JPH05273209A - Dna detecting kit - Google Patents

Abstract

PURPOSE:To obtain a kit used in a simple DNA detecting method using a primer or a probe. CONSTITUTION:A kit contains an anti-6-O-methyldeoxyguanosine antibody and is used in the detection of DNA using a primer or probe wherein 6-O- methyldeoxyguanosine is present in a base sequence. The primer, the probe and a reagent for ELISA may be added to the kit other than the antibody. Therefore, the detection of objective DNA can be performed with good sensitivity, for example, by the combination of the anti-6-O-methyldeoxyguanosine antibody with an enzyme labelled secondary antibody.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a simple kit for detecting a target DNA using an antigen / antibody reaction.

[0002]

2. Description of the Related Art Conventionally, in order to obtain a large amount of a desired DNA fragment, a method of producing a large amount of recombinant DNA by growing a host cell into which the recombinant DNA has been incorporated has been used. It was

However, in recent years, a target DNA was obtained from a small amount of cellular DNA by the PCR (polymerase chain reaction) method developed by Saiki et al.
It has become possible to amplify only a region approximately 100,000 times by automatic processing for 2-3 hours [Science]
239, pp. 487-491 (1988)].

In the PCR method, DNA primers (2
(0 to 30 nucleotides) are synthesized and annealed with single-stranded DNA by heat denaturation, and then DNA complementary strands are repeatedly synthesized with a DNA polymerase (for example, heat-resistant tack polymerase) to obtain 2
Amplified doubled, after n cycles is amplified 2 ⁿ times.

The base sequence and base mutation in the DNA region amplified using this can be identified by next labeling a specific synthetic oligonucleotide and using this as a probe. As a labeling method for DNA, a labeling method using a radioisotope is generally used because it requires high sensitivity.

As described above, the PCR method has been in the spotlight in recent years as a method of detecting a specific DNA from an extremely small amount of sample.

When a synthetic oligonucleotide is labeled with a radioisotope as a probe, it is highly sensitive, but it is not recently used because of its safety, stability, facilities, and disposal problems after use. Radiolabels have come to the fore.

[0008]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention As a method for detecting DNA using a non-radioactive label, the present inventors have proposed Japanese Patent Application No.
As described in Japanese Patent No. 112, a method for detecting a sulfonated deoxycytidine added to the 5′-terminal side of a primer or DNA amplified by the primer with an antibody is provided. However, in this method, Japanese Patent Laid-Open No.
As described in JP 1-7469 A, it is necessary to chemically modify cytidine after synthesizing a primer, and it has been desired to develop a method that is simpler in operation.

In view of the above situation, an object of the present invention is to provide a kit for use in a simple method for detecting DNA using a primer or a probe, which does not need to be chemically modified after the primer or the probe is prepared. is there.

[0010]

The present invention will be described in brief. The present invention relates to a kit for detecting DNA, which comprises 6-O in the base sequence.
A kit for detecting DNA using a primer or probe containing -methyldeoxyguanosine, characterized in that the kit contains an anti-6-O-methyldeoxyguanosine antibody.

The primer or probe containing 6-O-methyldeoxyguanosine (hereinafter abbreviated as 6-O-MG) used in the present invention is, for example, a commercially available 6-O-methyldeoxyguanosine phosphoamidite (American Bionetics). (Manufactured by the company) as a coupling unit and can be synthesized by a DNA synthesizer.

The number of 6-O-MG in the primer may be at least one, and its position may be any position that does not interfere with the annealing with the template and the elongation reaction of DNA, but it is usually the 5'end of the primer. Side is desirable. In the case of synthesizing a primer, for example, 6-O-MG may be inserted at the position of deoxyguanosine on the 5'side in the base sequence of the primer to synthesize the primer. A sequence having —O-MG may be added and synthesized. When two or more 6-O-MGs are included in the primer, their positions are not particularly limited, but it is preferable that they are separated from each other. For example, 5'of the primer
When two 6-O-MGs are added to the ends, a sequence such as a poly T sequence that does not easily take a secondary structure and does not prevent annealing with a template should be provided between the two 6-O-MGs. May be inserted.

