JP5218944B2 - Method for labeling nucleic acid in sequence-specific manner, and novel nucleic acid detection method using the same - Google Patents

Description

  The present invention relates to a method for labeling a nucleic acid, a method for producing a labeled nucleic acid, a method for detecting a nucleic acid using the same, and a kit and a system using them.

  Various labels have been used to detect nucleic acids. However, most of such labels label molecules that specifically interact with the detection target rather than labeling the detection target itself. Therefore, there is a drawback that the procedure is inevitably complicated.

  Since the beginning of the 1990s, in addition to the conventionally known rRNA, tRNA, and mRNA, the existence of a new type of RNA has become apparent. Such newly discovered types of RNA include small RNAs such as siRNA and miRNA. In recent years, it has been elucidated that such a small RNA plays an important role in the living body, and the development of its detection technology leads to the promotion of such elucidation. Therefore, there is a great demand for a technique for easily detecting nucleic acids such as various RNAs.

  An object of the present invention is to provide a technique capable of easily detecting a nucleic acid (particularly, small RNA).

(Disclosure of the Invention)
As a result of the successful application of the hybridization technique, the present inventors have unexpectedly developed that even a small RNA can be easily labeled and / or detected, thereby completing the present invention. Settled.

Small RNA plays an important role in gene regulation, including transcription, translation and mRNA stability. It is also thought to play an important role in maintaining chromosome structure. In addition, small RNAs also function as key factors in the self-defense system at the nucleic acid level in host cells, such as breaking down invading viral RNA or inactivating self-transposons. Thus, small RNA is an increasingly important field in animal and plant research. However, because of its small molecular weight (short nucleotide length and poor abundance), the method for detecting small RNA is complex and inefficient. In the present invention, there is provided a method for labeling small RNAs in a sequence-specific manner with dideoxynucleotides using a DNA template and DNA polymerase. The small RNA is hybridized with a template DNA that is substantially complementary to the target small RNA. The DNA-RNA complex is then incubated with a polymerase, such as the Klenow fragment of DNA polymerase I, and a deoxynucleotide or dideoxynucleotide labeled with a ³² P label or the like. The small RNA serves as a primer for DNA synthesis and the reaction is terminated by the incorporation of this dideoxynucleotide. This is called the primer halt method (Primer Halt). The labeled RNA can be detected by denaturing gel electrophoresis separation and autoradiography. Labeling is performed in a sequence-specific manner and is completely dependent on the absence of DNA polymerase. Using the primer termination method, at (10 ^-18 ) molar level of synthetic RNA and biological RNA (for example, mouse miRNA, transgenic plant siRNA, etc.) can be detected. Genes such as GFP gene, luciferase gene, miR16, miR171, etc. can be used for detection purposes such as experiments to find detection limits.

Accordingly, the present invention provides the following.
(1)
A method for producing a nucleic acid into which a label has been introduced, comprising the following steps:
A) Step of providing a nucleic acid to be labeled and a template nucleic acid, wherein the template nucleic acid is complementary to at least a part of the nucleic acid to be labeled, and the template nucleic acid is a complementary portion of the nucleic acid to be labeled Having a nucleotide sequence that is longer by at least one nucleotide in a portion of at least one tip of the nucleic acid to be labeled when the template nucleic acid is hybridized with the template nucleic acid;
B) A step of hybridizing the labeled target nucleic acid with the template nucleic acid to generate a labeled target nucleic acid-template nucleic acid complex;
C) A step of adding a nucleic acid synthase and a labeled nucleotide to the labeled target nucleic acid-template nucleic acid complex to perform an extension reaction, wherein at least one labeled nucleotide is added to the labeled target nucleic acid as the template nucleic acid And D) removing the extended nucleic acid to be labeled from the resulting mixture after the extension reaction;
Including the method.
(2)
Item 2. The method according to Item 1, wherein the nucleic acid to be labeled is DNA or RNA or a combination thereof.
(3)
Item 2. The method according to Item 1, wherein the nucleic acid to be labeled is RNA.
(4)
Item 2. The method according to Item 1, wherein the nucleic acid to be labeled is RNA selected from the group consisting of siRNA and miRNA.
(5)
Item 2. The method according to Item 1, wherein the template nucleic acid comprises a sequence that is complementary to all the nucleic acids to be labeled.
(6)
Item 2. The method according to Item 1, wherein the template nucleic acid has a nucleotide sequence that is at least one nucleotide longer at both ends of the nucleic acid to be labeled.
(7)
Item 2. The method according to Item 1, wherein the template nucleic acid has a nucleotide sequence that is at least about 10 nucleotides longer than the tip of at least one of the nucleic acids to be labeled.
(8)
Item 2. The method according to Item 1, wherein the long nucleotide sequence of the template nucleic acid is located on the 5 ′ end side of the template nucleic acid.
(9)
Item 7. The long nucleotide sequence of the template nucleic acid on the 3 'end side of the template nucleic acid has a different length from the long nucleotide sequence of the template nucleic acid on the 5' end side of the template nucleic acid. the method of.
(10)
2. The method according to item 1, wherein the long nucleotide sequence of the template nucleic acid is located on the 5 ′ end side of the template nucleic acid, and the 5 ′ end portion of the nucleic acid to be labeled is longer than the 3 ′ end of the template nucleic acid. (11)
Item 6 wherein the long nucleotide sequence of the template nucleic acid at the 3 ′ end of the template nucleic acid is longer than the long nucleotide sequence of the template nucleic acid at the 5 ′ end of the template nucleic acid by a distinguishable length. The method described in 1.
(12)
12. The method of item 11, wherein the distinguishable length is at least about 10 nucleotides.
(13)
Item 2. The method according to Item 1, wherein the nucleic acid synthase is a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase.
(14)
2. The method according to item 1, wherein the labeled nucleotide comprises a nucleotide labeled with a label selected from the group consisting of fluorescence, radioactivity, phosphorescence, biotin, digoxigenin (DIG), enzyme, and chemiluminescence.
(15)
Item 2. The method according to Item 1, wherein the labeled nucleotide comprises radioactivity.
(16)
Item 2. The method according to Item 1, wherein the step of taking out the elongated nucleic acid to be labeled includes a denaturation step.
(17)
Furthermore, the method of item 1 including the process of removing an unreacted substance.
(18)
Item 18. The method according to Item 17, wherein the removal of the unreacted material is achieved by ethanol precipitation or gel filtration chromatography.
(19)
Item 18. The method according to Item 17, wherein the unreacted substance removing step further includes a step of separating the extended nucleic acid to be labeled.
(20)
20. A method according to item 19, wherein the separation is achieved by electrophoresis or chromatography.
(21)
Item 2. The method according to Item 1, wherein the step of taking out the elongated nucleic acid to be labeled includes the step of detecting the label.
(22)
Item 22. The method according to Item 21, wherein the detection includes detecting the label directly or indirectly.
(23)
Item 22. The method according to Item 21, further comprising the step of separating the elongated nucleic acid to be labeled.
(24)
24. A method according to item 23, wherein the detection is performed during the separation.
(25)
25. A method according to item 24, wherein the separation and detection is achieved by autoradiograph. (26)
Furthermore, the method of item 1 including the process of stopping the said extension reaction.
(27)
27. The method according to item 26, wherein the stopping step is achieved by primer stop (Primer Halt method) or primer run-off method.
(28)
Item 2. The method according to Item 1, wherein the nucleic acid to be labeled is used as a probe.
(29)
Item 2. The method according to Item 1, wherein the nucleic acid to be labeled is a nucleic acid to be detected.
(30)
Item 2. The method according to Item 1, wherein the template nucleic acid is modified at a terminal nucleotide.
(31)
Item 2. The method according to Item 1, wherein the terminal nucleotide of the template nucleic acid is modified at the 3 'end.
(32)
Item 2. The method according to Item 1, wherein the terminal nucleotide of the template nucleic acid is modified so as not to incorporate a labeled nucleotide.
(33)
The modification is a modification selected from the group consisting of the dideoxy chain termination method, fluorescein isothiocyanate (FITC), biotinylation, amination, phosphorylation, tetramethylrhodamine (TAMRA), thiolation and rhodamineation. 31. The method according to item 30.
(34)
The 3 'terminal nucleotide of the template nucleic acid is selected from the group consisting of the dideoxy chain termination method, fluorescein isothiocyanate (FITC), biotinylation, amination, phosphorylation, tetramethylrhodamine (TAMRA), thiolation and rhodamineation Item 2. The method according to Item 1, wherein the selected modification is made.
(35)
A method for introducing a label into a nucleic acid comprising the following steps:
A) a step of providing a nucleic acid to be labeled and a template nucleic acid, the template nucleic acid being complementary to at least a part of the nucleic acid to be labeled, and a complementary part of the nucleic acid to be labeled and the template nucleic acid And having a nucleotide sequence that is at least one nucleotide longer in the portion relative to the tip of at least one of the nucleic acids to be labeled;
B) a step of hybridizing the nucleic acid to be labeled with the template nucleic acid to produce a nucleic acid to be labeled-template nucleic acid complex; and C) a nucleic acid synthase and a labeled nucleic acid to be labeled with the nucleic acid to be labeled-template nucleic acid complex. Adding a nucleotide to perform an extension reaction, and at least one labeled nucleotide is incorporated into the nucleic acid to be labeled based on the template nucleic acid,
Including the method.
(36)
A method for detecting a target nucleic acid comprising the following steps:
A) providing a target nucleic acid and a template nucleic acid, wherein the template nucleic acid is complementary to at least a part of the target nucleic acid and further hybridizes the complementary part of the target nucleic acid with the template nucleic acid. Having a nucleotide sequence that is at least one nucleotide longer in the portion relative to the tip of at least one of its target nucleic acids when soyed;
B) hybridizing the target nucleic acid with the template nucleic acid to produce a target nucleic acid-template nucleic acid complex;
C) adding a nucleic acid synthesizing enzyme and a labeled nucleotide to the target nucleic acid-template nucleic acid complex to perform an extension reaction; and D) detecting whether an extension product corresponding to the target nucleic acid is present. The presence of the extension product is an indicator of the presence of the target nucleic acid,
Including the method.
(37)
The method according to item 36, further comprising the step of separating the unreacted product and the extension product of the labeled nucleotide from the resulting mixture after the extension reaction.
(38)
37. The method according to item 36, wherein the target nucleic acid is DNA or RNA or a combination thereof.
(39)
The method according to item 36, wherein the target nucleic acid is RNA.
(40)
The method according to item 36, wherein the target nucleic acid is RNA selected from the group consisting of siRNA and miRNA.
(41)
40. The method of item 36, wherein the template nucleic acid comprises a sequence that is complementary to all of the target nucleic acids.
(42)
38. The method of item 36, wherein the template nucleic acid has a nucleotide sequence that is at least one nucleotide longer at both ends of the target nucleic acid.
(43)
38. The method of item 36, wherein the template nucleic acid has a nucleotide sequence that is at least about 10 nucleotides longer than the tip of at least one of the target nucleic acids.
(44)
The method according to item 36, wherein the long nucleotide sequence of the template nucleic acid is located on the 5 ′ end side of the template nucleic acid.
(45)
45. The item 42, wherein the long nucleotide sequence of the template nucleic acid on the 3 ′ end side of the template nucleic acid has a different length from the long nucleotide sequence of the template nucleic acid on the 5 ′ end side of the template nucleic acid. the method of.
(46)
The method according to item 36, wherein the long nucleotide sequence of the template nucleic acid is located on the 5 ′ end side of the template nucleic acid, and the 5 ′ end portion of the nucleic acid to be labeled is longer than the 3 ′ end of the template nucleic acid. .
(47)
The long nucleotide sequence of the template nucleic acid on the 3 ′ end side of the template nucleic acid is longer than the long nucleotide sequence of the template nucleic acid on the 5 ′ end side of the template nucleic acid by a distinguishable length, Item 42 The method described in 1.
(48)
48. The method of item 47, wherein the distinguishable length is at least about 10 nucleotides.
(49)
The method according to item 36, wherein the nucleic acid synthase is a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase.
(50)
40. The method of item 36, wherein the labeled nucleotide comprises a nucleotide labeled with a label selected from the group consisting of fluorescence, radioactivity, phosphorescence, biotin, digoxigenin (DIG), enzyme and chemiluminescence.
(51)
40. The method of item 36, wherein the labeled nucleotide comprises radioactivity.
(52)
The method according to item 36, wherein the separation of the extension product and the template nucleic acid complex is achieved by denaturation.
(53)
38. The method of item 36, wherein the unreacted product is separated from the extension product by removing the unreacted product by ethanol precipitation and filtration.
(54)
40. The method of item 36, wherein the detection comprises detecting the label directly or indirectly.
(55)
38. The method according to item 36, further comprising the step of removing an extension product corresponding to the target nucleic acid.
(56)
38. The method according to item 37, wherein the separation is performed by electrophoresis or chromatography.
(57)
38. The method of item 37, wherein the detection is performed during the separation.
(58)
58. The method of item 57, wherein the separation and detection is accomplished by autoradiograph.
(59)
The method according to item 36, further comprising the step of stopping the extension reaction.
(60)
60. The method according to Item 59, wherein the stopping step is achieved by a primer halt method or a primer run-off method.
(61)
The method according to item 36, wherein the detection is performed based on the sequence of the extended portion.
(62)
38. The method according to item 36, wherein the detection is performed using an agent that specifically binds to the extended target nucleic acid.
(63)
63. The method according to item 62, wherein the detection is performed by detecting a label attributed to the labeled nucleotide and a label attributed to the drug.
(64)
63. The method of item 62, wherein the agent that specifically binds to the extended target nucleic acid does not bind to the non-extended target nucleic acid or the labeled nucleotide.
(65)
The method according to item 36, wherein the detection is performed based on the length of the extended target nucleic acid. (66)
37. The method according to item 36, wherein the template nucleic acid is modified at a terminal nucleotide.
(67)
The method according to item 36, wherein the terminal nucleotide of the template nucleic acid is modified at the 3 ′ end.
(68)
The method according to item 36, wherein the terminal nucleotide of the template nucleic acid is modified so as not to incorporate the labeled nucleotide.
(69)
The modification is a modification selected from the group consisting of the dideoxy chain termination method, fluorescein isothiocyanate (FITC), biotinylation, amination, phosphorylation, tetramethylrhodamine (TAMRA), thiolation and rhodamineation. The method according to item 66.
(70)
The 3 'terminal nucleotide of the template nucleic acid is selected from the group consisting of the dideoxy chain termination method, fluorescein isothiocyanate (FITC), biotinylation, amination, phosphorylation, tetramethylrhodamine (TAMRA), thiolation and rhodamineation 40. The method of item 36, wherein the selected modification has been made.
(71)
A kit for producing a nucleic acid introduced with a label, comprising the following means:
A) Complementary to at least a part of a nucleic acid to be labeled as a target for introduction of a label, and when a complementary part of the target nucleic acid is hybridized with the template nucleic acid, at least one tip of the target nucleic acid A template nucleic acid having a nucleotide sequence that is at least one nucleotide longer in the part to; and B) a nucleic acid synthase;
A kit comprising:
(72)
C) The kit according to item 71, further comprising a labeled nucleotide.
(73)
73. A kit according to item 72, further comprising means for separating the unreacted product of the labeled nucleotide and the extended product of the extension from the resulting mixture.
(74)
73. A kit according to item 72, further comprising means for detecting a labeled nucleotide.
(75)
72. The kit according to Item 71, further comprising a standard target nucleic acid as a control.
(76)
The kit according to Item 75, wherein the standard nucleic acid includes a nucleic acid selected from the group consisting of a nucleic acid for identifying the template nucleic acid and a nucleic acid for identifying the nucleic acid to be labeled.
(77)
72. The kit according to item 71, further comprising a reagent for the reaction of the nucleic acid synthase.
(78)
Item 73. The kit according to Item 72, wherein the labeled nucleotide has an action of stopping the reaction of a nucleic acid synthase.
(79)
72. The kit according to item 71, further comprising a support, wherein the template nucleic acid is fixed to the support.
(80)
80. The kit according to Item 79, wherein the support is glass or a membrane.
(81)
80. The kit according to Item 79, wherein the template nucleic acid immobilized on the support is arranged in an array.
(82)
72. The kit according to item 71, wherein the template nucleic acid is modified at a terminal nucleotide.
(83)
72. The kit according to Item 71, wherein the terminal nucleotide of the template nucleic acid is modified at the 3 ′ end.
(84)
72. The kit according to Item 71, wherein the terminal nucleotide of the template nucleic acid is modified so as not to incorporate the labeled nucleotide.
(85)
Item 82 is a modification selected from the group consisting of dideoxylation, fluorescein isothiocyanate (FITC), biotinylation, amination, phosphorylation, tetramethylrhodamine (TAMRA), thiolation and rhodamineation. The kit according to 1.
(86)
The nucleotide at the 3 ′ end of the template nucleic acid is selected from the group consisting of dideoxylation, fluorescein isothiocyanate (FITC), biotinylation, amination, phosphorylation, tetramethylrhodamine (TAMRA), thiolation and rhodamineation 72. The kit according to item 71, wherein the kit is modified.
(87)
A method for producing a support on which a nucleic acid introduced with a label is immobilized, comprising the following steps:
A) a step of providing a labeled nucleic acid and a template nucleic acid immobilized on a support, the template nucleic acid being complementary to at least a part of the labeled nucleic acid and further complementary to the labeled nucleic acid Having a nucleotide sequence that is at least one nucleotide longer in the portion relative to the tip of at least one of the nucleic acids to be labeled when the portion and the template nucleic acid are hybridized;
B) A step of hybridizing the labeled target nucleic acid with the template nucleic acid to generate a labeled target nucleic acid-template nucleic acid complex;
C) A step of adding a nucleic acid synthase and a labeled nucleotide to the labeled target nucleic acid-template nucleic acid complex to perform an extension reaction, wherein at least one labeled nucleotide is added to the labeled target nucleic acid as the template nucleic acid Incorporated on the basis of a process;
D) after the extension reaction, removing the support containing the nucleic acid to be labeled extended from the resulting mixture,
Including the method.
(88)
90. The method of item 87, wherein the removal step is accomplished by washing the support to remove reactants.
(89)
90. The method of item 87, wherein the removing step is accomplished by removing the label from the support.
(90)
A kit for detecting a target nucleic acid comprising :
A) Complementary to at least a part of the target nucleic acid, and when the complementary part of the target nucleic acid and the template nucleic acid are hybridized, at least one nucleotide is longer in the part with respect to at least one tip of the target nucleic acid A template nucleic acid having a nucleotide sequence;
B) Nucleic acid synthase;
C) labeled nucleotides;
D) means for detecting the labeled nucleotide;
A kit comprising:
(91)
91. The kit according to item 90, further comprising means for separating the unreacted product of the labeled nucleotide and the extended product of extension from the resulting mixture.
(92)
Item 91. The kit according to Item 90, wherein the template nucleic acid is modified at a terminal nucleotide.
(93)
A method for detecting a target nucleic acid on a support on which a nucleic acid is immobilized, comprising the following steps:
A) a step of providing a template nucleic acid and a nucleic acid to be labeled immobilized on a support, wherein the template nucleic acid is complementary to at least a part of the nucleic acid to be labeled, and the template nucleic acid is further to be labeled Having a nucleotide sequence that is at least one nucleotide longer in the portion of at least one tip of the nucleic acid to be labeled when hybridizing the complementary portion of the nucleic acid and the template nucleic acid;
B) A step of hybridizing the labeled target nucleic acid with the template nucleic acid to generate a labeled target nucleic acid-template nucleic acid complex;
C) A step of adding a nucleic acid synthase and a labeled nucleotide to the labeled target nucleic acid-template nucleic acid complex to perform an extension reaction, wherein at least one labeled nucleotide is added to the labeled target nucleic acid as the template nucleic acid Incorporated on the basis of a process;
D) after the extension reaction, a step of determining whether or not the extended nucleic acid to be labeled is present, and the presence of the extended nucleic acid to be labeled indicates that the target nucleic acid is present; Process,
Including the method.
(94)
94. The method according to Item 93, wherein the nucleic acid to be labeled is siRNA.
(95)
94. The method (96) of item 93, wherein the support is an array.
A method for detecting a target nucleic acid on a support on which a nucleic acid is immobilized, comprising the following steps:
A) a step of providing a labeling target nucleic acid and a template nucleic acid immobilized on the support, wherein the template nucleic acid is complementary to at least a part of the labeling target nucleic acid and further complementary to the labeling target nucleic acid Having a nucleotide sequence that is at least one nucleotide longer in the portion relative to the tip of at least one of the nucleic acids to be labeled when the portion is hybridized with the template nucleic acid;
B) A step of hybridizing the labeled target nucleic acid with the template nucleic acid to generate a labeled target nucleic acid-template nucleic acid complex;
C) A step of adding a nucleic acid synthase and a labeled nucleotide to the labeled target nucleic acid-template nucleic acid complex to perform an extension reaction, wherein at least one labeled nucleotide is added to the labeled target nucleic acid as the template nucleic acid Incorporated on the basis of a process;
D) after the extension reaction, a step of determining whether or not the extended nucleic acid to be labeled is present, and the presence of the extended nucleic acid to be labeled indicates that the target nucleic acid is present; Process,
Including the method.
(97)
99. The method according to Item 96, wherein the template nucleic acid is mRNA.
(98)
99. A method according to item 96, wherein the support is an array.

