JP4105294B2 - Oligonucleotide labeling method and use thereof - Google Patents

Description

【図面の簡単な説明】
【図１】オリゴヌクレオチドのテイリング反応を模式的に示す図。オリゴヌクレオチドはＡＴ繰り返し配列の部分で自己アニーリングし、続いてＤＮＡポリメラーゼによってｄＡＴＰ及びｄＴＴＰ（ｄＵＴＰ）の付加が行われる。この付加によってオリゴヌクレオチドのＡＴ繰り返し配列の部分は伸長した後解離し、以降自己アニーリングと伸長、解離を繰り返すことを示す。
【図２】オリゴヌクレオチドがセルフプライミングで伸長することを示す写真。
【図３】オリゴヌクレオチドの濃度とテイリング効率との関係を示す写真。
【図４】付加されるテイルの長さと反応時間との関係を示す写真。
【図５】Ｔａｑ ＤＮＡポリメラーゼ及びＴｄＴのオリゴヌクレオチドへの標識効率を示す写真。
【図６】オリゴヌクレオチドの５’末端にジゴキシゲニンを標識する従来の標識法と本発明の標識法によりマウス白血病ウイルスＲＮＡを検出した結果を示す写真。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for labeling a synthetic oligonucleotide. More specifically, an arbitrary base sequence on the 5 ′ side and a repetitive sequence represented by (XY) n at the 3 ′ end (where X and Y are a combination of adenine-thymine or uracil, or guanine-cytosine). , N represents the number of bases), and relates to a method for labeling the oligonucleotide, which comprises adding a nucleotide labeled with a radioisotope (RI) or modified with a labeled compound and extending with a DNA polymerase.
[0002]
[Prior art]
Synthetic oligonucleotides have recently been applied in all fields of molecular biology. In particular, labeled synthetic oligonucleotides are widely used as probes that can be most easily prepared when detecting a gene having a specific sequence. The detection sensitivity of a gene using a probe is uniquely determined by the intensity of a signal generated by each labeled compound incorporated into the probe and the number of labeled compounds. Therefore, in order to detect a specific gene present in a minute amount in a living body or in a cell, it is necessary to efficiently operate a highly sensitive probe.
[0003]
RI shows the strongest signal as a labeling compound, but lacks versatility for reasons such as “requires special equipment to generate radiation” or “short lifetime”. As non-RI labeling compounds, fluorescently labeled compounds such as fluorescein and rhodamine (Dirks et al. (1991) Exp. Cell Res. 194, 310-315) and biotin that reacts with antibodies (Biotin) are used. And the like (Langer et al. (1981) Proc. Natl. Acad. Sci. USA 78, 6633-6737) and the like, but these signals are not as strong as RI. In recent years, digoxigenin (DIG) has attracted attention as a non-RI labeling compound whose signal intensity is comparable to RI (Kessler (1990) BioTechnology Int. 183-194).
[0004]
Conventionally, synthetic oligonucleotides are labeled using a labeling compound such as an enzyme or a dye, or a method of chemically modifying an oligonucleotide using an RI-labeled amidite (Pieless et al. (1990) Nucleic Acids Res. 18, 4355-4360), tailing that adds a deoxynucleotide triphosphate (dNTP) or dideoxynucleotide triphosphate (ddNTP) label to the 3 ′ end of the oligonucleotide by enzymatic reaction of terminal deoxynucleotidyl transferase (TdT). (Schmiz et al. (1991) Anal. Biochem. 192, 222-231) and the photobiotin method (Forster et al. (1 85) Nucleic Acids Res.13, 745-761) and the like have been used.
[0005]
In general, the detection sensitivity when the tailing method is used depends on the number of labeled molecules to be incorporated. For example, TdT adds tails (tails) that are on average several tens of bases in length (Schmitz et al. (1991) Anal. Biochem. 192, 222-231). When Northern blot hybridization or Southern blot hybridization is performed using the oligo probe obtained by this method, target DNA or RNA can be detected up to 1 pg.
