JPH0591896A - Assay of nucleic acid hybrid - Google Patents

Abstract

PURPOSE:To shorten time for assay of DNA hybrid and improve throughput by subjecting a fragment of the objective gene substance nicked by a restriction enzyme to hybridization with a probe having a length equivalent to the fragment. CONSTITUTION:The objective gene substance 1 is nicked with one or plural restriction enzymes and subjected to hybridization reaction 7 with a DNA having length equivalent to the target DNA. Hybrid body 8 and unreacted DNA probe 9 are separated by electrophoresis, etc., and fluorescent strength emitted from a labeled fluorescent substance 3 of the probe is measured.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for assaying a target genetic material such as DNA or RNA by hybridization with a DNA probe which is a polynucleotide having a sequence complementary thereto. In particular, the present invention relates to a diagnostic method for detecting the presence or absence of an exogenous genetic material such as a virus or bacterium causing a disease, or a genetic material that carries specific genetic information of an organism such as a human, and a mutation.

[0002]

2. Description of the Related Art Either a target nucleic acid sample or a nucleic acid probe which is a DNA or RNA fragment containing a base sequence complementary to the target nucleic acid sample is immobilized on a solid phase, and a hybridization reaction is carried out with this. The conventional method for detecting genetic material is described in Proceedings of the National Academy of Sciences of USA, 80 (1983), pages 278 to 282.
Page (Proc. Natl. Acad. Sci. USA 80 (1983) pp. 27.
8-282).

In this method, first, a DNA fragment sample whose molecular weight has been separated by electrophoresis is transferred and fixed on a nitrocellulose membrane, and then this membrane is immersed in a solution containing a RI (radioisotope) labeled DNA probe. Perform a hybridization reaction. After the reaction, the unreacted DNA probe is washed from the membrane by washing. Thereafter, the DNA probe remaining bound to the membrane is detected by autoradiography to determine the presence or absence of the target genetic material. Also,
In the hybrid body formed by the above-mentioned hybridization reaction, the higher the complementarity of the base sequences, the stronger the binding between the DNA fragment sample and the DNA probe, and the less dissociation occurs even at a high temperature. Therefore, when the DNA fragment sample has complete complementarity with the DNA probe, it is not dissociated, and when the complementarity is incomplete, washing is performed at a temperature at which it dissociates, whereby the DNA fragment sample becomes the DNA probe. Whether or not it has perfect complementarity can be determined. DN if complementarity is perfect
The A probe remains bound to the membrane and is detected, otherwise the DNA probe is washed off the membrane and is not detected.

[0004]

In the above-mentioned conventional techniques, the hybridization reaction between the target genetic material and the nucleic acid probe was carried out on the membrane. In addition, R is used for labeling the nucleic acid probe.
I was using. Therefore, the transfer of the genetic material to the membrane, the hybridization reaction, and the detection by autoradiography required a long time such as overnight. In addition, there is a problem that it is not suitable for an automatic device because the reaction and detection are performed on the film. In addition, there is a problem that workers are exposed to radiation. Moreover, since the completeness of complementarity between the target genetic material and the DNA probe is determined by the washing temperature of the membrane, there is a problem that sufficient accuracy cannot always be obtained.

An object of the present invention is to provide a method for detecting a genetic material suitable for automation, which can carry out a hybridization reaction and detection of a hybrid in a short time. Another object of the present invention is to realize labeling of the nucleic acid probe by means other than RI, to free the operator from radiation exposure, and to eliminate restrictions on the work place without the need to shield radiation. Another object of the present invention is to provide a highly accurate means for determining the completeness of complementarity between a DNA probe and a target genetic material.