  In the following, the present invention is described by means of preferred embodiments. It will be understood by those skilled in the art that the embodiments of the present invention can be appropriately implemented based on the description of the present invention and well-known and common techniques in the field. The operation and effect of the present invention can be easily understood by those skilled in the art.

  According to the present invention, a nucleic acid to be labeled (for example, DNA, RNA) is hybridized with a template nucleic acid (for example, a template DNA in the primer stop method and a target nucleic acid in the primer array method), and then labeled. DNA synthesis using nucleic acid as a primer is performed. A method for labeling a target nucleic acid is provided by using a nucleotide to be newly added in the DNA synthesis reaction as a labeling compound. In this case, the labeled compound is used as a new nucleotide added to DNA synthesis. In this way, a method for labeling a target nucleic acid can be provided. The target nucleic acid may be RNA or DNA, but the present invention is particularly useful as a method for labeling short RNA. The label may be a radioisotope, a fluorescent dye, or the like, and is demonstrated in this specification by a method using a radioisotope. The labeled nucleic acid can be detected using the labeled compound as an index. In the case of a radioactive label, autoradiography and the like can be used. The DNA synthesis reaction for labeling can be stopped at a limited length by the dideoxy chain termination method. A method based on the dideoxy chain termination method is referred to as a primer halt method. The template DNA needs to have a sequence portion complementary to the target nucleic acid, but when the target nucleic acid is a small RNA of known sequence such as miRNA, the length and sequence before and after can be arbitrarily selected. . By using this, the DNA synthesis reaction using the target nucleic acid as a primer can be limited to a limited length (limited DNA synthesis). This is called a primer run-off method.

  The labeled target nucleic acid can be detected after separation by gel electrophoresis. By combining the limited DNA synthesis reaction and gel electrophoresis, the length (base pair) of the target nucleic acid can be known. This technology can also be applied to detection methods using fluorescent labels and capillary sequencers. This technique can be applied as a method for detecting siRNA and miRNA regardless of plants, animals and fungi. The present invention is not limited to short RNA, and can be applied as a method for specifically labeling general nucleic acid sequences. For example, sequence-specific end labeling of ssDNA is possible. In situ labeling is also possible. Various other applications are possible.

  As a technique for incorporating a fluorescent dye into a nucleotide, for example, an amino group introduction reagent (2- (4-monomethoxytritylamino) ethyl- (2-cyanoethyl)-(N, N-diisopropyl) -phosphoramidite Or a thiol group introduction reagent ((S-trityl-6-mercaptohexyl)-(2-cyanoethyl)-(N, N-diisopropyl) -phosphoramidite, (S-trityl-2-mercaptoethyl)-( 2-cyanoethyl)-(N, N-diisopropyl) -phosphoramidite etc.) and the like; a phosphoric acid bond at the labeling site of a fluorescent dye in a nucleic acid to a phosphonic acid bond having a functional group In this method, a linker is introduced and a fluorescent agent is bonded using this functional group (Japanese Patent Laid-Open No. 61-443). 3), and the like, but are not limited to.

  The method of the present invention is a novel nucleic acid detection method, and has novelty and utility in that a nucleic acid can be directly labeled in a sequence-specific manner. Further, the length to be labeled can be defined (limited to 1 to several bases) by selection of nucleotide or template. In particular, it is effective as a method for detecting short RNAs such as siRNAs involved in RNA silencing that have been attracting attention in recent years and miRNAs that have been noted as part of the gene expression regulation mechanism after translation.

Compared with the conventional method, the present invention is, for example:
1) Protocol operation is simple and can be detected in a short time. (Nucleic blots can be directly labeled with nucleic acids in the same procedure as probe synthesis, and can be directly detected by electrophoresis after labeling. No operations such as electrophoresis transfer, membrane hybridization, or washing are required. );
2) The nucleic acid to be labeled can be directly labeled unlike the Northern blot method, etc .;
3) There is a level of detection sensitivity that can be compared with a method that is conventionally considered to be high sensitivity or higher. (Up to the atmol level. This is the same level as the detection limit of commercial kits based on RNase protection {that is, so-called champion data}. In addition, since RNase is used in RNase protection, there is a possibility that the detected RNA is an artifact. The method is faster and simpler than this method.); And 4) Detection of siRNA or miRNA is often impossible unless a small RNA fraction is purified. Then, it is possible with total RNA.
It is considered that there are advantages and effects.

FIG. 1 is a diagram illustrating the principle of the primer halt method. Small RNA: A short RNA targeted for detection. Template DNA: DNA used as a template. In the examples, synthetic ssDNA is used, but the present invention is not limited to this. DNA pol. : DNA polymerase. Although Klenow fragment is used in the examples, the present invention is not limited to this. In principle, reverse transcriptase is also possible. (1) A short RNA is hybridized with a template DNA. (2) Add DNA polymerase and dideoxy NTP (or dideoxy ATP only) to room temperature. The dideoxyNTP is labeled with ³² P or the like. (3) The short RNA is labeled by a DNA synthesis reaction with DNA polymerase using the short RNA hybridized with DNA as a primer. At this time, since dideoxyNTP is incorporated, the reaction is stopped only by adding one base. (4) The labeled RNA is denatured and dissociated from the DNA. (5) It can be easily detected by autogram after gel electrophoresis. FIG. 2 shows the sequences of synthetic DNA and RNA used in the model experiment shown in the examples. DNA 1 and 2 are part of the ORF in the sense direction of the GFP gene. The RNA has a sequence complementary to a part of the 21 nucleotide DNA. The hybridized state is shown in the figure. When a single base is added to 3 'of RNA by a reaction using RNA as a primer, dATP is incorporated in the combination of DNA1-RNA1 and dTTP is incorporated in the combination of DNA2-RNA2. There is no homology between DNA1 and RNA2, and DNA2 and RNA1. FIG. 3 is a model experiment of the primer halt method and the primer run-off method. pol. : Klenow fragment: sRNA (synthetic RNA): synthetic RNA 1 or 2: Temp (template DNA). : Synthetic DNA 1 or 2. DNA and RNA, respectively 1 pmol and ³³ P-dideoxy NTP, were mixed in a Klenow reaction solution, and then hybridized by returning to a denaturation-normal temperature at 95 ° C. Klenow enzyme was added and a DNA synthesis reaction was performed using sRNA as a primer. Klenow enzyme was added thereto and a DNA synthesis reaction was performed using sRNA as a primer. No nucleotides other than ³³ P-labeled dideoxyNTPs were added. The labeled RNA was detected by autogram after denaturing acrylamide gel electrophoresis. DNA1-RNA1, since the DNA 2-RNA2 combination of polymerase-dependent manner ³³ P uptake is observed, it was shown this method by a possible indicator of sequence-specific short RNA. FIG. 4 shows the detection limit of primer halt (Primer Halt) and the detection of miRNA from organisms by primer halt (Primer Halt). Detection sensitivity was determined by serially diluting small RNAs to 1/10. It has been found that the method of the present invention can detect at least up to 5 atmole (5 × 10 ⁻¹⁸ ) levels. The left lane is as follows. (A) The detection of a synthetic RNA dilution series, the two lanes on the right are those detected miRNA contained in total RNA prepared from a plant. Luc: Reaction using a part of the sequence of luciferase gene luc not encoded by plants as a template as a negative control. miR171: A primer halt method using a sequence complementary to miR171 which is a plant endogenous microRNA (miRNA) + a sequence containing a single base T. In this way, plant endogenous miRNA can be detected. FIG. 4B shows that mouse miRNA can be confirmed by the primer run-off method of the present invention. FIG. 5 shows the principle of the primer stopping method. FIG. 6 shows the principle of the primer run-off method. FIG. 7 shows the principle of the primer array method.

  The invention will now be described by way of example, with reference to the accompanying drawings, where appropriate. The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

  The embodiments provided below are provided for a better understanding of the present invention, and the scope of the present invention should not be limited to the following description. It will be apparent to those skilled in the art that modifications can be made as appropriate within the scope of the present invention in consideration of the description in the present specification.

(the term)
As used herein, “polynucleotide”, “oligonucleotide”, “nucleic acid” and “nucleic acid molecule” are used interchangeably herein and refer to a polymer of nucleotides of any length. Examples of such nucleic acid molecules include, but are not limited to, cDNA, mRNA, and genomic DNA. The term also includes “oligonucleotide derivatives” or “polynucleotide derivatives”. As used herein, the term “oligonucleotide derivative” or “polynucleotide derivative” refers to an oligonucleotide or polynucleotide that includes a derivative of a nucleotide or that has an unusual linkage between nucleotides, and is used interchangeably. Specific examples of such an oligonucleotide include, for example, 2′-O-methyl-ribonucleotide, an oligonucleotide derivative in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphate in an oligonucleotide. Oligonucleotide derivatives in which diester bonds are converted to N3′-P5 ′ phosphoramidate bonds, oligonucleotide derivatives in which ribose and phosphodiester bonds in oligonucleotides are converted to peptide nucleic acid bonds, uracil in oligonucleotides Is an oligonucleotide derivative substituted with C-5 propynyluracil, an oligonucleotide derivative wherein uracil in the oligonucleotide is substituted with C-5 thiazo-ruuracil, and cytosine in the oligonucleotide is substituted with C-5 propynylcytosine Oligonucleotide derivatives, oligonucleotide derivatives in which cytosine in the oligonucleotide is substituted with phenoxazine-modified cytosine, oligonucleotides in which the ribose in DNA is substituted with 2'-O-propyl ribose Examples thereof include nucleotide derivatives and oligonucleotide derivatives in which the ribose in the oligonucleotide is substituted with 2′-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence is also a conservatively modified variant thereof (eg, a degenerate codon substitute), similar to a sequence explicitly shown in the sequence listing. , Complementary sequences, and corresponding sequences (eg, mouse sequences for human sequences, etc.) are contemplated.

  As used herein, “small RNA” refers to small-sized RNA, specifically including RNA of about 50 nucleotides or less, more preferably about 30 nucleotides or less. Can be used. Examples of small RNAs include, but are not limited to, miRNA and siRNA. Small RNA has attracted attention in recent years and is said to play an important role in gene regulation including transcription, translation and mRNA stability.

  In the present specification, the “nucleotide” may be natural or non-natural. “Nucleotide derivative” or “nucleotide analog” refers to a substance that is different from a naturally occurring nucleotide but has a function similar to that of the original nucleotide. Such nucleotide derivatives and nucleotide analogs are well known in the art. Examples of such nucleotide derivatives and nucleotide analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral methylphosphonate, 2-O-methylribonucleotide, peptide-nucleic acid (PNA). .

  When used in an extension reaction, the nucleotide can take the form of a triphosphate. Examples of nucleoside triphosphates include deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), and deoxyuridine. Examples thereof include, but are not limited to, triphosphate (dUTP) and deoxyinosine triphosphate (dITP). In the extension reaction, nucleotides for stopping the reaction include dideoxyadenosine triphosphate (ddATP), dideoxyguanosine triphosphate (ddGTP), dideoxycytidine triphosphate (ddCTP), dideoxy, as used in the dideoxy chain terminator method. Examples thereof include, but are not limited to, thymidine triphosphate (ddTTP), dideoxyuridine triphosphate (ddUTP), dideoxyinosine triphosphate (ddITP), and the like. Unless otherwise stated, it is understood that “nucleotide” and “nucleotide triphosphate” are used interchangeably in the present invention.

  As used herein, a nucleotide can be “labeled”. Examples of such a label include, but are not limited to, any label using fluorescence, radioactivity, phosphorescence, biotin, DIG, enzyme, and chemiluminescence.

