JPS61162752A - Detection of dna and/or rna - Google Patents

Abstract

PURPOSE:To facilitate the detection of DNA or RNA in a sample, by hybridizing DNA or RNA in the sample using a specific base repetitive configuration as probe. CONSTITUTION:A vivo-derived sample containing DNA or RNA is supported on a carrier. At least DNA among DNA and RNA in the sample is denaturalized to obtain a single-chain DNA or RNA. Then, a probe comprising one chain of DNA or r-RNA having a specific base repetitive configuration existing in DNA or RNA to be detected is converted to a label. The single-chain DNA or RNA obtained is hydridized with the label-converted probe. Then, the presence of DNA or RNA in the sample is detected by checking the label on the carrier.

Description

[Detailed description of the invention] (Industrial application field) The present invention is a method for detecting DNA and/or RNA.

In particular, at least one type of repetitive sequence gene group present in DNA and Metsenger RNA precursor is used as a probe, and this and DNA and/or RNA in the sample are used as probes.
The present invention relates to a method for detecting DNA and/or RNA in a specimen by hybridization with A.

(Prior Art) At present, the only method for detecting DNA or RNA in a specimen derived from a living body with relatively high accuracy is a method that utilizes an antigen-antibody reaction. According to this method, anti-DNA antibodies present in the serum of autoimmune disease patients are labeled with a radioactive iodine compound.

Using this, the DNA concentration in the sample 9, for example, serum can be measured (Davis G, L, et a
l, +Arthritis Rheum, 16.52
-58 (1973). However, since anti-DNA antibodies exist only in the serum of patients with autoimmune diseases, it is difficult to obtain them in large quantities, and it is difficult to make them into reagents from the standpoint of securing supply sources.

A method for detecting a special type of DNA is described in Japanese Patent Application Laid-open No. 1704-1983.
It is disclosed in Japanese Patent Laid-open No. 96 and Japanese Patent Application Laid-open No. 130000/1983. The method disclosed in Japanese Unexamined Patent Publication No. 170496/1980 is to extract hepatitis B virus in serum by its DNA.
This is a method of detection by detecting A. According to this method, the sample serum is first supported on a nitrocellulose filter or the like, and then treated under conditions to denature the DNA and make it a single strand. Next, a hepatitis B virus-derived DNA probe labeled with 32P or the like is allowed to act on this. If hepatitis B virus is present in the sample, the above DNA will be added to each denatured iDNA of the virus.
A probe hybridizes. Therefore, by detecting the label on the carrier using autoradiography or a scintillation counter, it is possible to determine the presence or absence of hepatitis B virus-derived DNA in the sample supported on the carrier, or in other words, the presence or absence of hepatitis B virus in the sample. It is confirmed. Furthermore, Japanese Patent Application Laid-Open No. 130000/1983 discloses a method for determining the type of HLA (human leukocyte antigen), of which five types exist in each individual. This detection method is also performed on the same principle as the above method, and the DNA of known types of HLA is
A probe is used. Hybridization occurs if a single strand of HLA DNA corresponding to the probe DNA is present in the sample. Therefore, the HLA type can be easily determined. As a probe, approximately 1.100 to 1
Since a single strand of DNA with a long base sequence of .,200 is used, it is possible to accurately determine the HLA type. These methods are highly specific analytical methods using DNA probes.

Although special types of DNA can be detected with high accuracy, DNA of unknown origin cannot be detected.

By the way, it has recently been reported by Avis G, L et al. that DNA is present in large amounts in the serum of patients with malignant tumors (Arthritis Rheua+, 16.
52-58 (1973)).

5hapiro B, et al., DN in the serum of patients with malignant tumors
It has been reported that the average amount of A is 2'lOng/la 12, which is higher than the average of 100 ng/mA or less in healthy people (Cancer 51.21
16-2120 (j983)), on the other hand, Leon
S, A, et al. reported that the activity of DNA degrading enzyme (Dljase) in the blood of patients with malignant tumors was low (1 Enrop, J, Cancer 17 (1).
5) 533-538 (1981)), thus [
This is because the activity of 1Fase is low.