[0006]
Furthermore, when detecting a trace amount thing, the polymerase chain reaction (PCR) method (Saiki et al. (1988) Science 239, 487-491) is used. The PCR method is a method for clarifying the presence of a target gene by amplifying a gene fragment of a target gene present in a trace amount by using one or more primers and detecting the amplified gene fragment. Therefore, it can be said that it is a very effective method for detecting a small amount of a target gene present in a tissue or the like. However, in this method, in order to detect a released gene fragment, the localization of the target gene is clarified, unlike, for example, Northern blot hybridization or Southern blot hybridization in which the probe directly acts on the tissue. I can't. As for the PCR method, depending on the site of the gene to be amplified, amplification efficiency may be low and high sensitivity may not be obtained, and in order to prove the presence of a specific RNA by the PCR method, a reverse transcription reaction is required and indirect The problem is pointed out that only proof can be made.
[0007]
As described above, in order to detect a target gene with high sensitivity using a non-RI labeled probe, it is necessary to incorporate a large amount of the labeled compound into the oligonucleotide. However, when such a probe is used, there is a problem that the ratio of the sequence site involved in the hybridization becomes small and the hybridization efficiency is lowered due to steric hindrance. In particular, when detecting a specific gene in a tissue by in situ hybridization, the size of the probe is extremely important because the probe must penetrate into the tissue. .
[0008]
In addition, the conventional method of labeling oligonucleotides using an enzyme reaction has a high non-specific reaction and is not suitable for in situ hybridization. That is, after hybridizing the oligonucleotide, if, for example, dNTP or ddNTP to which a labeled compound is bound using TdT is added to the 5 ′ or 3 ′ side of the oligonucleotide, this enzyme is a nucleic acid having 3 or more bases. Since it has an activity to add dNTP or ddNTP nonspecifically, it also labels nucleic acid fragments other than the target present in tissues or cells, resulting in a nonspecific reaction.
[0009]
[Problems to be solved by the invention]
As described above, since the conventional method uses a pre-labeled oligonucleotide as a probe, there is a contradiction between the amount of the labeled compound incorporated for increasing the sensitivity and the hybridization reactivity. Particularly, in-situ hybridization has a problem of reduced reactivity.
[0010]
The present invention has an oligonucleotide complementary to the gene to be detected on the 5 ′ side and a repetitive sequence represented by (XY) n on the 3 ′ side (where X and Y are adenine-thymine or A method of labeling an oligonucleotide having uracil or a combination of guanine and cytosine, wherein n represents an integer of 1 or more, using a DNA polymerase and a nucleotide labeled with RI or modified with a labeled compound, in a template-independent manner The purpose is to provide.
[0011]
Another object of the present invention is to provide a method for detecting a specific gene such as a tissue, a cell or a virus by using the method for labeling the above-mentioned oligonucleotide.
[0012]
Still another object of the present invention is to provide a labeled oligonucleotide labeled by the above-described method and a method for detecting a specific gene such as a tissue, cell or virus using the labeled oligonucleotide.
[0013]
Still another object of the present invention is to provide a method for preparing a probe for detecting a specific gene by PCR using the labeled oligonucleotide labeled by the above method as a primer.
[0014]
[Means for Solving the Problems]
As a result of intensive studies to achieve the above object, the present inventors have found that a DNA polymerase derived from Thermus aquaticus has an enzymatic activity that extends an oligomer consisting of a repeat sequence of AT in a short time. In addition, a DNA polymerase and digoxigenin-labeled deoxyuracil triphosphate having an oligonucleotide having a partial base sequence of the pUC19 plasmid on the 5 ′ side and the repetitive sequence on the 3 ′ side By reacting at a constant temperature in a mixed solution containing (DIG-dUTP), it was found that a long chain consisting of a repeating sequence of labeled AU was added to the 3 ′ side of the oligonucleotide, and the present invention was completed. It was.
[0015]
Therefore, the present invention includes a method of labeling an oligonucleotide having a partial base sequence of pUC19 and an AT repeat sequence with DIG-dUTP and a labeled oligonucleotide obtained by the method.
[0016]
The present invention also includes a method for detecting a specific gene by detecting pUC19 introduced into a tissue, cell, virus or the like or pUC19 containing a foreign gene using the above method and labeled oligonucleotide.
[0017]
The present invention also includes a method for detecting a gene having a sequence complementary to the sequence by using an oligonucleotide having an arbitrary base sequence on the 5 'side.
