JPH0561920B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for quantifying certain nucleic acid molecules using sandwich hybridization or solution hybridization, in particular genes and/or corresponding genes.
This invention relates to a method for quantifying the degree of amplification of RNA molecules and reagents used therefor. The copy number of individual genes in the genome is usually constant. In some cases there is only one gene per haploid genome, and in other cases there may be several. Copy number can change under certain circumstances. Amplification of certain genes, for example, has been found to be associated with the development of cancer. It is also known that external factors such as pharmaceuticals and metals can amplify genes. For disease development, incomplete or increased expression levels of genes, such as oncogenes,
In other words, the amount of messenger RNA in cells is very important. Some genetic diseases or other disorders result from an increase in the number of certain chromosomes, whereas some genetic diseases result only from the duplication of a single recessive gene. In all such instances it is important to determine the number of chromosomes or genes present. The number of DNA molecules, for example the degree of amplification of a given gene, is currently determined by digesting the extracted DNA to be investigated with restriction enzymes and by agarose gel electrophoresis. It is determined by separating nucleotide fragments according to the Next, a single strand
The DNA is transferred to a nitrocellulose filter and fixed, where hybridization is performed using the gene to be investigated or a portion of the gene as a probe. The results are obtained by autoradiography (Southern, Journal of Molecular Biology, Vol. 98, 503).
- p. 517 (1975)”). The amount of DNA in the cells from each parallel analysis is the same. The intensities of the hybridization bands, or signals, are compared and the ratio between copy numbers of the gene under consideration in the test sample is estimated. This method gives only approximate results. Similarly, RNA is measured using the Northern or Dot-plotting method. These methods are quantitatively very imprecise (Thomas, Methods in Enzymology).
100, pp. 255-266 (1983)). Known methods such as Southern plotting and Northern plotting are time consuming and difficult to perform. Since they only give approximate results, their diagnostic value is questionable in cases where it is important to know a certain number of nucleic acid molecules per certain unit, such as a cell. US Patent No. 4,486,539 and UK Patent Application No.
The Sand-Germany hybridization method or solution hybridization method described in GB 2169403 is quantitative (Virtanen et al., Lancet, vol. 1).
(See pages 381-383 (1983)). Furthermore, in the method of the present invention, cells with a constant copy number,
A standard nucleic acid is required that allows measurement of the number of related nucleic acid molecules per given unit, such as a nucleus, ribosome or chromosome. An object of the present invention is to provide an accurate and rapid method for quantifying nucleic acid molecules, which is simpler and less time consuming than currently used methods. The present invention is useful for cancer and prenatal diagnostics as well as for measuring Methsenjar RNA expression levels.
In addition, it can be used to detect factors that amplify genes and to indicate, for example, the development of drug resistance. At least two measurements are required in the present invention. One measures a nucleic acid molecule, ie, a test nucleic acid, which may be present in several copies. The other measures constitutive nucleic acid molecules, ie standard nucleic acids, which are advantageously present in a constant number. In the method according to the invention, the nucleic acid molecule represents a certain nucleotide sequence with 10-12 nucleotides or a gene containing several thousand nucleotides. It can also refer to Messenger RNA or a nucleotide sequence significantly longer than a single gene, amplicon. Measurement of test and reference nucleic acids can be carried out using the otherwise standard sandwich hybridization method described, for example, in U.S. Pat. No. 4,486,539 or as described in British Patent Application No. GB 2,169,403. It is carried out using a solution hybridization method. The invention also provides at least one
The invention relates to a reagent containing a nucleic acid reagent consisting of one test probe pair and at least one standard probe pair, ie at least four probes. Preferably, such reagents are used as a reagent kit. The reagent or probe used in the method of the present invention is