When DNA elutes into the blood, in a healthy person, DNAs
DNA is degraded by e, but in the case of patients with malignant tumors, DNA remains and stored in the blood without being degraded, resulting in DNA
A concentration increases. Most of the DNA present in serum is thought to be derived from malignant tumor cells and abnormal cells that have changed from normal cells such as lymphocytes due to interaction with viruses and malignant tumor cells. However, the form of its DNA (
A simple method for detecting not only unique types of DNA, such as hepatitis B virus DNA, whose chain length (whether it exists as two strands or single strands) is unknown, but also general DNA is available. If so, it would be useful for early detection of cancer. Incidentally, methods for detecting malignant tumors that are currently in practical use include blood α-fetoprotein and carcinoembryonic antigen (Carcino-embryonic antigen).
There are methods for detecting fetal proteins such as en; CEA). α-Fetoprotein is a protein synthesized in the fetal body in place of adult albumin, and is absent in healthy individuals. However, α-fetoprotein is present in the fluid of liver cancer patients. Therefore, by measuring α-fetoprotein, it becomes possible to determine the disease course, therapeutic effect, prognosis, etc. of liver cancer. Furthermore, by measuring carcinoembryonic antigen, the presence of cancer in the large intestine, colon, and digestive organs can be detected.

However, these cancer discrimination methods can only detect the above-mentioned types of cancer, and are not a general cancer detection method. Further, since some cancer patients show low values of the above-mentioned α-fetoprotein and carcinoembryonic antigen, it cannot be said that the accuracy as a cancer discrimination method is high.

(Problem that the invention attempts to solve) The present invention solves the above-mentioned conventional drawbacks.

The purpose is to provide a method that allows DNA or RNA to be detected with high accuracy using simple operations.

Another object of the present invention is to provide a method for easily detecting DNA or RNA of unknown origin or shape using a DNA or RNA probe.

An object of the present invention is to provide a method for detecting NA, which allows DNA present in body fluids such as serum to be easily detected using easily available reagents, and can be used as a criterion for determining the presence or absence of a malignant tumor.

(Means for solving the problems) The present invention provides DNA and mesenger RNA precursors (
r-RNA) has a unique base repeat sequence depending on each species, so if this repeat sequence is used as a probe, the probe and DNA or RNA in the sample can be
DNA and/or RNA in a specimen can be easily detected by hybridizing with It was completed based on the inventor's knowledge. Therefore, (1) the method for detecting DNA and/or RNA of the present invention includes: (a) DNA and/or RNA contained in a specimen derived from a living body;
The step of supporting A on a carrier, the DN in the sample
A step of denaturing at least DNA of A and the RNA to obtain single-stranded DNA and/or RNA, (C)
A step of labeling a probe consisting of a base repeat sequence in DNA and/or RNA, cd) the single-stranded DNA and/or RNA obtained in step (c) and the labeled probe obtained in step (C). and (e) detecting the presence of DNA and/or RNA in the sample by detecting the label on the carrier, thereby achieving the above objective.

When detecting DNA or RNA by the method of the present invention, first, a biological sample containing DNA or RNA is supported on a carrier. for example.

When detecting DNA contained in serum, the serum is spotted on a carrier as a specimen, and the DNA contained therein is supported. DNA generally contains adenine (A), respectively.
, guanine (G), cytosine (C) and thymine (T)
A double-stranded chain molecule consisting of a phosphodiester bond of a deoxynucleotide containing one of the four types of bases, A and G are bonded by hydrogen bonds to the DNA base that specifically corresponds to T and C. For example, AA(:CGT
The sequence AG corresponds to the sequence TTCGCATC.

It exists in the form of two strands. RNA is a polynucleotide in which uracil (U) is attached instead of T, and usually exists as a single strand. DNA carried on the above carrier
is denatured into single-stranded DNA by boiling or alkali treatment. Since RNA exists as a single strand, 1 it usually does not require denaturation treatment, but 2 it may be partially bent to form hydrogen bonds, so it is denatured if necessary. Because RNA is degraded by alkali.

The denaturation treatment is performed without grooves, and single-stranded DNA or RNA that has been denatured in advance may be supported on the carrier.