[0006]

In order to achieve the above object, in the present invention, the target genetic material 1 is cleaved with one or more restriction enzymes, and a nucleotide sequence complementary to a specific fragment among the cleaved fragments is obtained. And has a length relatively close to the fragment,
A hybrid 8 is formed by forming a hybrid 8 with the single-stranded DNA probe 2 to which a labeling substance other than RI is bound.
The unreacted single-stranded DNA probe 9 is separated, and the hybrid 8 and the unreacted probe 9 are labeled and detected. In order to carry out the hybridization reaction 7 in a short time, the hybrid body 8 is formed in an aqueous solution. Therefore, in order to efficiently form the hybrid body 8,
By the restriction enzyme cleavage reaction 4, the target genetic material 1 is cleaved with a restriction enzyme so as to have the same length as the DNA probe 2. Hereinafter, this is referred to as target DNA5. Further, electrophoresis is used as a means for separating and detecting the hybrid body 8 and the unreacted probe 9. In order to perform detection in a short time, electrophoresis is performed in a capillary. The fluorescent substance 3 is used as the labeled substance, and the hybrid substance 8 is detected by fluorescence detection.

In order to determine the completeness of complementarity between the target genetic material 1 and the DNA probe 2, the electrophoretic medium is provided with a temperature gradient in the present invention. As another means, the electrophoresis gel is provided with a denaturant concentration gradient.

[0008]

Since the hybridization reaction 7 between the target genetic material 1 and the DNA probe 2 is carried out in an aqueous solution, formation of a hybrid can be carried out in a short time. On the other hand, there is a limit to the length of the DNA probe that can be synthesized, and practically the upper limit is about 100 bases. In order to uniquely specify the genome sequence of higher organisms such as humans, a DNA probe of about 20 bases is statistically sufficient. However, in a hybridization reaction between a DNA probe having several tens of bases and a genomic DNA, the recombination reaction between the genomic DNAs proceeds faster because the collision frequency is much higher. Therefore, it is difficult to increase the efficiency of hybridization between the target genetic material 1 and the DNA probe 2. Further, even if the hybrid body 8 is once formed, dissociation of the hybrid body may occur due to the substitution reaction by the genomic DNA bound to a site other than the hybrid formation site.

Therefore, in the present invention, the target genetic material 1 is cleaved with one or more restriction enzymes to fragment the region containing the target site in the target genetic material 1 into shorter fragments. By carrying out the hybridization reaction 7 with the DNA probe 2 having a relatively close length to the target DNA 5 thus obtained, the recombination reaction of the target genetic material and the hybridization reaction 7 with the DNA probe 2 can be performed at the same rate. Can be Moreover, the substitution reaction with the genomic DNA can be substantially ignored.

In the present invention, separation by electrophoresis was used in order to separate and detect the unreacted DNA probe 9 and the hybrid 8 formed in the aqueous solution. Single-stranded and double-stranded DNA fragments of several hundred bases or less can be easily separated by electrophoresis in polyacrylamide gel. In particular, in electrophoresis in a capillary tube having an inner diameter of 100 μm or less, the applied voltage per unit length can be increased and the time required for separation can be shortened.

In order to detect the hybrid 8 of the target genetic material 1 and the DNA probe 2 without using RI, fluorescence detection is used in the present invention. For that purpose, the synthetic DNA probe may be labeled with a fluorophore in advance.

When the electrophoretic medium for separating the hybrid body 8 and the unreacted probe 9 is irradiated with light such as laser light,
When the hybrid body 8 and the unreacted probe 9 pass therethrough, they emit fluorescence. By detecting this fluorescence, the hybrid body 8 and the unreacted probe 9 can be separated and detected. Furthermore, by measuring the fluorescence intensity from the hybrid 8, the amount of the hybrid 8, that is, the amount of the target genetic material 1 can be quantified.

By giving the electrophoretic medium a temperature gradient or by giving the electrophoretic gel a denaturant concentration gradient,
The migration rate of the hybrid differs depending on whether the nucleotide sequences are completely complementary or incomplete. By discriminating this difference, the completeness of complementarity can be determined. That is, the presence or absence of mutation such as point mutation in the target genetic material can be determined.

