WO2002002814A1

WO2002002814A1 - Highly sensitive method of detecting nucleic acid

Info

Publication number: WO2002002814A1
Application number: PCT/JP2001/005783
Authority: WO
Inventors: Junichi Mineno; Edy Meiyanto; Norihiro Ishida; Tatsuo Takeya; Kiyozo Asada; Ikunoshin Kato
Original assignee: Takara Bio Inc.
Priority date: 2000-07-05
Filing date: 2001-07-04
Publication date: 2002-01-10
Also published as: AU2001271017A1

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

Description Highly sensitive nucleic acid detection method Technical field

The present invention relates to a method for preparing a labeled probe useful in the field of genetic engineering, and a highly sensitive detection method using the probe. Background art

The hybridization method is a commonly used method for identifying the type of mRNA expressed in cells or tissues or for quantifying the molecular weight of each mRNA. Therefore, hybridization technology for identification and quantification of mRNA is of great importance in the fields of biology and medicine.

As the above hybridization method, for example, detection is performed by immobilizing total RNA or purified RNA on a porous carrier such as a nylon membrane and labeling with a fluorescent substance or a radioisotope (RI). There is a method that mixes with a probe complementary to the gene (RNA) to be hybridized. Technologies belonging to this category include the Northern Hybridization method.

Alternatively, a mixture containing the gene to be detected (RNA), or RNA purified from the mixture, is labeled with a fluorescent substance or a radioisotope and immobilized on a membrane. There is a method of hybridizing with a probe. As a technique belonging to this category, there is an inverted dot plot method. These two methods have been used in various experiments in research in the field of genetic engineering.

In the conventional hybridization method, nucleic acids immobilized on a membrane have usually been used. Recently, however, the above-mentioned hybridization technology has been applied to a non-porous hard carrier such as glass. In addition, a hybridization technology has been developed which mainly uses a known DNA fragment immobilized. In this technique, many types of DNA fragments can be immobilized at a very high density in a predetermined region on the surface of a carrier. Such a carrier on which DNA fragments are immobilized at high density is It is commonly called a DNA microarray (DNA chip). By using the DNA microarray, it became possible to identify and quantify many types of mRNA in a small amount of sample at once.

When the above-mentioned DNA microarray is used, it is common to detect a nucleic acid in a sample by labeling it with, for example, a fluorescent substance, and hybridizing it with a DNA fragment immobilized on a carrier. That is, when mRNA in a sample is detected by a DNA microarray, for example, the sample can be labeled by the following method.

1. A method of preparing a fluorescently labeled c'DNA by adding a fluorescently labeled nucleotide during cDNA synthesis using mRNA in the sample as a type II, or

2. A method of synthesizing cDNA from mRNA in a sample, and then performing a transcription reaction with RNA polymerase using the cDNA as a mirror in the presence of a fluorescently labeled nucleotide to prepare a fluorescently labeled RNA probe.

The labeled DNA or RNA prepared by the above method has a base sequence derived from the mRNA in the sample or a base sequence complementary thereto, and is placed on a microarray on which an appropriate DNA fragment is immobilized. Can be detected.

However, when a labeled nucleic acid having a base sequence derived from mRNA in a sample is prepared by the method described above, the number of labeled compounds incorporated into the labeled nucleic acid is proportional to the length of the nucleic acid. A long labeled nucleic acid has a disadvantage that the number of labeled nucleotides incorporated per molecule is smaller than that of a long labeled nucleic acid. Therefore, when a labeled nucleic acid having the same number of molecules is hybridized with a carrier, for example, a DNA fragment immobilized on a microarray, the intensity of the labeled signal derived from the labeled compound obtained for each array is the nucleic acid strand to be hybridized. It will be greatly influenced by the length. This is a very important issue in experiments that require quantification.

On the other hand, as a nucleic acid labeling method that is not affected by the chain length of the nucleic acid to be labeled, there is a terminal labeling method described in US Pat. No. 6,004,755. However, this method is a method of synthesizing cDNA using an end-labeled oligo dT primer, and the number of labeled compounds added to the primer is limited. So try to detect If the amount of mRNA derived from the gene is small, the signal intensity may be too low to detect.

Numerous types of genes are expressed in cells, and the polypeptides encoded by each gene have different structures and functions, so the mRNAs corresponding to these polypeptides also have longer chain lengths. It is widely distributed. Therefore, there is a need for a method for detecting mRNA or a nucleic acid having a nucleotide sequence derived from mRNA, which provides a signal with excellent quantification for detecting and quantifying such mRNA as accurately and with high reproducibility. I was Purpose of the invention

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

The present inventors have conducted intensive studies on the detection and quantification of nucleic acids by the hybridization method, and have identified mRNA that hybridizes with the immobilized probe, at the 3 'end of the mRNA. By using the A sequence for detection, we have found a method capable of detecting mRNA with excellent quantitative properties and detection sensitivity. In addition, the present inventors have found a method capable of detecting a target DNA in the same manner as described above even for DNA to which a polynucleotide has been artificially added, thereby completing the present invention.