Also, the number of 6-O-MG in the probe may be one or more, and its position may be a position that does not prevent hybridization with the target, but normally, the position is 5 of the probe. The'side and / or 3'side is desirable. When synthesizing the probe, 6-O-MG may be inserted at the position of deoxyguanosine in the nucleotide sequence of the probe to synthesize the probe, but it is preferable to newly add to the end of the probe and synthesize. When two or more 6-O-MGs are contained in the probe, it is desirable that the positions are separated, as in the case of the primer. For example, 2
The above-mentioned poly T sequence may be inserted between the 6-O-MGs and added to the ends of the probe for synthesis. Moreover, for example, random primers (dN6 to 20, N = A, G, C, T)
You may synthesize | combine the primer which added at least 1 6-O-MG to the 5'side, and may adjust a probe by the random priming method. If 6-O-methyldeoxyguanosine triphosphate is prepared, it is used as a substrate to enzymatically convert DNA fragment into 6 by nick translation method, ordinary random priming method, PCR method, or the like.
-O-MG can be incorporated. The enzyme used at this time is not particularly limited as long as it is a DNA polymerase. It is also possible to prepare a DNA fragment containing 6-O-MG by 3'tailing with terminal deoxynucleotidyl transferase, and these DNA fragments can be used as a probe.

6 in the molecule synthesized as described above
By carrying out, for example, the above-mentioned PCR method using a primer containing -O-MG, the target DNA can be labeled simultaneously with the amplification of DNA. As a method for amplifying DNA using primers, this PCR is used.
Although the method is the simplest and most efficient method, the method for amplifying DNA in the present invention is not limited to the PCR method, and any method can be used as long as it can amplify DNA.

In the molecule prepared by the above method, 6-
The DNA fragment having O-MG is, for example, 6-O.
-By using an antibody that specifically recognizes MG,
It can be detected immunologically. That is, the antibody used in the present invention may be any antibody that does not show antigen recognition property to guanosine but specifically shows antigen recognition property to 6-O-MG, and may be, for example, a polyclonal antibody or a monoclonal antibody.

The anti-6-O-MG antibody is, for example, 6-OM
It can be obtained from the serum of an animal by cross-linking G with a carrier protein and immunizing an experimental animal several times. Alternatively, by cross-linking 6-O-MG with a carrier protein, immunizing a mouse, and fusing the spleen cells with mouse myeloma cells to obtain a hybridoma producing the desired antibody, and using the monoclonal antibody produced by the hybridoma, It doesn't matter.

The anti-6-O-MG antibody that can be used in the present invention is, for example, the hybridoma A created by the present inventors.
MD17-2 [Microtechnology Research Institute, Microbiology No. 12527 (FERM
P-12527)] produced by the anti-6-O-MG monoclonal antibody AMD17-2, also hybridoma AM
D-15-9 [Microtechnology Research Institute Microbiology No. 12528 (FERMP
-12528)] produced by the anti-6-O-MG monoclonal antibody AMD15-9.
-It is possible to detect DNA fragments labeled with MG.

The DNA fragment labeled with 6-O-MG can be used as it is as a probe for dot blot hybridization, Southern blot hybridization, and screening from a library, and it is determined whether or not the target DNA is present. It is also effective when doing. That is, in general, in the analysis of amplified DNA fragments, pattern analysis by electrophoresis or detection with a probe labeled with an isotope or biotin fixed to a filter or membrane is performed, whereas in the present invention, amplified DNA is amplified. Since the fragment has already been labeled with a modified base that can be detected by an antibody, rather than immobilizing the sample DNA, the probe side is immobilized on some solid phase carrier for hybridization, and after washing, the antibody is used. The target DNA can be detected. Examples of solid phase carriers include multi-plates for ELISA, various beads, filters, glass plates, sample tubes, and vials. Immobilization can be adjusted not only by covalent bonding via a suitable spacer, but also by binding of the 5'-terminal biotinylated probe and an avidin-ized solid phase carrier. According to this method, not only immobilization of sample DNA can be omitted, but also immobilization of the probe facilitates recovery after washing, simplification of detection, and simultaneous treatment of a large number of samples becomes possible.