In this specification, the “label” refers to a presence (for example, a substance, energy, electromagnetic wave, etc.) for distinguishing a target molecule or substance from others. Examples of such labeling methods include, but are not limited to, RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method and the like. When both the nucleic acid fragment and the complementary oligonucleotide are labeled by the fluorescence method, the labeling is performed with fluorescent substances having different fluorescence emission maximum wavelengths. The difference in the maximum fluorescence emission wavelength is preferably 10 nm or more. Any fluorescent substance can be used as long as it can bind to the base moiety of the nucleic acid. However, cyanine dyes (eg, CyDye ^™ series Cy3, Cy5 etc.), rhodamine 6G reagent, N-acetoxy-N2-acetyl Aminofluorene (AAF), AAIF (iodine derivative of AAF) or the like is preferably used. Examples of the fluorescent substance having a difference in fluorescence emission maximum wavelength of 10 nm or more include a combination of Cy5 and rhodamine 6G reagent, a combination of Cy3 and fluorescein, a combination of rhodamine 6G reagent and fluorescein, and the like. In the present invention, by using such a label, the target object can be modified so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can appropriately carry out such methods depending on the label and the target object. In one preferred embodiment, the label is radioactively labeled.

  As used herein, the terminal nucleotide of the template nucleic acid can be “modified”. Such a modification of the end of the nucleic acid is preferably a modification that does not incorporate other nucleotides (for example, labeled nucleotides). Although it may be 5 'end or 3' end, it is preferably 3 'end. Such modifications can include, but are not limited to, dideoxylation, fluorescein isothiocyanate (FITC), biotinylation, amination, phosphorylation, tetramethylrhodamine (TAMRA), thiolation and rhodamineation. Not. Such modifications such as fluorescein isothiocyanate (FITC), biotinylation, amination, phosphorylation, tetramethylrhodamine (TAMRA), thiolation and rhodamine incorporation are incorporated into the 3 ′ end to extend by polymerase. The reaction can be stopped.

  As used herein, “denaturation” refers to a phenomenon in which a nucleic acid loses its natural three-dimensional structure without accompanying covalent bond breakage. It is understood that inclusion of a denaturation step facilitates separation of the nucleic acid and facilitates removal of the elongated labeled target nucleic acid.

  In this specification, “removal of unreacted substances” refers to removal of unreacted materials (for example, labeled nucleotides) in a nucleic acid synthesis reaction. The unreacted material to be removed in particular advantageously contains labeled nucleotides. This is because, by removing the labeled nucleotide, detection can be performed using only the extended label as a clue. Unreacted substances can be removed by, for example, alcohol precipitation (for example, precipitation with 2-propanol, ethanol or the like) and filtration after the reaction. Removal of unreacted substances in the array can be performed by washing.

  As used herein, “separation of nucleic acids” refers to separating a set of nucleic acids from a set of nucleic acids having different properties. Such separation can be carried out based on molecular weight, labeling, affinity and the like. When the molecular weight is separated by a clue, it can be separated by electrophoresis. Separation by molecular weight can also be performed using molecular sieves.

  The removal of the nucleic acid may be performed simultaneously with the detection of the label.

  In the present invention, the detection of the label can be performed directly or indirectly. When performing directly, for example, radioactivity can be detected with a Geiger counter or the like, or fluorescence can be detected directly with the naked eye or a camera. In the case of performing indirectly, for example, another label is bound to the label to detect the other label, or a factor that activates the label is added to activate the label (biotin, The detection can be performed by detecting the use of streptavidin or the like).

As used herein, “autoradiograph” and “autoradiography” refer to the distribution of in vivo substances by observing the uptake of radioactive substances (tracers) in the living body using an X-ray photographic film or a dry plate. This refers to a method for examining migration and metabolism cytochemically or histochemically. Examples of the autoradiograph include macro autoradiograph method (visible autoradiograph method), micro autoradiograph method (light microscope autoradiograph method), and ultra micro autoradiograph method (electron microscope autoradiograph method). . In the present invention, one of these can be arbitrarily selected and used according to the purpose.

  In the method of the present invention, various detection methods and detection means can be used as long as information of biomolecules or information resulting from substances interacting with the biomolecules can be detected.

  As used herein, “specifically interacts” with a biomolecule (eg, a polynucleotide or polypeptide, etc.) means that the affinity for the biomolecule is other unrelated (especially polynucleotide, For a polypeptide or the like, it means that it is typically equivalent or higher, or preferably significantly higher than its affinity for a polynucleotide or polypeptide (for example, less than 30% identity). Such affinity can be measured, for example, by hybridization assays, binding assays, and the like.

  In the present specification, the “target nucleic acid” refers to a nucleic acid to be subjected to a reaction (for example, labeling or detection). Therefore, when the purpose is labeling, the target nucleic acid and the target nucleic acid can overlap.

  As used herein, “labeled nucleic acid” refers to any nucleic acid that is the target of the labeling method of the present invention. Any nucleic acid can be used as such a nucleic acid. Examples of such nucleic acids to be labeled include, but are not limited to, PNA that can perform an extension reaction with DNA, RNA, and nucleic acid synthase. Particularly interesting examples of target nucleic acids include small RNAs such as miRNA, siRNA and the like. The nucleic acid to be labeled becomes a detection target in the primer stop method and the primer run-off method, and becomes a probe in the primer array method.

In the present specification, the “template nucleic acid” refers to any nucleic acid that serves as a template to be used when labeling a nucleic acid to be labeled in the method of the present invention. The template nucleic acid itself is not labeled in the method of the present invention. The template nucleic acid becomes a probe in the primer stop method and the primer run-off method, and becomes a target in the primer array method. The template nucleic acid used in the present invention is usually complementary to at least a part (preferably all) of the nucleic acid to be labeled, and is labeled when the complementary part of the nucleic acid to be labeled and the template nucleic acid are hybridized. It has a nucleotide sequence that is at least one nucleotide longer in the portion of at least one tip of the subject nucleic acid. Therefore, the template nucleic acid is usually longer than the nucleic acid to be labeled, but is not limited thereto, and may be a template nucleic acid that is long only in the extending direction (often 5 ′ side). Examples of the template nucleic acid include, but are not limited to, DNA, RNA, and PNA that can be a template for an extension reaction by a nucleic acid synthase. The difference in length between the template nucleic acid and the nucleic acid to be labeled can preferably be about 5 nucleotides or more, about 10 nucleotides or more, or about 15 nucleotides or more.

  In the present specification, the length difference of “a degree that can be discriminated” refers to a length that can easily identify a difference when detecting, and when electrophoresis is used, for example, at least about 5 When there are differences in nucleotides, preferably at least about 10 nucleotides.

  In the present specification, “complementary” refers to a base sequence (nucleic acid) in which each base is replaced with a base paired by a Watson-Crick bond in the base sequence of the nucleic acid when referring to the nucleic acid. Usually, A and T (U) and C and G are said to be complementary to each other.

  Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by a generally recognized one letter code.

The character codes are as follows.
3 letter code of amino acid 1 letter code Meaning Ala A alanine Cys C cysteine Asp D aspartic acid Glu E glutamic acid Phe F phenylalanine Gly G glycine His H histidine Ile I isoleucine Lys K lysine Leu L leucine Met M methionine Asl G Glutamine Arg R Arginine Ser S Serine Thr T Threonine Val V Valine Trp W Tryptophan Tyr Y Tyrosine Asx Asparagine or Aspartate Glx Glutamine or Glutamate Xaa Unknown or other amino acids.
Base symbol Meaning
a adenine g guanine c cytosine t thymine u uracil r guanine or adenine purine y thymine / uracil or cytosine pyrimidine m adenine or cytosine amino group k guanine or thymine / uracil keto group s guanine or cytosine w adenine or thymine / uracil b guanine or cytosine Thymine / uracil d adenine or guanine or thymine / uracil h adenine or cytosine or thymine / uracil v adenine or guanine or cytosine n adenine or guanine or cytosine or thymine / uracil, unknown or other base.

  In this specification, the comparison of similarity, identity and homology between amino acid sequences and base sequences is calculated using default parameters using BLAST, which is a sequence analysis tool.

  As used herein, “at least one tip” of a nucleic acid means at least one of the 3 ′ end or the 5 ′ end of the nucleic acid.

In the present specification, “hybridizes under stringent conditions” refers to well-known conditions commonly used in the art. Such a polynucleotide can be obtained by using colony hybridization method, plaque hybridization method, Southern blot hybridization method or the like using a polynucleotide selected from among the polynucleotides of the present invention as a probe. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques was immobilized, and then 0.1 to 2 times the concentration. SSC (saline-sodium citrate) solution (composition of SSC solution of 1-fold concentration is 150 mM
The filter is washed at 65 ° C. with sodium chloride, 15 mM sodium citrate). A polynucleotide identified by such a method is referred to as a “polynucleotide that hybridizes under stringent conditions”. Hybridization was performed in Molecular Cloning 2nd ed. , Current Protocols in Molecular Biology, Supplements 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford Universe.

  As used herein, a “hybridizable” nucleic acid or polynucleotide refers to a nucleic acid or polynucleotide that can hybridize to another nucleic acid or polynucleotide under the above hybridization conditions. Specifically, the hybridizable nucleic acid is a nucleic acid having at least 60% or more homology with a DNA base sequence encoding a polypeptide having the amino acid sequence shown in the sequence listing, preferably 80% or more of homology. And more preferably nucleic acids having a homology of 95% or more. Nucleic acid sequence homology is shown in score by using, for example, a search program BLAST using an algorithm developed by Altschul et al. (J. Mol. Biol. 215, 403-410 (1990)).

As used herein, “highly stringent conditions” are designed to allow hybridization of DNA strands having a high degree of complementarity in nucleic acid sequences and to exclude hybridization of DNA having significant mismatches. Say conditions. Hybridization stringency is primarily determined by temperature, ionic strength, and the conditions of denaturing agents such as formamide. Examples of “highly stringent conditions” for such hybridization and washing are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65-68 ° C., or 0.015 M sodium chloride, 0.0015 M citric acid. Sodium, and 50% formamide, 42 ° C. For such highly stringent conditions, see Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory (Cold Spring Harbor 9th Edition, Cold Spring 9th Edition, 198th Edition). 2001); and Anderson et al. Nucleic Acid Hybridization: A Practical Approach, IV, IRL Press Limited (Oxford, England). See Limited, Oxford, England. If necessary, more stringent conditions (eg, higher temperature, lower ionic strength, higher formamide, or other denaturing agents) may be used. Other agents can be included in the hybridization and wash buffers for the purpose of reducing non-specific hybridization and / or background hybridization. Examples of such other agents include 0.1% bovine serum albumin, 0.1% polyvinyl pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate (NaDodSO ₄ or SDS), Ficoll, Denhardt solution, and dextran sulfate, but other suitable agents can also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is almost pH independent. Anderson et al. , Nucleic Acid Hybridization: a Practical Approach, Chapter 4, IRL Press Limited (Oxford, England).

Factors affecting the stability of DNA duplex include base composition, length, and degree of base pair mismatch. Hybridization conditions can be adjusted by one of ordinary skill in the art to apply these variables and to allow different sequence related DNAs to form hybrids. The melting temperature of a perfectly matched DNA duplex can be estimated by the following equation:
Tm (° C) = 81.5 + 16.6 (log [Na ⁺ ]) + 0.41 (% G + C) −600 / N−0.72 (% formamide)
Where N is the length of the duplex formed, [Na ⁺ ] is the molar concentration of sodium ions in the hybridization or wash solution, and% G + C is the (guanine + Cytosine) base percentage. For imperfectly matched hybrids, the melting temperature decreases by about 1 ° C. for each 1% mismatch.

  When RNA, PNA or the like is used, those skilled in the art can use a relational expression appropriate for the nucleotide.

  It will be appreciated that those skilled in the art can find appropriate conditions for labeling or detection, etc. using the relational expressions as described above.

  As used herein, the percentage of “identity”, “homology” and “similarity” of sequences (such as amino acids or nucleic acids) is determined by comparing two sequences that are optimally aligned in a comparison window. Is also possible.

  As used herein, the term “probe” refers to a substance that is used in biological experiments such as screening in vitro and / or in vivo, etc., and serves as a means for capturing a search target. Examples thereof include, but are not limited to, a nucleic acid molecule containing or a peptide containing a specific amino acid sequence.

  Examples of nucleic acid molecules that are usually used as probes include those having a nucleic acid sequence of at least 8 consecutive nucleotides that is homologous or complementary to the nucleic acid sequence of the target gene. Such a nucleic acid sequence is preferably at least 9 contiguous nucleotides long, more preferably 10 contiguous nucleotides long, even more preferably 11 contiguous nucleotides long, 12 contiguous nucleotides long, 13 14 contiguous nucleotide lengths, 14 contiguous nucleotide lengths, 15 contiguous nucleotide lengths, 20 contiguous nucleotide lengths, 25 contiguous nucleotide lengths, 30 contiguous nucleotide lengths, 40 contiguous nucleotide lengths It can be a nucleic acid sequence that is 50 contiguous nucleotides in length. Nucleic acid sequences used as probes include nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably 90% homologous, 95% homologous to the sequences described above. It is.

  In the present specification, the “primer” refers to a substance necessary for initiation of a reaction of a polymer compound to be synthesized in a polymer synthase reaction. In the synthesis reaction of a nucleic acid molecule, a nucleic acid molecule (for example, DNA or RNA) complementary to a partial sequence of a polymer compound to be synthesized can be used.

  Examples of nucleic acid molecules that are usually used as primers include those having a nucleic acid sequence of at least 8 consecutive nucleotides that is complementary to the nucleic acid sequence of the target gene. Such a nucleic acid sequence is preferably at least 9 contiguous nucleotides long, more preferably 10 contiguous nucleotides long, even more preferably 11 contiguous nucleotides long, 12 contiguous nucleotides long, 13 19 contiguous nucleotide lengths, 14 contiguous nucleotide lengths, 15 contiguous nucleotide lengths, 16 contiguous nucleotide lengths, 17 contiguous nucleotide lengths, 18 contiguous nucleotide lengths, 19 contiguous lengths It can be a nucleic acid sequence of nucleotide length, 20 contiguous nucleotide lengths, 25 contiguous nucleotide lengths, 30 contiguous nucleotide lengths, 40 contiguous nucleotide lengths, 50 contiguous nucleotide lengths. The nucleic acid sequence used as a primer is a nucleic acid that is at least 70% homologous, more preferably at least 80% homologous, more preferably 90% homologous, most preferably 95% homologous to the sequence described above. Contains an array. A sequence suitable as a primer may vary depending on the nature of the sequence intended for synthesis (amplification), but those skilled in the art can appropriately design a primer according to the intended sequence. Such primer design is well known in the art, and may be performed manually or using a computer program (eg, LASERGENE, PrimerSelect, DNAStar).

  As used herein, “substitution, addition or deletion” of a polypeptide or polynucleotide is a substitution of an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, with respect to the original polypeptide or polynucleotide. , Adding or removing. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. The number of substitutions, additions or deletions may be any number as long as it is one or more, and such number is a function (for example, information on hormones, cytokines) in a variant having the substitution, addition or deletion. As long as the transmission function is maintained, it can be increased. For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.

  Molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the art, and are described in, for example, Sambrook J. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F .; M.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associate ES and Wiley-Interscience; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F .; M.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F .; M.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, “Experimental Methods for Gene Transfer & Expression Analysis”, Yodosha, 1997, etc., which are related in this specification (may be all) Is incorporated by reference.

  For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M. et al. J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRLPpress; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approac, IRL Press; Adams, R .; L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T.A. (I996). Bioconjugate Technologies, Academic Press, etc., which are incorporated herein by reference in the relevant part.

  In order to confirm the presence of the nucleic acid in the present specification, it can be evaluated by any appropriate method including a molecular biological measurement method such as radioactivity method, fluorescence method, Northern blot method, dot blot method, PCR method and the like.

  As used herein, “screening” refers to selecting a target such as a target organism or substance having a specific property from a large number of populations by a specific operation / evaluation method. For screening, the detection / labeling techniques of the present invention can be used.

  In the present specification, the “complex” refers to a state in which two or more molecules associate with each other through an interaction and behave as if they were one molecule. Thus, the hybridized nucleic acid-nucleic acid is a complex.

