JPH0666791A - Nuclei acid base detector - Google Patents

Abstract

PURPOSE:To highly sensitively and safely detect a nuclei acid component to be labeled by introducing a specific element to a nuclei acid or nuclei acid mixture in advance. CONSTITUTION:A nuclei acid sample is labeled in advance with an element which is not contained in natural nuclei acids or another element which is contained in natural nuclei acids by an extremely small amount. For example, nucleotide in which the oxygen element at the phosphoric ester bonding part is substituted by sulfur atoms is biochemically bonded in advance with the ends of cut pieces of deoxyribonuclei acid. When a labeled nuclei acid solution in a sample solution tank 1 is transferred to a filler 14 together with an eluent in an eluent tank 15 by means of a liquid pump 2, the cut pieces of nuclei acid successively form segregated zones 16 in order from that having the smallest mass number and the zones 16 move to a labeled element detecting section 9. Therefore, the zones 16 can be continuously detected at the section.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a nucleic acid base detection apparatus, and more particularly to a nucleic acid base detection apparatus suitable for improving the accuracy of a nucleic acid detection method.

[0002]

2. Description of the Related Art Conventional nucleic acid detection methods use either one of the following two methods, but each of them has the following drawbacks. That is, one is that a nucleic acid labeled with a radioisotope such as ³² P or ³⁵ S is added to an X-ray film, G.
M. This is a method of detecting with a radiation detector such as a tube or a scintillation counter. Although this method has high sensitivity (<0.1 pg), it is inconvenient to handle because of safety regulations. On the other hand, a method of irradiating a nucleic acid labeled with a fluorescent dye such as ethidium bromide, acridine orange, and proflavin with ultraviolet rays to generate fluorescence and detecting the fluorescence (for example, protein / nucleic acid / enzyme separate volume: fluorescence measurement The principle and application to biological systems, pp.206-231) are safe but lack sensitivity (1/1 of the former method).
It is not practical when the amount of nucleic acid is extremely small (<1 ng).

[0003]

The above-mentioned prior art has a problem in safety in the method of detecting a nucleic acid labeled with a radioisotope with a radiation detector, and the nucleic acid labeled with a fluorescent dye is detected using ultraviolet rays. The method had a problem of poor sensitivity.

An object of the present invention is to provide a nucleic acid base detection apparatus using a highly sensitive (~ 1 pg) and highly safe nucleic acid detection method.

[0005]

SUMMARY OF THE INVENTION The present invention provides a nucleic acid or a mixture of nucleic acids to be detected which is not previously contained in the natural nucleic acid or has a very small content, and the introduction of the nucleic acid results in the chemistry of the entire nucleic acid. It is characterized in that it is labeled with a specific element that does not significantly change its biological properties, the specific element in the nucleic acid fragment separated by molecular weight is identified, and the nucleic acid fragment is detected by this.

As the above-mentioned specific element, for example, S, B
non-metallic elements such as r and I, Ag, Au, Pt and O
There are metallic elements such as s and Hg.

Atomic absorption spectrometry, plasma emission spectrometry, or mass spectrometry is used as a method for identifying these specific elements.

[0008]

According to the present invention, by introducing such a specific element into a nucleic acid or a nucleic acid mixture, the nucleic acid can be separated even after various chemical operations on the nucleic acid. By extracting the component and identifying the specific labeling element,
The labeled nucleic acid component can be detected with high sensitivity and safely.

[0009]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT An embodiment of the present invention will be described below with reference to FIGS.

In this example, the nucleic acid sample was previously
Label with an element that is not contained in the natural nucleic acid or has a very small content. For example, as shown in FIG. 1, instead of a normal substrate, a nucleotide (dideoxyadenosine-3- [α-S] phosphate) in which the oxygen element in the phosphate ester binding site is replaced with a sulfur atom is deoxyliponucleic acid (DNA). Biochemically bound to the end of the fragment.

FIG. 2 shows the entire apparatus of an embodiment to which the present invention is applied when molecular weight separation is carried out by electrophoresis. The apparatus includes a sample solution tank 1, a liquid feed pump 2, a buffer solution tank 3 for electrophoresis, an acrylamide gel (hereinafter referred to as a gel) 4 for electrophoretic separation, a labeling element detection unit 9, and a buffer solution tank 12 for eluting an electrophoretic separation zone. Is the main component.