It is prepared using recombinant DNA technology from nucleic acids that are sufficiently homologous to a test nucleic acid and a reference nucleic acid. Sufficiently homologous nucleic acids can also be prepared synthetically and semi-synthetically. Test and reference nucleic acids may be isolated directly from cells and identified by various hybridization techniques. However, such test and standard nucleic acids are available commercially and from various gene banks. The test nucleic acid and standard nucleic acid may be either DNA or RNA. Probe pairs suitable for sandwich hybridization are prepared from nucleic acids sufficiently homologous to the test and reference nucleic acids by recombinant DNA technology. The relevant nucleic acids are digested with appropriate restriction enzymes and at least two of the resulting restriction enzyme fragments located relatively close to each other are cloned into at least two suitable vectors. One of the fragments is a probe for the detection of a hybrid formed with the nucleic acid of interest (hereinafter referred to as a "detector probe") and is labeled with a suitable detectable label, such as 125I or 32P.
The other one is a probe for immobilizing the target nucleic acid (hereinafter referred to as "capturing probe"), and is immobilized on a suitable carrier, for example, a nitrocellulose filter. Capturing probes that allow separation of hybrids obtained from hybridization mixtures by other substances, such as affixed or complementary components of an affinity pair. Fixed to. A probe pair consists of a detector probe and a capturing probe. The test probe pair and the standard probe pair are suitable reagents in which both the test probe pair and the standard probe pair are DNA or RNA, or the test probe pair is DNA and the standard probe pair is RNA, or vice versa. Can be combined as Therefore, pretreatment and further treatment of the sample before hybridization is necessary.
treatment) and hybridization conditions should be according to the probe pair used for the measurement test. The method of the invention is particularly suitable for determining the number of nucleic acid molecules directly from cell homogenates. Of course, the method of the invention can also be used to measure purified or pure nucleic acids. However, before carrying out the method of the invention, a suitable pretreatment of the nucleic acid sample should be selected. Using the method of the invention it is possible to perform both DNA and RNA measurements. The deoxyribonucleic acid is denatured if necessary to obtain single strands. Single-stranded messenger RNA molecules that can interfere with hybridization tests are
Hydrolysis can be carried out, for example, by alkaline boiling. In addition, double-stranded deoxyribonucleic acid
Since it does not interfere with RNA measurements, the sample is not denatured for ribonucleic acid measurements. Of course, decomposing DNA with deoxyribonuclease,
Alternatively, the DNA can be chemically or mechanically altered so that it cannot participate in the hybridization reaction. For the determination of DNA and RNA, an appropriate method must therefore be chosen for the secondary processing of the sample, or this secondary processing may be omitted. The selection of an appropriate method for secondary processing will, of course, depend on the method used to pre-process the nucleic acid sample. Numerous methods for pre-treatment and secondary treatment of nucleic acid samples are described in the literature, with which one can choose the appropriate method in each case. When both the test nucleic acid and the standard nucleic acid are DNA or RNA, the measurement can be performed using the sample as it is. The test nucleic acid
Measurements where DNA is the standard nucleic acid and RNA is the standard nucleic acid, or vice versa, require different methods for secondary processing, such as separating the sample into solutions containing DNA and solutions containing RNA. must be done separately. The samples may of course be separated even if the test and standard nucleic acids are of the same nucleic acid type. The hybridization test itself is performed by simultaneously contacting an unseparated sample solution with at least one test probe pair and one standard probe pair. If the sample solution is separated, it is contacted separately with at least one test probe pair and at least one standard probe pair. In such cases, the amount of test nucleic acid is measured in one reaction vessel and the amount of standard nucleic acid is measured in the other vessel. Regardless of whether the sample is split or not, hybridization is carried out in each case under the most advantageous conditions and times. Once the reaction has occurred, the resulting test and standard hybrids are separated from the hybridization mixture by a carrier and washed, or transferred to an isolating agent, such as a complementary member of an affinity pair. It is separated by twisting. The labels added to the test and standard hybrids are measured and the results compared to a standard curve. In this way the number of nucleic acid molecules to be investigated can be determined per selected unit. The method of the invention has practical diagnostic significance, particularly in the detection of certain types of cancer. In small cell lung carcinoma, the c-myc gene is often amplified and its expression level is much higher than in normal tissue.