As used herein, “nucleic acid synthase” refers to any enzyme having the ability to synthesize nucleic acids. In this specification, it is also called a polymerase. This is a general term for enzymes that catalyze the reaction of condensing nucleotides into polynucleotides. Examples of such a nucleic acid synthase include, but are not limited to, DNA polymerase (eg, DNA-dependent DNA polymerase, RNA-dependent DNA polymerase), RNA polymerase, and the like. Examples of the polymerase include DNA-dependent DNA polymerase and RNA-dependent DNA polymerase. In addition, it is understood that both polymerases may detect DNA or RNA. The only difference is which mold is either (or both). (RNA-dependent DNA polymerases also use DNA as a template. Some of the DNA-dependent DNA polymerases sold also have RNA-dependent activity). Examples of such polymerase include Escherichia coli-derived DNA polymerase I, DNA polymerase I Klenow fragment, Taq polymerase, KLA-Taq polymerase, KOD polymerase, Vent polymerase, AMV reverse transcriptase, Pfu polymerase, T4 DNA polymerase and the like. But are not limited to these.

  As used herein, “extension reaction” refers to any reaction in which a nucleic acid extends at least one nucleotide. When an extension reaction using a polymerase is performed, since a nucleotide is usually introduced based on a template, a specific sequence is extended in a nucleic acid to be extended such as a nucleic acid to be labeled.

  The extension reaction comprises (i) a hybridization (annealing) reaction between the target nucleic acid and its complementary strand and a nucleic acid primer and a nucleic acid probe, and (ii) a primer strand extension reaction and a probe degradation reaction by a nucleic acid synthase. (I) and (ii) can be carried out in an aqueous solution containing a suitable buffer such as, for example, Tris-HCl buffer. This hybridization reaction can be typically performed at 55 to 75 ° C., but is not limited thereto. The enzyme reaction can be typically performed at 37 ° C., but is not limited thereto.

  The denaturation of the primer extension product can be carried out, for example, by subjecting the solution containing the primer chain extension product obtained by the extension reaction to a heat treatment at 94 to 95 ° C. for 0.5 to 1 minute, for example.

For amplification of the target nucleic acid, for example, the above-described extension reaction and denaturation may be repeated a plurality of times under the same conditions as described above until the target nucleic acid (labeled nucleic acid) reaches a desired amount. That is, the above extension reaction and denaturation are repeated a plurality of times under similar conditions. If the above process is performed n times, the amount of target nucleic acid is theoretically amplified to 2 ^n-1 times the original.

  Such an extension reaction can be carried out simply and efficiently by using a commercially available apparatus for PCR (polymerase chain reaction) (for example, sold by Applied Biosystems).

  Nucleic acid can be removed from the mixture using any method in the art. Examples of such extraction include, but are not limited to, electrophoresis, chromatography, denaturation, alcohol precipitation, affinity purification, and antibodies.

(Description of preferred form)
A description of the preferred embodiment is set forth below, which is illustrative of the invention. Accordingly, the scope of the invention is not limited except as by the appended claims.

  In one aspect, the present invention is a method for producing a nucleic acid into which a label has been introduced, the following steps: A) providing a nucleic acid to be labeled and a template nucleic acid, wherein the template nucleic acid comprises: A nucleotide sequence that is complementary to at least a part of the nucleic acid to be labeled and that is longer by at least one nucleotide in a portion of at least one tip of the nucleic acid to be labeled when the complementary portion of the nucleic acid to be labeled and a template nucleic acid are hybridized B) a step of hybridizing the labeling target nucleic acid with the template nucleic acid to produce a labeling target nucleic acid-template nucleic acid complex; C) a nucleic acid synthase in the labeling target nucleic acid-template nucleic acid complex. And a step of performing an extension reaction by adding a labeled nucleotide, wherein at least one labeled nucleotide is added to the nucleic acid to be labeled. Plastid is taken based on the template nucleic acid, steps; D) after the extension reaction, comprising the steps, to retrieve the elongated nucleic acid to be labeled from the resulting mixture, a method. For the first time, this method provides a method for freely labeling a target nucleic acid itself, and it has become possible to produce such a labeled nucleic acid.

  In another aspect, the present invention is a method for introducing a label into a nucleic acid, which comprises the following steps: providing a nucleic acid to be labeled and a template nucleic acid, wherein the template nucleic acid is the nucleic acid to be labeled A nucleotide sequence that is complementary to at least a part of the target nucleic acid and has a nucleotide sequence that is longer by at least one nucleotide in the part of at least one tip of the target nucleic acid when hybridizing the complementary part of the target nucleic acid to the template nucleic acid. B) a step of hybridizing the nucleic acid to be labeled with the template nucleic acid to produce a nucleic acid to be labeled-template nucleic acid complex; and C) a nucleic acid synthase and a labeled nucleic acid to be labeled with the nucleic acid to be labeled-template nucleic acid complex; A step of performing an extension reaction by adding a nucleotide, wherein at least one labeled nucleotide is added to the template nucleic acid. Taken in Zui, a method comprising steps a. This method provides for the first time a method for freely labeling a target nucleic acid itself.

  In another aspect, the present invention is a method for detecting a target nucleic acid, comprising the following steps: A) providing the target nucleic acid and a template nucleic acid, wherein the template nucleic acid is the target nucleic acid. Having a nucleotide sequence that is complementary to at least a portion and has a nucleotide sequence that is at least one nucleotide longer in the portion of at least one tip of the target nucleic acid when the complementary portion of the target nucleic acid is hybridized with the template nucleic acid; B) A step of hybridizing the target nucleic acid with the template nucleic acid to produce a target nucleic acid-template nucleic acid complex; C) an extension reaction by adding a nucleic acid synthesizing enzyme and a labeled nucleotide to the target nucleic acid-template nucleic acid complex. And D) detecting whether an extension product corresponding to the target nucleic acid is present, wherein the presence of the extension product is Is indicative of the presence of the target nucleic acid, the process including, provides methods. The present invention has significantly facilitated the detection of nucleic acids of interest whose sequence is partially or completely known or whose sequence is partially or completely unknown. This is because the unknown nucleic acid itself is labeled. Therefore, it is understood that the method using a labeled probe as in the prior art is remarkably different in terms of sensitivity and ease of detection. Preferably, the detection may further include a step of separating the unreacted product and the extension product of the labeled nucleotide from the resulting mixture after the extension reaction. This is because detection becomes easy.

  The step of providing a nucleic acid used in the method of the present invention can be performed using any technique known in the art (for example, chemical synthesis, utilization of genetic engineering, extraction from a living body).

  The production of the complex used in the method of the present invention can be performed using any technique known in the art. For example, it is understood that complex formation can be performed by appropriately selecting hybridization conditions that are normally used as described elsewhere in this specification.

  The extension reaction used in the method of the present invention can be performed using any technique known in the art. The extension reaction includes an extension reaction catalyst (for example, a nucleic acid synthase), a substance that provides conditions for the reaction of the catalyst (for example, a buffer solution), and a nucleotide to be entered during the extension (labeled, unlabeled, etc.) ) Can be considered. Moreover, in order to control reaction conditions, you may use the apparatus (for example, thermostat) which adjusts temperature etc.

  After the extension reaction, the step of removing the nucleic acid with the label introduced from the mixture can also be performed using any technique known in the art. Such a removal step can be performed by any technique that is commonly used for purifying or separating nucleic acids.

  In the method of the present invention, in the step of detecting the presence of an extension product corresponding to the target nucleic acid, the detection is carried out by any method directly or indirectly using any technique well known in the art. It is understood that you can. Here, it is understood that the presence of the extension product is an indicator of the presence of the target nucleic acid. This is because the extension product is not detected unless there is an unknown nucleic acid before extension. However, since it is considered that there are a plurality of types of unknown nucleic acids before being extended, more detailed detection can be performed by determining the sequence of the extension product. Such extension products can be sequenced using any technique known in the art.

  In one embodiment, the nucleic acid to be labeled used in the present invention is DNA or RNA or a combination thereof, and is preferably small RNA (for example, siRNA and miRNA).

  It is preferable that the complementarity between the template nucleic acid and the nucleic acid to be labeled is sufficient to allow the extension reaction to proceed. The degree of complementarity varies depending on the length, extension reaction conditions, and the like, and the vicinity of the 3 'end is preferably suitable for the extension reaction. In particular, it may be preferred that 1-2 nucleotides at the 3 'end have perfect complementarity. Alternatively, it is usually preferable that the nucleic acid has at least 50% homology based on the shorter nucleic acid, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably It may further be preferred that have a homology of at least 90%, more preferably at least 95%. Alternatively, it may be complementary to all the nucleic acids to be labeled.

  In another embodiment, the template nucleic acid used in the present invention has at least one nucleotide-long nucleotide at both ends of the nucleic acid to be labeled, and more preferably at least one nucleotide-long nucleotide added in the template nucleic acid. Is at least about 2 nucleotides longer, preferably at least about 3 nucleotides longer, more preferably at least about 4 nucleotides longer, more preferably at least about 5 nucleotides longer, more preferably at least about 6 nucleotides longer, more preferably May be at least about 7 nucleotides longer, more preferably at least about 8 nucleotides longer, more preferably at least about 9 nucleotides longer, and about 10 nucleotides longer.

  In another embodiment of the invention, the template nucleic acid can be modified. Such modification is preferably performed at the 3 'end. This is because accidental incorporation of external nucleotides (eg, labeled nucleotides) at the 3 'end can be prevented. Therefore, it is preferable that the terminal nucleotide of the template nucleic acid is modified so as not to incorporate the labeled nucleotide.

  In one preferred embodiment, such modifications are from the group consisting of dideoxylation, fluorescein isothiocyanate (FITC), biotinylation, amination, phosphorylation, tetramethylrhodamine (TAMRA), thiolation and rhodamineation. It can be the modification chosen.

  Such a modification may result from the nucleotide at the 3 ′ end of the template nucleic acid being dideoxylated, fluorescein isothiocyanate (FITC), biotinylated, aminated, phosphorylated, tetramethylrhodamine (TAMRA), thiolated and rhodamined. Preferably, the modification selected from the group consisting of

  In another embodiment, it is preferable that the template nucleic acid and the nucleic acid to be labeled are designed to have different expected lengths after the extension reaction. It will be appreciated that such designs will vary depending on the detection method used for detection (eg, primer stop method, primer on-off method, primer array method, etc.). The primer termination method utilizes the ability of the dideoxynucleotide to be used to stop the extension by the polymerase, and therefore the design can be varied by controlling the position where the dideoxynucleotide to be used is incorporated.

  In another preferred embodiment, at least one nucleotide longer nucleotide added in the template nucleic acid is added at least 5 'to the end. Although the extension reaction usually extends at the 3 'recessed end, the present invention is not limited thereto.

  In still another embodiment, the present invention provides that the nucleotide sequence added at least one nucleotide longer in the template nucleic acid has a different length on the 3 ′ end side and the 5 ′ end side as viewed from a portion complementary to the nucleic acid to be labeled. Only that is added. This is because the target nucleic acid can be easily distinguished from the template nucleic acid.

  In another preferred embodiment, the present invention is such that at least one nucleotide-long nucleotide sequence added in the template nucleic acid is added 5 'longer than the portion complementary to the nucleic acid to be labeled, but 3' The ends of the template nucleic acid are shorter. That is, the 5 'end portion of the nucleic acid to be labeled extends from the 3' end of the template nucleic acid. In such a case, in the primer run-off method, in particular, it is ensured that the template nucleic acid is shorter, and the labeling target nucleic acid can be distinguished from the template nucleic acid. Even in the primer stop method, the template nucleic acid can be easily distinguished from the nucleic acid to be labeled by being short.

  In another embodiment, the nucleotide sequence added at least 1 nucleotide longer in the template nucleic acid is added longer at the 3 'end than at the 5' end as viewed from the portion complementary to the labeled nucleic acid. This is because when the extension product is identified by electrophoresis or the like, identification with the naked eye is also possible.

  In another embodiment, the nucleotide sequence added at least 1 nucleotide longer in the template nucleic acid is added so that the 5 ′ terminal side is distinguishably longer than the 3 ′ terminal side from the part complementary to the nucleic acid to be labeled. Yes. Such distinguishable length varies depending on the detection method, but for the naked eye, the distinguishable length can be 1-5 nucleotides, but should be at least about 10 nucleotides. preferable.

  In another embodiment, the nucleic acid synthase can be a DNA dependent DNA polymerase or an RNA dependent DNA polymerase. It is understood that any synthetic enzyme may be used as long as the extension reaction is achieved.

  Examples of the label used in the labeling method of the present invention include, but are not limited to, fluorescence, radioactivity, phosphorescence, biotin, DIG (digoxigenin), enzyme, and chemiluminescence. It will be understood that one or more (or multiple types) of signs may be used. For dyes, fluorescence, etc., multiple colors can be used. In a preferred embodiment, radioactivity is used as the label. This is because radioactivity enables detection of atmolar levels. However, the present invention is not limited to this.

  In another embodiment, the step of removing the elongated labeled target nucleic acid can include a denaturation step. This is because denaturation makes it easier to identify the extended nucleic acid to be labeled and facilitates removal.

  In a preferred embodiment, the method of the present invention further comprises removing unreacted material. This is because the unreacted material can be a noise when the labeled nucleic acid is subsequently used. Here, any available technique can be used to remove unreacted material, but preferably it can be achieved by alcohol precipitation, such as 2-propanol precipitation or ethanol precipitation, and filtration. This is because this method is simple.

  In removing unreacted substances, a step of “separating” the extended nucleic acid to be labeled may be further included. This is because the purity is increased by the separation. For such separation, any technique in the art can be used, but electrophoresis or chromatography is preferred.

  In another embodiment, the step of removing the elongated labeled target nucleic acid in the method of the present invention includes the step of detecting the label. This detection step may detect the label directly or indirectly.

  In another embodiment, the method of the present invention further comprises a step of separating the extended nucleic acid to be labeled. For such separation, any technique in the art can be used, but electrophoresis or chromatography is preferred. These techniques are known in the art, and it can be understood that any of the forms described above can be used.

  In another embodiment, the method of the invention may perform detection “on separation”. This is because the detection accuracy can be increased by performing the separation, and the efficiency of detection and separation is increased.

  In other embodiments, separation and detection may use an autoradiograph. This is because separation and detection can be performed automatically.

  In yet another embodiment, the method of the invention includes the step of terminating the extension reaction. The termination step can be achieved by primer termination (Primer Halt method) or primer run-off method, but is not limited thereto. Since such a stopping step could not be considered by the conventional labeling method, it is understood that highly specific labeling can be realized.

  The nucleic acid to be labeled is usually a target nucleic acid whose sequence is partially or completely known or unknown, but is not limited thereto, and may be used as a probe. In the case of a nucleic acid of interest, it is understood that it is highly effective in detecting the nucleic acid of interest even if its sequence is partially or completely unknown.

  In one exemplary embodiment, DNA synthesis is performed by hybridizing a nucleic acid to be labeled with a template DNA or RNA, adding a DNA polymerase and a free nucleotide thereto, and using the nucleic acid to be labeled (possibly a probe) as a primer. By performing an (extension) reaction, the nucleic acid is labeled in a sequence-specific manner. Therefore, conventional sequence-specific nucleic acid detection methods such as Southern blotting, Northern blotting, primer extension method, Southern blot, Northern blot, primer extension method, RNase protection, microarray, and macroarray, which are conventionally used, are used for hybridization. The probe is labeled before, but in the method of the present invention, after the nucleic acid to be labeled and the nucleic acid to be probe are hybridized, a DNA synthesis reaction is performed using either one as a primer, so that only the hybridized nucleic acid is obtained. It is a technique of putting a sign. In the method of the present invention, the label is usually placed on the nucleic acid used as the target nucleic acid, but the present invention is not limited thereto. Therefore, the nucleic acid used as a probe may be labeled. Since only the hybridized nucleic acid is labeled in the method of the present invention, the point that the nucleic acid to be labeled is labeled in a sequence-specific manner is remarkable compared to the method of labeling a conventional probe (that is, indirect). Has a different effect. The template nucleic acid plays the role of the probe in the conventional method. Therefore, the label enters the nucleic acid to be labeled, not the template nucleic acid. Conversely, the nucleic acid to be labeled can also be used as a probe. In this case, a method of putting a label on the probe side is also included. Such a form is necessary when the present method is applied to a microarray using mRNA.

  This method takes advantage of the fact that template-dependent DNA polymerase can use both DNA and RNA as primers. The template-dependent DNA polymerase is preferably a DNA-dependent DNA polymerase (Klenow fragment, DNA polymerase I, etc.), but can also be an RNA-dependent DNA polymerase (for example, reverse transcriptase).