[0014]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A first embodiment according to the present invention will be described below with reference to FIG. In this example, double-stranded M13mp8RFDNA, which is a kind of phage vector, is used as the target genetic material 1. As the DNA probe 2, 5'-TGGCTGCAGG corresponding to the part sandwiched by the restriction enzymes EcoRI and HindIII in the restriction enzyme recognition site of M13mp8.
TCGACGGATCCCCCGGGAATT-3 '3
One consisting of 0 bases is synthesized and used.

As the labeling fluorescent substance 3, fluorescein isothiocyanate (FITC) which can be efficiently excited by an argon ion laser (oscillation wavelength 488 nm) is used to label the single-stranded DNA. The fluorescent substance 3 is labeled by Japanese Patent Laid-Open No. 61-
The method disclosed in 44353 can be used. This method replaces the phosphate bond at the fluorophore labeling site of the polynucleotide with a phosphonic acid bond having a functional group, and realizes the fluorophore labeling by binding this functional group and the fluorophore. This is a method that can introduce a labeled substance into. In the present example, the labeling position was the phosphorus portion between the third G and the fourth C from the 5'end.

First, M13mp8RFDNA was added at a concentration of 10
mM Tris-HCl (pH 7.5), concentration 10 mM Mg
Hind III in a buffer solution consisting of Cl ₂ , Dithiothreitol at a concentration of 1 mM and NaCl at a concentration of 50 mM at 37 ° C.
Then, the concentration of Tris-HCl (pH 7.5) is changed to 50 mM and the concentration of NaCl is changed to 100 mM, and EcoR I is added for digestion. Due to this two-step restriction enzyme cleavage reaction 4, a fragment consisting of the target DNA 5 and other DNA 6 is produced in the reaction solution.

Next, the DNA probe 2 is added to this reaction solution. After raising the temperature of the reaction solution to 65 ° C. and leaving it for 10 minutes, lowering it to 25 ° C. and allowing it to stand for another 10 minutes, the hybridization reaction 7 is carried out. The above reaction is continuously performed in the same container, for example, in a test tube. By the hybridization reaction 7, a hybrid 8 of the target DNA 5 and the DNA probe 2, an unreacted probe 9, a recombined product 10 of the target DNA and other DNA 6 not involved in the reaction are present in the reaction solution. next,
This reaction solution is injected into the sample injection port 24 in the electrolytic solution tank 12 on the negative electrode side of the electrophoresis device in which a gel is filled in a thin tube having an inner diameter of 100 μm. 6% polyacrylamide is used as the gel. The electrophoretic voltage is set to 50 V / cm, and electrolysis is supplied by the high-voltage DC power supply 14 through the electrodes in the negative electrode side electrolytic solution tank 12 and the positive electrode side electrolytic solution tank 13. The length of the capillary is 20 cm, and fluorescence is detected at a migration distance of 10 cm.

Output 1 as a light source 15 for exciting fluorescence
An air cooled argon laser of 0 mW is used. The laser beam was condensed by the lens 16 and was applied to the gel 11 in the thin tube.
The fluorescence is measured from the direction of 90 ° with respect to the incident beam. The fluorescence is collected by the lens 17 to form a parallel beam, and only the fluorescence component is selectively transmitted by the optical filter 19, and is detected by the photomultiplier tube 20 via the lens 18. The detected signal is processed by the signal processing device 21, analyzed by the data processing device 22, and output by the output device 23. An example of the observed results is shown in FIG. 2. The peak 26 based on the hybrid 8 and the unreacted DNA probe 9 are shown.
It is possible to identify and observe the peak 25 based on. By comparing the intensities of the peak 26 based on the hybrid 8 and the peak 25 based on the unreacted probe 9, the amount of the hybrid 8, that is, the amount of M13mp8DNA can be quantified.