DNA can be easily detected by preparing a kit containing the anti-6-O-MG antibody used in the present invention. Each reagent for ELISA, a primer having 6-O-MG in the molecule, a probe and the like may be contained in this kit. The reagents contained in the kit may be solutions or lyophilized products.

[0020]

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

Reference Example 1 Preparation of conjugate of 6-O-MG and carrier protein 5 mg of KLH (keyhole limpet hemocyanin:
400 μl of A solution (0.1M NaH)
CO ₃ , 2 mM EDTA), then 400 μl
15 mg of BS ₃ (bissulfosuccinimidyl suberate: manufactured by Pierce) dissolved in the solution A was mixed and shielded from light and left at room temperature for 10 minutes. Reaction liquid is Centricon 1
The mixture was placed in a 00 Micro Concentrator (manufactured by Amicon), centrifuged at 1000 g for 30 minutes, and the residue was washed 3 times with 1 ml of sterilized water. The residue is then recovered and 500 μl
Was mixed with 3'amino-6-O-methyldeoxyguanosine dissolved in the solution A, protected from light, and allowed to stand at room temperature for 2 hours. In addition, 3'amino-6-O-methyldeoxyguanosine is 3'amine-on CPG (manufactured by Clontech) and 6
-O-Methyldeoxyguanosine phosphoramidite (manufactured by American Bionetics) was used to synthesize with an ABI 380B DNA automatic synthesizer (1 μmol scale), and half of the crude product was used. The reaction product after standing for 2 hours was recovered by Centricon 100 in the same manner as above to obtain 1.8 mg of the desired bound product.

Reference Example 2 Preparation of Antiserum against 6-O-MG A conjugate was prepared by the method described in Reference Example 1 and used as an antigen, 0.5 mg of which was dissolved in 0.5 ml of physiological saline. An equal amount of Freund's adjuvant was added, emulsified, and then subcutaneously injected into a rabbit. Every 2 weeks, the same amount of the antigen emulsified with incomplete Freund's adjuvant was subcutaneously injected 4 times, 10 days after the final immunization, the whole blood was collected, left at room temperature for 60 minutes, and then centrifuged. By doing so, an antiserum containing an anti-6-O-MG antibody was obtained. The specificity and titer of the antibody were confirmed by methods well known to those skilled in the art, that is, the ELISA method and the dot blotting method.

Reference Example 3 A conjugate was prepared by the method described in Reference Example 1 and used as an antigen. 100 μg of the antigen was dissolved in 0.1 ml of physiological saline and an equal amount of complete Freud's adjuvant was added to emulsify the Balb.
/ C mice were injected intraperitoneally. Antigen 100 after 4 weeks
Only μg was intraperitoneally injected into the same mouse. Three days later, spleen cells were obtained from the spleen extracted from the mouse, mixed with mouse myeloma cells (SP2 / 0) at a cell number ratio of 10: 1, and mixed with 1% in the presence of 50% polyethylene glycol and 20% dimethyl sulfoxide. After allowing to stand for a minute, cell fusion was performed. After serum-free DMEM medium was added and diluted, the supernatant was removed by centrifugation and 10% fetal bovine serum-containing DME was added.
The cells were suspended in M medium and dispensed into a 96-well microtiter plate at 2 × 10 ⁴ cells per well. After that, half the amount of the medium is replaced with HAT medium every 1 to 3 days, and after 10 to 20 days, the culture supernatant in which the fused cells (hybridomas) have grown is collected, and the presence or absence of antibody production is determined by ELI.
By the SA method or the like, three strains of hybridoma producing an antibody against 6-O-MG were selected. Cloning was performed twice on these hybridomas by the limiting dilution method, and two clones of hybridomas producing high titers of antibodies were selected.
7-2 and hybridoma AMD15-9.
It was confirmed by dot blotting that the monoclonal antibody produced by these hybridomas specifically reacts with 6-O-MG. Next, in order to obtain a large amount of these monoclonal antibodies, about 2 × 10 ⁷ hybridomas were intraperitoneally injected into Balb / c mice to form ascites tumors, and ascites were collected 10 days later. Anti-6-O-MG monoclonal antibody was purified by ammonium sulfate salting out and DEAE cellulose chromatography according to the method, and the anti-6-OM produced by the hybridoma AMD17-2 strain was produced.
G monoclonal antibody is AMD17-2 monoclonal antibody, and anti-6 produced by hybridoma AMD15-9 strain
The -O-MG monoclonal antibody was named AMD15-9 monoclonal antibody, respectively. These clonal strains are Hybridoma AMD17-2 and H, respectively.
It is displayed as ybridoma AMD15-9, and is sent to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, respectively.
Deposited as No. 527 (FERM P-12527) and Micromachine Research Institute No. 12528 (FERM P-12528).