  Both types of polymerases can be used as primers for both DNA and RNA as described above, and catalyze the reaction of adding a base complementary to the hybridizing nucleic acid to the 3 'side of the primer. The reaction usually takes place only at the 3 '"recessed end". The reaction usually does not occur because there is no template at the 3 'protruding end. A “reverse reaction” may be possible if the reaction of adding a base to the 5 ′ end can be catalyzed.

(Reaction stopped)
In the present specification, “stopping” an extension reaction means stopping a nucleic acid synthesis reaction. The extension reaction can be typically stopped by using the following two methods (primer stop method and primer run-off method). Here, another feature is that the number of bases incorporated into the labeled nucleotide can be defined (limited).

  In this specification, “primer halt method” means that nucleotide triphosphate added in the DNA synthesis (elongation) reaction is not a deoxyribonucleotide (of course, not a ribonucleotide) because it is normally used for DNA synthesis. ) Nucleotide triphosphate or the like that is not subject to the subsequent extension reaction by a nucleic acid synthase such as dideoxyribonucleotide (a mixture of ddATP, ddCTP, ddTTP, ddGTP or any one) but can be incorporated. In this method, the reaction is stopped after one base, several bases, or several tens of bases are incorporated. In principle, the same principle as the dideoxy chain termination method used for DNA sequencing reactions can be used. 1 and 5 can be referred to for the principle of primer stop.

  In this specification, “primer run-off method” means that a template (probe) nucleic acid is 1 to several bases longer than the 3 ′ side (5 ′ side when the probe is seen) of the nucleic acid to be labeled. In this case, the DNA synthesis reaction is stopped only by the length (since the template is eliminated). For the principle of primer run-off, FIG. 6 can be referred to.

  The primer stop method can be used in any case. The primer run-off method is effective when the sequence of the nucleic acid to be labeled is a known miRNA (microRNA), but in the case of siRNA in gene inactivation, a mixture of small RNAs complementary to the inactivated gene. The primer termination method is preferred because it contains various molecular species.

  In the case of the primer halt method, the length of the template is not important, and an arbitrary length can be used. When siRNA is detected by this method, since siRNA is an assembly of various molecular species, it is not possible to define the next base, and therefore, there are four types of labeled nucleotides (ddATP, ddCTP, ddTTP, ddGTP) if possible. It is desirable to include all, but is not limited thereto. Those skilled in the art will understand that even one type is possible if the sensitivity is set to 1/4.

  When miRNA is detected by the primer run-off method, it is preferable that the 5 'side (3' side when viewed from the target) of the template should have a predetermined length (usually protruding by one base). The portion that hybridizes with the target is preferably complementary, but the sequence of the protruding portion can be freely selected. Therefore, the template can be designed so that one of the four labeled nucleotides (ie, dATP, dCTP, dTTP, dGTP) is incorporated, and only one necessary type needs to be added to the reaction.

  In the present specification, the “primer array method” is a method for simultaneously detecting and quantifying many types of nucleic acids. In the primer array method, a probe nucleic acid is immobilized on a support, and a nucleic acid is labeled by incorporating a labeled nucleotide when a DNA synthesis reaction is performed using a complex of the immobilized probe and a nucleic acid to be detected. is there. A plurality of types of nucleic acids can be simultaneously quantified by immobilizing multiple types of probes at different sites on the support. The nucleic acid to be detected may be mRNA or small RNA, and may be DNA in some cases. Compared with the conventional microarray technology, a specific labeling reaction is carried out on the support, so that it is possible to carry out efficient detection. A method capable of exhaustively analyzing small RNA has not been known so far. FIG. 7 can be referred to for the principle of the primer array method.

  The primer termination method is typically suitable for detecting siRNA. The primer run-off method is typically suitable for detection of miRNA and SnRNA (small nuclear RNA =). The primer array method is suitable for detection of mRNA.

  In the primer stopping method, the nucleic acid to be labeled becomes a target nucleic acid (detection target; for example, siRNA), and corresponds to a primer in a polymerase reaction. Therefore, the detection target itself is labeled. On the other hand, the template nucleic acid is a “provided” nucleic acid such as a synthetic DNA and corresponds to a probe. For example, ssDNA, dsDNA, etc. can be used. Thus, the template nucleic acid is not labeled. In the primer termination method, the extension reaction is stopped by using a nucleotide triphosphate having an action of stopping the extension reaction such as dideoxynucleotide.

  In the primer run-off method, the target nucleic acid to be labeled becomes a target nucleic acid (detection target; for example, miRNA, SnRNA) and corresponds to a primer in a polymerase reaction. Therefore, the detection target itself is labeled. On the other hand, the template nucleic acid is a “provided” nucleic acid such as a synthetic DNA and corresponds to a probe. For example, ssDNA, dsDNA, etc. can be used. Thus, the template nucleic acid is not labeled. In the primer run-off method, the extension reaction is stopped by the fact that the corresponding template nucleic acid does not extend any more because there is no nucleotide sequence in the portion to be extended.

  In the primer array method, the nucleic acid to be labeled is now preferably immobilized on a solid support, and thus becomes a primer. Since the nucleic acid to be labeled serves as a primer, a nucleic acid such as a synthetic DNA that is labeled can be used. In this case, the template nucleic acid corresponds to mRNA and is not labeled. In the primer array method, the termination reaction of the extension reaction is not always necessary.

  The “method for specifically labeling a nucleic acid to be labeled by a DNA synthesis reaction using the nucleic acid to be labeled as a primer” of the present invention and the “number of extended bases by the primer stop method or primer run-off method during the DNA synthesis reaction” of the present invention. In combination with the “method for limiting the number of”, it becomes a novel nucleic acid detection method (especially effective for small RNA). Therefore, it can be said that such a labeling method has an exceptional effect that could not be detected conventionally. The former alone is a novel nucleic acid labeling / detection method.

  Various combinations of denaturation, electrophoresis, and detection can be considered. By implementing the “sequence-specific nucleic acid labeling method that can define the number of bases” of the present invention as described in the present specification, known techniques can be applied. Can be implemented. However, it is understood that there are several applications in various combinations.

In one embodiment, detection can be performed by examining the sequence of the extended portion. In this case, a drug that specifically binds to the extended target nucleic acid may be used. Such agents can include, but are not limited to, another nucleic acid or antibody having a sequence that specifically interacts with the extended target nucleic acid. Alternatively, an agent that specifically binds to the extended target nucleic acid does not bind to the non-extended target nucleic acid or the labeled nucleotide.
In another embodiment, detection may be performed by detecting a label attributed to the labeled nucleotide and a label attributed to the agent.

  In another embodiment, detection can be based on the length of the extended target nucleic acid.

(device)
The present invention can be applied to labeling and detection in devices such as arrays.

  In this specification, “device” refers to a part that can constitute part or all of the apparatus, and is composed of a support (preferably a solid support) and a target substance to be supported on the support. Is done. Examples of such devices include, but are not limited to, chips, arrays, microtiter plates, cell culture plates, petri dishes, films, beads, and the like.

  The array uses nucleic acids bound to a support, and can be broadly divided into two applications.

1) siRNA, miRNA detection micro (macro) array A template nucleic acid for small RNA (siRNA, miRNA) is immobilized on glass, membrane, etc., and hybridization, labeling and detection are carried out on this support. It is expected that the abundance of small RNA can be comprehensively analyzed, and no other method for comprehensively analyzing small RNA is known.

  2) A nucleic acid having a sequence complementary to mRNA is immobilized on a support, and hybridization, labeling and detection are performed on the support. As with conventional microarrays, it can be used to comprehensively analyze the abundance of mRNA. Compared with the conventional method, the reaction system is simple.

  For both 1) and 2), a method, a nucleic acid itself immobilized on a support, and a kit are considered as embodiments.

  Or conversely, small RNA may be used as a template. Alternatively, it is possible to label at both 5 'and 3' ends.

  From the viewpoint of the nucleic acid to be labeled, the 3 ′ side (5 ′ on the template side) has a length that is most likely to protrude by one base, but is not limited thereto. Apart from this, it is desirable that the 5 'side is about 10 bases and the template is longer. This causes the addition of one base to the target. The reason why the template is lengthened on the 5 'side is to allow the template to be distinguished from the target in terms of length even if the template is labeled nonspecifically.

  As used herein, “support” refers to a material that can immobilize a substance such as a biomolecule. Support materials can be either covalently bonded or non-covalently bonded to a substance such as a biomolecule used in the present invention or derivatized to have such characteristics. Any solid material to be obtained is mentioned.

  As such a material for use as a support, any material capable of forming a solid surface can be used, for example, glass, silica, silicon, ceramic, silicon dioxide, plastic, metal (including alloys) ), Natural and synthetic polymers such as, but not limited to, polystyrene, cellulose, chitosan, dextran, and nylon. The support may be formed from a plurality of layers of different materials. For example, an inorganic insulating material such as glass, quartz glass, alumina, sapphire, forsterite, silicon oxide, silicon carbide, or silicon nitride can be used. Polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate Organic materials such as polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide, and polysulfone can be used. In the present invention, a membrane used for blotting, such as a nitrocellulose membrane, a nylon membrane, or a PVDF membrane, can also be used. When the material constituting the support is a solid phase, it is particularly referred to herein as “solid support”. In the present specification, it may take the form of a plate, microwell plate, chip, slide glass, film, bead, metal (surface) and the like. The support may be coated or uncoated.

  As used herein, “substrate” refers to the material (preferably solid) on which the chips or arrays of the invention are constructed. Therefore, the substrate is included in the concept of a plate. The material of the substrate can be any solid, either covalently or noncovalently, that has the property of binding to the biomolecules used in the present invention or that can be derivatized to have such properties. Materials.

  Such materials for use as plates and substrates can be any material that can form a solid surface, for example, glass, silica, silicon, ceramic, silicon dioxide, plastics, metals (including alloys) Natural and synthetic polymers such as, but not limited to, polystyrene, cellulose, chitosan, dextran, and nylon. The substrate may be formed from a plurality of layers of different materials. For example, inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, and silicon nitride can be used. Polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin Organic materials such as polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide, and polysulfone can be used. A preferable material for the substrate varies depending on various indicators such as a measuring instrument, and those skilled in the art can appropriately select an appropriate material from the various materials described above.

  As used herein, “coating”, when used on a solid support or substrate, refers to the formation of a film of material on the surface of the solid support or substrate and such a film. The coating is performed for various purposes, for example, improving the quality of the solid support and the substrate (for example, improving the lifetime, improving the environmental resistance such as acid resistance), the substance to be bonded to the solid support or the substrate. In many cases, the purpose is to improve the affinity. Various materials can be used as the material for such coating. In addition to the materials used for the solid phase support and the substrate itself, biological materials such as DNA, RNA, protein, and lipid, polymers ( For example, poly-L-lysine, hydrophobic fluororesin), silane (APS (eg, γ-aminopropylsilane)), metal (eg, gold, etc.) can be used, but is not limited thereto. The selection of such materials is within the skill of the artisan and can be selected on a case-by-case basis using techniques well known in the art. In one preferred embodiment, such coatings are poly-L-lysine, silane (eg, epoxy silane or mercapto silane, APS (γ-aminopropyl silane)), MAS, hydrophobic fluororesin, gold and the like. It may be advantageous to use a simple metal.

  In this specification, “chip” or “microchip” is used interchangeably and refers to a micro integrated circuit that has various functions and is a part of a system. Examples of the chip include, but are not limited to, a DNA chip and a protein chip.

In this specification, an “array” refers to a substrate (eg, a chip) having a pattern or a pattern in which compositions (eg, DNA, RNA, etc.) containing one or more (eg, 1000 or more) target substances are arranged and arranged. ) Itself. Among the arrays, those patterned on a small substrate (eg, 10 × 10 mm) are referred to as microarrays. In this specification, microarrays and arrays are used interchangeably. Accordingly, even a pattern that is larger than the above-described substrate may be called a microarray. For example, an array consists of a set of desired nucleic acids that are themselves immobilized on a solid surface or membrane. Array preferably comprises at least 10 ^two identical or different nucleic acid, at least 10 ³ and more preferably, and more preferably at least 10 ^4, even more preferably at least 10 ⁵ a. These antibodies are preferably arranged on a surface of 125 × 80 mm, more preferably 10 × 10 mm. As the format, a microtiter plate of a size such as a 96-well microtiter plate or a 384-well microtiter plate or a size of a slide glass is contemplated. The composition containing the target substance to be immobilized (for example, nucleic acid or the like) may be one kind or plural kinds. The number of such types can be any number from 1 to the number of spots. For example, a composition containing about 10, about 100, about 500, or about 1000 types of target substances (for example, nucleic acids) can be immobilized.

Any number of target substances (eg, nucleic acids, etc.) can be placed on a solid surface or membrane, such as a substrate, as described above, but typically up to 10 ⁸ biomolecules per substrate and others In embodiments, up to 10 ⁷ biomolecules, up to 10 ⁶ biomolecules, up to 10 ⁵ biomolecules, up to 10 ⁴ biomolecules, up to 10 ³ biomolecules, or 10 ² biomolecules. Up to 10 biomolecules can be arranged, but a composition containing a target substance exceeding 10 ⁸ biomolecules may be arranged. In these cases, the size of the substrate is preferably smaller. In particular, the spot size of a composition (eg, nucleic acid, etc.) containing the target substance can be as small as the size of a single biomolecule (this can be on the order of 1-2 nm). The minimum substrate area is determined in some cases by the number of biomolecules on the substrate. In the present invention, a composition containing a target substance intended to be introduced into a cell is usually arrayed and fixed in a spot shape of 0.01 mm to 10 mm by covalent bonding or physical interaction.

  A “spot” of biomolecules can be placed on the array. As used herein, “spot” refers to a certain set of compositions containing a target substance. As used herein, “spotting” refers to producing a spot of a composition containing a target substance on a substrate or plate. Spotting can be done by any method, for example, it can be accomplished by pipetting or the like, or it can be done by automated equipment, such methods are well known in the art.

  As used herein, the term “address” refers to a unique location on a substrate and may be distinguishable from other unique locations. The address is suitable for associating with a spot with that address, and can take any shape, with entities at all each address being distinguishable from entities at other addresses (eg, optical). The shape defining the address can be, for example, a circle, an ellipse, a square, a rectangle, or an irregular shape. Therefore, “address” indicates an abstract concept, and “spot” can be used to indicate a specific concept, but in the present specification, if it is not necessary to distinguish between the two, “Spot” may be used interchangeably.

  The size of each address, among others, is the size of the substrate, the number of addresses on a particular substrate, the amount and / or available reagents of the composition containing the target substance, the size of the microparticles and the array thereof Depends on the degree of resolution required for any method. The size of each address can range, for example, from 1-2 nm to a few centimeters, but can be any size consistent with the application of the array.

  The spatial arrangement and shape defining the address is designed to suit the particular application in which the microarray is used. The addresses can be densely distributed, widely distributed, or subgrouped into a desired pattern appropriate for a particular type of analyte.

  Microarrays are widely reviewed in the genome function research protocol (experimental medicine separate volume, experimental lecture 1 in the post-genomic era), genomic medicine science and future genomic medicine (experimental medicine extra number).

  Since the data obtained from the microarray is enormous, data analysis software for managing the correspondence between clones and spots, data analysis, etc. is important. As such software, software attached to various detection systems can be used (Ermolaeva O. et al. (1998) Nat. Genet. 20: 19-23). The database format includes, for example, a format called GATC (Genetic Analysis Technology Consortium) proposed by Affymetrix.

  For microfabrication, see, for example, Campbell, S .; A. (1996). The Science and Engineering of Microelectronic Fabrication, Oxford University Press; Zaut, P. et al. V. (1996). Micromicroarray Fabrication: a Practical Guide to Semiconductor Processing, Semiconductor Services; Madou, M .; J. et al. (1997). Fundamentals of Microfabrication, CRC1 5 Press; Rai-Chudhury, P .; (1997). Handbook of Microlithography, Micromachining, & Microfabrication: Microlithography, etc., which are incorporated by reference in the relevant portions herein.

  As a detection technique, various detection methods and detection means can be used as long as information related to the labeling of the nucleic acid can be detected. Examples of such detection methods and detection means include visual observation, optical microscope, confocal microscope, fluorescence microscope, reader using a laser light source, surface plasmon resonance (SPR) imaging, electrical signal, chemical or biochemical Although the method and means using any or several types of markers can be mentioned, it is not limited to them. Examples of such a detection apparatus include, but are not limited to, a fluorescence analysis apparatus, a spectrophotometer, a scintillation counter, a CCD, and a luminometer, and any means capable of detecting a biomolecule can be used. It may be a thing.

