 5

Designed oligonucleotide primer to amplify a portion of human pRB gene SEQ ID NO: 6 Designed oligonucleotide primer to amplify a portion of human pRB gene SEQ ID NO: 7

Designed oligonucleotide primer to amplify a portion of human pRB gene SEQ ID NO: 8

Designed oligonucleotide primer to amplify a portion of human pRB gene

SEQ ID NO: 9

Designed oligonucleotide primer to amplify a portion of human GAPDH gene

SEQ ID NO: 10

Designed oligonucleotide primer to amplify a portion of human GAPDH gene

SEQ ID NO: 11

Designed oligonucleotide primer to amplify a portion of human pl30 gene

SEQ ID NO: 12

Designed oligonucleotide primer to amplify a portion of human pl30 gene

SEQ ID NO: 13

Designed oligonucleotide primer to amplify a portion of human pl30 gene

SEQ ID NO: 14

Designed oligonucleotide primer to amplify a portion of human pl30 gene

SEQ ID NO: 15

Designed oligonucleotide primer to amplify a portion of human pl6 gene

SEQ ID NO: 16

Designed oligonucleotide primer to amplify a portion of human pl6 gene SEQ ID NO: 17

Designed oligonucleotide primer to amplify a portion of human pl6 gene SEQ ID NO: 18

Designed oligonucleotide primer to amplify a portion of human pl6 gene SEQ ID NO: 19

Designed oligonucleotide primer to amplify a portion of human pl9 gene SEQ ID NO: 20

Designed oligonucleotide primer to amplify a portion of human pl9 gene SEQ ID NO: 21

Designed oligonucleotide primer to amplify a portion of human pl9 gene SEQ ID NO: 22

Designed oligonucleotide primer to amplify a portion of human pl9 gene

SEQ ID NO: 23

Designed oligonucleotide primer to amplify a portion of human p27 gene SEQ ID NO: 24

Designed oligonucleotide primer to amplify a portion of human p27 gene SEQ ID NO: 25

Designed oligonucleotide primer to amplify a portion of human p27 gene SEQ ID NO: 26

Designed oligonucleotide primer to amplify a portion of human p27 gene SEQ ID NO: 27

Designed oligonucleotide primer to amplify a portion of human PCNA gene

SEQ ID NO: 28

Designed oligonucleotide primer to amplify a portion of human PCNA gene

SEQ ID NO: 29

Designed oligonucleotide primer to amplify a portion of human Ras oncogene

SEQ ID NO: 30

Designed oligonucleotide primer to amplify a portion of human Ras oncogene

SEQ ID NO: 31

Designed oligonucleotide primer to amplify a portion of human pl8 gene SEQ ID NO: 32

Designed oligonucleotide primer to amplify a portion of human pl8 gene

SEQ ID NO: 33

Designed oligonucleotide primer to amplify a portion of human pl8 gene SEQ ID NO: 34

Designed oligonucleotide primer to amplify a portion of human pl8 gene SEQ ID NO: 35

Designed oligonucleotide primer to amplify a portion of human p21 gene SEQ ID NO: 36

Designed oligonucleotide primer to amplify a portion of human p21 gene SEQ ID NO: 37

Designed oligonucleotide primer to amplify a portion of human pl4 gene

SEQ ID NO: 38

Designed oligonucleotide primer to amplify a portion of human pl4 gene SEQ ID NO: 39

Designed oligonucleotide primer to amplify a portion of human pl4 gene SEQ ID NO: 40

