JP3001919B2 - Preparation method and kit for fluorescently labeled DNA - Google Patents

Description

The present invention relates to a method for preparing a fluorescently labeled DNA and a kit thereof.

[Conventional technology]

Conventionally, in order to obtain a large amount of a target DNA fragment, a technique of producing a large amount of recombinant DNA by growing host cells into which the recombinant DNA has been incorporated has been used.

However, recently, PCR developed by Saiki et al.
(Polymerase chain reaction) method,
It has become possible to amplify only a target DNA region from a small amount of cell DNA by about 1,000,000 times by automatic processing for 2 to 3 hours [Science, Vol. 239, pp. 487-491 (198
8)].

In the PCR method, a DNA primer (20 to 30 nucleotides) for the plus and minus strands across the DNA region to be amplified
After annealing with a single-stranded DNA by heat denaturation, the synthesis of a complementary DNA strand by a DNA polymerase (for example, a heat-resistant tack polymerase) is repeated, whereby the DNA is amplified twice in each cycle, 2 after n cycles
Amplified ^n- fold.

The base sequence or base mutation in the DNA region amplified by using this can then be identified by labeling a specific synthetic oligonucleotide and using this as a probe. In addition, DN
As a labeling method for A, a labeling method using a radioisotope is generally used because high sensitivity is required.

As described above, the PCR method has recently been spotlighted as a method for detecting a specific DNA from a very small amount of sample.

[Problems to be solved by the invention]

When a synthetic oligonucleotide is labeled with a radioisotope as a probe, it has high sensitivity, but it has limited safety, stability, equipment, and disposal problems after use. Non-radioactive labels are attracting attention.

Therefore, a method of labeling a primer with a non-radioactive substance has been developed (Japanese Patent Application Laid-Open No. 1-252300). In this case, it is necessary to synthesize a labeled substance for each primer used. There is also a method of using an aminohexyl group for a nucleotide used as a substrate, incorporating the nucleotide into amplified DNA, and then labeling the aminohexyl group to achieve labeling of DNA.・ Biochemistry (Analytical Biochemistry), Volume 157,
199-207 (1986)], in which case the operation of labeling the amplified DNA is complicated.

An object of the present invention is to provide a method for preparing DNA which does not have the above-mentioned problems and a kit therefor.

[Means for solving the problem]

In summary, the first invention of the present invention relates to a method for preparing a fluorescently labeled DNA, which comprises preparing a DNA by amplification, wherein the nucleotide used as a substrate includes a fluorescently labeled nucleotide. Further, the second invention of the present invention is a kit used for preparing a fluorescence-labeled DNA by amplification, wherein the kit comprises a fluorescence-labeled substrate nucleotide and another substrate nucleotide. It relates to a kit for preparation.

As a result, the present inventors have conducted intensive studies and have found a method for labeling DNA that does not require a radioisotope, and have completed the present invention.

As a method for amplifying DNA, the PCR method is simple, and therefore, the present invention will be described below by using the PCR method as an example.However, the present invention is not limited to the PCR method, and the method used for DNA amplification is not limited thereto. Anything is available.

In the PCR method, four types of substrate nucleotides, dATP, dCT
Among P, dGTP and TTP, one of the substrates is replaced with a fluorescently labeled nucleotide (preferably 7 to 75%), and the other is a gene amplification kit containing tack polymerase and an automatic kit commercially available from Takara Shuzo Co., Ltd. The amplification reaction of a specific DNA region is performed using a gene cycler thermal cycler. The substrate nucleotide containing the fluorescently labeled nucleotide may be used as a substrate in the PCR method during the entire PCR cycle, or at any time during the cycle, or may be performed intermittently. Only the late cycle is needed. When a complementary DNA strand is synthesized from a primer by the method described above, it is labeled by incorporating a fluorescently labeled nucleotide, so that a new 5′-terminal fluorescently labeled primer is synthesized and used without using it. Fluorescently labeled DNA can be prepared. In addition, it is not a detection method using an antibody as in the case of labeling with a biotin-avidin system, but can directly detect a target DNA.