The carrier materials used here include nitrocellulose, nylon, diazobenzyloxymethylcellulose (DBM), and diazomethaminobenzyloxymethylcellulose, which easily adsorb DNA and RNA, and the carriers can be shaped like filter canes, textiles, etc. can be used.

For example, a nitrocellulose filter with a pore size of 0.2-0.45 μm; a nylon filter (e.g. Ze
ta Probesie+5brane HBio R
(manufactured by AD) is preferably used.

In addition, microspheres such as latex can also be used as carriers.

Next, the probe is prepared. As a probe, a single strand and/or
Or r-RNA is used.

Here, the unique base repeat sequence is a base repeat sequence that is unique to nine species present in the DNA of higher organisms.

Since r-RNA is synthesized using DNA as i-type, bases with a sequence complementary to DNA are specifically present therein.

For repetitive sequences in DNA, for example, C, M,
Rubin et al. Nature 284+372 (19
80), C, L Mouck et al. J, Mol
.

Biol, 132.289 (1979), in Human D
Reports on repetitive sequences present in NA. This repetitive sequence in human DNA is called Alu Faa+ily, and consists of about 300 base sequences.* Alu F
There are 300,000 to 500,000 copies of aa+ily in the entire human gene sequence. If there are 300,000 copies in all human genes (3 x 10" base pairs), there will be one Alu Family for every 104 base pair gene. In other words.

The Alu Family accounts for 3% of all human genes. It is unknown whether these 300,000 Alu Family base sequences are exactly the same, but they are extremely similar, and 90 of the 300 base sequences
% or more are considered to be identical sequences.

When detecting DNA in human serum, for example, by the method of the present invention, the above-mentioned Alu Fa
mily is used. Alu Family reduces the molecular weight of DNA obtained from humans to approximately 500 to 10,000 base pairs, denatures it into a single strand, recombines it under certain conditions, and purifies it using methods such as electrophoresis. The length of the repeat sequence varies depending on the species of organism, but if the probe is 15 to 20 bases long, the hybridization described below will occur in a specific and stable state. /or RNA can be detected.

Generally, such probe DNA can be extracted from a living body in a small amount, so it is also possible to prepare a large amount by cloning it using genetic recombination technology. By.

Cloned repeat sequence DNA can be obtained using recombinant DNA techniques using Escherichia coli and the like.

Probe DNA or RNA thus obtained
After being labeled, the DNA is denatured into single-stranded DNA by boiling or alkali treatment, and hydrogen bonds within the RNA molecules are removed. Labeling may be performed after denaturation.

To label 2Usually, “Hl”CI”PI
A labeling method using a radioactive substance containing 2% S, Its 1 , I@l H, etc. is used. In addition, labeling can be performed using fluorescent substances, chemiluminescent substances, immunofluorescent substances, enzymes, and the like. 3 in the labeling method using radioactive substances! N1ck translat using P
ion method is preferably used e N1ck tran
When labeled with sap using the slation method, DNA is labeled at 108 to 10' cp/μg, and the DNA in the sample is isolated by hybridization described below.
can be detected in units of pg (10-''g).The above-mentioned labeling method using fluorescent substances, enzymes, etc.2 For example, biotin is bound to the probe, and the fluorescent substance is added after hybridization as described below. This is a method in which avidin to which a photosubstance or an enzyme is bound is applied to biotin, and avidin is bound to biotin, and detection is performed by luminescent two-color development or the like.

Next, a hybridization solution containing the labeled probe described above is applied to the single-stranded DNA or RNA on the carrier to hybridize the single-stranded DNA or am RNA and the probe. Prior to hybridization, single-stranded DNA or RNA is typically treated with a prehybridization solution. The prehybridization solution and the hybridization solution may be an aqueous solution or a solution containing an organic solvent such as formamide. Examples of compositions of hybridization solutions are shown in Tables 1 and 2, and examples of compositions of hybridization solutions are shown in Tables 3 and 4. Kokote SSC means that NaCl is contained in a proportion of 0.15M and sodium citrate (pH 7.0) is contained in a proportion of 0.015M. Therefore, 3XSSC is Na
C1 is 0.15X 3M, sodium citrate (p
H7,0) is contained in a proportion of 0.015X 3 M.