  For radioactivity, techniques well known in the art such as a Geiger counter and a scintillation counter can be used.

  In the case of fluorescence, it is necessary to measure the excitation wavelength and the detection wavelength when measuring fluorescence polarization. However, those skilled in the art appropriately select according to the type of fluorescent label used (for example, fluorescein isothiocyanate as a fluorescent label). The excitation wavelength and the detection wavelength can be 490 nm and 520 nm, respectively).

(kit)
As used herein, the term “kit” refers to a unit provided with a portion (eg, reagent, enzyme, template nucleic acid, standard, etc.) to be provided, usually divided into two or more compartments. This kit form is preferred when it is intended to provide a composition that should not be provided in a mixed form but is preferably used in a mixed state immediately prior to use. Such kits preferably do what is provided with the moieties (eg, reagents, enzymes, nucleotides, labeled nucleotides, nucleotides that stop the extension reaction, etc. (and their triphosphates), template nucleic acids, standards, etc.) It is advantageous to have instructions describing what should be processed. In the present specification, when a kit is used as a reagent kit, the kit usually includes reagent components, a buffer solution, a salt concentrate, auxiliary means for use, instructions describing the method of use, and the like.

  In the present specification, the “instruction” describes the usage method, reaction method and the like of the reagent of the present invention to the user. This instruction manual describes a word indicating a procedure such as an enzyme reaction of the present invention. This instruction is prepared according to the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.) in the country where the present invention is implemented as necessary. It will be clearly stated that it has been approved. The instruction sheet is a so-called package insert, and is usually provided as a paper medium, but is not limited thereto. For example, a film attached to a bottle, an electronic medium (for example, a home page provided on the Internet) (Website), e-mail, etc.).

  In one aspect, the present invention is a kit for producing a nucleic acid into which a label has been introduced, and is complementary to at least a part of the nucleic acid to be labeled as a target for introduction of the label: A) And a template nucleic acid having a nucleotide sequence that is at least one nucleotide longer in the portion of at least one tip of the nucleic acid to be labeled when hybridizing the complementary portion of the nucleic acid to be labeled and the template nucleic acid; and B) a nucleic acid synthase; C) a labeled nucleotide is provided.

  In one embodiment, the kit of the present invention comprises a DNA synthase, a control template, a target (primer), a reaction buffer, etc., and the template for the actual target nucleic acid is prepared by itself or ordered as oligo DNA. You may comprise as follows. Apart from this, one containing a set of templates for the major small RNAs is also conceivable. Alternatively, a set of templates and a kit in which the templates are arranged as described above are also considered.

  In another embodiment, the kit of the present invention may further include a means for separating the unreacted product of the labeled nucleotide and the extended product of the labeled nucleotide from the resulting mixture.

  In another embodiment, the kit of the present invention may further comprise a means for detecting a labeled nucleotide.

  In a preferred embodiment, the kit of the present invention further comprises a standard nucleic acid as a control. A standard nucleic acid serving as a control can be used as an index for detection in electrophoresis or the like.

  The standard nucleic acid that can be used here may include a nucleic acid selected from the group consisting of a nucleic acid for identifying the template nucleic acid and a nucleic acid for identifying the nucleic acid to be labeled.

  In another embodiment, the kit of the present invention further comprises a reagent for the reaction of a nucleic acid synthase. Such reagents can include, but are not limited to, buffers, necessary ion concentrates, salt concentrates, pH adjusters, and the like.

  In a preferred embodiment, the kit of the present invention further comprises a support, and the template nucleic acid is immobilized on the support. In such a case, the support may be glass or a membrane, but is not limited thereto.

  In a preferred embodiment, the template nucleic acid immobilized on a support contained in the kit of the present invention is arranged in an array. Such a thing can also be called a DNA chip or the like. A method for producing such an array is also within the scope of the present invention.

  In the kit of the present invention, it should be understood that the various constituent requirements should be in any form detailed in the labeling method and detection method of the present invention and the method of producing a nucleic acid into which a label has been introduced. The

  In another aspect, the present invention is a kit for detecting a target nucleic acid, comprising the following steps: A) complementary to at least part of the target nucleic acid, and further complementary to the target nucleic acid and template A template nucleic acid having a nucleotide sequence that is at least one nucleotide longer in the portion relative to the tip of at least one of the target nucleic acids when hybridized with the nucleic acid; B) a nucleic acid synthase; C) a labeled nucleotide; D) a labeled nucleotide A kit comprising: means for detecting.

  It can be understood that each component (eg, template nucleic acid, nucleic acid synthase, labeled nucleotide, detection means) and the like can take any form detailed in the description of the method of the present invention.

  In one embodiment, the kit of the present invention may further comprise means for separating the unreacted product of the labeled nucleotide from the extended product from the resulting mixture. Examples of such means include, but are not limited to, electrophoresis and chromatography.

(Array manufacturing method)
In another aspect, the present invention provides a method for producing a support on which a nucleic acid into which a label has been introduced is immobilized, comprising the following steps: A) a nucleic acid to be labeled and a template nucleic acid immobilized on a support. The template nucleic acid is complementary to at least a part of the nucleic acid to be labeled, and at least the nucleic acid to be labeled is hybridized with the complementary portion of the nucleic acid to be labeled and the template nucleic acid. A step having a nucleotide sequence that is at least one nucleotide longer in a portion to one tip; B) a step of hybridizing the nucleic acid to be labeled with the template nucleic acid to generate a nucleic acid to be labeled-template nucleic acid complex; C) the label A step of adding a nucleic acid synthase and a labeled nucleotide to a target nucleic acid-template nucleic acid complex to perform an extension reaction, and At least one labeled nucleotide is incorporated based on the template nucleic acid; and D) after the extension reaction, removing the support containing the nucleic acid to be labeled extended from the resulting mixture. Provide a way to do it. In the production method described above, any technique as described elsewhere in this specification can be applied to the provision of nucleic acids, the formation of complexes, the extension reaction, and the removal of the support. Regarding the provision of nucleic acid, once information on the target nucleic acid is provided, any technique such as chemical synthesis, application of genetic engineering, extraction from cell components, etc. can be used. Formation of the complex can be performed by placing the two nucleic acids at a distance capable of complex formation and exposing them to conditions that cause complex formation. The extension reaction can be performed, for example, by adding a nucleic acid synthase after the complex has been formed and exposing the mixture of the complex and the nucleic acid synthase to conditions such that the nucleic acid synthase functions. The support after the reaction can be preferably taken out by separating the support from other reagents used in the reaction. As needed, you may wash | clean (for example, using a physiological saline, a phosphate buffered saline, etc.) on the conditions which the fixed labeled nucleic acid is not destroyed.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  Hereinafter, although an example explains the composition of the present invention in detail, the present invention is not limited to this. The reagents used in the following were those commercially available from Wako Pure Chemicals, Sigma, Toyobo, New England Biolabs, Amersham, Invtrogen, Funakoshi, Nippon Gene, etc., unless otherwise specified. Synthetic DNA was commissioned for production by Hokkaido System Science Co., Ltd., and synthetic RNA was commissioned for synthesis by Nippon Bioservice Co., Ltd., but those skilled in the art could use such techniques to know such DNA and RNA. It is understood that they can be synthesized.

Example 1
In this example, it was demonstrated whether the labeling technique of the present invention is applicable to RNA and DNA.

(Materials and methods)
(Nucleic acid)
Synthetic small RNA complementary to the GFP gene and sense strand DNA corresponding to the GFP gene are listed in the table below. Further, FIG. 2 shows an overview of the complementarity of RNA1, RNA2, DNA1, and DNA2. The right side of FIG. 3 ((i) to (v)) shows an overview of the mutual complementarity of the nucleic acids shown in Table 1.
(Table 1)
Representative synthetic DNA and RNA sequences used in the Examples Small RNA
RNA1
5'-CUC AUC AUG UUG UUU GUA UAG UUC-3 '(SEQ ID NO: 1) RNA2
5′-AUC GCC AAU UGG AGU AAU UUG-3 ′ (SEQ ID NO: 2)
Template DNA
DNA1 (m-GFP5-ER-S 725-812)
5′-GAG AGA CCA CAT GGT CCT TCT TGA GTT TGT AAC AGC TGC TGG GAT TAC ACA TGG CAT GGA TGA ACT ATA CAA ACA TGA TGA GCT TTA 3 ′
DNA2 (m-GFP5-ER-S 572-670)
5′-CAA CTT CAA GAC CCG CCA CAA CAT CGA AGA CGG CGG CGT GCA ACT CGC TGA TCA TTA TCA ACA AAA TAC TCC AAT TGG CGA TGG CCC-3 TGT CCT TCT CCT-3
LUC (2113-LUC 2603-2702)
5'-TTG TAA TAT TAT ATG CAA ATT GAT GAA TGG TAA TTT TGT AAT TGT GGG TCA CTG TAC TAT TTT AAC GAA TAA TAA AAT CAG GTA TAG
Ntab miR171
5′-TGA TAT TGG CGC GGC TCA ATC ATG ATA TTG GCG CGG CTC AAT CA-3 ′ (SEQ ID NO: 6)
Ntab miR167
5′-TTA GAT CAT GCT GGC AGC TTC ATG ATA TTG GCG CGG CTC AAT CA-3 ′ (SEQ ID NO: 7)
Mmus miR181 T
5′-TAC TCA CCG ACA GCG TTG AAT GTT TGA TAT TGG CGC GGC TCA ATC A-3 ′ (SEQ ID NO: 8)
Mmus miR16 T
5′-TCG CCA ATA TTT ACG TGC TGC TAT GAT ATT GGC GCG GCT CAA TCA-3 ′ (SEQ ID NO: 9)
GFP5-ER G10 + 786-812
5′-GGG GGG GGG GGA ACT ATA CAA ACA TGA TGA GCT TTA ATG ATA TTG GCG C-3 ′ (SEQ ID NO: 10)
GFP5-ER G + 786-812
5′-GGA ACT ATA CAA ACA TGA TGA GCT TTA ATG ATA TTG GCG CGG CTC AAT CA-3 ′ (SEQ ID NO: 11)
Total RNA was extracted from adult female mice and tobacco plants. Extraction was performed using concert reagents (mouse; Invitrogen) or Trizol reagents as described in the manufacturer's instructions (tobacco, Invitrogen).

(Hybridization and labeling)
A solution (total amount 4 μl) containing the small RNA and template DNA (0.05 pmol) was mixed in a hybridization buffer (150 mM KCl, 10 mM Tris-HCl (pH 7.5), 0.5 mM EDTA). Hybridized and denatured at 95 ° C. for 5 minutes. Subsequently, it was allowed to incubate at 64 ° C. for 6 hours unless otherwise specified. Then 4 μl of this solution was mixed with 5 units of DNA polymerase I Klenow fragment and incubated at 37 ° C. for 1-2 hours. The reaction was terminated by adding formamide solution (20 mM EDTA and 0.05% BPB) and denatured at 95 ° C. for 5 minutes. The mixture was then mixed with an acrylamide gel (15% acrylamide (acrylamide: bisacrylamide 19: 1) and 7M urea (urea 12.75 g), 40% bisacrylamide 11.25 ml, 10 × TBE 3 ml, H ₂ O. A 15% denaturing acrylamide gel was prepared by adding 3 μl of N ′, N ′, N ′, N′-tetramethylethylenediamine (TEMED) 30 μl, 10% APS 300 μl, TBE 0.445 mol / l. Tris-boric acid, 10 mmol / l EDTA was prepared as a 5-fold solution) and electrophoresis was performed for 30 minutes (the gel plate 10 cm was used and the effective gel length was about 8 cm).

As another method, 1 pmol of template DNA and small RNA are denatured, and a reaction buffer (10 × hybridization buffer (150 mM KCl, 10 mM Tris-HCl (pH 7.5), 0.5 mM EDTA) is added for 10 minutes. Of α- ³³ P-labeled dideoxyadenosine triphosphate, cytosine triphosphate, uracil triphosphate, and guanosine triphosphate), followed by addition of DNA polymerase for nucleotide extension. After doing so, the reaction was terminated. Termination was carried out by denaturation at 95 ° C. for 5 minutes, or spontaneous termination.

  As another method, a solution (small volume 4 μl) containing small RNA and template DNA (0.05 pmol) is mixed with a hybridization buffer (150 mM KCl, 10 mM Tris-HCl (pH 7.5), 0.5 mM EDTA). Hybridized in, and denatured at 95 ° C. for 5 minutes. Subsequently, the mixture was incubated at 64 ° C. for 6 hours, 4 μl of this solution was added with 5 units of DNA polymerase (Toyobo), and incubated at 37 ° C. for 2 hours. Reactions were performed using formamide / EDTA / BPB (0.05% bromophenol blue (BPB) and 20 mM EDTA dissolved in formamide) or reaction stop / loading stain (0.05% (bromophenol blue (BPB), 0. The solution was terminated by adding a solution of 05% xylene cyanol (XC) and 20 mM EDTA in formamide) and subjected to denaturing acrylamide electrophoresis, by using the Klenow fragment of DNA polymerase I. Was able to detect a sequence-specific extension of a specific length (here 1 nucleotide).

  The result is shown in FIG. FIG. 3 (a) shows the sequence-specific labeling by combining the presence or absence of polymerase, the type of synthetic RNA (sRNA) (1 or 2) as the target nucleic acid, and the type of template nucleic acid (DNA) (1 or 2). It shows how it is. As shown, the signal was observed significantly in the combination with complementarity and in the presence of polymerase. The detailed description is as follows.

First, “ ³³ P] ddATP, ddTTP, ddGTP, and ddCTP (all are Amersham AH9539, 55.5 TBq / mmol = 1,500 Ci / mmol) are mixed in a single tube to obtain a ddNTP mixture.

The reaction was performed using 3 μl of 10 × M buffer (100 mM Tris-HCl (pH 7.5), 500 mM NaCl, 100 mM MgCl ₂ , and 10 mM DTT were used as 10 × M buffer), primer (0.1 pmol / l). ) 0.5 μl, 0.5 μl of template (0.1 pmol / l), and 10.5 μl of H ₂ O were added to 14.5 μl, which was reacted at 95 ° C. for 5 minutes and then allowed to stand at room temperature for 6 hours . To this, 0.5 μl of 5 U / μl Klenow fragment (available from Toyobo) was added and allowed to react, 3 μl of which was added 3 μl of ddNTP mixture, and reacted at 37 ° C. for 30 minutes. To this was added 4 μl of stop / loading buffer or STOP solution, the reaction was stopped at 95 ° C. for 2 minutes and placed on ice. This was subjected to electrophoresis by 15% denaturing PAGE (using a gel plate of 10 cm, and the effective gel length was about 8 cm) for analysis after separation. Electrophoresis is MiniGel
Pre Run was performed at 400V × 30 minutes, and then performed at 400V × 30 minutes. The gel was then dried and detected by autoradiogram.

As described above, the position of the signal labeled with ³² P is somewhat higher than that of the original small RNA of 21 nucleotides in length, indicating that the incorporation of one nucleotide is reflected. Therefore, it has been proved that the present invention can directly label nucleic acids in a sequence-specific manner.

(Example 2: Improvement of detection conditions)
Next, in order to enable stronger signal detection, the present inventors examined hybridization conditions and reaction conditions. Instead of the ³³ P labeling used in Example 1, ³² P labeled dideoxyadenosine triphosphate (Amersham Pharmacia) was used. Other conditions were substantially the same.

  Furthermore, hybridization and reaction steps were performed separately to attempt to improve hybridization efficiency.

  Template DNA1 (mGFP, SEQ ID NOs: 3 and 4) and small RNA (siRNA1) were mixed in hybridization buffer and denatured at 95 ° C. for 5 minutes. This was then incubated at 70-55 ° C. for 6 hours or cooled to room temperature. 4 μl of the above mixture was removed, mixed with dideoxynucleotide, added with Klenow enzyme and incubated for 1 hour.