In general, in the analysis of amplified DNA fragments, pattern analysis by electrophoresis or detection by a probe labeled and fixed to a filter or membrane by Southern blotting or dot blotting is performed. Is already fluorescently labeled, instead of immobilizing the sample DNA, the probe is immobilized on a solid-phase support for hybridization.After washing, the fluorescence is measured and the target DNA is directly detected. Can be done. Examples of the solid phase carrier include a multiplate for EIA, various beads, a filter, a glass plate, a sample tube, and a vial. Immobilization can be prepared by covalent bonding via an appropriate spacer or by bonding a 5'-terminal biotinylated probe to an avidinated solid support. According to the method of the present invention, not only the immobilization of the sample DNA can be omitted, but also the immobilization of the probe facilitates recovery after washing, simplification of detection, and simultaneous processing of a large number of samples.

〔Example〕

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

Example 1 Synthesis of fluorescein-labeled dUTP Nucleic Acids Research
Research) Vol. 15, pp. 4857-4876 (1987), according to a method modified from 5-iododeoxyuridine (IdU:
From [1]), a protected derivative of 5- (aminoprop-1-ynyl) -deoxyuridine [2] was synthesized. next,
After deprotection of the 3 '-, 5'-hydroxyl protecting group [3], and synthesis with monophosphoric acid derivative [4] by reaction with phosphorus oxychloride, this is treated with N, N'-carbonyldiimidazole (CDI). Activated and reacted with pyrophosphoric acid to synthesize a triphosphate derivative [5]. Finally, after deprotection of the amino group-protecting group, a fluorescent compound fluorescein isothiocyanate (FITC) which easily reacts with the amino group was added to obtain fluorescein-labeled dUTP (FTC-AP-dUTP: [6]).

An example of the above reaction process is shown in FIG. 1 as a process diagram.
In FIG. 1, Tol represents a toluoyl group and Tfa represents a trifluoroacetyl group.

Example 2 Preparation of fluorescently labeled DNA by PCR (2-1) PCR in the presence of fluorescently labeled nucleotide
Amplification 100 ng of human placenta-derived DNA (Clontech) 0.5 m
Transfer to a l l tube (Biovic) and heat-treat at 95 ° C for 10 minutes.
Kit) [Takara Shuzo Co., Ltd.] 10 µ buffer for 10-fold concentration [100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15
mM MgCl ₂ , 0.1% (w / v) gelatin], 16 μm of the dNTP mixed solution containing the fluorescently labeled nucleotide described in Table 1, 1 μm
0μMc-Ki-ras / 61 forward primer, 1μ1
00 μM c-Ki-ras / 61 reverse primer (both from Takara Shuzo) and 0.5 μ of 2.5 units / μ tack polymerase were added, and sterile water was further added to make a 100 μ solution.

Add 100μ mineral oil (Sigma) to the reaction mixture.
After that, an amplification reaction was performed using an automatic gene amplifying device, Thermal Cycler (Takara Shuzo Co., Ltd.). As the reaction conditions, 40 cycles of 94 ° C. for 1 minute (denaturation) → 55 ° C. for 2 minutes (primer annealing) → 72 ° C. for 1 minute (synthesis reaction) were performed.

After the reaction, after removing the upper layer of mineral oil, 10 μl of the reaction solution was taken and 3% Nu-ssieve GTG agarose / 1% Sea-Kem agarose (FMC
Perform gel electrophoresis and DN with ethidium bromide.
A was stained and the amplified DNA was analyzed. As a result, 128b
A single band of p was identified.

(2-2) Detection of Fluorescence Intensity of Amplified DNA About TTP, 20 to 40% of the reaction was performed by substituting FTC-AP-dUTP. To 90 µ of each reaction solution, 9 µ of 3 M sodium acetate, pH 5.2 was added, and further, 200 µ of cold ethanol,-
Add 20 ° C and mix, then let stand at -20 ° C for 1 hour or more. After centrifuging at 4 ° C. at 12,000 g for 5 minutes to dry the precipitate, the precipitate is dissolved in 50 μl of sterilized water to obtain a sample solution.

The 1 μm sample solution was analyzed by an automatic base sequencer (Hitachi Electronics). A peak indicating the presence of fluorescence intensity was confirmed at a position corresponding to a migration degree of 128 bp (second
Figure). When no fluorescein-labeled dUTP was contained, no fluorescence intensity was observed.