Table 1 Aqueous hybridization solution (3XSSC
:Table 2 Prehybridization solution containing organic solvent (5XSSC) Table 3 Hybridization aqueous solution (4X5SC) □ (blank below) Table 4 Hybridization solution containing organic solvent (5XSSC) Hybridization solution is almost neutral .

This solution mainly contains salt to adjust the ionic strength, and also contains Denhardt's solution and transfer RNA (tRNA). Denhardt's solution and tRNA contain denatured single-stranded DNA and RNA that are not attached to a carrier. It works to prevent specific binding. In addition to this, the hybridization solution contains a surfactant such as sodium dodecyl sulfate (SDS) at a concentration of 0.1 to 0.5
It may be contained in a proportion of w/v%. Hybridization is carried out at 30-75°C under neutral conditions of pH 6-9.
This is carried out over a period of 2 to 48 hours. 60-70℃ when using an aqueous solution as a hybridization solution
When using melting that partially contains an organic solvent, it is effective to carry out hybridization at 35 to 50° C. under relatively high ionic strength.

When using an aqueous solution, the temperature is within the above range and 5 to 10°C higher than the melting temperature of the hybrid to be produced.
The line can be heated for a few seconds at a moderately low temperature.

When incubated in an aqueous hybridization solution, if a DNA single strand or r-RNA of a complementary sequence corresponding to the probe is present on the carrier, A-T
(or A-, U), C, and -C hydrogen bonds specifically capture all or part of the base sequence constituting the probe, producing a hybrid. After washing, removing, and drying the probe specifically adsorbed on the carrier, the specific base sequence in the sample is detected by detecting the label on the carrier.
In other words, the presence of DNA and RNA in the specimen is confirmed. If the probe is labeled with a radioactive substance, it is detected, for example, by autoradiography. When the dried carrier is kept at -70° C. and subjected to autoradiography, the X-ray film is exposed to light due to β decay of 3 tp, for example.

The area where the probe is captured is exposed to light and turns black.

When the carrier is visually observed, the portion where the specimen was spotted is found to be black on the X-ray film. By measuring the degree of blackening using a densitometer, for example, the DN in the specimen can be determined.
It is possible to calculate the amount of A and/or RNA. The DNA and RNA in the sample can also be quantified by cutting the carrier corresponding to the photosensitive portion into pieces and measuring with a liquid scintillation counter or the like.

According to the method of the present invention, species-specific DNA and/or r-
Since the base repeat sequences in RNA are used as probes, DNA and RNA (especially r-
RNA) can be detected with high accuracy with simple operations. According to the conventional RIA (radioimienoassay) method using an anti-DNA antibody, the detection limit of DNA in a specimen is 25
ng/wβ, but according to the method of the present invention, it is possible to detect even amounts of 0.02 ng (20 pg) or less per 1 spot.

Therefore, if the spot volume per spot is 100 μl, it is possible to detect up to a concentration of 0.2 Tng/la 1.

Using the method of the present invention, DNA in serum can be detected and quantified with high accuracy, and therefore the presence or absence of a malignant tumor can be detected with simple operations and with high accuracy. Furthermore, by detecting and quantifying r-RNA using the method of the present invention, it becomes possible to measure the efficiency of gene transcription from DNA to r-RNA, in other words, the efficiency of gene expression. For example, when measuring the production of insulin in a living body, the expression efficiency of the insulin-producing gene can be checked by measuring r-RNA instead of measuring the amount of insulin, which is the final product. In addition, DN
When detecting or quantifying either DNA or RNA from a mixture of A and RNA, first separate them by ultracentrifugation or use DNase or RNase.
You can disassemble the other part that is not intended for use.