In the case of neuroblastoma, the N-myc gene is amplified. The methods of the invention can also be used to explain the mutagenic or carcinogenic effects of certain drugs or the development of drug resistance. It is known that external pressures of selection lead to increased expression of certain genes. In the treatment of cancer, cancer cells exhibit resistance to administered drugs by amplifying genes, and the expression products of the genes inactivate the drugs. One such case is methotrexate, which induces amplification of the gene for dihydrofolate reductase (DHFR). Another example is the amplification of genes for metallothionine under the influence of cadmium. The expression level of a gene is important from the viewpoint of cellular phenotype and function. The expression level of the gene is
The correlation with the amount of protein encoded by Messenger RNA can be determined by measuring the amount of Messenger RNA.
The oncogene transcription product ultimately determines how it is expressed. Oncogene expression levels vary depending on cell type, level of differentiation, and phase of cell development. For example, at some stages of fetal development the c-myc oncogene is copied rapidly, but at other stages it is copied very slowly. Although the expression level of a gene may increase significantly without amplification, the degree of amplification often correlates with the expression level of the gene. In such cases, the role of an oncogene is often determined by measuring its expression level rather than its copy number. In some cases, quantification of messenger RNA is simpler and more convenient than quantification of the gene product itself. An example of such is c
- Myc oncogene, an unstable protein that is easily coagulated by heat. The method of the invention can also be used to confirm numerical chromosomal abnormalities such as Down syndrome. Prenatal diagnosis can also determine whether a fetus has a defect, ie, is homozygous for a certain recessive gene. Next, the quantitative method of the present invention and the nucleic acid reagent used therein will be explained in more detail using Examples.
The present invention is not limited to such embodiments. Example 1 Quantification of amplified cancer genes (a) Nucleic acid reagent and its preparation (1) Standard probe (cell standard nucleic acid) The c-Ki-ras gene is present in all human cells. A probe pair for sandwich hybridization was prepared by subcloning the Hind fragment of the c-Ki-ras gene and measuring 3.8 kb in length. Restriction enzyme maps were published by Chang et al. in the Proceedings of the National Academy of Sciences.
Signs (PNAS) vol. 79, pp. 4848-4852 (1982).”
It is described in. The fragment is available, for example, cloned into plasmid pBR322 (ATCC 41032), and can be obtained, for example, from the American Type Culture Collection (ATCC). (Secondary treatment of cell standard nucleic acid) The pBR322 clone described above was treated with the restriction enzyme Bgl.
and Hind, and the resulting fragments were isolated from agarose gel. Purified fragments located in close proximity to each other were subcloned into two appropriate vectors for detector probe and capturing probe preparation. (Standard Detector Probe) A 1.1 kb long Bgl-Bgl fragment was subcloned into the BamH restriction enzyme site of plasmid pBR322 and labeled by nick translation using 125-labeled dCTP. (Standard chap-ring probe) Bgl-Hind fragment 0.5kb with M13mp10 and
Restriction enzyme BgmH of M13mp11 phage vector
and Hind restriction enzyme sites, and then fixed on a nitrocellulose filter at 150 ng DNA/1 cm in diameter. (2) Test Probe (Test Nucleic Acid) A probe pair for sandwich hybridization was prepared from the cloned c-myc gene, which can be obtained from, for example, ATCC (ATCC41010). A restriction enzyme map of the gene is described in Watt et al., Proceedings of the National Academy of Sciences, Vol. 80, pp. 6307-6311 (1983). (Secondary treatment of test nucleic acid) The c-myc gene was treated with restriction enzymes Hind and Xba.
and Pst, the fragment was isolated from agarose gel, purified and subcloned into appropriate vectors to prepare detector and capturing probes. (Test Detector Probe) The 3.7 kb single-stranded tails of the Hind-Xba restriction enzyme fragment of the c-myc gene were made into double strands using DNA polymerase. Add a Hind linker to the resulting blunt-ended DNA fragment.
- Inserted using ligase. After phenol extraction, the DNA was treated with the restriction enzyme Hind. The DNA fragment was then cloned into plasmid pBR322 at the restriction enzyme site Hind and labeled by nick translation using 125-labeled dCTP. (Test capturing probe) 1.1kb Xba-Pst fragment of c-myc gene
Clone between the restriction enzyme sites of the restriction enzymes Xba and Pst in the M13mp10 and M13mp11 phage cloning vectors and then
150 ng DNA/1 cm diameter was fixed on a nitrocellulose filter. (b) Measurement of standard curve The sample used for measurement of the standard curve consisted of an alkali-denatured pBR322 clone of the c-myc gene. The sandwich hybridization solution to which the test probe was added was 4x SSC, 1x Denhardt's solution, and herring sperm.
It consisted of 200 μg/ml DNA and 0.2% SDS. Hybridization was carried out at 65°C for 17-19 hours and the filters were then washed at 50°C in wash solution (0.1x SSC and 0.2% SDS). Next, the label added to the Sand-Germany hybrid was measured in a gamma counter. The standard curve obtained is shown in Table 1.