Example 1 1-1 Preparation of Primer Containing 6-O-MG 6-O- in the sequence shown in SEQ ID NOS: 1 to 6 of Sequence Listing
The primer having MG is 380BDNA manufactured by ABI.
They were synthesized using an automatic synthesizer and purified by ion exchange chromatography by a conventional method. That is, the primer A represented by SEQ ID NO: 1 in the sequence listing is R manufactured by Takara Shuzo Co., Ltd.
V-primer is a primer in which 6-O-MG is added to the 5'end, and the primer B shown in SEQ ID NO: 2 is RV.
A primer having a heptamer in which a poly T sequence is located between two 6-O-MGs was added to the 5'end of the primer, and the primer C shown in SEQ ID NO: 3 was 6 at the 5'end of the RV primer. -O-MG octamer-added primer, which is the primer D shown in SEQ ID NO: 4.
Is 6-O- at the 5'end of M4 primer manufactured by Takara Shuzo.
The primer E to which MG is added, and the primer E shown in SEQ ID NO: 5 is a primer in which a heptamer in which a poly T sequence is located between two 6-O-MGs is added to the 5 ′ end of the M4 primer. Yes, the primer F shown in SEQ ID NO: 6 has 6-O-MG at the 5'end of the M4 primer.
Is an octamer-added primer.

1-2 Amplification Reaction with 6-O-MG-Containing Primer At the HincII site of pUC18 plasmid (Takara Shuzo), v-Ki-ras probe (Takara Shuzo) 3 was added.
A recombinant having a 70 bp DNA fragment incorporated therein was prepared, and then 1 ng / μl of this recombinant was placed in a 0.5 ml tube (manufactured by Elecon Co.) and 10 μl of the gene amp kit (manufactured by Takara Shuzo) was used. 10x concentration amplification buffer [100m
M Tris-HCl buffer, pH 8.3, 500 mM MgCl
₂ , containing 0.1% (w / v) gelatin], 16 μl of d
NTP mixture (A, G, C, T each 1.25 mM), 20
pmol of primer A and M4 primer, 0.5μ
1 unit of 5 units / μl Taq polymerase was added, and sterile water was further added to make a 100 μl solution. Similarly, R
In addition to the combination of the V primer and the primer D, the primer pairs having the combinations shown in Table 1 were prepared.

[0026]

[Table 1] Table 1 ─────────────────────── RV Primer D RV Primer E RV Primer F Primer A Primer D Primer A Primer E Primer A primer F primer B M4 primer primer B primer D primer B primer E primer B primer F primer C M4 primer primer C primer D primer C primer E primer C primer F ─────────────── ────────

After overlaying 100 μl of mineral oil (manufactured by Sigma) on these reaction solutions, an amplification reaction was carried out by an automatic gene amplifier / thermal cycler (manufactured by Takara Shuzo). The reaction conditions are 94 ° C. for 30 seconds (denaturing step),
2 minutes at 55 ° C (step of primer annealing),
25 cycles of 1 minute at 72 ° C. (synthesis reaction step) were performed. After the reaction, remove the upper layer mineral oil,
Next, 10 μl of the reaction solution was sampled, and 4% Nusibe (Nusive
ieve) GTG agarose gel electrophoresis was performed, stained with ethidium bromide, and the amplified DNA was analyzed. As a result, 49 for all combinations of samples
A single band was confirmed around 0 bp.

