JPH04135498A - Detection of gene - Google Patents

Abstract

PURPOSE:To enable easy and quick detection of gene in high sensitivity by reacting a single-stranded denaturated DNA extracted from a specimen with a labeled gene probe, reacting with nuclease and separating the gene-containing fraction. CONSTITUTION:A double-stranded DNA extracted from a specimen such as human blood containing a hepatitis virus gene, etc., is cut with restriction enzymes and denaturated with an alkali at >=70 deg.C to obtain a single-stranded DNA (A). A hybrid (C) is prepared by reacting the component A with a labeled gene probe (B) labeled with a substance (e.g. biotin) capable of specifically bonding with the component A. The unreacted single-stranded DNA or RNA remaining in the component C is decomposed with S1 nuclease, etc., to obtain a digested mixture (D). The enzymatic reaction of the component D is stopped by adding EDTA, etc., and the product is fractionated and separated by get- filtration, etc., based on the molecular weight difference to obtain the objective fraction (E). The objective gene can be detected by detecting the labeled part (e.g. biotin) of the component E.

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] (Industrial Application Field) The present invention relates to a gene detection method for specifically detecting a specific gene sequence present in a sample.

(Prior Art) Genetic information encoded by genes (DNA) is expressed as proteins such as enzymes via messenger RNA, and living organisms exist as a collection of various compounds produced by the actions of these proteins. The total number of such genes is said to be 50,000 to 1,100,000 in humans, and some abnormality or change (for example, deletion, duplication, etc.) may occur among them.

In that case, the characteristics, type, amount, etc. of the produced proteins change, resulting in an imbalance in the biological system and disease. Therefore, conversely, by detecting the gene causing the disease, it becomes possible to identify and prevent the disease. With recent advances in genetic engineering, diagnosis based on the genes themselves (called genetic diagnosis)
has become possible.

Considering the mechanism of gene expression, it is presumed that most changes at the biochemical level are preceded by genetic changes. Therefore, genetic diagnosis allows for diagnosis and prediction prior to the phenotypic change of the disease (ie, before the onset of the disease, during the latent period of the disease, or at the very early stage).

Furthermore, since the genes of all cells within a living body are the same, the organ or tissue that is the target of analysis regarding hereditary diseases cannot be specified. In particular, fetuses can be diagnosed simply by collecting amniotic fluid from a pregnant woman and examining fetal cells floating in the fluid, which is a very important diagnostic method.

Generally, genetic diagnosis is performed as follows.

First, a gene is extracted from a sample and, if necessary, cut with an appropriate restriction enzyme. Next, this gene is subjected to electrophoresis, and then a Southern blot is performed.

Next, a gene probe (usually labeled with a radioactive isotope) corresponding to the gene of interest is hybridized, followed by autoradiography at low temperatures and the presence or absence of the gene of interest is confirmed on X-ray film. do.

(Problems to be Solved by the Invention) However, in the above method, it takes at least 2 to 30 days to detect a gene, and the measurement operation is also quite complicated.

Therefore, an object of the present invention is to provide a simple and highly sensitive gene detection method that can obtain results in a short time.

[Structure of the invention]

(Means for Solving the Problem) The gene detection method of the present invention includes a step of extracting a gene from a measurement sample, a step of denaturing the extracted gene to make it a single strand, and a step of making the single strand of the gene. A step of reacting with a labeled gene probe that specifically binds to the gene to be detected; and a step of reacting the unreacted single-stranded gene and the unreacted gene probe with a method that specifically acts on single-stranded DNA or RNA. The method is characterized by comprising the following steps: a step of digesting the mixture with a nuclease, a step of separating the mixture after being digested with the nuclease based on a difference in molecular weight, and a step of detecting a labeling agent in the separated fractions.

Hereinafter, the gene detection method of the present invention will be explained in more detail with reference to the drawings.