In the present example, the separation of the hybrid body 8 and the unreacted probe 9 was performed by the gel 11 in the thin tube, but flat plate slab gel and columnar gel were also excited by fluorescence in the same manner as described above. It goes without saying that the hybrid 8 and the unreacted probe 9 can be separated and detected by the structure for detecting fluorescence. Further, the electrophoresis can also be performed by using an electrolyte solution in a thin tube containing no gel as a medium to separate and detect the hybrid body and the unreacted probe. Especially, inner diameter 100μm
In electrophoresis in the following thin tube, the applied voltage per unit length can be increased, so that the time required for separation can be shortened.

Furthermore, in this embodiment, the laser beam is applied to the gel in the thin tube, but the position of the light irradiation for exciting the fluorescence does not necessarily have to be limited to the electrophoretic medium. It may be detected in the electrolytic solution. Moreover, the measurement of fluorescence does not necessarily have to be performed during electrophoresis. The object of the present invention can also be achieved by scanning the electrophoretic medium after electrophoresis with light and measuring the fluorescence emitted therefrom. In this example, the case where the fluorophore label is bonded to the phosphorus atom of the nucleotide skeleton was described, but the labeling position is not limited to this, and Science 238 (1987) pages 336 to 341 (Sci
ence, 238 (1987) pp.336-341), it is also possible to directly label the base.

A second embodiment according to the present invention will be described with reference to FIG. The present embodiment is an example in which the target genetic material 1 has a point mutation. In this embodiment, D
M13mp8RFDNA containing mutation as NA sample 1
Was used.

M13mp8RFDNA containing the mutation is
Prepare as follows. First, both ends are the restriction enzymes EcoR I and Hi.
It has a cut end when cut with nd III, except for one part in between, EcoR I and Hi of M13mp8RF DNA
A double-stranded DNA oligomer having the same sequence as the sequence between the cleavage sites of nd III was prepared. The mutation site is
At the 3'-most position of the BamHI site of the (+) chain, C is A
To create. Next, this is M13mp8RFDNA
A clone inserted between the EcoR I and Hind III cleavage sites is prepared, and the presence or absence of the insertion is confirmed by sequencing the DNA base sequence. Cleavage reaction 4 with a restriction enzyme and hybridization reaction 7 are performed in the same procedure as in the first embodiment of the present invention.

Separation and detection of the hybrid body 8 and the unreacted probe 9 are also carried out by using the same apparatus as in the first embodiment, but in this embodiment, the temperature of the gel 11 in the capillary has a gradient. The gel temperature is set to 60 ° C. at the maximum, and is low on the sample injection side 24 and high on the fluorescence detection side, that is, on the positive electrode side electrolytic solution tank 13 side. A heating block 28 for temperature gradient for giving this temperature gradient is arranged so as to enclose the capillary tube for migration, and is controlled by the controller 29 for heating block so as to maintain a constant temperature gradient. FIG. 4 shows an example of the observed results. Figure 4
(a) is the case where the DNA containing the mutation is used as a sample.
(b) is the case of using DNA without mutation as a sample,
The presence or absence of mutation can be detected at the time when each signal appears. The peaks 30 and 32 based on the unreacted probe 9 appear at almost the same time from the start of the migration, and the peak 31 based on the hybrid 8a containing the mutation 27 is different from the peak 33 based on the hybrid 8 not containing the mutation 27. Appears in.

In this embodiment, an example in which the temperature of the electrophoretic medium has a gradient is shown, but the same effect can be achieved by mixing a denaturing agent such as urea in the electrophoretic gel. That is, the concentration of the denaturant in the gel may be low on the sample injection side 24 and high on the opposite side.

In the first and second embodiments described above, an example in which phage DNA is used as the target genetic material 1 is shown. However, the present invention is a genomic DNA of eukaryote such as human. , Or DNA of bacteria, viruses, etc.
Needless to say, it can be applied to.

