JP2647676B2 - Labeling of biotin to nucleic acids using glutaraldehyde as a cross-linking agent - Google Patents

Description

The present invention relates to a novel method for labeling a nucleic acid with biotin, and uses the obtained labeled compound as a probe for nucleic acid hybridization to a diagnostic method in the clinical field. It facilitates the use of.

[Prior Art] In researching genetic problems, it is very important to label a specific nucleic acid and recognize it.
Conventionally, the most widely used method is to use a radioisotope as an element constituting a nucleic acid.However, in consideration of the effect on the human body in the production and measurement of labeled compounds, handling is considered. Strict attention was required and it was troublesome. Thus, the use of non-radioactive probes has begun to be studied, and a method for labeling nucleic acids with biotin has recently been developed. As a method for labeling biotin on a nucleic acid, 2
Enzymatic method by nick translation using '-deoxyuridine triphosphate 5-allylamine-biotin (biotin-11-dUTP) as a substrate and photobiotin [Photobiotin: N- (4-azido-2) as an activating reagent
-Nitrophenyl) -N- (N-d-biotinyl-3-
[Aminopropyl) -N-methyl-1,3-propanediamine] has been developed.

[Problems to be Solved by the Invention] Photobiotin has been increasingly used as a non-radioactive labeling reagent, but lacks stability to light and the like.
The handling is troublesome. The labeling method is complicated in the enzymatic method using biotin-11-dUTP. Therefore, development of a method for labeling using a simple and stable reagent has been desired.

[Means for Solving the Problems] The present inventors have conducted intensive studies to satisfy the above-mentioned demands, and completed the present invention with the idea of binding a stable biotin derivative to a nucleic acid with a crosslinking agent.

That is, a feature of the present invention is a method of labeling a nucleic acid with biotin using glutaraldehyde as a crosslinking agent by reacting the nucleic acid with biotin hydrazide in the presence of glutaraldehyde.

In carrying out the method of the present invention, the reaction is preferably carried out in an aqueous medium in consideration of the solubility of the nucleic acid, and the nucleic acid may be either DNA or RNA.

The present invention can be easily carried out by dissolving a nucleic acid in water and adding biotin hydrazide and glutaraldehyde to react with it, but in the case of double-stranded DNA, prior to the reaction, heat and boil. Cool to a single strand,
Thereafter, it is subjected to the reaction.

After completion of the reaction, the target labeled probe is collected and stored by a conventional method. For example, after completion of the reaction, the labeled probe precipitated in the cooled reaction solution is isolated by a separation method such as centrifugation, washed, dried, and stored. When the reaction is performed quickly, separation and purification can be performed using a Sephadex G-25 column or the like.

After hybridization, the thus obtained probe can be quantified by reacting it with an avidin-bound substance to be measured, such as an enzyme. EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

Example 1 Preparation of Probe (Labeling of DNA with Biotin) 25 μl of an aqueous solution of DNA (1 μg / μl) is boiled at 95 ° C. for 5 minutes, and then cooled on ice for 5 minutes. To this, 12.5 μl of an aqueous solution of biotin hydrazide (10 mg / ml) and 15 μl of an aqueous solution of glutaraldehyde (5%) were added and reacted at 37 ° C. for 10 minutes. Next, 5.83 μl of 3M sodium acetate and ethanol 14
6 μl was added and left at −40 ° C. overnight. The resulting precipitate is centrifuged (small tabletop centrifuge, 10,000 rpm, 10 minutes), washed with 80% ethanol, and dried by vacuum drying in a desiccator under reduced pressure, or dissolved by adding 50 μl of 0.01 M EDTA aqueous solution. Stored at ° C.

Biotin-labeled λ-phage (DNA) according to the above method
Was manufactured.

Reference Example Hybridization of λ-phage DNA Hybridization was carried out by a microtiter plate method which was simple and quantified.

1) Preparation of prehybridization solution 10 × SSC 5.2 ml, 0.06% PVP & 0.06% Ficoll (swelling agent) 2.6 ml, 0.32% bovine serum albumin 2.6 ml, water 2.47 m
and 0.13 ml of denatured bovine thymus DNA (D-1501) (final concentration: 50 μg / ml).

2) Preparation of hybridization solution 5.2 ml of 10 × SSC, 2.6 ml of 0.06% PVP & 2.6 ml of Ficoll,
2.6 ml of 0.32% bovine serum albumin, 2.6 ml of 50% dextran sulfate, 0.13 ml of denatured bovine thymus DNA (final concentration 50 μg / m
l) and 25 μl of the biotin-labeled λ-phage DNA probe solution obtained in Example 1 were mixed.

4) Hybridization Dissolve λ-phage DNA in water, boil it for 5 minutes to make a single strand, dilute with phosphate buffer containing 0.1 M magnesium chloride, add 200 μl to each well, and leave at 4 ° C. overnight. To fix the phage to the wells.

The unfixed λ-phage was removed from the wells, and 200 μl of the above prehybridization solution was added thereto.
Cover the plate with a plate seal, place in a plastic bag and float on a 65 ° C water bath for 1 hour. After the reaction, the prehybridization solution was removed by suction, 200 μl of the hybridization solution was added, and the reaction was allowed to proceed at 65 ° C. overnight. Washing is performed at 65 ° C in 200 µl of 2xSSC and 1 in 30 minutes
And three times with 200 μl of 0.05M phosphate buffer.

5) Quantification of DNA To the hybridized biotin-labeled DNA obtained as described above, 100 μl of avidin-bound alkaline phosphatase solution (manufactured by Bio-Yeda) (a 20000-fold diluted solution) was added, and reacted at room temperature for 2 hours. Washed three times with saline. 100 μl of NADP solution (3 mg dissolved in 20 ml of 0.05 M DEA buffer) was added thereto, and the mixture was reacted at room temperature for 1 hour. Next, an enzyme solution [ADH 0.08 mg / ml in 0.15 M phosphate buffer, ethanol 0.375%, 1-MPMS 1.25 × 10 ⁻⁵ M) was added, and the mixture was added at room temperature for 2 hours.
The reaction was performed for 0 minutes. The reaction was stopped with 50 μl of 0.2 M sulfuric acid.
Chemiluminescence was measured using 10 μl of the reaction solution.

Transfer 10 μl of the reaction solution to a glass tube and add IL / m-
POD mixture (2.4 × 10 ^-4 M / 1 × 10 ^-6 M, 0.4 M carbonate buffer pH 9.
5) 200 μl was added, and the generated luminescence was measured with an alloluminescence reader (wait time 15 seconds, integration time 6 seconds).

The calibration curve of the λ-phage DNA thus obtained was as shown in FIG. 1, and the calibration range was 10 pg to 5 ng.

[Effects of the Invention] The present invention is a novel method for labeling a nucleic acid with biotin, whereby a stable biotin-labeled probe was obtained. When this probe was used to quantify nucleic acids by a chemiluminescence assay, very sensitive measurements could be performed.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a biotin-labeled λ obtained by the method of the present invention.
-Calibration curve of λ-phage DNA detected using a phage DNA probe, the vertical axis indicates the luminescence intensity (count / 6
Second) and the horizontal axis is the amount of λ-phage DNA (pg / well)
Is shown.

Claims (1)

(57) [Claims] 1. A method for labeling biotin on a nucleic acid using glutaraldehyde as a cross-linking agent, wherein the nucleic acid is reacted with biotin hydrazide in the presence of glutaraldehyde.