Dot blot detection of fragment amplified with primer containing 1-3-6-O-MG Each DN amplified with combination of M4 primer and each primer of primer A, primer B and primer C
The A fragment was added to TE buffer (10 mM Tris buffer p
H8.0, containing 1 mM EDTA) 1 ng, 100 p
g, 50 pg, 10 pg, 5 pg, 1 pg, 0.5 p
g, 0.1 pg / μl, and 1 μl each was spotted on a high bond N nylon membrane (Amersham), and then fixed by UV irradiation. Immerse the membrane in blocking solution (0.2% purified casein-dissolving phosphate buffer, containing 0.1% Twin 20) and let it stand at room temperature for 30 minutes.
AMD1 as described in Reference Example 3 after incubation for minutes
Incubation with 7-2 monoclonal antibody or AMD15-9 monoclonal antibody at 37 ° C. for 30 minutes.
After that, wash the membrane with washing solution A (phosphate buffer, 0.3%
After rinsing lightly with Twin 20), wash 3 times with the same wash solution for 10 minutes, and then conjugate buffer (0.2
30% at 37 ° C. with alkaline phosphotase-labeled anti-mouse IgG antibody (reagent in Chemiprobe kit manufactured by Takara Shuzo) diluted 2500 times with 1% purified casein-dissolved phosphate buffer)
Incubated for minutes. After rinsing the membrane lightly with the washing solution A, the membrane was washed three times for 10 minutes with the washing solution A, and the assay buffer solution (50 mM NaHCO ₃ / Na ₂ CO _{2) was used.
3} in 1 mM MgCl ₂ , pH 9.5 for 5 minutes each 2
Washed twice. After removing excess water from the membrane, the chemiluminescent substrate, 3- (2'-spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl, was prepared to 100 μg / ml with the assay buffer. -1,
2-Dioxetane (AMPPD: manufactured by Tropics)
The solution was added on a membrane with the spotted side facing up, and allowed to stand at room temperature for 20 minutes while protected from light. After removing excess water, the membrane was sandwiched between plastic bags and sealed, and XAR films (manufactured by Kodak Co.) were stacked and exposed for 10 minutes or 30 minutes to detect amplified DNA. The detection limits are shown in Table 2.

[0029]

[Table 2] Table 2 ───────────────────────────────── Primer used Exposure time 10 minutes 30 minutes ── ──────────────────────────────── M4 primer, primer A 10 pg 1 pg M4 primer, primer B 5 pg 0.5 pg M4 Primer, primer C 10 pg 1 pg ──────────────────────────────────

Both AMD17-2 and AMD15-9 monoclonal antibodies sensitively recognized the DNA containing 6-O-MG and were spotted as a control, M4 primer,
No amplification reaction was detected that did not contain 6-O-MG amplified with the RV primer.

Example 2 Preparation of Probe Containing 2-6-O-MG The probes for detecting HPV (human papillomavirus) DNA shown in SEQ ID NOS: 7 to 9 in the sequence listing were synthesized in the same manner as in Example 1, It was then purified by reverse phase chromatography. The base sequence of the probe is HP for isotope label
V detection probe, HPVb16, HPVb18, HPV
It is quoted from b33 (manufactured by Takara Shuzo). That is, nucleotides 4 to 33 of probe A shown in SEQ ID NO: 7 in the sequence listing are H
Sequence identical to PVb16, base numbers 1-3 and 34-3
6 is a sequence containing 6-O-MG newly added, and base numbers 4 to 33 of probe B shown in SEQ ID NO: 8 are HPV.
The same sequence as b18, a sequence containing 6-O-MG with base numbers 1 to 3 and 34 to 36 newly added, and base numbers 4 to 33 of probe C shown in SEQ ID NO: 9 are HPVb3.
It is the same sequence as 3 and a sequence containing 6-O-MG with base numbers 1 to 3 and 34 to 36 newly added.