In summary, the first invention of the present invention relates to a labeled polynucleotide probe which hybridizes to a polyadenine nucleotide portion in a target nucleic acid. In the first invention of the present invention, the polyadenine nucleotide portion to which the labeled polynucleotide probe hybridizes may be a polyriboadenine nucleotide tail of mRNA. In the present invention, as the labeled polynucleotide probe, a probe having a chain length of 50 nucleotides or more can be suitably used. The probe is preferably a polynucleotide consisting of deoxyperacyl nucleotide and Z or deoxythymine nucleotide, and contains a labeled deoxyperacyl nucleotide and Z or deoxythymine nucleotide. Preferably. Furthermore, as the labeled nucleotide, those labeled with a substance selected from the group consisting of radioisotopes, chemical fluorescent substances, fluorescent substances, ligands and receptors can be suitably used. Although not particularly limited, for example, a substance selected from the group consisting of fluorescein, cascade buoy / ray, Oregon green, BODIPY, rhodming lean, Alexa F1 uor, Texas red, Cy3 and Cy5. A labeled polynucleotide probe that is labeled can be suitably used, and particularly preferably, dUTP or dTTP having the above label is exemplified.

The second invention of the present invention comprises a step of performing a nucleotide extension reaction using a polyriboadenine nucleotide or a polydoxyadenine nucleotide as a 鎳 -form and oligo dT as a primer, wherein the first invention comprises a step of: And a method for preparing a labeled polynucleotide probe. In the second invention of the present invention, the nucleotide extension reaction may be a reverse transcription reaction using a polyriboadenine nucleotide as a type III. Further, in the present invention, the chain length of the polylipoadenine nucleotide or polydeoxyadenine nucleotide having a type III is preferably 50 nucleotides or more. In the present invention, the nucleotide extension reaction may be a deoxynucleotide polymerization reaction. Further, in the present invention, the chain length of the labeled polynucleotide probe is preferably 50 nucleotides or more, and the nucleotide extension reaction or the deoxynucleotide polymerization reaction is preferably performed using the labeled deoxydilacil nucleotide triphosphate. And / or a reaction carried out using labeled deoxythymine nucleotide triphosphate. In this case, the molar ratio of labeled deoxyduracil nucleotide triphosphate and Z or labeled deoxythymine nucleotide triphosphate to unlabeled deoxydilacil nucleotide triphosphate and / or unlabeled deoxythymine nucleotide triphosphate is It is preferably carried out in a probe preparation reaction solution of 1: 1 to 1: 3, and the labeling substance used is a group consisting of a radioisotope, a chemiluminescent substance, a fluorescent substance, a ligand and a receptor. More selected substances can be suitably used. Although not particularly limited, for example, selected from the group consisting of fluorescein, Cascade Bull—, Oregon Green, BOD IPY, Rhodamine Green, A1eXa Fluor, Texas Red, Cy3 and Cy5. Preferably, they are labeled with a substance of choice.

The third invention of the present invention includes a step of hybridizing a labeled polynucleotide probe that hybridizes to a polyadenylate nucleotide portion with a carrier on which the target nucleic acid is immobilized together with a sample that may contain the target nucleic acid. The present invention relates to a method for detecting a target nucleic acid, characterized by the following. In the third invention of the present invention, the polyadenine nucleotide portion may be a polyriboadenine nucleotide tail of mRNA, and any one obtained by artificially adding a polyadenine nucleotide portion afterwards is preferably used. it can. That is, there is no particular limitation as long as the labeled polynucleotide probe of the first invention of the present invention efficiently and specifically hybridizes. In the third invention of the present invention, a labeled polynucleotide probe containing a labeled deoxydilacil nucleotide and / or a labeled deoxythymine nucleotide can be suitably used. The labeled nucleotide is preferably labeled with a substance selected from the group consisting of a radioisotope, a chemiluminescent substance, a fluorescent substance, a ligand, and a receptor, and is not particularly limited, but includes, for example, fluorescein, cascade pull, Oregon Green, BODIPY, Rhodamine Green, A

And a labeled polynucleotide probe labeled with a substance selected from the group consisting of 1 eXaFluor, Texas Red, Cy3 and Cy5. The fourth invention of the present invention comprises: a) a step of annealing a first sample which may contain a target nucleic acid with a polynucleotide probe labeled with a first label which hybridizes to a polyadenine nucleotide portion; b ) A polynucleotide labeled with a second label different from the first label, which hybridizes a second sample different from the first sample, which may contain the target nucleic acid, to the polyadene nucleotide portion. C) a step of hybridizing a sample, which may contain the target nucleic acid annealed by the labeled polynucleotide probe obtained in steps a and b, with a carrier having the target nucleic acid immobilized thereon; d) on the carrier Detecting the hybridized first and second labels; and e) comparing the presence or absence or degree of the first and second labels detected. And a method for analyzing target gene expression. In the fourth invention of the present invention, for example, but not limited to, fluorescein, cascade pull, Oregon green, BODIPY, A labeled polynucleotide probe labeled with a substance selected from the group consisting of Rhodamine Green, Alexa F1 uor, Texas Red, Cy3 and Cy5 can be suitably used.