In FIG. 2, lane 1 shows the base sequence peak of M13mp18 used as a molecular weight marker, and lane 2 shows FTC
-Shows AP-dUTP-labeled amplified DNA. The horizontal axis indicates the migration time, and the vertical axis indicates the relative intensity of the fluorescence.

Example 3 Detection of Base Difference in Amplified DNA Region Using Immobilized Probe (3-1) Aminopropylation of 96-well Multiplate for Fluorescence 5 μm of 46 μm 3-aminopropyltriethoxysilane
1 μl of absolute ethanol, dispensed 200 μl into each well, sealed and gently shaken overnight at room temperature. After the solvent was removed by pipetting, washing with water was performed three times, and washing with methanol was performed once, followed by drying at 50 ° C. or less.

(3-2) Immobilization of probe on aminopropylated plate 1 ml of dimethylformamide solution of 21 mg of disuccinimidyl suberate in 400 μm of 0.1 M NaHCO ₃ , 2 mM EDTA solution
, Mix 100 μl into each well, shield from light, and
Let stand for 0 minutes. After removing the solution by pipetting and washing three times with water, a probe having an amino spacer added to the 5 'end [Takara Shuzo Co., Ltd., Oncogene Probe for detection of point mutation ras gene (Oncogene Probe) c-Ki
Ras / 61Gly, Ser, Val 3 types)
M NaHCO ₃ , dissolved in 300 μm of 2 mM EDTA solution, per well
50 μl was dispensed, sealed and gently shaken overnight at room temperature. After removing the solution by pipetting, it was washed with water three times and air-dried.

(3-3) Hybridization and detection of sample DNA 300 μl of 10 mM pH 7.4 phosphate buffer (PBS) plus 5% SDS was added per well, and gently shaken at 37 ° C. for 3 hours (Pre-high Hybridization), and after removing the solution, the PCR reaction product precipitated with ethanol and dried in the same manner as in (2-2).
μ, and heat denatured at 95 ° C. for 10 minutes, and dispensed 100 μl into each well in which each probe was fixed.

After gentle shaking at 37 ° C overnight, remove the solution and add
The plate was washed with PBS containing 5% SDS (5 times at 37 ° C. for 15 minutes), and then washed with PBS. Finally, 100 μl of PBS was added per well, and the fluorescence intensity was measured with Fluoroscan II (manufactured by Titertec) (excitation: 485 nm). , Measurement 538 nm).

At the expected position of the K61 Gly type, a fluorescence intensity having a significant difference from the Ser and Val types was confirmed.

〔The invention's effect〕

As described in detail above, the amplification method according to the present invention,
In particular, in the PCR method, direct fluorescent labeling can be performed by using a fluorescent substrate.Furthermore, by fluorescently labeling the sample DNA, hybridization can be performed without preparing a DNA filter for the sample, thereby simplifying detection. Could be provided.

[Brief description of the drawings]

FIG. 1 is a process diagram showing an example of the synthesis of fluorescein-labeled dUTP, and FIG. 2 is a diagram showing detection of the fluorescence intensity of FTC-AP-dUTP-labeled amplified DNA.

Continuation of front page (72) Inventor Ikunoyuki Kato 3-4-1, Seta, Otsu City, Shiga Prefecture Inside Takara Shuzo Co., Ltd. Central Research Laboratory (56) References Prac, Natl. Acad. ScI USA 86 (1989) p. 9178-9182 Bioscience and Industry 47 [10] (1989) p. 1063-1067 (58) Fields surveyed (Int. Cl. ⁷ , DB name) WPI (DIALOG) BIOSIS (DIALOG)

Claims (2)

(57) [Claims] 1. A method for preparing a fluorescence-labeled DNA, wherein a nucleotide used as a substrate in the preparation of the DNA by amplification contains a nucleotide labeled with a fluorescence. 2. A kit for preparing a fluorescently labeled DNA by amplification, comprising at least the following a and b: a. Fluorescently labeled substrate nucleotide b. Substrate nucleotide other than a above