[Table] (c) Measurement of the number of genes The samples are (1) human placenta-derived cells, and
(2) For example, obtained from ATCC (ATCC−
CCC220) Colo320 cells were used. DNA was isolated from both samples and an equal amount of cellular DNA denatured by alkaline boiling was sampled for testing. Alkaline denaturation hydrolyzed any RNA present in the sample. The test uses c-myc filter and c-Ki-
ras filter and standard DNA for each sample
This was done by adding to each sample two labeled reagents that allowed both the test DNA and the test DNA to be measured. Based on c-Ki-ras measurements, each sample was found to contain the same amount of DNA, and c-myc in Colo320 cells.
It can be estimated that the gene is present in approximately 16-20 times higher copy number than in normal conditions. The results are shown in Table 2.

[Table] (Note) *: The reading value from the blank filter was subtracted from each reading value.
Example 2 Quantification of propagated genes (a) Nucleic acid reagent and its preparation (1) Standard probe (cell standard nucleic acid) The control nucleic acid is located in the promoter region of metallothionin in mice, that is, the MT gene DNA is present immediately upstream. The structure of the MT gene is described in Pavlakis and Hamer, Proceedings of the National Academy of Sciences, Vol. 80, pp. 397-401.
(1983)”. The reference nucleic acid fragment is
An example is vector pBPV-MMneo (432-12)
(ATCC37224) and can be obtained from ATCC, for example. (Secondary treatment of cell standard nucleic acid) Restriction enzymes Kpn and Bgl were added to subclon the MT gene into plasmid pAT153.
and treated with EcoR. Kpn tail was converted to Hind tail using a linker. (Standard detector probe) EcoR-Kpn-(Hind) located upstream of the promoter region of the metallothionin gene.
The 1.2 kb fragment was cloned into plasmid pAT153 between the restriction enzyme sites EcoR and Hind and then labeled by nick translation using 32P-labeled nucleoside triphosphate. (Standard capturing probe) Kpn-Bgl fragment consisting of the metallothionin gene promoter region and its upstream region
0.8 kb between the restriction enzyme sites of Kpn and BamH in the phage vector M13mp18 and
M13mp19 was cloned and then fixed on a nitrocellulose filter. (2) Test probe (test nucleic acid) A commercially available plasmid is used as a probe pair for sandwich hybridization test.
pMTVdhfr (Bethesda Research Laboratories),
Product No. 5369SS). The structure of the plasmid is described in Lee et al., Nature 294, 228-232 (1981). (Secondary treatment of test nucleic acid) Plasmid pMTVdhfn containing cDNA of dihydrofolate reductase (DHFR) gene was treated with restriction enzymes Hind and Bgl. (Test detector probe) Hind-Bgl fragment 0.75kb corresponding to the region encoding the DHFR gene of plasmid pMTVdhfr
was inserted into the plasmid vector pAT153 between the restriction enzyme sites Hind and BamH, which was then labeled by nick translation with 32P-labeled nucleoside triphosphate. (Test capture probe) A 1.4kb Hind fragment taken from the MMTV gene region of plasmid pMTVdhfr was used as M13mp18 and
Cloned into M13mp19 phage vector,
It was then immobilized on a nitrocellulose filter at 150 ng DNA/1 cm diameter. (b) Measurement of standard curve The test sample is plasmid pMTVdhfr
It was purified DNA from The test itself was carried out as described in Example 1(b), except that a liquid scintillation counter was used for the measurements. The standard curve obtained is shown in Table 3.