A fifth invention of the present invention provides a labeled polynucleotide probe characterized in that it hybridizes to a polyadenylate nucleotide portion and a labeled polynucleotide probe characterized in that it hybridizes to a portion other than the polyadenylate nucleotide of an expressed gene. And a step of hybridizing with a sample that may contain an expressed gene by combining the expression gene. In the fifth invention of the present invention, examples include, but are not limited to, foresin, cascade blue, Oregon green, BODIPY, rhodamine green, Alea F 1 uor, Texas red, Cy3 and Cy5 A labeled polynucleotide probe labeled with a substance selected from the group can be suitably used. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing the specificity in hybridization of the labeled polynucleotide probe of the present invention.

FIG. 2 is a diagram showing the detection sensitivity of the labeled polynucleotide probe of the present invention. FIG. 3 is a diagram showing detection of an expressed gene using the labeled polynucleotide probe of the present invention.

FIG. 4 is a diagram showing the specificity in the hybridization of the labeled polynucleotide probe of the present invention.

FIG. 5 is a diagram showing detection on a DNA chip using the labeled polynucleotide probe of the present invention.

FIG. 6 is a diagram showing gene expression analysis on a DNA chip using two types of labeled polynucleotide probes having different labels of the present invention. Detailed description of the invention

Hereinafter, the present invention will be described in detail.

In the present specification, nucleotide means deoxyribonucleotide or ribonucleotide. Refers to nucleotides. For example, in the case of deoxyliponucleotides, refers to nucleotides in which the saccharide moiety is composed of D-2-dexoxylipose. For example, has adenine, cytosine, guanine, thymine, and peracil in the base moiety. Things. In the case of liponucleotides, it refers to a nucleotide in which the sugar moiety is composed of D-ribose, and includes those having adenine, cytosine, guanine, and peracyl in the base moiety. In addition, the nucleotide includes a modified deoxyliponucleotide or a modified ribonucleotide, such as a modified ribonucleotide [(Hi-S) ribonucleotide, in which the oxygen atom of the phosphate group at the position is replaced with a sulfur atom, α-S)}] and other derivatives. In the present specification, a polyadenine nucleotide refers to a polymer of lipoadenine nucleotide or deoxyadenine nucleotide. The polymer includes both artificially prepared and naturally occurring polymers. Although not particularly limited, for example, a polytail (polyriboadenine nucleotide tail) of eukaryotic mRNA is exemplified.

As used herein, the term “labeled nucleotide” refers to a nucleotide labeled with an identifying substance. The labeling substance includes any of those linked directly to the above-mentioned deoxynucleotide or ribonucleotide or via a linker or the like. As used herein, the term “labeled polynucleotide” refers to a labeled nucleotide polymer, and the polymer is preferably a polymer composed of labeled nucleotides and / or unlabeled nucleotides.

In the present specification, the nucleotide extension reaction is not particularly limited as long as it is a reaction for extending the above nucleotide, and a reverse transcription reaction using a polyriboadenine nucleotide as a 鎳 type, a DNA polymerase reaction, or a terminal deoxynucleotide transfer reaction. Any of the deoxynucleotide polymerization reactions described above can be suitably used.

As used herein, a target nucleic acid refers to a nucleic acid sequence to be detected, for example, a nucleic acid sequence of any gene. The nucleic acid sequence may be either DNA or RNA.

(1) The labeled polynucleotide probe of the present invention

The labeled polynucleotide probe of the present invention has a polyadenylate nucleotide portion. Any polynucleotide probe can be used as long as it can hybridize, and is not particularly limited. A polymer of deoxyperidine nucleotide (dUTP), a polymer of deoxythymine nucleotide (dTTP), or a mixture of dUTP and dTTP Any of the polymers can be suitably used. As used herein, the term “polyadenine nucleotide portion” refers to a portion in a target nucleic acid to which the labeled polynucleotide probe of the present invention hybridizes and which comprises a polyadenine nucleotide. The chain length of the labeled polynucleotide probe of the present invention is not particularly limited, but preferably 50 nucleotides or more, more preferably 100 nucleotides or more can be suitably used. The labeled nucleotide probe of the present invention is not particularly limited as long as it contains a substantially constant amount of a labeled compound per probe molecule, and includes, for example, a nucleotide to which a labeled compound is added (labeled nucleotide). There can be. The labeled nucleotide is not particularly limited, but is preferably, for example, labeled deoxyperacyl nucleotide or labeled deoxythymine nutretide, and these may be used as a mixture. Further, as the labeled nucleotide, any of a radioisotope, a chemiluminescent substance, a fluorescent substance, a ligand or a nucleotide labeled with a receptor can be suitably used. Although not particularly limited, for example, radioisotopes such as ³² P and ³³ P, chemical luminescent substances such as AM PPD, fluorescein, cascade blue, Oregon green, BOD I PY, rhodamine green, and Alexa Fluo For r, Texas red and Cy 3, a fluorescent substance such as Cy5, a ligand such as biotin, avidin, digoxigenin or a nucleotide labeled with a receptor can be suitably used. In the present invention, a fluorescent substance, particularly preferably dUTP labeled with A1eXaF1uor, Cy3 or Cy5 can be suitably used.