FIG. 3B is an experiment in which the temperature conditions for the primer-template complex system in the present invention were verified. Its detailed description is as follows:
Add 8 μl of 10 × hybridization buffer, 40 μl of primer (0.1 pmol / l), 20 μl of template (0.1 pmol / l), and 12 μl of H ₂ O to make a total volume of 80 μl. These were reacted at 95 ° C. for 5 minutes and 75-55 ° C. or room temperature for 6 hours. Take 4 μl from each tube and transfer to a new reaction tube, 1.25 μl 10 × Klenow buffer, 0.5 μl Klenow fragment, [α- ³² P] ddATP (3000 Ci / mmol = 110 TBq / mmol, Amersham, PB10233 (This specific activity is used unless otherwise stated.) 4 μl and 2.75 μl of H ₂ O were added and reacted at 37 ° C. for 1 hour. To this mixture was added 8.5 μl of stop / loading buffer. Thereafter, the reaction is stopped by placing it at 95 ° C. for 2 minutes, placing it on ice, and separating a portion (for example, 10 μl) by electrophoresis from 15% denaturing polyacrylamide gel electrophoresis (PAGE) for analysis. went.

  Radiolabeled oligonucleotides were detected after separation on a denatured agarose gel. From room temperature to about 75 ° C., the temperature was easy to detect, and the strongest signal was detected at around 64 ° C., but observation was possible even at temperatures outside this range.

  Thus, heat denaturation and slow cooling were sufficient for hybridization, but longer incubations around 64 ° C improved hybridization efficiency.

FIG. 3 (c) is an experiment in which, in addition to the conditions of FIG. 3 (a), the presence / absence of radiolabeled ddATP and the presence / absence of G + T (dGTP and dTTP) as dNTP are examined. Its detailed description is as follows:
Add 10 μl hybridization buffer 0.8 μl, primer (0.1 pmol / l) 2 μl, template (0.1 pmol / l) 2 μl, and H ₂ O to a total volume of 8 μl for 5 minutes at 95 ° C., and The reaction was performed at 64 ° C. for 30 minutes. Take 4 μl of this, transfer to a new reaction tube, use 1.25 μl of 10 × Klenow buffer, 0.5 μl of Klenow fragment, [α- ³² P] (3000 Ci / mmol, use this specific activity unless otherwise stated) ) 4 μl of ddATP and 2.75 μl of H ₂ O were added and reacted at 37 ° C. for 1 hour. Only lane 7 was used 5 μm dTTP 1 μl, 5 mM dCTP 1 μl, H ₂ O 0.75 μl. To this mixture was added 8.5 μl of stop / loading buffer. Thereafter, the reaction was terminated at 95 ° C. for 2 minutes, and placed on ice. Subsequently, this is separated by electrophoresis by 15% denaturing PAGE and analyzed. The primer-template combinations actually used, polymerase, ddNTP, etc. are described in FIG. 3 (c).

This reaction shows that it is closely dependent on the polymerase used, the dideoxyadenosine used, the primers used and the level of homology between the small RNA and the template DNA used. After priming primer 1 in template 1, the first nucleotide was adenosine, so a strong signal was detected in combination. Since primer 2 in template 2 was thymine, primer 1 is not labeled with ³² P-labeled dideoxyadenosine. However, addition of deoxy TTP and GTP resulted in a strong signal around 24 nucleotides long and an additional signal at higher molecular weights. The 24 nucleotide long signal band corresponds to the product of primer 2 with a 3 nucleotide extension (deoxy T and deoxy G and deoxy A added), and the upper band is an incorrect nucleotide to primer RNA and template DNA. It is thought to be the result of uptake and elongation. When the priming of RNA 1 in template 1 is not terminated by dideoxy ATP, the reaction takes A, T, C, C, G in order.

  The inventors then prepared a reaction mixture containing deoxy ATP, deoxy TTP and deoxy CTP without deoxy GTP.

The left side of FIG. 3 (d) is a diagram showing the experimental effect of the presence or absence of polymerase in the presence of labeled dCTP without the labeled ddATP and in the presence of dATP and dTTP. The detailed description is as follows: The primer should be labeled with ³² P deoxyCTP and the absence of deoxyGTP should terminate the extension to the 5 nucleotide side. However, this reaction produces multiple higher molecular weight bands, indicating that inappropriate nucleotides are mistakenly incorporated into primer RNA and template DNA.

The detailed experimental procedure in FIG. 3 (d) is as follows:
Add 10 μl hybridization buffer 0.8 μl, primer (0.1 pmol / l) 2 μl, template (0.1 pmol / l) 2 μl, and H ₂ O to a total volume of 8 μl for 5 minutes at 95 ° C., and The reaction was performed at 64 ° C. for 30 minutes. Take 4 μl of this, transfer to a new reaction tube, 10 × Klenow buffer 1.25 μl, Klenow fragment 0.5 μl, [α- ³² P] (3000 Ci / mmol, use this specific activity unless otherwise stated) D) 4 μl of dCTP, 1 μl of dATP, 1 μl of dTTP, and 2.75 μl of H ₂ O were added and reacted at 37 ° C. for 1 hour. To this mixture was added 8.5 μl of stop / loading buffer. This was subjected to isopropanol precipitation, and 10 μl of stop / loading buffer was added to the precipitated pellet. Then react at 95 ° C. for 5 minutes and place on ice. A part (for example, 10 μl) is separated by electrophoresis by 15% denaturing PAGE for analysis. Lane 1 shows the presence of Klenow fragment, and lane 2 shows no Klenow fragment (instead H ₂ O).

  The right side of FIG. 3D is an experiment for confirming the effect of adding 1 nucleotide or 10 nucleotides to the template DNA. The detailed description is as follows.

First, primer 1 (0.1 pmol / μl), template DNA1 (0.1 pmol / μl), DNA1-G (0.1 pmol / μl), or DNA-1G10 (0.1 pmol / μl) is used. The reaction was performed by adding 0.8 μl of 10 × hybridization buffer, 2 μl of primer (0.1 pmol / l), 2 μl of template (0.1 pmol / l) and adding 3.2 μl of H ₂ O to 8 μl at 95 ° C. for 5 minutes. The reaction was carried out at 64 ° C. for 30 minutes. To 4 μl of this, 1.25 μl of 10 × Klenow buffer, 0.5 μl of Kure nou fragment, 4 μl of [α- ³² P] ddATP, and 2.75 μl of H ₂ O are added and reacted at 37 ° C. for 1 hour. To this is added 8.5 μl of stop / loading buffer. This was analyzed by electrophoresis through 15% denaturing PAGE.

  Thus, we can label small RNAs by DNA synthesis primed by small RNAs. The reaction is sequence specific and depends on the presence of labeled dideoxynucleotides and DNA polymerase. Labeled nucleotides could be easily detected by denaturing acrylamide gel electrophoresis and autoradiography. The dideoxy ATP can be used to determine the length of the labeled small RNA.

The reaction primed with small RNAs is terminated directly by incorporation of dideoxynucleotides (also referred to herein as the “Primer Halt method”). In order to evaluate the detection limit of primer termination, a 10-fold serial dilution of the synthesized small RNA was made and labeled as described above. In order to reduce the background derived from unincorporated dideoxy ATP, isopropanol precipitation was performed followed by electrophoresis. Using the primer termination method, we were able to detect at least 5 × 10 ¹⁸ molecules (5 atmole) of small RNA by overnight exposure. This is significantly higher than conventional methods based on ribonuclease protection assays or Northern blotting.

(Example 3: Verification with siRNA)
In order to further verify the possibility of the primer stopping method, siRNA (small interfering RNA) was used to conduct the same experiment as in the above example.

  This siRNA was detected from transgenic tobacco using a luciferase gene that was silenced after translation. The procedure is shown below.

  siRNAs are known to be found in organisms (including animals and plants) where the transgene is inactivated by post-transcriptional gene silencing (RNA silencing). . As a model of an organism in which a foreign gene is inactivated by RNA silencing, a tobacco plant (NW7-24-4) that highly expresses the luciferase gene described in Genetics 160: 343-352 (2002) and An inactivated tobacco plant (NW7-13-10) was used. As template DNA, plasmid pT3 / T7-luc (Clonetech) having the same sequence as the introduced luciferase gene was used. This plasmid was cleaved with a restriction enzyme SmaI that cleaves only at one site, and recovered by ethanol precipitation was used as a template DNA. The closed circular plasmid was linearized by the restriction enzyme treatment, and easily dissociated into a single strand by heat denaturation.

Total RNA was prepared from a wild-type tobacco plant (Nicotiana tabacum cv. Sumsun NN) and the above-described transformed plant using a Trizol reagent manufactured by Invitrogen. RNA extraction and preparation methods followed Invitrogen instructions and manual. The template DNA (0.5 pmol) and 10 μg of the prepared total RNA (10 × hybridization buffer) were mixed, sterilized water was added to make a total of 8 μl, and heat denaturation was performed at 95 ° C., followed by hybridization at 64 ° C. for 90 minutes. Take 4 μl of this, add 1.25 μl of 10 × Klenow reaction solution, 0.5 μl of Klenow fragment (5 units / μl), 4 μl of [α- ³² P] ddATP and 2.75 μl of sterilized distilled water at 37 ° C. Let react for 1 hour. The reaction is precipitated with isopropanol and 10 μl of stop / loading buffer is added to the precipitated pellet. This is separated by 15% denaturing acrylamide gel electrophoresis and separated for analysis.

  In the Luc gene, a strong signal is detected in the 21-25 nucleotide long portion from the inactivated plant, but no signal is detected from the wild type plant and the plant that highly expresses the luc plant.

(Example 4: Verification with miRNA)
Furthermore, the same experiment was conducted with miRNA. In this example, miRNAs as shown in SEQ ID NOs: 6 to 9 were selected as miRNAs, and it was verified whether they could be detected by the primer stop method and the primer run-off method.

  Template DNA complementary to plant miRNA and template DNA complementary to mouse miRNA were synthesized. The synthesis was carried out substantially according to the above examples. This DNA further contained a T residue at the 5 'end, allowing labeling with dideoxy ATP at the 3' end.

  Labeling and detection were performed substantially according to the procedure of the above example. Except that the RNAs used were those of SEQ ID NOs: 6 to 9, experiments were performed according to the above-described examples to perform the primer termination method.

  FIG. 4 shows the result. FIG. 4 shows the detection limit of primer halt (Primer Halt) and the detection of miRNA from organisms by primer halt (Primer Halt).

  The left figure of FIG. 4 is as follows.

As shown in FIG. 3 (a), the left figure is a detection of a dilution series of synthetic RNA. The two on the right are those in which miRNA contained in total RNA prepared from plants was detected.
Luc: Reaction using a part of the sequence of luciferase gene luc not encoded by plants as a template as a negative control.
miR171: A primer halt method using a sequence complementary to miR171 which is a plant endogenous microRNA (miRNA) + a sequence containing a single base T. In this way, plant endogenous miRNA can be detected.

  In the following, a method for detecting tobacco miRNA is carried out.

  Tobacco total RNA is extracted by 5 μg by Trysol method. Use miR171 or LUC as template.

The reaction was 0.8 μl of 10 × hybridization buffer, 2 μl of primer (0.1 pmol / l), 2 μl of template (0.1 pmol / l), and 8 μl with H ₂ O, at 95 ° C. for 5 minutes, and The reaction was performed at 64 ° C. for 30 minutes. Of these, take 4 μl, 10 × Klenow buffer 1.25 μl, Klenow fragment 0.5 μl, [α- ³² P] ddATP (3000 Ci / mmol, use this specific activity unless otherwise stated), and H 2.75 μl of ₂ O is added and reacted at 37 ° C. for 1 hour. To this mixture is added 8.5 μl of stop / loading buffer. This is subjected to isopropanol precipitation and 10 μl of stop / loading buffer is added to the precipitated pellet. This is analyzed by electrophoresis through 15% denaturing PAGE.

As shown in FIG. 4, when the detection sensitivity was determined by serially diluting small RNAs to 1/10, it was clear that at least 5 atmole (5 × 10 ⁻¹⁸ ) can be detected by the method of the present invention. Became.

  Using the primer termination method, we were able to detect the endogenous miRNA of the organism.

  Next, it was confirmed whether the primer run-off method works.

  The mouse used was a C57 mouse female, and RNA isolated from the brain and heart of this mouse was used.

Concert RNA extraction reagent (manufactured by Invitrogen) was used as RNA. As templates, DNA1G (= 5′-GGA ACT ATA CAA ACA TGA TGA GCT TTA ATG ATA TTG GCG CGG CTC AAT CA-3 ′ (SEQ ID NO: 12); GFP sense DNA negative control), miR16G (for mouse miRNA detection) Mmus miR16 = GCG CCA ATA TTT
ACG TGC TGC TAT GAT ATT GGC GCG GCT CAA
TCA-3 ′ (SEQ ID NO: 13)) was used.

The reaction was performed by adding 0.8 μl of 10 × hybridization buffer, 2 μl of primer (0.1 pmol / l), 2 μl of template (0.1 pmol / μl), and 8 μl of H ₂ O as appropriate. The reaction was allowed to proceed for 30 minutes at 64 ° C. 4 μl of this was taken and transferred to a new reaction tube, 10 × Klenow buffer 1.25 μl, Klenow fragment 0.5 μl, [α- ³² P] dCTP (Amersham, AA0005, 3000 Ci / mmol) 4 μl and H ₂ 2.75 μl of O was added and reacted at 37 ° C. for 1 hour. This was ethanol precipitated and 20 μl of stop / loading buffer was added to the resulting pellet. Thereafter, this was subjected to electrophoresis by 15% denaturing PAGE (pre-run at 400V × 30 minutes, execution at 400V for 30 minutes) for separation and analysis. The result is shown in FIG. 4B. FIG. 4B shows that mouse miRNA can be confirmed by the primer run-off method.

  Thus, the present invention provides a novel method (primer stop method) for detecting small RNAs. This is a simple but highly sensitive method that takes less time. The method of the present invention has the possibility of being widely used in place of the conventional Northern blot.

(Example 5: Study of other condition improvements)
In order to increase the sensitivity, the hybridization reaction system was improved and experiments using ³² P-labeled dideoxy ATP were conducted. The temperature of hybridization was mainly examined.

After denaturing synthetic DNA1 and RNA1 (each 1 pmol) in a small volume of hybridization buffer, hybridization was carried out by incubating at 75 ° C. to 55 ° C. or returning to room temperature. The hybridized DNA-RNA hybrid was added to Klenow buffer containing dideoxy ATP but no other nucleotides (10-fold solution, 0.5 M Tris-HCl (pH 7.5), 0.1 M MgCl ₂ , 10 mM DTT, And 0.5 mg / ml bovine serum albumin was prepared and diluted 10-fold), and a DNA synthesis reaction was performed. The highest uptake is observed when the temperature is kept at around 65 ° C., but strong radioactivity uptake is also observed by slowly returning to room temperature.

(Control experiment of primer stop method)
After a small amount of denaturation at −64 ° C. as described above, a DNA synthesis reaction was performed under the conditions shown above.

(I) ³² P incorporation depends on the polymerase used, the small RNA used, the template DNA used, the ddATP used and the homology between the DNA and RNA used. From the fact that a band can be seen at a position (polymeric) having a slightly lower mobility than the inserted RNA, it was confirmed that a single base was taken up.

(Ii) In the case of the combination of DNA2 and RNA2, since the base to be incorporated next to RNA2 is TTP, the incorporation did not occur only with ddATP. However, the addition of dCTP and dTTP allowed the DNA synthesis reaction to proceed to A 4 bases ahead, so that uptake could be observed. In this case, a band appears at a higher polymer position reflecting the synthesis reaction of 4 bases. In addition, some non-specific reaction is observed, but it does not seem to affect the detection. Non-specific detection can be demonstrated, for example, by using it as a control indicating that the result (band) of a certain reaction is dependent on the interaction between the template and the target. Therefore, it is possible to confirm that the reaction is a specific reaction by using two lengths such as DNA1-G and DNA1-G10.

(RNA labeling reaction with dCTP)
(Iii) In the combination of DNA1-RNA1, the following bases are A, T, C, C, A, and T, and the reaction is performed in the presence of ³² P-dCTP and dATP, dTTP (all of which are deoxy, not dideoxy). It was expected that the reaction would stop at 6 bases while incorporating ³² P-dCTP (due to the absence of dGTP), but at a relatively high molecular position (possibly due to non-specific elongation). Uptake was seen.

(Primer termination reaction using ddATP as a combination of DNA1-RNA1)
(Iv) In the combination of DNA1G-RNA1, dCTP was used instead of ddATP. DNA1G has a sequence complementary to RNA1 and a single base G. In this reaction, the extension termination by dideoxynucleotide did not occur, but the template disappeared when C was added, so the reaction by polymerase was terminated.