[Table] (c) Measurement of the number of genes Various amounts of mRNA corresponding to the DHFR gene
Mouse fibroblasts transfected with cDNA and available from ATCC
An established cell line derived from NIH3T3 (ATCC CRL1658) was cultured on a cell culture plate and used as a sample. The cells were lysed using sodium dodecyl sulfate and the DNA was then cut by squeezing it out of a syringe through a hypodermic needle. about
A 250 μ sample corresponding to 10 ⁶ cells was taken from the homogenate to which 50 μ NaOH was added. Samples were boiled and neutralized with acetic acid and hybridization mixture. The total volume is 0.5ml
It was hot. All the probes were added at the same time and a so-called blank filter was added as a background control. Hybridization, washing, and label measurement were carried out in the same manner as in Example 1(b) except that a liquid scintillation counter was used for measurement. The results are shown in Table 4.

[Table] (Note) *: Each reading value is the value obtained by subtracting the reading value of the blank filter.
The MT gene is an internal marker that measures the number of cells present in a sample. The results obtained show that a number of 10 ⁶ cells gave a MT-specific signal of 165+20 cpm in this test. The DHFR reagent measures the amount of DHFR-cDNA.
Cell numbers were estimated from MT-specific signals.
In this way, DHFR-
Measurement of cDNA copy number is possible as shown in Table 4. Example 3 Quantification of Messenger RNA Using the test probe described in Example 2,
It is also possible to measure the amount of mRNA derived from DHFR-cDNA. The structure of plasmid pMTVdhfr is such that transcription of the DHFR gene begins at the MMTV promoter. The resulting message is approximately 1.0kb in length. Of this, approximately 0.25kb
is derived from the promoter MMTV region,
The remainder is from DHFR-cDNA (see Lee et al., Nature, 294, 228-232 (1981)). (1) Standard Probe The cell standard nucleic acid, standard detector probe, and standard capturing probe were the same as described in Example 2. (2) Test probe The test nucleic acid, test detector probe, and test capture probe were the same as described in Example 2. (b) Measurement of standard curve Methsenjar RNA corresponding to dihydrofolate reductase prepared by in vitro transcription.
A sample consisting of was used for standard curve measurement. The plasmid contains the DNA required for its transcription.
Hind fragment 1.4kb and Hind-Bgl fragment of MMTV promoter of pMTVdhfr (DHFR-
cDNA) 0.75kb adjacent to each other and restriction enzymes
It was prepared by subcloning into plasmid pSP64 between the Hind and BamH restriction enzyme sites. Sample RNA was stored in 0.2% SDS aqueous solution. Example 1(b) except that the sandwich hybridization test was not denatured.
and as described in Example 2(b). The standard curve obtained is shown in Table 5.

[Table] (c) Measurement of the number of Methsenjar RNA molecules Methsenjar RNA corresponding to the DHFR gene
The number of molecules was determined from the cell line described in Example 2. Cells were lysed using sodium dodecyl sulfate and the DNA was excised by squeezing it out of a syringe through a fine hypodermic needle. Approximately 5×
A 250 μ sample of homogenate corresponding to 10 ⁶ cells was taken. The homogenate was then subjected to a sandwich hybridization test without denaturation. Sand German hybridization was carried out in Example 2 except that only the DHFR probe was added to the hybridization solution.
(c) and as described in Example 1(b). In parallel test samples of 250μ of homogenate, cell numbers were determined using the MT probe as described in Example 2(c). The results are shown in Table 6.