In another embodiment, a label is prepared by preparing a polymer of the modified nucleotide using a modified nucleotide having a functional group such as an amino group or a thiol group, and then adding the labeling substance using the functional group. Polynucleotides are also included in the labeled polynucleotide probes of the present invention.

Although not particularly limited, the labeled polynucleotide probe of the present invention hybridizes to a polyadenine nucleotide portion under stringent conditions. Is preferred. As the stringent conditions, for example, those described in Molecular Cloning, A laboratory manual, 2nd edition (1989), Molecular Cloning, A laboratory manual can be suitably used.

The signal intensity obtained in the detection of mRNA using the labeled polynucleotide probe of the present invention depends solely on the number of copies of each mRNA, regardless of the size of mRNA. Also, there is no need to prepare a probe for each mRNA to be detected, and the probe can be used in common for different samples.

Further, the probe of the present invention is labeled at a plurality of positions in its nucleotide, and gives a stronger signal than an end-labeled polynucleotide or a cDNA probe. Therefore, provided that the continuous binding of labeled dUTP does not affect the nucleotide extension reaction or does not cause steric hindrance to the extension product, the labeled polynucleotide probe of the present invention may be, for example, labeled dUTP. By adding four, a signal about four times stronger than an end-labeled cDNA probe of the same length obtained by one type of nucleotide labeling can be obtained.

(2) Method for preparing labeled polynucleotide probe of the present invention

The method for preparing the labeled polynucleotide probe of the present invention is not particularly limited.For example, the labeled polynucleotide probe may be prepared by a nucleotide extension reaction using a polylipoadenine nucleotide or a polydeoxyadenine nucleotide as a 铸 -type and oligo dT as a primer. Can be. In the nucleotide extension reaction, the enzyme to be used is not particularly limited as long as the labeled polynucleotide probe of the present invention can be efficiently prepared, and may be any of room temperature, thermophilic, and hyperthermostable enzymes. Good. For the nucleotide extension reaction, a reverse transcription reaction using a polyriboadenine nucleotide as a 铸 -type can be suitably used. In this case, there is no particular limitation. For example, reverse transcriptase derived from avian myeloblastosis virus (AMV RTase), reverse transcriptase derived from Moro-mouse leukemia virus (M-MLV RTase), Rouss-related virus 2 Reverse transcriptase (RAV-2), DNA polymerase derived from Bacillus canoledotenax (Bca: Bacilluscardotenax), DNA polymerase derived from Thermothothermophilus (Tth), and the like. Further, the nucleotide extension reaction is carried out by a DNA polymerase. May be a complementary strand synthesis reaction. In this case, although not particularly limited, for example, tarenow fragment, Bca DNA polymerase, DNA polymerase derived from Bacillus stearothermophilus (B st: Bacillusstearothermo philus), Thermus aquaticus (Taq: Ther mu saquaticus) DNA polymerases derived from Pyrococcus furiosus (P fur Pyrococcus furiosus), DNA polymerases derived from Thermococcus kodakaraensis (KOD), and the like. Also preferably used are those obtained by chemically synthesizing a short-chain nucleotide into a polynucleotide by a ligation reaction.The chain length of the above-mentioned type II polylipoadenine nucleotide is preferably 50 nucleotides And more preferably 100 nucleotides or more. When type I is used, the range of the labeled polynucleotide to be prepared is 50 nucleotides or more, and another embodiment of the nucleotide extension reaction may be a deoxynucleotide polymerization reaction. A commercially available modifying enzyme such as Deoxynucleotidylyl transferase (TdT) can be suitably used, in which case the reaction conditions are set such that the chain length of the probe is 50 nucleotides or more. Is preferred.

In any of the above nucleotide extension reactions, a labeled polynucleotide is easily prepared by performing the reaction in the presence of labeled deoxydilacil nucleotide triphosphate and / or labeled deoxythymine nucleotide triphosphate. can do. In the above-mentioned nucleotide extension reaction, labeled deoxydilacil nucleotide triphosphate and / or labeled deoxythymine nucleotide triphosphate and unlabeled deoxydilacil nucleotide triphosphate and Z or unlabeled deoxythymine nucleotide in the reaction solution The molar ratio of triphosphoric acid is preferably in the range of 1: 1 to 1: 3, particularly preferably 1: 2. In addition, the labeled nucleotide triphosphate used in the preparation method of the present invention can be selected from a radioisotope, a chemiluminescent substance, a fluorescent substance, a ligand or a nucleotide triphosphate labeled with a receptor. Labeled nucleotide triphosphate that does not inhibit the extension reaction or the labeled nucleoside It may be an analog of tide triphosphate.

Further, as a method for preparing the labeled polynucleotide probe of the present invention, a method of incorporating a labeled nucleotide to which the above-mentioned labeled compound is added at the time of a nucleotide extension reaction is exemplified, but in another embodiment, an unlabeled nucleotide is used. The method may be a method in which the labeled compound is used in a nucleotide extension reaction, and after completion of the reaction, a labeled compound is further introduced enzymatically or indirectly, and finally, the labeled polynucleotide probe of the present invention is prepared. Any of the labeling methods that can be used can be suitably used.