Since the base repeat sequences in the DNA corresponding to the human Alu Family also exist in other organisms, it is possible to detect and quantify the DNA and /*゛ or RNA of the organisms using this sequence. It is known that base repeat sequences in DNA are relatively similar among higher organisms. Therefore, using the method of the present invention, DNA 1 constituting human Alu Faawily
When bovine DNA was detected using this strand as a probe, a hybrid was quantitatively generated under a temperature condition of 35 to 40°C. Since the optimal temperature for hybridization when detecting human DNA is 60-68°C, no hybrid was generated when the temperature condition was 50-60°C. This is due to the fact that the human and bovine base-□ repeat sequences have a certain degree of similarity (homology), and under appropriate temperature conditions a hybrid will form due to hydrogen bonding, but if these conditions are removed, a hybrid will form. This is thought to be due to the fact that it no longer occurs. The method of the present invention can also serve as a standard for determining the close relationship between biological species from the perspective of biological evolution. Furthermore, according to the method of the present invention, it is also possible to diagnose diseases in livestock such as cattle.

(Margin below) (Example) The invention will be explained below with reference to examples.

(A) Extraction of human Alu Family DNA: Human Alu Family DNA was extracted using the method of C, M, Houck et al. (J, Mol.

Btol, +132.289-306 (1979))
It was extracted by the following method based on the following.

DNA extracted from human placenta is reduced to about 500 to 10,000 bases using a homogenizer, and then boiled to denature it.
It was made into a full-stranded DNA. The obtained one [0 in the DNA aqueous solution]
．． NaCl was added to make 3M. By incubating this at 60° C. for 2 days, most of the repetitive sequence DNA was renaturated.

゛Next, 0.5a+H zinc chloride (ZnC1) was added to this solution.
g) and after separating equal volumes of 50+sM sodium acetate buffer (pH 4,5) containing 10mM mercaptoethanol.

Nuclease S1 was added and incubated for 1 hour at 37°C to degrade unrecombined single-stranded DNA. The obtained DNA solution was charged to a hydroxylapatite column chromatography.

After flowing 0.1 M sodium phosphate to flow out and remove single-stranded DNA, double-stranded DNA was eluted with 0.3 M sodium phosphate. The obtained double-stranded DNA was concentrated and purified by ethanol precipitation. 5% polyacrylamide gel electrophoresis was performed using the purified DNA, and 250 to 35
A portion having a chain length of 0 base pairs was cut out from the gel. This was electroeluted to concentrate fragments consisting mostly of 250 to 350 base pairs. As a result of fluorescence analysis of the obtained DNA fragment by ethidium bromide staining, it was found that the main component was 300 base pair DNA.

(B) Labeling of human Alu Family DNA:
Approximately 300 base pairs of human Alu F obtained in section (A)
amily DNA was extracted using the method of Rigby et al. (J, Mo.
1. Biol, 113.237-251 (197
9) Labeled with high activity according to ).

10mM melemercaptoethanol 0mM Tris
-HCI (pH 7,5), 5a+M MgC
1g, 15#I'l dATP, 15μM dGTP
.

15 μM dTTP, 100 μC1(7)α-”P
-0.1 gg of human^lu Fa+wilyDN in an Eppendorf tube (trade name) containing dCTP (having a specific activity of 3,000 Ci/mmol or more)
Added A. DN diluted to 1100n/+ol to this
AseI lμ! , and DNA polymerer-tX 4
) (add 1 μl to make a total volume of 50 μl, 15 μl)
The reaction was incubated for 2 hours at 0.degree. C. and then EDTA was added to a final concentration of 50 mM to stop the reaction. This is extracted with phenol to remove the proteins contained in it.
10mM Tris-HCI (pH 7,5) -I
A 5 ephadex G100 (trade name) column (0
, 50IX15al).

Purified by gel filtration with TE.

□In this way, Alu Fami13T DNA is
Labeled 2 x 10''cpa+ per μg.

'(C) DNA detection: liver cancer patients, gastric cancer patients.

Venous blood of rectal cancer patients, lung cancer patients, and healthy people was collected at 10'm.
1 of each sample was placed in a test tube and allowed to coagulate at room temperature.