[0018]
The present invention also includes a method for preparing a probe for detecting a specific gene by PCR using the labeled oligonucleotide obtained by the above method as a primer. The present invention is described in further detail below.
[0019]
The method and labeled oligonucleotide of the present invention have an arbitrary base sequence on the 5 ′ side and a repeating sequence represented by (XY) n on the 3 ′ side (where X and Y are adenine-thymine or uracil, Or a guanine-cytosine combination, where n represents an integer of 1 or more), a DNA polymerase lacking 5 ′ → 3 ′ exonuclease activity, and a nucleotide label.
[0020]
The oligonucleotide constituting the present invention is synthesized by a chemical synthesis method, for example, using a DNA synthesizer (Applied Biosystems) and a method according to the protocol of the apparatus. The resulting oligonucleotide is deprotected and cut out from the resin by the ammonium method. For purification of the crude oligonucleotide cut out from the deprotected resin, a method usually used for purifying a nucleic acid, such as electrophoresis, liquid chromatography, gel filtration chromatography, affinity chromatography, etc., is used as appropriate. You can choose to do it.
[0021]
Oligonucleotides can be prepared by general gene cloning methods described by Sambrook et al. (Molecular Cloning, A Laboratory Manual Second Edition. Cold Spring Harbor Press, NY, 1989) or PCR. It can be prepared by a gene recombination technique.
[0022]
The 5'-side sequence of the oligonucleotide for specifically binding to the target gene may have any chain length as long as it specifically hybridizes. Preferably, an oligonucleotide having 15 or more bases on the 5 'side is used.
[0023]
(XY) The repetitive sequence represented by n (wherein X and Y consist of a combination of adenine-thymine or uracil, or guanine-cytosine, and n represents the number of bases) requires at least 4 bases or more To do. Preferably, an oligonucleotide having 5 to 20 bases, more preferably 20 bases or more is used.
[0024]
The method of labeling the oligonucleotide of the present invention is used for detecting a target gene in Southern blot hybridization, Northern blot hybridization, dot blot hybridization, in situ hybridization, and the like.
[0025]
That is, in the method of the present invention, a target gene is hybridized with an oligonucleotide having a base sequence complementary to the target gene on the 5 ′ side and an AT or AU repeat sequence on the 3 ′ side according to a conventional method, It is used in a method for detecting a target gene by extending a labeled AT or AU repeat sequence at the 3 ′ end of the hybridized oligonucleotide in the presence of a nucleotide label and a DNA polymerase.
[0026]
The labeled oligonucleotide obtained by the method of the present invention is used as a probe for detecting a specific gene as in the conventional method.
[0027]
DNA polymerase is deficient in 5 '→ 3' exonuclease-selective polymerase from DNA derived from Thermus thermophilus, Thermococcus litoralis, Pyrococcus furiosus, etc. Preferably, however, the DNA polymerase from Thermus thermophilus is used.
[0028]
As the labeled nucleotide, a nucleotide labeled with a radioisotope selected from 32P, 3H, 35S or the like or modified with a labeling compound selected from fluorescein, rhodamine, Texas red, etc. is used. Preferably, digoxigenin labeled nucleotides are used.
[0029]
For the nucleotide, when the 3'-side of the oligonucleotide is an AT repeating sequence, labels A and T or U are used, and when the GC is a GC repeating sequence, labels G and C are used.
[0030]
The DNA polymerase reaction is performed at a constant temperature of 30 to 75 ° C. Preferably, it is performed at a constant temperature of 50 to 70 ° C. In addition, the extension reaction can also be performed by repeating the treatment at an annealing temperature of 55 ° C. and an extension reaction temperature of 72 ° C. used in normal PCR. The chain length of the labeled repetitive sequence can be changed by appropriately adjusting the reaction time according to the amount of the target gene or the reaction system to be used.
[0031]
【The invention's effect】
According to the present invention, there are provided a method for labeling the 3 'side of an oligonucleotide, a labeled oligonucleotide, and a method for detecting a specific gene by using the labeling method and the labeled nucleotide.