Designed oligonucleotide primer to amplify a portion of human pl4 gene

Claims

The scope of the claims
1. labeled polynucleotide probe, which comprises hybridizing to Poriade two down nucleotide moiety in the target nucleic acid.
2. labeled polynucleotide probe according to claim 1, wherein the poly adenine nucleotide moiety is a polyribonucleotide adenine Nucleotidyl Doteru of mRNA.
3. labeled polynucleotide probe according to claim 1, wherein the chain length is 50 nucleotides or more.
4. De O carboxymethyl © La sill nucleotide and / or de-O alkoxy claim 1, wherein the labeled Porinutare Ochidopurobu which is a thymine nucleotide or Ranaru polynucleotide.
5. labeled de O carboxymethyl © La sill nucleotides and / or labeled de O carboxymethyl thymine Nutare Ochido, characterized in that it contains the claim 1, wherein the labeled polynucleotide probe.
6. radioisotopes, chemiluminescent substances, fluorescent substances, labeled polynucleotide probe according to claim 1, characterized in that it is labeled with a material selected from the ligand and receptor or Ranaru group.
7. Full old Resin, cascade one Dobunore, Oregon Green, BOD IPY, Rhodamine Green, A lexa F luor, Tekisasuretsudo, claims, characterized in that it is labeled with a substance selected from the group consisting of Cy 3 and Cy 5 item
Labeled polynucleotide probe 6, wherein.
8. Poly-lipoic adenine nucleotide or Poridokishiade - the N'nukureochi de and 鍚型 method made adjustment of the labeled polynucleotide probe according to claim 1, characterized by comprising the step of performing nucleotide extension reaction using oligo dT as primer.
Process for the preparation of labeled polynucleotide probe according to claim 8, wherein the 9 nucleotide extension reactions is reverse transcription reaction was 鐯型 the polyribonucleotide adenine nucleotides.
10. 铸型 become poly lipoic adenine nucleotide or Poridokishiade - claim 8 process for the preparation of labeled polynucleotide probe, wherein the chain length of the emission nucleotides is 50 nucleotides or more.
11. The nucleotide extension reaction, a process for the preparation according to claim 8, wherein the labeled polynucleotide probe, which is a de-O alkoxy nucleotide polymerization reaction.
12. labeled polynucleotide preparation process of claim 8 wherein the labeled polynucleotide probes chain length of the probe and wherein the this is 50 nucleotides or more.
Process for the preparation of 13. nucleotide extension reactions labeled de O carboxymethyl © La sills nucleotide and Z or labeled de O carboxymethyl claim 8 Symbol mounting of labeled polynucleotide probe, characterized in that is carried out using a thymine nucleotides.
14. label de O carboxymethyl © La sill nucleotide triphosphates and / or labeled Dokishichi Min nucleotide triphosphates and unlabeled de O carboxymethyl © La sill nucleotide triphosphate San及Pi
! Molar ratio of Z or unlabeled de O carboxymethyl thymine nucleotide triphosphate is 1: to 1:
13. process for the preparation of labeled polynucleotide probe, wherein the carried out in a probe preparative reaction is 3.
Preparation 15. labeled polynucleotide probe is a radioisotope, chemiluminescent substance, a fluorescent substance, labeled polynucleotide Puropu of claim 8, wherein that you have been labeled with a substance selected from the group consisting of ligand and receptor Method.
16. labeled polynucleotide probe Furuoresein, Cascade Blue, Oregon Green, BOD I PY, Rhodamine Green, Al e xa F l uo r, labeled with a substance selected from the group consisting of Texas Red, Cy 3 and Cy 5 15. process for the preparation of labeled polynucleotide probe, wherein the are.
17. Poriade - labeled Porinukureo Chidopuropu of High Priestess soybeans to down nucleotide portion of the target nucleic acid, characterized in that the target nucleic acid with the sample that may contain target nucleic acid encompasses fixed I spoon has been carrier and High Priestess step of soybean detection method.
18. Poly adenine nucleotide moiety, the method for detecting a target nucleic acid according to claim 17, which is a polyribonucleotide adenine j Kure Ochidoteru of mRNA.
19. labeled polynucleotide probe is labeled de O carboxymethyl © La sill nucleotide 及 beauty / or labeled de O carboxymethyl method for detecting a target nucleic acid according to claim 17 or claim 18, characterized in that it comprises a thymine nucleotides.
20. radioisotope, I arsenide chemiluminescent substance, a fluorescent substance, a target nucleic acid of claim 17, wherein the use of labeled polynucleotide probe that is labeled with a material selected from the ligand and receptor mosquito ^ group consisting the method of detection.
21. Funoreoresein, Cascade Blue, Oregon Green, BOD I PY, Rhodamine Green, Al exa F l uo r, Tekisasuretsudo, labeled polynucleotide probe that is labeled with a substance selected from the group consisting of Cy 3 and C y 5 method for detecting a target nucleic acid of claim 20, wherein the use of.
22. a) step of the first sample that may contain the target nucleic acid, is first labeled with a labeled polynucleotide probe and Aniru that Haiburidizu poly adenine nucleocapsid tide moiety;
b) potentially containing the target nucleic acid, the second sample is different from the first sample, to hybridize to Polya de Nin nucleotide moiety, labeled with different second-labeled the first label step of polynucleotide probes and Aniru;
c) labeled polynucleotide probe obtained in step a and b were Aniru, a sample possibly containing the target nucleic acid, the step of fixing I inhibit the carrier and Haipuridai's target nucleic acid;
Step for detecting the first and second of label Haiburidizu on d) a fixed I 匕担 body; and
The first and second step of comparing the presence, absence or degree of labeling, characterized in that it comprises a method of analyzing a target gene expression e) detection.
23. Furuoresein, Cascade Blue, Oregon Green, BOD I PY, Rhodamine Green, Al exa F l uo r, Tekisasuretsudo, labeled polynucleotide probe that is labeled with a substance selected from the group consisting of Cy 3 and C y 5 analysis method for target gene expression according to claim 22, wherein the use of.
24. Poriade two emissions nucleotide moiety High Priestess characterized by soybean labeled polynucleotide probe set labeled polynucleotide probes characterized by High Priestess soybean in a portion other than poly adenine nucleotide expressed genes viewed together expressed genes analysis method of expressed gene, wherein a step of the sample that hybridizes to packaging containing that may contain.
25. Funoreoresein, cascade one Dobunore, Oregon Green, BOD I PY, Rhodamine Green, Al exa F l uo r, Tekisasuretsudo, labeled polynucleotide flop rope that is labeled with a substance selected from the group consisting of Cy 3 and Cy 5 analysis method of a target nucleic acid of claim 24, wherein the use of.
PCT/JP2001/005783 2000-07-05 2001-07-04 Highly sensitive method of detecting nucleic acid WO2002002814A1 (en)

Priority Applications (4)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
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Publication number Priority date Publication date Assignee Title
US5853993A (en) * 1996-10-21 1998-12-29 Hewlett-Packard Company Signal enhancement method and kit
WO1999051772A1 (en) * 1998-04-07 1999-10-14 Incyte Pharmaceuticals, Inc. Quantitative microarray hybridization assays

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GOLDSWORTHY S.M. ET AL.: "Variation in expression of genes used for normalization of northern blots after induction of cell proliferation", CELL. PROLIF., vol. 26, 1993, pages 511 - 518, XP002940686 *
MASAAKI MURAMATSU ET AL.: "Saibou kougaku bessatsu; genome science series (1), DNA maicro array to saishin PCR-hou", KABUSHIKI KAISHA HOUJUNSHA, 16 March 2000 (2000-03-16), pages 43 - 54, XP002940685 *

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