Serum was obtained by separation using a centrifuge at 3.00 rpm. 10 μl each of this serum was spotted onto a carrier in a circular pattern on a trocellulose filter (pore size: 0.45 μm+m). After air drying this filter,
Whatman impregnated with 0.5M sodium hydroxide
The filter was placed on a 3M Fl filter paper (trade name) with the spot side up and left at room temperature for 2 to 5 minutes.The filter was then diluted with lM Tris-HCI (pH
7,5) and neutralized for 5 minutes. Next, add the filter 1.
5M NaC1-I M Tris-HCI (pH 7,5
) was transferred onto a hatmann filter paper impregnated with 100% chlorine, left for 5 minutes, and then air-dried at room temperature. The air-dried filter was immersed in a proteolytic solution containing 0.2 μ/all of pronase and 4×SSC (pH 7.0) at 37° C. for 60 minutes. After immersion, the serum spot on the filter was no longer visible. After soaking, the filter was washed with 4XSSC aqueous solution and air-dried.
Then, it was heated and dried at 80° C. for 2 hours.

Labeled Alu Family D obtained in section (B)
NA (labeled probe DNA) was dissolved in 50 μl of TE buffer, boiled at 100° C. for 5 minutes, and then rapidly cooled with ice water to denature. Hybridization solutions shown in Table 3 were prepared using the labeled probe DNA thus obtained. Next, the filter after heat drying and Table 1
The prehybridization solution IQm1 shown in Figure 1 was placed in a heat-sealable plastic bag, sealed, and then incubated at 65°C for 4 hours.

Remove the hybridization solution from the bag.

Instead, 10 mA of the above hybridization solution was added. The bag was resealed and partially incubated at 65°C. Remove the hybridization solution, take out the filter from the bag, and incubate twice with 3X5SC-0,1% SDS solution at 60°C, and then with IX5SC-0,1% SDS solution.
The plate was washed twice with S solution at 60°C to remove non-specific adsorption or bound substances. The filter was dried and subjected to autoradiography using an intensifying screen at -76°C overnight.
The X-ray film was developed the next day.

All areas where cancer patient serum was spotted turn black.
It was determined to be positive. On the other hand, no changes were observed in all areas where serum from healthy individuals was spotted, and the results were determined to be negative.

Separately, a sample containing a known amount of DNA extracted from human placenta was spotted, and the amount of label due to hybridization was measured using a liquid scintillation counter.

The results are shown in Table 5. A calibration curve was created from this result. The amount of label after hybridization of the spot portions of the serum of the above-mentioned cancer patients, patients, and healthy individuals was measured using a liquid scintillation counter. The non-spot area was also measured and subtracted as a blank, and the DNA concentration in the serum was calculated from the obtained value and the above calibration curve. The results are shown in Table 6. From Table 6, blood DNA of cancer patients11
The level is clearly higher than that of healthy people. There was found.

Table 5 Table 6 (A) Extraction of human^lu FamilV DNA: Same as Example 1 (A).

(B) Cloning of human Alu Family DNA: Alu Faaifl obtained in section (A) of this example
Bag old linker was ligated to the y DNA using T4 ligase. This was treated with restriction enzyme Ba-old, and A
A fragment having Ba+iHI restriction enzyme sites at both ends of the lu Family DNA was obtained. This fragment was separated by 5% polyacrylamide gel electrophoresis and then purified by electroelution.

On the other hand, plasmid pBR322 was digested with restriction enzymes Baa+HI
The DNA was treated with and cut into linear DNA1i. By treating this with alkaline phosphatase derived from bacteria, the phosphate group at the 5' end is removed.

The above DNA fragment having the BamHI site was ligated to this using T4 ligase. Through this operation, a library of plasmids in which the above DNA fragment was inserted into the Bam+HI site of plasmid pBR322 was obtained. The library of the plasmid was injected into E. coli 1!
The 118101 strain of E. coli (K12 strain) was transduced, and this was cultured on a 1% agar medium (LB medium) containing the antibiotic ampicillin. Table 7 shows the composition of LB medium.
A nitrocellulose membrane was brought into close contact with this agar medium, as shown in Figure 2, and a portion of the E. coli in the colony was transferred onto the nitrocellulose filter. This filter was brought into close contact with a 1% agar medium (LB medium) containing tetracycline to replicate E. coli, which was then cultured overnight. By screening for colonies that grew on ampicillin-containing medium but could not grow on tetracycline-containing medium, a clone in which the DNA fragment was inserted into plasmid B1322 was obtained. Approximately 30 ampicillin-resistant and tetracytalline-sensitive colonies were randomly selected and inserted into a plasmid using the Sanger method.
Sequence analysis of 0 base pair DNA was performed. As a result, 3 out of 10 had sequences very similar to human repeat sequences described in the following literature @C, M,
Rubin et al.