[0032]
For example, according to the oligonucleotide labeling method of the present invention, the amount of the labeled compound incorporated into the oligo probe is several to several tens of times better than the commonly used TdT, and the Northern blot hybridization method, It is possible to increase the gene detection sensitivity by the Southern blot hybridization method several times to several tens of times compared with the conventional method.
[0033]
In addition, according to the method of the present invention, since an oligo probe can be labeled in a tissue in in situ hybridization, after binding the probe to a target gene without interfering with the tissue permeability of the probe, It is possible to label with high sensitivity. Compared with the in situ polymerase chain reaction (in situ PCR) method, the reaction can be carried out at a constant temperature, so no special equipment is required. Also, since the oligo probe can be labeled independently of the template, the primer design differs from the Primed In Situ Labeling method (Boehringer Mannheim) in which the oligo primer is annealed to the target gene and then the labeled compound is incorporated in a template-dependent manner. The labeling efficiency does not change depending on the condition. Even when the presence of RNA having a specific sequence is clarified, an oligo probe can directly bind to RNA, and thus does not require a reverse transcription reaction. The method of the present invention is suitable for examining the localization of genes in tissues because the target gene and oligo probe remain bound. Hereinafter, the present invention will be described in more detail with reference to examples.
[0034]
【Example】
Example 1: Demonstration that AT repeat sequence self-extends
It was examined using the oligonucleotides described in SEQ ID NOs: 1 to 3 in the Sequence Listing that the oligonucleotide was extended by self-priming. 5 μl of the oligonucleotide described in SEQ ID NO: 1 (1 pmole / μl) or 5 μl each of the oligonucleotides described in SEQ ID NO: 2 and 3 (each 1 pmole / μl) was added to 10 μl in total, dATP and dTTP (2 pmole / μl each, Perkin Elmer) 5 ml, AmpliTaq DNA Polymerase (Perkin Elmer) which is a DNA polymerase derived from Thermus Aquatics, AmpliTaq DNA Polymerase, Stoffel Fragment (Perkin Elmer), Tarms Thermophile DNA Polymerase (TOYOBO), ΔTth DNA Polymerase (TOYOBO), Thermococcus Either 2.5 units (U) of Vent DNA Polymerase (NEW ENGLAND BIOLABS) or Vent (exo-) DNA Polymerase (NEW ENGLAND BIOLABS) derived from Laris and 5 μl of 10 × buffer attached to each enzyme Water was added to the reaction solution added to make the total volume 50 μl and reacted at 30 ° C. for 30 minutes. The reaction product was electrophoresed on a 2% agarose gel, and the length of the oligonucleotide was examined by ethidium bromide staining (FIG. 2). In the mixture of SEQ ID NO: 2 and 3, the primer does not extend, and in SEQ ID NO: 1, the primer extends, so that the primers do not expand by annealing, extension, or frame shift, but may extend by self-priming. It was revealed.
[0035]
Example 2: Addition of AT repeat sequence to oligonucleotide
The addition reaction (tailing reaction) of the AT repeat sequence of the oligonucleotide probe was examined by changing the concentration of the oligonucleotide. The oligonucleotide used has the base sequence set forth in SEQ ID NO: 4 in the Sequence Listing. 10 pmole / μl of the oligonucleotide was prepared in 10-fold dilution series with distilled water, and each 5 μl of 0.2 mM each of dATP, dCTP, dGTP and dTTP (Perkin Elmer), 2.5 mM magnesium chloride, 10 mM Tris-HCl A total of 50 μl containing 45 μl of a reaction solution containing 2.5 U of AmpliTaq DNA Polymerase, Stoffel Fragment (Perkin Elmer), a DNA polymerase derived from Thermus Aquatics, dissolved in a mixture of (pH 8.0) and 50 mM potassium chloride The reaction was carried out at 6 ° C. for 6 hours. After the reaction product was electrophoresed on a 2% agarose gel, the tailing efficiency due to the oligonucleotide concentration difference was examined by ethidium bromide staining (FIG. 3). The lower the oligonucleotide concentration, the better the tailing efficiency of the oligonucleotide. AT repeat sequences of about 2,000 bases at 50 pmole / 50 μl and about 10,000 bases or more at 5 pmole / 50 μl were added.