Preparation of fluorescently labeled polydeoxydilacil nucleotides by reverse transcription in the presence of polyriboadenine nucleotides and oligodeoxythymine nucleotides indicated as type I and primers Can be done. The labeled polynucleotide probe of the present invention containing labeled deoxydilacil nucleotides and unlabeled deoxythymine nucleotides prepared by the method of the present invention was spotted on a membrane or glass surface (DNA chip). It can be used to detect mRNA that hybridizes with a DNA sequence.

(3) The method for detecting a target nucleic acid and the method for analyzing an expressed gene of the present invention

In the target nucleic acid detection method of the present invention, the detection can be performed by hybridizing a labeled polynucleotide probe to the polyadenine nucleotide portion of the target nucleic acid. The polyadenine nucleotide portion of the target nucleic acid may be one obtained by artificially adding a polylipadenine nucleotide or polydeoxyadenine nucleotide afterwards, or may be a natural mRNA polylipoadenine nucleotide tail. . That is, there is no particular limitation as long as the labeled polynucleotide probe of the present invention efficiently and specifically hybridizes. The labeled nucleotide is labeled with a radioisotope, a chemiluminescent substance, a fluorescent substance, a ligand or a receptor. Preferably, the label is selected from any of the following nucleotides. For example, the label is fluorescein, cascade blue, Oregon green, BOD IPY, mouth-damine green, Alexa Fluor, Texas red, Cy3 or Cy5. And a labeled nucleotide selected from the group consisting of: In this method, for example, target nuclei in a sample hybridized to nucleic acids immobilized on a carrier Acids can be detected using methods optimal for labeling polynucleotide probes.

Another embodiment of the detection method of the present invention includes a method of analyzing an expressed gene. In this method, the expression of various types of genes in two different types of samples can be compared by using at least two types of labeled polynucleotide probes having different types of labels. Specifically, a sample that may contain the target nucleic acid is annealed with a labeled polynucleotide probe that hybridizes to the polyadenine nucleotide portion, and another sample that may contain the target nucleic acid is a polyadenine nucleotide portion. Annealed with a polynucleotide probe labeled with another label that hybridizes to the sample, and the resulting labeled polynucleotide probe is used to immobilize a sample that may contain the annealed target nucleic acid, for example, immobilize the target nucleic acid The expression of the target gene can be analyzed by detecting each of the labels hybridized on the carrier after hybridization with the carrier and comparing the presence or absence or the degree of each of the detected labels. The labeled polynucleotide probe is not particularly limited, but for example, a polynucleotide probe labeled with Cy3 and Cy5, respectively, can be suitably used.

Further, as another embodiment of the method of analyzing an expressed gene, a labeled polynucleotide probe that hybridizes to a polyadenylate nucleotide portion and a labeled polynucleotide probe that hybridizes to a portion other than the polyadenylate nucleotide of an expressed gene (c DNA-labeled polynucleotide probe). In this case, different labels can be used. A probe that hybridizes to a portion other than the polyadenine nucleotide of the above expressed gene can be prepared by a known method, for example, a cDNA synthesis reaction using mRNA in a sample as a mirror type. Although the method of the present invention is not particularly limited, for example, it can be used in the expression of a specific gene that is controlled by polyadenylation or deadenylation at the 3 ′ end, and the aforementioned control can be detected. .

In addition, in the detection method of the present invention, since the labeled polynucleotide probe can be used in common, there is no need to prepare a cDNA probe in accordance with a conventional target nucleic acid, and detection can be performed at low cost and in a short time. It can be carried out. Example

The present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1

(1) Preparation of labeled polynucleotide probe

Labeled polynucleotide probes were prepared using the reverse transcription Z labeling reaction. The reaction was performed with the following reaction solution composition. That is, the synthesis template of lO Opmol-Poly (rA)-p (dT) ₁₂ _ ₁₈ (Amersham 'Falmacia' Biotech) of primers was type III, and 0.5 mM dATP, dCTP, dGT P, 40 / M d TTP and 20 μΜ A 1 exa—dUTP (Molecular Probe), 1 OmM DTT, 25U liponucleotide inhibitor (Gibco BRL), 1.5 U superscript reversal Transcriptase (manufactured by Gibco BRL) was added to adjust the reaction solution volume to 50 IX. The reaction mixture was kept at 30 ° C. for 60 minutes, then heated at 94 ° C. for 3 minutes to inactivate the enzyme, and cooled on an ice bath. The reaction solution was purified using a Microspin S-300HR column (manufactured by Amersham Pharmacia Biotech). Next, the purified product is treated with 0.3N NaOH at 37 ° C for 20 minutes to decompose type I poly (rA), and then treated with hydrochloric acid and 1 M Tris-monohydrochloride buffer (pH 7.0). Neutralized. This solution was used as a fluorescence-labeled poly dU probe solution.