Nature, 284.372-374 (198
0); W, R, Jelineket al, P
roc, Natl, Acad, Sci, USA
? ? +1398-1402 (1980) One of the clones was named HAF801.

Table 7 LB medium (C) Alu Family from HAF801
DNA isolation: Bacterial colony HAF801 cloned in section (B) of this example was incubated at 5011 g/s+ overnight.
The cells were incubated in 20-1 LB medium containing J. ampicillin.

This culture was transferred to 21 M9 medium containing 50 μg/all ampicillin. The composition of M9 medium is shown in Table 8. The cultured bacteria was grown at 550 n■. D. After growing until the value reached 0.9, 100 μg of chloramphenicol per liter was added to stop bacterial growth, and plasmids were amplified overnight at 37°C. Cool the culture flask and centrifuge the bacteria to 20++M Tria-HCI.
(pH 7,5) Wash with aqueous solution 9 then 25 mjl
The cells were suspended in 50mM Tris-HCI (pH 7,5). Add 5 tsll of lysozyme solution (50 d
110 l1/lI+1 lysozyme in Tris-HCI (pH 8,0) was added and kept on ice for 10 minutes. Add 3I of 0.5M EDTA (pH 8,0) to this.
IIt was added and kept on ice for an additional 10 minutes. Then 2% T
2 tsll of riton X-100 (trade name) was added and slowly stirred. After that, the lysate was heated to 1B at 4℃ for 1 hour.
, 000 rp- to obtain a cell extract containing the plasmid. Solid CsC1 was added to this extract at a final concentration of 0.9 g/mA, and ethidium firomide was added at a final concentration of 1/-β.

The obtained solution was placed in an ultracentrifuge at 40 + 000 r.
The plasmid was purified by centrifugation at pm+ for 20 hours.

The plasmid-containing solution was extracted three times with isopropanol at iM in CsC1 and then dialyzed against TH.
Further purification was performed by ethanol precipitation.

As a result, 9 recombinant plasmids pHAP801 were hydrolyzed with 7.0% pHAF801100 #g + Ram old, and a DNA fragment consisting of approximately 300 base pairs was separated by 5% polyacrylamide gel electrophoresis, and this portion was recovered by electroelution and ethanol precipitation. did. As a result, 6 μg of a DNA fragment containing no vector plasmid was obtained. This DNA fragment was the Alu Fao+ily
This is DNA that has the same sequence as .

This DNA fragment was named HAF-DNA.

(Margin below) Table 8 M9 medium (D) Labeling of 11AF-DNA: Using the HAF-DNA obtained in section (C), labeling was performed in the same manner as in section (B) of Example 1. Result 1 (AF
- Labeled at 3 x 10'' cpm per μg of DNA.

(E) Detection of DNA: The labeled HAF-DNA obtained in section (D) of this example was used as a probe.

DNA in serum was detected in the same manner as in Section (C) of Example 1. Table 9 shows the DNA concentrations in the serum of cancer patients and healthy individuals. The results in Table 9 are in good agreement with the results in Table 6,
It is clear that the repetitive sequence DNA obtained by cloning can also be used as a probe for growth effects.

Table 9 (A) Extraction of human Alu Family DNA: Same as Example 1 (A).

(B) Cloning of human Alu Family DNA: Same as Example 2 (B).

(C) Alu Family from HAF801
Separation of DNA: Same as in Example 2 (C).

(D) Labeling of HAF-DNA: Example 2 (C)
Same as section. .

(E) RNA extraction: guanidine isothiocyanate 6
M, sodium citrate 5-M, mercaptoethanol 0.1 M, and sodium lauryl sarcosylate (
A guanidine isothiocyanate solution containing 0.5% Sarkosyl) was prepared. Dissolve ~30 human cells (approximately 106 cells) in this guanidine isothiocyanate solution, and add 0.4 g of CsC1.
and dissolved. The resulting solution was added to 5.7M CsC
l - 1 mg of 0,1M EDTA (pH 7,5) solution
was added to the centrifuge tube containing CsC1-BDTA and layered on top of the CsC1-BDTA solution. This centrifuge tube was set in a swing rotor, and ultracentrifugation was performed at 20° C. and 35.00 rpm overnight (12 to 16 hours). DNA and proteins derived from human cells form layers and float in a solution depending on their specific gravity.