Next, the detection procedure will be described. The labeled nucleic acid sample from the sample solution tank 1 was placed on the negative electrode side of the gel 4 by the liquid feed pump 2 so that both ends of the gel 4 were in contact with the electrophoresis buffer tank 3 and the high voltage DC power supply 13 was applied to 50V.
Electrophoresis at a voltage of about / cm. Then, the nucleic acid components having the same molecular weight move toward the positive electrode from the negative electrode while forming the electrophoretic separation zone 6, and migrate with a mobility substantially inversely proportional to the logarithm of the molecular weight. On the other hand, through the window 7 provided in the electric insulating plate 5 near the negative electrode end rather than the positive electrode end of the gel 4, the same composition as the gel solution from the migration separation zone elution buffer tank 12 is obtained. The buffer solution is allowed to flow slowly, and a voltage that does not disturb the potential gradient in the migration direction is applied to the buffer solution by the low-voltage DC power supply 11. As a result, the separated electrophoretic particles are collected as the eluted electrophoretic separation band 8,
It is sent to the labeling element detection unit 9. In the labeling element detection unit 9,
For example, an atomic absorption spectrometer (sensitivity ~
ppt), plasma emission spectrometer (sensitivity to ppb), iodine and sulfur used in this example are oxidized to sulfur dioxide (in the case of sulfur), and then mass spectrometer (sensitivity <ppb) Thus, since the labeling elements can be identified respectively, it is possible to detect the labeled nucleic acid fragments that are eluted in order from the one having the smallest molecular weight.

FIG. 3 shows the whole apparatus of another embodiment to which the present invention is applied when molecular weight separation is carried out by a high speed gel filtration method or a liquid chromatography method. The apparatus mainly comprises an eluent tank 15, a liquid feed pump 2, a sample solution tank 1, a packing material 14 which is a packing material for high-speed gel filtration agent or ion exchange chromatography, and a labeling element detection part 9. The detection procedure is as follows: the labeled nucleic acid solution in the sample solution tank 1 is used together with the eluent in the eluent tank 15.
When it is transferred into the packing material 14 by the liquid feed pump 2, the nucleic acid fragments form a separation band 16 in order from the one having the smallest mass number due to the molecular sieving effect or the distribution effect of the packing material 14. Move to the side. These separation bands 16 can be continuously detected by the labeling element detection unit 9 by the same method as described in the previous embodiment.

[0014]

According to the present invention, the following effects can be obtained as compared with the conventional nucleic acid detection device.

(B) A highly safe labeled substance can be used with almost no change in the chemical or biochemical properties of nucleic acid, and safe and highly sensitive detection can be performed.

(B) Since the molecular weight separation operation is performed in a flow type, the measurement time can be shortened and the amount of the sample to be measured can be reduced as compared with the conventional electrophoresis method.

[Brief description of drawings]

FIG. 1 is an explanatory diagram showing an example of labeling with a specific element according to the present invention.

FIG. 2 is a configuration diagram showing a configuration of an apparatus according to an embodiment of the present invention.

FIG. 3 is a configuration diagram showing a configuration of an apparatus according to another embodiment of the present invention.

[Explanation of symbols]

 1 sample solution tank 2 liquid feed pump 3 electrophoresis buffer tank 4 acrylamide gel for electrophoretic separation 5 electrical insulating plate 6 electrophoretic separation zone 7 window 8 eluted electrophoretic separation zone 9 labeled element detector 11 low-voltage DC power supply 12 electrophoretic separation zone Elution buffer tank 13 High-voltage DC power supply 14 Filler 15 Eluent tank 16 Separation zone

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI Technical display location C12N 15/00 8931-4B (72) Inventor Jiro Tokita 1-280 Higashi Koigakubo, Kokubunji, Tokyo Hitachi Central Research Laboratory (72) Inventor Keiichi Nagai 1-280 Higashi Koigakubo, Kokubunji City, Tokyo Hitachi Central Research Institute Co., Ltd. In-house

Claims (1)

[Claims] 1. A nucleobase detection device for labeling a nucleic acid, and using a detection means to detect a nucleic acid fragment whose molecular weight has been separated by electrophoresis, liquid chromatography, or high-speed gel filtration, in a natural nucleic acid. Is not contained or has a very small content, and the chemical or biochemical introduction of a specific element that does not significantly change the chemical properties of the entire nucleic acid due to its introduction is used as a sample. A nucleic acid base detection apparatus, characterized by comprising means for identifying the specific element in the nucleic acid fragment whose molecular weight has been separated by atomic absorption spectrometry, plasma emission spectrometry or mass spectrometry.