(2) Cell culture, RNA extraction, cDNA probe preparation

Human HeLa cells and WI38 cells were obtained from the Human Science Research Resource Bank (HSRRB), and Dulbecco's modified Ea containing 10% fetalcalf serum was obtained. Cultured in glemedium. Total RNA was extracted from the above cultured cells by the guar-dimuphenol / chloroform method. Genomic DNA that may have been contaminated was removed using RNase-free DNase (RQ 1 DNase, pro-glasses: h $ mRNA). MRNA was removed, if necessary, using mRNA Purification on Kit (Amersham Fa Purified using Lumasia Biotechne ± M). 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 Fluorescence Labeling Core Kit (Takara Shuzo). The reaction conditions are

After incubating at 37 ° C for 60 minutes, 10/1 1N sodium hydroxide solution was added and incubated at 37 ° C for 10 minutes to degrade the mirror RNA. The reaction product was neutralized by adding 25 μl of 1 M Tris-HCl buffer (ρΗ8.0), and then precipitated with ethanol. The precipitate was redissolved in 101 high-buffer buffer.

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

Human GAP DH (glycera 丄 dehyde 3-phosphate dehydrogenase), PCNA (proliferating cell nuclear antigen) p53, Rb (retinoblastoma) _Λ R as oncogene, pl 30, pl 4, pl 6, pl 8, pl 9 The primers described in SEQ ID NOs: 1 to 40 in the sequence listing and TaKaRaRNA PCR Kit (AMV) Ver. 2.1 (manufactured by Takara Shuzo Co.) Was used to perform RT-PCR. The genes for p53, RB, pi30, pi6, pl9, p27, pl8, and pi4 were subjected to nested PCR after the reverse transcription reaction. For type I, the RNA obtained in Example 1 (2) was used. The obtained amplified fragment was purified and then subcloned into a pT7B1ueT cloning vector (Novagen), and DNA sequence analysis was confirmed. After confirming the insertion base sequence, the above-mentioned insertion fragment was immobilized on Hybond N + (Amersham Pharmacia Biotechne; fc ^) using Bio-Dot (manufactured by Bio-Rad). The DNA microarray used was a commercially available Te starray (Takara Shuzo).

(4) Hybridization 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% (SDS) using 3 Prehybridization was performed at 7 ° C for 1 hour. First, add 10 ^ 1 of the labeled poly dU probe solution prepared in Example 1 (1) to 10 μl of either total RNA or purified RNA obtained in Example 1 (2) above, A hybridization buffer was added to 200 µl, heated at 70 ° C for 10 minutes, and then hybridized with the above membrane filter at 30 ° C for 6 to 10 hours. The membrane filters are gently shaken in Washing Buffer I (1 XSSC, 0.1% SDS) for 15 minutes at room temperature, and then washed for 10 minutes in Washing Buffer II (0.1 XSSC, 0% (1% SDS) and shaken. After washing, the membrane filter was detected with a fluorescence image analyzer FMB IOII (Takara Shuzo). On the other hand, hybridization of the DNA microarray was performed as instructed in the attached instruction manual. That is, prehybridization was performed using 14 μl of prehybridization solution (6 XSSC, 0.1% SDS, 5 X Denhardt's solution, 0.1 μg / μ1 salmon sperm DNA). One hour at 65 ° C. For hybridization with the poly-dU probe, 1〃1 of poly-dU was added to 600 ng of the mRNA obtained in Example 1 (2) above, and the volume was adjusted to 14 μl. The solution was heated at 70 ° C for 10 minutes. In addition, a cDNA probe was prepared using the same amount of mRNA. The above reaction solution was added to the microarray, and the array was then incubated at 65 ° C for 18 to 20 hours, and further incubated at 30 ° C for 3 hours. After hybridization, the microarray was washed with a 2XSSC solution at 60 ° C for 15 minutes, and further with a washing solution (2XSSC, 0.1% SDS) at 60 ° C for 15 minutes, and finally washed at room temperature for 2 minutes. Wash twice with 2XSSC. The signal on the microarray was detected using an array scanner-418 (manufactured by Affimetrix).

As an example of the labeled polynucleotide probe of the present invention in a membrane filter, the hybrid-specific production of a labeled polynucleotide probe labeled with Alexa-dUTP is shown in FIG. 1A. Figure 1-B shows the spotted position of each polynucleotide. As shown in FIG. 1-A, it was confirmed that the labeled polynucleotide probe specifically hybridized to polylipoadenine nucleotide (po1yA) and did not hybridize to other nucleotides. Furthermore, the detection sensitivity was examined using a membrane in which polyriboadenine nucleotides were spotted in a concentration range of 1 ng (3 fmol) to lg (3 pmo1). Figure 2 shows the results. As shown in FIG. 2, it was confirmed that a clear fluorescent signal was obtained even when 1 ng (3 fmo 1) was fixed at 12 mm ² . In addition, using a filter in which ljug of the GAPDH DNA fragment and pUC18 plasmid DNA were spotted on the same membrane, 10 to 40 μg of total RNA derived from HeLa cells was used as described in Example 1 (1). The labeled poly-dU probe solution 10 ^ 1 prepared in the above was annealed and hybridized. Figure 3 shows the results. As shown in FIG. 3, a signal was detected only on the spot of GAPDH DNA, not on pUC18 DNA.