2-2 Detection of DNA Using 6-O-MG-Containing Probe HPV DNA was detected in cervical cancer precancerous lesion tissue using the above probe. Approximately 10 μg of genomic DNA was obtained from approximately 10 mg of lesion tissue biopsies of 27 cases according to a conventional method. After heat denaturing these at 94 ° C. for 10 minutes, 1 μg of 0.5 ml tube (manufactured by Elecon)
5 μl of 10x concentration amplification buffer and 8 μl
DNTP mixed solution (1.25 mM for each of A, G, C, and T),
10 pmol HPVpF primer (manufactured by Takara Shuzo),
10 pmol HPVp16R primer (manufactured by Takara Shuzo), 10 pmol HPVp18R primer (manufactured by Takara Shuzo), 10 pmol HPVp33R primer (manufactured by Takara Shuzo), 1.25 units of tack polymerase was added, and sterilized water was further added to 50 μl of the solution. did. After overlaying 50 μl of mineral oil on these reaction solutions, amplification reaction was carried out by the above-mentioned automatic gene amplification device. The reaction conditions were as described in Example 1, and 30 cycles were performed. After the reaction, remove the upper layer mineral oil, and then take 10 μl of the reaction solution and add 4% Nucive G.
TG agarose gel electrophoresis was performed, stained with ethidium bromide, and the amplified DNA was analyzed. As a result, a single amplified DNA band was confirmed around 140 bp in 19 of 27 cases (70%). The remaining reaction solution was heat-denatured, spotted in 1 μl each on 3 sheets of high bond N nylon membrane, and then immobilized, to prepare 3 membranes on which each sample was spotted. 6 × SSC (0.9 M NaCl, 9) containing 100 μg / ml heat-denatured herring DNA for each type of membrane.
0 mM sodium citrate, pH 7.0, 5 × Denhardt's, immersed in 0.1% SDS solution, incubated at 37 ° C. for 2 hours (prehybridization), and newly added in the same solution at a ratio of 50 ng / ml. The probe containing 6-O-MG prepared in Example 2-1 was added and incubated at 37 ° C. for 2 hours (hybridization). Membrane 2 x SSC, 0.1% S
0.2xS after washing twice in DS for 10 minutes each at room temperature
Washed twice in SC, 0.1% SDS at 55 ° C. for 20 minutes each. The membrane was treated with a blocking solution, and then the same operation as in Example 1-3 was performed, and the membrane was exposed for 30 minutes. Two
16 types of HPV DNA were detected in all 19 cases in which bands were confirmed by PCR in 7 cases, of which 6 cases were simultaneously detected as 18 type and 4 cases were detected as 33 type simultaneously. In 18 cases in which no band was confirmed in 27 cases, 16, 18, 3
Neither type 3 HPV DNA was detected.
Moreover, this result was in agreement with the result of the experiment using the probe of isotope label.

Example 3 Preparation of DNA Detection Kit As a kit containing anti-6-O-MG antibody, 6-O-
Hybridization was performed using a probe containing MG, and a kit for detecting this with the antibody was prepared as shown in Table 3.

[0034]

[Table 3] Table 3 ─────────────────────────────────── DNA detection kit Detection capacity 4000 cm ² A Agent AMD17-2 monoclonal antibody solution 200 μl Agent B alkaline phosphatase labeled anti-mouse IgG antibody solution 80 μl Agent C purified casein 2 g Agent D AMPPD substrate solution 2500 μl ───────────────────── ───────────────