The RNA was precipitated in the form of a pellet at the bottom of the centrifuge tube.

After removing the supernatant, the pelleted RNA was incubated at 4 x ss.
c 100111 to obtain an RNA solution.

(F) Measurement of r-RNA concentration: R obtained in section (A)
0.22 μl of NA1 solution was spotted onto a nitrocellulose filter in a circular manner. Separately 1.1 μL
2.2 μl, 11 μl, and 22 μl of the RNA solution were spotted to obtain spots with five different concentrations (here, concentration refers to the amount of RNA per spot). This filter was washed with a 4XSSC solution, air-dried, and then heated and dried at 80° C. for 2 hours.

Using this filter and the labeled HAP-DNA obtained in this example (section D>), hybridization was performed according to the method in section (C) of Practical Example 1.

The amount of label was measured using a liquid scintillation rate counter. Separately, r-RNA of known concentration was measured in the same manner, a calibration curve was created, and the concentrations of RNA at the above five concentrations were calculated. R of the RNA solution obtained in Section (E) of this example
The concentration of NA was measured separately by ultraviolet spectroscopy, and the amount of RNA contained in each spot was calculated. The respective results are shown in Table 10.

Table 10 From Table 10, the concentration of r-RN measured by the method of this example is in good agreement with the RNA concentration measured by ultraviolet spectroscopy.
It is clear that r-RNA can be detected and quantified by the method of the present invention.

(Effect of the invention) .

According to the method of the present invention, DNA and/or RNA in a sample can be detected and quantified with high accuracy with simple operations! J<7. In particular, when the 41u Family, which is a repetitive sequence in human DNA, is used as a probe, DNA in human serum can be detected and quantified with high accuracy, and can serve as an effective criterion for determining the presence or absence of malignant tumors. Furthermore, by detecting and quantifying r-RNA using the method of the present invention, it is possible to measure the gene expression efficiency by changing the gene transcription efficiency from DNA to r-RNA.

The DNA used for the probe can be propagated in large quantities by cloning, for example, using a vector plasmid. Therefore, probes as detection reagents can be obtained at low cost, and DNA and/or RNA detection and quantification according to the present invention can be performed at low cost.

Claims (1)

[Claims] 1. (a) A step of supporting DNA and/or RNA contained in a living body-derived specimen on a carrier; (b) at least DNA of the DNA and RNA in the specimen;
(c) labeling a probe consisting of a base repeat sequence in the DNA and/or RNA; (d) 1 obtained in step (b); Double-stranded DNA and/or RNA (c
), and (e) detecting the presence of DNA and/or RNA in the sample by detecting the label on the carrier. Detection method. 2. DNA in which the repetitive sequence probe constitutes the AluFamily gene group in human DNA
The detection method according to claim 1, wherein A is a single strand. 3. Detection according to claim 1, wherein the repetitive sequence probe is a single strand of DNA constituting a gene obtained by cloning at least one member of the Alu family gene group in human DNA. Law. 4. The detection method according to claim 1, wherein the specimen is an aqueous solution or suspension derived from a living body. 5. Labeling of the probe is ^3H, ^1^4C, ^
3^2P, ^3^5S, ^1^2^5I and ^1^3
The detection method according to claim 1, which is carried out using a radioactive substance containing at least one selected from the group consisting of ^1I. 6. The detection method according to claim 1, wherein the probe is labeled using at least one selected from the group consisting of a fluorescent substance, a chemiluminescent substance, and an immunofluorescent substance. 7. The detection method according to claim 1, wherein the probe is labeled using an enzyme. 8. Claim 4, wherein the carrier is a filter, thin film, or fabric made of nitrocellulose, diazomethaminobenzyloxymethylcellulose, or nylon.
Detection method described in Section.