Next, using the membrane on which the DNA fragment prepared in (4) above was spotted, 20 μg of total RNA derived from HeLa cells and WI138 cells and the poly dU prepared in Example 1 (1) above were used. Annealed probe solution 101 was hybridized. Fig. 4 shows the results. FIG. 4-11 shows the results obtained using HeLa cell-derived samples, and FIG. 4-2 shows the results obtained using WI138-derived samples. Fig. 43 shows the location of each spotted gene. From FIGS. 411 and 412, it was confirmed that a signal was detected depending on the gene to be expressed. Furthermore, comparison with the results of the conventional Northern plot confirmed that the same expression pattern was detected.

The examination was performed using a DNA microarray and mRNA derived from WI38 cells. Figure 5 shows the results. In Fig. 5, the DNA microarray used was one in which two sets of 96 DNAs were spotted (duplicated and spotted) in order to reproduce the data. Arrows indicate the top and bottom of each la. As shown in FIG. 5A, it was shown that mRNA labeled with WI38 cells annealed with the labeled polynucleotide probe of the present invention hybridized to a specific target derived from mammalian cells. On the other hand, none of the Cyanobacteria gene was detected. This is because cyanobacteria are prokaryotes and have completely different DNA sequences from eukaryotic mammals, so no hybridization occurs and this is the result. From this, it was confirmed that the labeled polynucleotide probe of the present invention can be used in a DNA microarray. Again the same Fig. 5B shows the results obtained by examining the samples using cDNA probes prepared by the conventional method from the same mRNA preparation. From the comparison between A and B in FIG. 5, it was confirmed that the detection method 1S of the present invention showed basically the same results as the conventional method.

Example 2

(1) Preparation of labeled polynucleotide probe

Example 1 (1) 1 6 3 — (11; Ding ?. 3— (11; Ding, Cy 5 -dUTP (Amersham 'Pharmacia Biotech Co., Ltd.)) Nucleotide probes were prepared and used as a Cy3-labeled poly-dU probe and a Cy5-labeled poly-dU probe, respectively.

(2) Cell culture, RNA extraction

Mouse RAW247 cells were obtained from Human Science Research Resource Punk (HumanScienceReseSearcnResoeurcesBank: HSRRB). Cell culture and mRNA extraction were performed in the same manner as in Example 11- (2). RAW247 cells were divided into osteoclasts by the method of Hsu H. et al. (Proc. Natl. Acd. Sci. USA. 96: p3540—354,

(1992 years) We used Osteoc, Osteoc, Last, Differen, Tia, tion, Factoor (ODF).

(3) NO, IB, and detection

MRNA extracted from undifferentiated RAW247 cells was annealed with Cy3-labeled poly-dU probe. That is, 600 ng of mRNA and 101 of Cy3-labeled poly-dU probe were mixed and treated at 70 ° C for 10 minutes. In addition, mRNA extracted from RAW247 cells induced into osteoclasts and Cy5-labeled poly-dU probe were annealed in the same manner as described above.

Hybridization was carried out using IntelliGene Mouse CH IPS et I ver. 1.0 (manufactured by Takara Shuzo) as instructed in the attached instruction manual. That is, prehybridization was performed using a 14 ^ 1 prehybridization solution (6 XSSC, 0.1% SDS, 5 X Denhardt's solution, 0.1 μg / μ1 salmon sperm DNA). Performed at 65 ° C for 1 hour. Cy3 and Cy5 labeled poly-dU probe and mRNA as probe The annealed solution was mixed to 141 and added to the microarray, then the array was incubated at 65 ° C for 18-20 hours, then further incubated at 30 ° (:, 3 hours. After the dilution, the microarray was washed with a 2XSSC solution at 60 ° C for 15 minutes, and further washed with a pre-cleaning solution (2XSSC, 0.1% SDS) at 60 ° C for 15 minutes. Finally, the plate was washed twice at room temperature with 0.2 XSSC, and the signals on the microarray were detected using a 418 array scanner (manufactured by Abimetrix) The results are shown in FIG.

As shown in Fig. 6, even when two types of labeled polynucleotide probes labeled with different fluorescent substances are combined, two colors using a conventional cDNA probe labeled with Cy3 or Cy5 are used. It was confirmed that gene expression analysis could be performed in the same manner as the method. Industrial applicability

According to the present invention, there are provided a labeled nucleic acid probe useful in hybridization, a method for preparing the probe, and a method for highly sensitive detection of a target nucleic acid using the probe. 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. A labeled polynucleotide probe that hybridizes to a polyadenylate nucleotide portion in a target nucleic acid.

2. The labeled polynucleotide probe according to claim 1, wherein the polyadenine nucleotide portion is a polyriboadenine nucleotide tail of mRNA.

3. The labeled polynucleotide probe according to claim 1, wherein the chain length is 50 nucleotides or more.

4. The labeled polynucleotide probe according to claim 1, wherein the probe is a polynucleotide comprising a deoxyperacyl nucleotide and / or a deoxythymine nucleotide.

5. The labeled polynucleotide probe according to claim 1, wherein the probe comprises a labeled deoxydilacil nucleotide and / or a labeled deoxythymine nutretide.

6. The labeled polynucleotide probe according to claim 1, which is labeled with a substance selected from the group consisting of radioisotopes, chemiluminescent substances, fluorescent substances, ligands and receptors.