FIG. 1 is a diagram illustrating one embodiment of the gene detection method of the present invention. In this method, first, sample DNA I is extracted from a sample according to a conventional method.

This sample DNA 1 is double-stranded DNA. Next, the sample DNA 1 is digested with restriction enzymes as necessary, and then single-stranded DNA 2 is cleaved, for example, by alkaline denaturation.
Make it. At this time, the reaction is preferably carried out at 70° C. or higher in order to prevent self-hybridization between the double-stranded DNAs.

Next, the solution containing the DNA was neutralized and the above denaturation was removed by 70
If the temperature was above ℃, cool to 50 to 60℃, then
A gene probe 3 complementary to the target gene is added and reacted with the single-stranded DNA 2. Here, "target gene" means a gene to be detected. At this time, if the base sequence of the target gene is present in the single-stranded DNA 2, a hybrid 5 is formed. The gene probe 3 may be made of RNA or DNA; if the probe is RNA, it may be a DNA-RNA hybrid;
A, a DNA-DNA hybrid is formed. Furthermore, the gene probe 3 is previously labeled with a suitable labeling agent 4, such as biotin. The labeling agent 4 used here is not particularly limited as long as it is commonly used, and the optimal one is selected in consideration of the detection means, device, sensitivity, etc. Further, the method for immobilizing the labeling agent onto the gene probe is not particularly limited, but it is preferable to use an appropriate crosslinking agent in order to form a stronger bond.

After forming hybrid 5, single-stranded DNA or R
An appropriate amount of a reaction solution of a nuclease that specifically acts on NA, such as S1 nuclease, is added and reacted to degrade unreacted single-stranded DNA and gene probes.

Ammonium acetate and ethylenediaminetetraacetic acid (ED
After stopping the enzymatic reaction by adding TA) solution, hybrid 5 and the nucleic acid obtained by decomposing the unreacted DNA or RNA are separated based on the difference in molecular weight.

This separation is preferably performed by subjecting the obtained reaction solution to gel filtration. At this time, the gel filtration agent used for gel filtration is not particularly limited, and commonly used commercially available gel filtration agents can be used.

Further, any commonly used gel filtration method may be used, but in order to shorten the time required for gel filtration, it is preferable to use span column type centrifugation in combination.

Finally, the presence or absence of the labeling agent in the separated fractions is confirmed using an appropriate method depending on the labeling agent used. As mentioned above, when biotin is used as a labeling agent, first, a goat anti-biotin antibody labeled with an antibody is added and reacted, and after washing, a substrate for the labeled antibody is added to check the presence or absence of an enzyme reaction. By measuring, the presence or absence of biotin can be confirmed.

As a result of detection of the labeling agent, if the presence of the labeling agent is confirmed in a fraction corresponding to the molecular weight of the hybrid, it can be determined that the target gene is present in the sample. Conversely, if the labeling agent is detected only in the nucleic acid fraction, this means that the target gene is not present in the sample.

The reaction conditions such as reaction time, temperature, p+1, buffer solution, etc. in the method of this invention vary depending on the test gene and the type of the corresponding gene probe (its base sequence, length, etc.), and the optimum conditions may vary depending on each case. It is preferable to set

(Function) When the genetic DNA of the test sample contains the base sequence of the target gene, a probe complementary to the target gene is added and reacted to form a hybrid and become double-stranded. Conversely, if the gene of the test sample does not contain the base sequence of the target gene, hybridization does not occur and the gene remains single-stranded. Under these circumstances, when a nuclease that specifically acts on single-stranded DNA or RNA, such as S1 nuclease, is applied, a double-stranded hybrid is generated if the target gene is present in the test sample. Otherwise, if the target gene is not present, all DNA or RNA will be degraded into nucleic acids. Therefore, if a solution containing these is separated based on the difference in molecular weight by gel filtration or the like and the labeling agent labeled on the probe is detected, the target gene will be detected only when the target gene is contained in the test sample. The presence of hybrids can be detected in a fraction other than the fraction containing nucleic acids in the sample.