[0036]
Example 3: Change in tailing reaction over time
The tailing reaction of the oligonucleotide probe was examined over time. The same oligonucleotide as used in Example 2 was used. A total of 50 μl of the above reaction solution (45 μl) added to 10 μmole / μl of oligonucleotide (5 μl) was reacted at 65 ° C. for an arbitrary time, sampled, and stored at room temperature until electrophoresis. After the reaction product was electrophoresed on a 2% agarose gel, the length of the oligonucleotide was examined by ethidium bromide staining (FIG. 4). The length of the oligonucleotide reached a chain length of 500 bases 30 minutes after the reaction.
[0037]
Example 4: Comparison of tailing efficiency of Taq DNA polymerase and TdT
The tailing efficiency of oligonucleotide probes was compared between Taq DNA polymerase and TdT. The same oligonucleotide as used in Example 2 was used. DIG was used as the labeling compound, and tailing efficiency was examined using a DIG nucleic acid detection kit (Boehringer Mannheim) according to the protocol. In the case of Taq DNA polymerase, 50 μl containing 50 pmole oligonucleotide, 0.2 mM dATP, 0.19 mM dTTP, 0.01 mM DIG-dUTP, 2.5 U AmpliTaq DNA Polymerase, Stoffel Fragment was reacted at 65 ° C. for 30 minutes.
[0038]
In the case of TdT, 15 U TdT (Gibco BRL) dissolved in 50 pmole oligonucleotide, 0.2 mM dATP, 0.19 mM dTTP, 0.01 mM DIG-dUTP, 2 mM cobalt chloride, 100 mM potassium cacodylate (pH 7), 0.2 mM DTT ) Was allowed to react at 37 ° C. for 90 minutes. After preparing a 10-fold dilution series for the reaction product, 5 μl of the reaction product was dot-blotted to detect a DIG-labeled oligonucleotide. The result is shown in FIG. The tailing efficiency was more than 10 times higher with Taq DNA polymerase than with TdT.
[0039]
Example 5: Labeling of oligonucleotides after hybridization
After hybridization between the oligonucleotide and the target gene, DIG labeling was performed, and the detection sensitivity of the labeled oligonucleotide was examined. The oligonucleotide used was the same as in Example 1, and plasmid pUC19 was used as the target gene. The labeled oligonucleotide was detected using a DIG nucleic acid detection kit according to the protocol. The plasmid pUC19 was made into a single strand by alkali denaturation and then dot blotted onto a membrane (Immobilon, Millipore). Pre-hybridization was performed at 40 ° C. for 60 minutes, and then hybridization was performed overnight at 40 ° C. with a hybridization solution containing 10 pmol / ml oligonucleotide. Wash the membrane twice with 2 × SSC, 0.1% SDS solution kept at 40 ° C., twice with 0.1 × SSC, 0.1% SDS solution kept at 40 ° C., and then equilibrate with Taq DNA Polymerase buffer Turned into. Subsequently, in a 1 ml reaction solution containing 100 μM dATP, 95 μM dTTP, 5 μM DIG-dUTP, 2.5 U AmpliTaq DNA Polymerase, Stoffel Fragment, 2.5 mM magnesium chloride, 10 mM Tris-HCl (pH 8.3) and 50 mM potassium chloride. The reaction was carried out at 3 ° C. for 3 hours.