7. A claim characterized by being labeled with a substance selected from the group consisting of full-aged Resin, cascading Venorey, Oregon Green, BOD IPY, Rhodamine Green, Alexa Fluor, Texas Red, Cy3 and Cy5. Term

7. The labeled polynucleotide probe according to 6.

8. The method for preparing a labeled polynucleotide probe according to claim 1, comprising a step of performing a nucleotide extension reaction using a polylipadenine nucleotide or a polydoxyadenine nucleotide as a type I and oligo dT as a primer.

9. The method for preparing a labeled polynucleotide probe according to claim 8, wherein the nucleotide extension reaction is a reverse transcription reaction using a polyriboadenine nucleotide as a 鐯 type.

10. Polylipadenine nucleotides or polydeoxyadenes that form 铸 9. The method for preparing a labeled polynucleotide probe according to claim 8, wherein the nucleotide length of the polynucleotide is 50 nucleotides or more.

11. The method for preparing a labeled polynucleotide probe according to claim 8, wherein the nucleotide extension reaction is a deoxynucleotide polymerization reaction.

12. The method for preparing a labeled polynucleotide probe according to claim 8, wherein the length of the labeled polynucleotide probe is 50 nucleotides or more.

13. The method for preparing a labeled polynucleotide probe according to claim 8, wherein the nucleotide extension reaction is carried out using a labeled deoxydilacil nucleotide and Z or a labeled deoxythymine nucleotide.

14. Labeled deoxyperacil nucleotide triphosphate and / or labeled deoxythyramine nucleotide triphosphate and unlabeled deoxyperacil nucleotide triphosphate and

When the molar ratio of Z or unlabeled deoxythymine nucleotide triphosphate is 1 ::! To 1:

14. The method for preparing a labeled polynucleotide probe according to claim 13, wherein the method is carried out in the probe preparation reaction solution of item 3.

15. The preparation of a labeled polynucleotide probe according to claim 8, wherein the labeled polynucleotide probe is labeled with a substance selected from the group consisting of a radioisotope, a chemiluminescent substance, a fluorescent substance, a ligand and a receptor. Method.

16. The labeled polynucleotide probe is labeled with a substance selected from the group consisting of fluorescein, Cascade Blue, Oregon Green, BODIPY, Rhodamine Green, Alexa Fluor, Texas Red, Cy3 and Cy5. The method for preparing a labeled polynucleotide probe according to claim 15, wherein

17. A process for hybridizing a labeled polynucleotide probe that hybridizes to a polyadenylate nucleotide portion together with a sample that may contain the target nucleic acid and a carrier having the target nucleic acid immobilized thereon, the method comprising: Detection method.

18. The method for detecting a target nucleic acid according to claim 17, wherein the polyadenine nucleotide portion is a polyriboadenine nucleotide tail of mRNA.

19. If the labeled polynucleotide probe is a labeled deoxydilacil nucleotide or 19. The method for detecting a target nucleic acid according to claim 17 or 18, comprising a labeled deoxythymine nucleotide.

20. The target nucleic acid according to claim 17, wherein a labeled polynucleotide probe labeled with a substance selected from the group consisting of a radioisotope, a luminescent substance, a fluorescent substance, a ligand, and a receptor is used. Detection method.

21. A labeled polynucleotide probe labeled with a substance selected from the group consisting of funolescein, Cascade Blue, Oregon Green, BOD I PY, Rhodamine Green, Alexa Fluo, Texas Red, Cy3 and Cy5. 21. The method for detecting a target nucleic acid according to claim 20, wherein:

22. a) annealing a first sample, which may contain the target nucleic acid, with a polynucleotide probe labeled with a first label that hybridizes to a polyadenine nucleotide moiety;

b) a second sample different from the first sample, which may contain the target nucleic acid, is hybridized to the polyadenine nucleotide portion, and is labeled with a second label different from the first label. Annealing with a polynucleotide probe;

c) hybridizing a sample, which may contain the target nucleic acid, to which the labeled polynucleotide probe obtained in steps a and b has been annealed, with a carrier on which the target nucleic acid is immobilized;

d) detecting the first and second labels hybridized on the immobilization carrier; and

e) a step of comparing the presence or absence or the degree of the detected first and second labels, and a method for analyzing target gene expression.

23. A labeled polynucleotide probe labeled with a substance selected from the group consisting of fluorescein, Cascade Blue, Oregon Green, BOD I PY, Rhodamine Green, Alexa Fluo, Texas Red, Cy3 and Cy5. 23. The method for analyzing target gene expression according to claim 22, wherein the method is used.

24. Combination of a labeled polynucleotide probe characterized by hybridizing to a polyadenylate nucleotide portion and a labeled polynucleotide probe characterized by hybridizing to a portion other than polyadenine nucleotide of an expressed gene A method for analyzing an expressed gene, which comprises a step of hybridizing with a sample which may contain the expressed gene in combination.

25. A labeled polynucleotide probe that is labeled with a substance selected from the group consisting of funoleoresin, cascading vine, Oregon green, BOD I PY, rhodamine green, Alexa Fluor, Texas red, Cy3 and Cy5. 25. The method for analyzing a target nucleic acid according to claim 24, wherein the method is used.