Agent A was prepared by dissolving AMD17-2 monoclonal antibody in a phosphate buffer containing 0.1% NaN ₃ at a concentration of 2 mg / ml, and Agent B was 5 mg of alkaline phosphatase-labeled anti-mouse IgG antibody. It was prepared by dissolving it in the same buffer solution as the agent A at a concentration of / ml. Also, D agent is AM
PPD was prepared by dissolving it in 50 mM sodium carbonate buffer (pH 9.5) at a concentration of 10 mg / ml. When detecting DNA, C agent of blocking reagent is dissolved in phosphate buffer at 0.2% concentration to prepare a conjugate buffer, and agent A is diluted 1000 times and agent B is diluted 2500 times with the buffer solution. And used as the primary antibody and secondary antibody, respectively. Also, A
MPPD substrate solution is 50 mM sodium carbonate buffer (p
Use it after further diluting it 100 times with H9.5). Using these, the DNA of the membrane corresponding to 4000 cm ² can be detected.

[0036]

INDUSTRIAL APPLICABILITY As described in detail above, the present invention provides a kit capable of simply and sensitively detecting a target DNA.

[Sequence list]

 SEQ ID NO: 1 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence characteristics: 1 6-O-methyldeoxyguanosine Sequence: NCAGGAAACA GCTATGAC 18 SEQ ID NO: 2 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence features: 16- O-methyldeoxyguanosine 7 6-O-methyldeoxyguanosine Sequence: NTTTTTNCAG GAAACAGCTA TGAC 24 SEQ ID NO: 3 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence features: 1 6-O-methyldeoxyguanosine 2 6-O-methyldeoxyguanosine 3 6-O-methyldeoxyguanosine 4 6-O-methyldeoxyguanosine 5 6-O-methylde Xyoxyguanosine 6 6-O-methyldeoxyguanosine 7 6-O-methyldeoxyguanosine 8 6-O-methyldeoxyguanosine Sequence: NNNNNNNNCA GGAAACAGCT ATGAC 25 SEQ ID NO: 4 Sequence length: 18 Sequence type: number of nucleic acid chains : Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence characteristics: 1 6-O-methyldeoxyguanosine Sequence: NGTTTTCCCA GTCACGAC 18 SEQ ID NO: 5 Sequence length: 24 Sequence type : Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence characteristics: 1 6-O-methyldeoxyguanosine 7 6-O-methyldeoxyguanosine Sequence: NTTTTTNGTT TTCCCAGTCA CGAC 24 SEQ ID NO: 6 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence characteristics: 16-O Methyldeoxyguanosine 2 6-O-methyldeoxyguanosine 3 6-O-methyldeoxyguanosine 4 6-O-methyldeoxyguanosine 5 6-O-methyldeoxyguanosine 6 6-O-methyldeoxyguanosine 7 6-O-methyldeoxy Guanosine 86-O-methyldeoxyguanosine Sequence: NNNNNNNNGT TTTCCCAGTC ACGAC 25 SEQ ID NO: 7 Sequence length: 36 Sequence type: Nucleic acid Strand number: Single strand Topology: Linear Sequence type: Other nucleic acid ( Synthetic DNA) Sequence characteristics: 1 6-O-methyldeoxyguanosine 36 6-O-methyldeoxyguanosine Sequence: NTTCATTTTA TGCACCAAAA GAGAACTGCA ATGTTN 36 SEQ ID NO: 8 Sequence length: 36 Sequence type: Nucleic acid Number of strands: 1 Main strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence characteristics: 16-O-methyldeoxyguano 36 36-O-methyldeoxyguanosine Sequence: NTTTGAGAAA CACACCACAA TACTATGGCG CGCTTN 36 SEQ ID NO: 9 Sequence length: 36 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (Synthetic DNA) Sequence characteristics: 1 6-O-methyldeoxyguanosine 36 6-O-methyldeoxyguanosine Sequence: NTTCATTTTG CAGTAAGGTA CTGCACGACT ATGTTN 36

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Ikuno Kato, Inventor Ikunoshin Kato 3-4-1 Seta, Otsu City, Shiga Prefecture

Claims (1)

[Claims] 1. A DNA using a primer or probe containing 6-O-methyldeoxyguanosine in the base sequence.
A kit for the detection of
-A kit for detecting DNA, which comprises a methyldeoxyguanosine antibody.