[0026]

According to the present invention, the hybridization reaction between the target genetic material and the DNA probe in an aqueous solution can be carried out with high efficiency, so that the automation of the DNA hybrid assay is facilitated. Further, since the hybridization reaction and the separation and detection of the hybrid and unreacted probe can be performed in a short time, the assay time can be shortened and the throughput can be significantly improved.
Further, according to the present invention, it is not necessary to use RI for detecting the hybrid body, the restriction of the work place is eliminated, and the risk of radiation exposure of the worker is also eliminated. Furthermore, according to the present invention, a gene mutation in a target genetic material can be detected by detecting the presence or absence of completeness of complementation.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a first embodiment according to the present invention.

FIG. 2 is a diagram showing an example of a result in the first embodiment according to the present invention.

FIG. 3 is a schematic diagram showing a second embodiment according to the present invention.

FIG. 4 is a diagram showing an example of a result in the second embodiment according to the present invention.

[Explanation of symbols]

1 ... Target genetic material, 2 ... DNA probe, 3 ... Labeled fluorescent substance, 4 ... Restriction enzyme cleavage reaction, 5 ... Target DNA, 6 ... Other DNA, 7 ... Hybridization reaction, 8 ... Hybrid body, 8a ... Mutation part A hybrid containing 9
... unreacted probe, 10 ... recombinant of target DNA, 11
... thin tube gel, 12 ... negative electrode side electrolytic solution tank, 13 ... positive electrode side electrolytic solution tank, 14 ... high voltage DC power supply, 15 ... argon laser, 16 ... condensing lens, 17, 18 ... lens, 19 ... optical filter, Reference numeral 20 ... Photomultiplier tube, 21 ... Signal processing device, 22 ... Data processing device, 23 ... Output device, 24 ... Sample injection port, 25, 30, 32 ... Unreacted probe peak, 26, 33 ... Hybrid body Peak, 27 ...
Mutation part, 28 ... Heating block for temperature gradient, 29 ... Controller for heating block, 31 ... Peak of hybrid body containing mutation.

Claims (11)

[Claims] 1. A method for assaying the target genetic material by detecting the presence or absence of hybrid formation with a single-stranded nucleic acid probe complementary to a part of the base sequence of the target genetic material, the target genetic material Cleaving with one or more restriction enzymes, having a base sequence complementary to a specific fragment in the cleaved fragment, and having a substantially equivalent length to the fragment,
A nucleic acid hybrid comprising a process of forming a hybrid with a single-stranded DNA probe bound with a label, and a process of separating, identifying and detecting the single-stranded DNA probe that has not reacted with the hybrid and Assay method. 2. The nucleic acid hybrid assay method according to claim 1, wherein the hybridization reaction is performed in an aqueous solution. 3. The nucleic acid hybrid assay method according to claim 1, wherein the restriction enzyme cleavage reaction and the hybrid body formation reaction are carried out successively in the same container. 4. The nucleic acid hybrid assay method according to claim 1, wherein the hybrid and the unreacted probe are separated by electrophoresis. 5. The nucleic acid hybrid assay method according to claim 4, wherein the electrophoresis is performed in a polyacrylamide gel. 6. The nucleic acid hybrid assay method according to claim 5, wherein the polyacrylamide gel has a columnar shape. 7. A polyacrylamide gel having an inner diameter of 100 μm
The nucleic acid hybrid assay method according to claim 6, which is formed in the following thin tube. 8. The nucleic acid hybrid assay method according to claim 4, wherein the electrophoresis is performed in an electrolytic solution in a capillary having an inner diameter of 100 μm or less. 9. The nucleic acid hybrid assay method according to claim 4, wherein the electrophoretic medium has a temperature gradient. 10. A denaturing agent is mixed in the gel to give a concentration gradient of the denaturing agent in the direction of electrophoresis.
The nucleic acid hybrid assay method according to any one of claims 5 to 7. 11. The nucleic acid hybrid assay method according to claim 1, wherein the labeling substance bound to the single-stranded DNA is a fluorophore.