  (V) A combination of DNA1G10-RNA1 and dCTP was used. DNA1G10 has a sequence complementary to RNA1 + 10 base G (5'-GGG GGG GGG GGA ACT ATA CAA ACA TGA TGA GCT TTA ATG ATA TTG GCG C-3 '(SEQ ID NO: 14)). In this reaction, the template was lost when 10 bases of C were added, and the reaction with the polymerase was stopped.

  As expected, when DNA1G was used as a template, a band that seems to have been added by one base was detected as in the primer termination reaction with ddATP, and when DNA1-G10 was used, a higher molecular band was detected. By using ssDNA having a sequence complementary to the nucleic acid to be + an arbitrary base of a limited length as a template, the length by run-off replication rather than run-off replication It was found that labeling limited to

(Example 6: Another labeling method)
Although the protocol illustrated in the above example has shown a radiolabeled nucleotide-based method, it can also be realized by other labeling methods. In this example, detection was performed using small RNA labeled with fluorescently labeled nucleotides. If such a fluorescently labeled nucleotide is used, it can also be used in capillary electrophoresis and related techniques. Therefore, the present invention can be applied also to sequencing techniques, and further, since it performs sequence-specific direct labeling, various applications can be considered for polynucleotides and oligonucleotides.

  Here, the case where a fluorescent label is used is demonstrated.

First, 0.8 μl of 10 × hybridization buffer (1.5 M KCl, 0.1 M Tris-HCl (pH 7.5), 5 mM EDTA); 2 μl of primer 1 (0.1 pmol / μl); template DNA 1a (0. 1 μmol / μl) 2 μl; and 3.2 μl H ₂ O are added to make an 8 μl reaction. This is reacted at 95 ° C. for 5 minutes, and then reacted at 64 ° C. for 30 minutes. 8.5 μl of the reaction solution (1.25 μl of 10 × Klenow buffer, 0.5 μl of Klenow fragment, 1.25 μl of 20 mM Cy5-dCTP, ₄ μl of H ₂ O) is added thereto, and incubated at 37 ° C. for 60 minutes. To this was added 8.5 μl of reaction termination / loading stain (0.05% (bromophenol blue (BPB), 0.05% xylene cyanol (XC) and 20 mM EDTA dissolved in formamide) at 95 ° C. After 2 minutes of reaction, cool on ice, which is separated by electrophoresis on 15% PAGE and scanned (FLA-8000, Fujifilm).

  This shows that the method of the present invention can be carried out even when a fluorescent dye is used.

(Example 7: Experiment using reverse transcriptase)
The protocol exemplified in the above example can be realized even when reverse transcriptase is used.

Here, the case where reverse transcriptase (Toyobo M-MLV reverse transcriptase RNaseH Minus = code number RTN-101) is used is demonstrated. PT3 / T7-LUC (Clonetech) is used as a plasmid. This plasmid is digested with SmaI to generate a digested product. This produces a blunt end product with LUC in parallel next to T3. Thereafter, a transcription reaction is performed in vitro. Transcription reaction is 10 μl of 5 × reaction buffer; 10 mM rNTP each; T3 polymerase (Toyobo code number SC600111) 1 μl: and 2 μl of DNA, 50 μl, and the reaction is allowed to proceed at 37 ° C. for 30 minutes. Thereafter, the ethanol precipitate is dissolved in 10 μl of TE (composition: 1 mmol / L EDTA-containing 10 mmol / L Tris buffer, pH 9.0). This is used as a template. Thereafter, 10 × hybridization buffer; template nucleic acid (LUC RNA) 2 μl; tobacco (wild type plant, LUC high expression plant or LUC silencing plant) total RNA 5 μl, total 8 μl, and reacted at 95 ° C. for 5 minutes, Thereafter, the mixture is reacted at 64 ° C. for 30 minutes. 10 × M-MLV reverse transcriptase buffer Among 4 [mu] l (available .5 × reaction buffer obtained from Toyobo 250mM Tris-HCl (pH8.3), 375mM KCl, 15mM MgCl 2, and 50 mM DTT, diluted appropriately .) 1.25 μl; M-MLV reverse transcriptase (source) 0.5 μl; RNase inhibitor 1 μl; [α- ³² P] ddATP 4 μl; H ₂ O 1.25 μl is added, and the reaction is performed at 37 ° C. for 60 minutes. . To this is added 8.5 μl of stop loading buffer, followed by isopropanol precipitation and electrophoretic analysis with 15% PAGE. This reveals that the principle of the present invention can be applied even when reverse transcriptase is used.

(Example 8: Construction of array)
Synthesize | combine DNA containing the specific arrangement | sequence used as a probe at 3 'terminal, and prepare it at 200 pmol / microliter. Take 1 μl of this DNA and fix it on a nylon membrane.

Prepare mRNA (2 μg) or total RNA (40 μg) to 0.48 ml with sterile MilliQ water. This RNA solution is poured into a hybridization bag containing a nylon membrane, annealed at 64 ° C. for 30 minutes, and then incubated at 42 ° C. for 10 minutes. Superscript II (200 units / μl, GIBCO BRL) 40 μl, 5 × Superscript II reaction buffer (GIBCO BRL) 150 μl, DTT (0.1M, GIBCO BRL) 80 μl, dNTP mixture (dATP, dGTP, dT, dT, 40, respectively) 20 mM, Amercham Pharmacia) 40 μl and Cy5-dCTP (20 mM, Amersham Pharmacia) 40 μl are prepared. This solution is poured into the hybridization bag and allowed to react at 42 ° C. for 2 hours and 30 minutes in the dark. The membrane was washed once with a primary washing solution (1 × SSC containing 0.2% SDS (15 mM sodium citrate, 150 mM NaCl, pH 7.0), 65 ° C.) under light shielding, and a secondary washing solution (0.2 Wash twice with 0.1 × SSC containing 65% SDS (65 ° C.). After removing moisture, the fluorescence of Cy5 is detected and measured using a fluorescence scanner (FLA-8000, manufactured by FUJIFILM).

  Thereby, it is understood that the present invention functions even in the primer array method.

  While the invention has been illustrated with certain preferred embodiments, the invention is not to be construed as limited to these embodiments. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  Since the present invention realizes simple detection of nucleic acids and is useful in any scene where nucleic acid detection is required (for example, various diagnoses), agriculture and life science are generally involved. It is useful in various situations such as industry and pharmaceutical manufacturing industry.

(Explanation of sequence listing)
Sequence number 1: RNA1
Sequence number 2: RNA2
Sequence number 3: DNA1 (m-GFP5-ER-S 725-812)
SEQ ID NO: 4: DNA2 (m-GFP5-ER-S 572-670)
Sequence number 5: LUC (2113-LUC 2603-2702)
SEQ ID NO: 6: Ntab miR171
SEQ ID NO: 7: Ntab miR167
SEQ ID NO: 8: Mmus miR181 T
SEQ ID NO: 9: Mmus miR16 T
SEQ ID NO: 10: GFP5-ER G10 + 786-812
SEQ ID NO: 11: GFP5-ER G + 786-812
SEQ ID NO: 12: DNA1-G
SEQ ID NO: 13: Mmus miR16 for detection of mouse miRNA
SEQ ID NO: 14: DNA1-G10
SEQ ID NO: 15: RNA1-C (Amplification product using DNA1-G (SEQ ID NO: 12))
SEQ ID NO: 16: RNA1-C10 (amplification product using DNA1-G10 (SEQ ID NO: 14))

Claims (54)

A method for producing a nucleic acid into which a label has been introduced, comprising the following steps:
A) Step of providing a nucleic acid to be labeled and a template nucleic acid, wherein the template nucleic acid is complementary to at least a part of the nucleic acid to be labeled, and the template nucleic acid is a complementary portion of the nucleic acid to be labeled Having a nucleotide sequence that is at least one nucleotide longer in the portion of at least one tip of the nucleic acid to be labeled when the template nucleic acid is hybridized with the template nucleic acid;
B) A step of producing a labeling target nucleic acid-template nucleic acid complex by hybridizing the labeling target nucleic acid with the template nucleic acid;
C) a step of adding a nucleic acid synthetase and a labeled nucleotide to the labeled target nucleic acid-template nucleic acid complex to perform an extension reaction, wherein at least one labeled nucleotide is added to the labeled target nucleic acid And D) taking out the nucleic acid to be labeled that has been extended from the resulting mixture after the extension reaction, and
Including the method. The method according to claim 1, wherein the nucleic acid to be labeled is DNA or RNA or a combination thereof. The method according to claim 1, wherein the nucleic acid to be labeled is RNA. The method according to claim 1, wherein the nucleic acid to be labeled is RNA selected from the group consisting of siRNA and miRNA. The method according to claim 1, wherein the template nucleic acid includes a sequence that is complementary to all the nucleic acids to be labeled. The method according to claim 1, wherein the template nucleic acid has a nucleotide sequence that is at least one nucleotide longer at both ends of the nucleic acid to be labeled. The method according to claim 1, wherein the template nucleic acid has a nucleotide sequence that is at least 10 nucleotides longer than the tip of at least one of the nucleic acids to be labeled. The method according to claim 1, wherein the long nucleotide sequence of the template nucleic acid is located on the 5 'end side of the template nucleic acid. The long nucleotide sequence of the template nucleic acid on the 3 ′ end side of the template nucleic acid has a different length from the long nucleotide sequence of the template nucleic acid on the 5 ′ end side of the template nucleic acid. The method described. The method according to claim 1, wherein the long nucleotide sequence of the template nucleic acid is located on the 5 'end side of the template nucleic acid, and the 5' end portion of the nucleic acid to be labeled is longer than the 3 'end of the template nucleic acid. The long nucleotide sequence of the template nucleic acid at the 3 ′ end of the template nucleic acid is at least one nucleotide longer than the long nucleotide sequence of the template nucleic acid at the 5 ′ end of the template nucleic acid. the method of. 12. The long nucleotide sequence of the template nucleic acid at the 3 ′ end of the template nucleic acid is at least 10 nucleotides longer than the long nucleotide sequence of the template nucleic acid at the 5 ′ end of the template nucleic acid. the method of. The method according to claim 1, wherein the nucleic acid synthase is a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase. The method of claim 1, wherein the labeled nucleotide comprises a nucleotide labeled with a label selected from the group consisting of fluorescence, radioactivity, phosphorescence, biotin, digoxigenin (DIG), enzyme, and chemiluminescence. The method of claim 1, wherein the labeled nucleotide comprises radioactivity. The method according to claim 1, wherein the step of taking out the extended nucleic acid to be labeled includes a denaturation step. The method according to claim 1, further comprising removing unreacted substances. 18. The method of claim 17, wherein removal of unreacted material is achieved by ethanol precipitation or gel filtration chromatography. The method according to claim 17, wherein the unreacted substance removing step further includes a step of separating the extended nucleic acid to be labeled. 20. A method according to claim 19, wherein the separation is achieved by electrophoresis or chromatography. The method according to claim 1, wherein the step of taking out the elongated nucleic acid to be labeled includes the step of detecting the label. The method of claim 21, wherein the detecting comprises detecting the label directly or indirectly. The method according to claim 21, further comprising a step of separating the elongated nucleic acid to be labeled. 24. The method of claim 23, wherein the detection is performed during the separation. 25. The method of claim 24, wherein the separation and detection is accomplished by autoradiograph. The method according to claim 1, further comprising the step of stopping the extension reaction. 27. The method according to claim 26, wherein the stopping step is achieved by primer stop (Primer Halt method) or primer run-off method. The method according to claim 1, wherein the nucleic acid to be labeled is used as a probe. The method according to claim 1, wherein the nucleic acid to be labeled is a nucleic acid to be detected. The method according to claim 1, wherein a terminal nucleotide of the template nucleic acid is modified. The method according to claim 1, wherein the terminal nucleotide of the template nucleic acid is modified at the 3 ′ end. The method according to claim 1, wherein the terminal nucleotide of the template nucleic acid is modified so as not to incorporate a labeled nucleotide. The modification is a modification selected from the group consisting of the dideoxy chain termination method, fluorescein isothiocyanate (FITC) conversion, biotinylation, amination, phosphorylation, tetramethyl rhodamine (TAMRA) conversion, thiolation and rhodamine formation. The method of claim 30. From the group consisting of the dideoxy chain termination method, fluorescein isothiocyanate (FITC), biotinylation, amination, phosphorylation, tetramethylrhodamine (TAMRA), thiolation and rhodamineation The method of claim 1, wherein the selected modification is made. A method for introducing a label into a nucleic acid comprising the following steps:
A) a step of providing a nucleic acid to be labeled and a template nucleic acid, wherein the template nucleic acid is complementary to at least a part of the nucleic acid to be labeled, and a complementary part of the nucleic acid to be labeled and the template nucleic acid And having a nucleotide sequence that is at least one nucleotide longer in the portion with respect to the tip of at least one of the nucleic acids to be labeled when
B) a step of hybridizing the nucleic acid to be labeled with the template nucleic acid to form a nucleic acid to be labeled-template nucleic acid complex; and C) a nucleic acid synthesizing enzyme and a labeled nucleic acid to the nucleic acid to be labeled-template nucleic acid complex A step of performing an extension reaction by adding a nucleotide, and at least one labeled nucleotide is incorporated into the nucleic acid to be labeled based on the template nucleic acid,
Including the method. A kit for producing a nucleic acid introduced with a label, comprising the following means:
A) At least one tip of the target nucleic acid when it is complementary to at least a part of the target nucleic acid to be labeled and further hybridized with the complementary part of the target nucleic acid and the template nucleic acid A template nucleic acid having a nucleotide sequence that is at least one nucleotide longer in the part to; and B) a nucleic acid synthase;
A kit comprising: 37. The kit of claim 36, further comprising a labeled nucleotide.   38. The kit of claim 37, further comprising means for separating the unreacted and extended products of labeled nucleotides from the resulting mixture.   38. The kit of claim 37, further comprising means for detecting the labeled nucleotide.   Furthermore, the kit of Claim 36 provided with the standard target nucleic acid used as a control. The kit according to claim 40, wherein the standard nucleic acid includes a nucleic acid selected from the group consisting of a nucleic acid for identifying the template nucleic acid and a nucleic acid for identifying the nucleic acid to be labeled. 37. The kit according to claim 36, further comprising a reagent for the reaction of the nucleic acid synthase. The kit according to claim 37, wherein the labeled nucleotide has an action of stopping a reaction of a nucleic acid synthase. The kit according to claim 36, further comprising a support, wherein the template nucleic acid is immobilized on the support. 45. The kit of claim 44, wherein the support is glass or a membrane. 45. The kit according to claim 44, wherein the template nucleic acids immobilized on the support are arranged in an array. The kit according to claim 36, wherein the template nucleic acid is modified at a terminal nucleotide. The kit according to claim 36, wherein the terminal nucleotide of the template nucleic acid is modified at the 3 'end. The kit according to claim 36, wherein the terminal nucleotide of the template nucleic acid is modified so as not to incorporate a labeled nucleotide. The modification is a modification selected from the group consisting of dideoxylation, fluorescein isothiocyanate (FITC), biotinylation, amination, phosphorylation, tetramethylrhodamine (TAMRA), thiolation and rhodamineation. 47. The kit according to 47. The nucleotide at the 3 ′ end of the template nucleic acid is selected from the group consisting of dideoxylation, fluorescein isothiocyanate (FITC), biotinylation, amination, phosphorylation, tetramethylrhodamine (TAMRA), thiolation and rhodamineation 37. The kit of claim 36, wherein the kit is modified. A method for producing a support on which a nucleic acid introduced with a label is immobilized, comprising the following steps:
A) a step of providing a labeling target nucleic acid and a template nucleic acid immobilized on a support, wherein the template nucleic acid is complementary to at least a part of the labeling target nucleic acid, and further complementary to the labeling target nucleic acid Having a nucleotide sequence that is at least one nucleotide longer in the portion relative to the tip of at least one of the nucleic acids to be labeled when the portion and the template nucleic acid are hybridized;
B) A step of producing a labeling target nucleic acid-template nucleic acid complex by hybridizing the labeling target nucleic acid with the template nucleic acid;
C) A step of performing an extension reaction by adding a nucleic acid synthetase and a labeled nucleotide to the nucleic acid to be labeled-template nucleic acid complex, wherein at least one of the labeled nucleotides is labeled with the template nucleic acid. Incorporated on the basis of a process;
D) A step of removing the support containing the nucleic acid to be labeled extended from the resulting mixture after the extension reaction,
Including the method. Step to output said up is accomplished by removing the unreacted reactants by washing the support method according to claim 52. Step, it includes the step of detecting the label of the labeled target nucleic acid, The method of claim 52, to output the up.