[0040]
Example 6: Comparison of the conventional method and the method of the present invention in the detection of murine leukemia virus RNA
RNA was extracted from fibroblast-derived NIH3T3 cells and non-infected NIH3T3 cells infected with murine leukemia virus (MLV) using ISOGEN (NIPPON GENE). The RNA extracted from the infected cells was prepared in a 3-fold dilution series with sterile distilled water not containing DNA and RNase, and was electrophoresed on a 1% formaldehyde-denaturing gel with RNA extracted from non-infected cells for 3 hours. After electrophoresis, the RNA on the gel was transferred to a positively charged nylon membrane (Boehringer Mannheim) overnight at room temperature by a commonly used Northern blotting method. After prehybridization at 65 ° C. for 3 hours with DIG easy hive (Boehringer Mannheim), 65 ° C. with DIG easy vibe containing 3 pmoles / ml 5′-end digoxigenin-labeled oligo probe consisting of the base sequence shown in SEQ ID NO: 5. Hybridized overnight. The filter was washed twice at 2 × SSC at 65 ° C. for 5 minutes and twice at 0.1 × SSC at 65 ° C. for 15 minutes. One piece of filter was washed and air-dried at room temperature, and one piece was ΔTth reaction buffer (10 mM Tris-HCl, pH 8.9, 1.5 mM magnesium chloride, 80 mM potassium chloride, 0.1% sodium deoxycholate, 0.1% Triton X-100, 0.5 mg / ml bovine serum albumin (TOYOBO)) and equilibrated at 65 ° C. for 15 minutes. The extension reaction was performed by immersing in a ΔTth reaction buffer containing 50 U / ml ΔTth polymerase (TOYOBO), 0.2 mM dATP and dTTP, 0.01 mM DIG-dUTP (Boehringer Mannheim) at 60 ° C. for 3 hours. . After completion of the reaction, the filter was washed twice with 2 × SSC at 65 ° C. for 5 minutes and once with PBS at 65 ° C. for 5 minutes. The washed filter was equilibrated with a DIG wash buffer (100 mM maleic acid, 150 mM sodium chloride, pH 7.5, 0.3% Tween 20; Boehringer Mannheim) for 1 minute together with the air-dried filter described above, and a blocking solution (Boehringer Mannheim). And soaked lightly at room temperature for 60 minutes. Next, it was immersed in a blocking solution containing 150 mU / ml alkaline phosphatase-labeled anti-DIG Fab fragment (Boehringer Mannheim) for 30 minutes at room temperature. The filter was washed twice with DIG wash buffer at room temperature for 15 minutes and equilibrated with detection buffer (100 mM Tris-HCl, pH 9.5, 100 mM sodium chloride; Boehringer Mannheim). Detection was performed using a DIG-labeled nucleic acid detection kit (Boehringer Mannheim).
[0041]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram schematically showing a tailing reaction of an oligonucleotide. The oligonucleotide self-anneales at the AT repeat sequence portion, followed by addition of dATP and dTTP (dUTP) by DNA polymerase. This addition indicates that the AT repeat sequence portion of the oligonucleotide is extended and then dissociated, and thereafter self-annealing, extension and dissociation are repeated.
FIG. 2 is a photograph showing that an oligonucleotide is extended by self-priming.
FIG. 3 is a photograph showing the relationship between oligonucleotide concentration and tailing efficiency.
FIG. 4 is a photograph showing the relationship between the length of the added tail and the reaction time.
FIG. 5 is a photograph showing the labeling efficiency of Taq DNA polymerase and TdT to oligonucleotides.
FIG. 6 is a photograph showing the results of detection of murine leukemia virus RNA by the conventional labeling method of labeling digoxigenin at the 5 ′ end of the oligonucleotide and the labeling method of the present invention.

Claims (7)

A target gene, a base sequence complementary to the target gene and a repetitive sequence represented by (XY) n at its 3 ′ end (where X and Y are adenine and thymine or uracil, or a combination of guanine and cytosine) And n represents an integer of 5 or more), and then a nucleotide label constituting the repetitive sequence and a DNA polymerase lacking 5 ′ → 3 ′ exonuclease activity are used. A method for labeling oligonucleotides, wherein the repetitive sequence is additionally extended.   The DNA polymerase is a DNA polymerase derived from a microorganism selected from the group consisting of Thermusaquaticus, Thermus thermophilus, Thermococcus litorlis and Pyrococcus furiosus. The method of claim 1 wherein:   2. The method according to claim 1, wherein the label is a nucleotide labeled with a radioisotope or a nucleotide modified with a labeling compound.   4. The method according to claim 3, wherein the labeling compound is a labeling compound selected from the group consisting of digoxigen, fluorescein, rhodamine, avidin and biotin.   The method according to claim 1, wherein the reaction for addition elongation is performed at a constant temperature of 30 to 75 ° C or 50 to 70 ° C.   2. The method according to claim 1, wherein the reaction for addition elongation is carried out by repeating the treatment at 55 ° C. and 72 ° C. A method for detecting a target gene, comprising: hybridizing a target gene with an oligonucleotide labeled by the method according to any one of claims 1 to 6 having a base sequence complementary to the target gene.

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