(Example) The present invention will be explained in detail below.

Example 1 Detection of Hepatitis Virus (HBV) Gene in Human Serum Using blood samples from normal people and hepatitis patients, HBV was detected in serum according to the following procedure.

First, a gene (double-stranded DNA) was extracted from human serum and treated with the restriction enzyme PslI according to a conventional method. A general treatment method using a restriction enzyme is described, for example, in "Clinical Examination J, Vol. 32, No., April 19th, page 19H." Next, the gene cut with this restriction enzyme is Single-stranded DNA was prepared by alkali denaturation.

Next, after neutralizing the alkali, an HBV RNA gene probe (manufactured by Daiichi Kagaku Co., Ltd.) was added and reacted at 60° C. for 1 hour. This RNA gene probe is a commercially available photo
Label with biotin in advance using a biotinylation kit.

After the reaction, add 100 U of S1 nuclease (manufactured by Sigma).
/- was added and incubated at 45°C, and after 30 minutes 4M ammonium acetate and O,II
The reaction was stopped by adding IM EDTA.

Immediately after the reaction was stopped, the mixture was centrifuged for 5 minutes at 3000 ppm using Dispo Column PD10 (manufactured by Pharmacia), and only the genes forming double strands were collected as a filtrate.

Finally, a 100-fold dilution of alkaline phosphatase-labeled goat anti-biotin antibody and p-nitrophenylphosphate (PNPP), a substrate for alkaline phosphatase, were added.
was added and incubated at 37°C for 30 minutes. Both the anti-biotin antibody and PNPP used here are manufactured by Cymet.

After the enzymatic reaction was completed, the absorbance of the reaction solution at 420 nm was measured using a spectrophotometer. The results are shown in Table 1. In this example, commercially available reagents other than S1 nuclease were used without purification, and all water used was ion-exchanged water.

In Table 1, #1 to 7 are blood samples from hepatitis patients, and #8 to 10 are blood samples from normal people. As is clear from Table 1, the method of the present invention was able to detect HBV genes only from hepatitis patients.

Furthermore, when the detection sensitivity of the gene detection method of the present invention was tested using a standard HBV gene, detection was possible from about 1 pg of DNA. This is approximately the same sensitivity as conventional detection methods that use radioisotope-labeled gene probes.

Furthermore, the total time required for detection was approximately 2 hours, which was significantly reduced compared to conventional detection methods.

[Effects of the Invention] Conventional gene detection methods using radioisotope-labeled gene probes require exposing a film for a long time to detect trace amounts of radioisotopes. However, in the detection method of the present invention, there is no need to expose the film for a long time, and each step is completed in a short time. Therefore, according to the detection method of the present invention, genes can be detected simply and in a short time.

Furthermore, in the detection method of the present invention, hybrids are formed only when the target gene is present, and all others are degraded into nucleic acids. Therefore, there is a large difference in molecular weight, and it is possible to accurately separate the molecules by gel filtration or the like and perform accurate measurements with a high S/N ratio.

[Brief explanation of drawings]

FIG. 1 is a diagram illustrating one embodiment of the gene detection method of the present invention. 1... Sample DNA, 2... Single-stranded DNA, 3...
Gene probe, 4... labeling agent. Applicant's agent Patent attorney Takehiko Suzue] 3

Claims (1)

[Claims] (1) A step of extracting a gene from a measurement sample, a step of denaturing the extracted gene to make it a single strand, and specifically binding the single stranded gene to the gene to be detected.
a step of reacting with a labeled gene probe; a step of digesting the unreacted single-stranded gene and the unreacted gene probe with a nuclease that specifically acts on single-stranded DNA or RNA; A gene detection method comprising: separating the resulting mixture based on a difference in molecular weight; and detecting a labeling agent in the separated fractions.