[Table] (Note): Each reading value is the value obtained by subtracting the reading value of the blank filter.
The results showed that the established cell line was 1 from the DHFR gene.
Approximately 100 Methsenjar RNA molecules were produced per cell, indicating that the established cell line produced approximately 500 Methsenjah RNA molecules per cell from the DHFR gene. Example 4 Quantification of amplified genes by solution hybridization (a) Nucleic acid reagent and its preparation (1) Standard probe The cell standard nucleic acid, standard detector probe, and standard capturing probe were the same as in Example 2. Using. EcoR of plasmid pAT153
The 1.2 kb -Kpn- (Hind) fragment was labeled with 125-labeled deoxycytidine by Nik translation. M13mp18 and M13mp19
Kpn-Bgl fragment 0.8kb of phage vector,
Photoprobe ^™ reagent (Vector Laboratories, California, USA, product number SP ⁾
-1000) and modified with biotin. (2) Test probe The same test nucleic acid, test detector probe, and test capture probe as in Example 2 were used. Hind-Bal of plasmid pAT153
The 0.75 kb fragment was labeled with 125-labeled deoxycytidine. The 1.4 kb Hind fragment of M13mp18 and M13mp19 phage vectors was biotinylated using the Photoprobe ^™ . (b) Determination of standard curve A cell standard curve was created using a known amount of cells, and a hybridization signal was measured from the curve using a standard probe that recognizes the MT-gene. A test nucleic acid standard curve was created using the plasmid pMTVdhfr and a test probe that recognizes this plasmid. 0.6M NaCl, 20mM
Hybridization was carried out in 200μ of a solution consisting of phosphate buffer (PH7.5), 1mM EDTA and 4% polyethylene glycol (PEG6000) at 70°C for 1.5 hours. After the reaction, streptavicin-agarose (Bethesda Research
Laboratories, Maryland, USA, product number 5942SA) 50μ and 1M NaCl, 10mM
Sodium phosphate (PH7.5) and 1mM EDTA
was added to a final volume of 500μ. Hybrids were plated on streptavidin-agarose for 15 minutes at 37°C.
Collected for minutes. Agarose was washed with 1M NaCl buffer at 37°C once for 5 min, then with 15mM NaCl at 55°C.
Washed twice for 2 minutes with 1.5mM sodium citrate. The amount of bound hybrid was determined by measuring the agarose in a gamma counter (Syva¨nen et al., Nucleic Acids Research).
(Res.), Vol. 14, pp. 5037-5048 (1986)). The results are shown in Tables 7 and 8.

【table】

[Table] (c) Measurement of the number of genes A sample of the established cell line described in Example 2 was prepared in the same manner as described in Example 2, except that the volume per sample, which corresponds to approximately 2 × 10 ⁶ cells, was 125μ. Processed. To determine the number of cells and test nucleic acid molecules, combine the cell sample, appropriate detector and capture probes, and components of the hybridization mixture in a final volume of 200 μl.
in separate vials by adding up to Control assays without cellular standard DNA or cellular test DNA were also performed. Hybridization, hybrid collection, washing and measurements were performed as described in Example 4(b).
Results were read from a standard curve prepared in parallel as described in Example 4(b). The results obtained are shown in Table 9.

[Table] (Note) *: Values from control assays without cell standards or cell test nucleic acids have been subtracted.

Claims (1)

[Claims] 1. Comparing the number of test nucleic acid molecules that may be present in several copies in a unit with a selected standard number of nucleic acid molecules that are conveniently present in constant number in the unit. (a) a sample containing nucleic acids is in a form in which the nucleic acids cannot undergo a hybridization reaction; If the nucleic acid is present from the beginning, it is in a form that allows the hybridization reaction to occur, or if the nucleic acid is likely to interfere with the hybridization reaction, it is hybridized. (b) one test probe pair sufficiently homologous to the test nucleic acid molecule that the nucleic acid may be present in several copies; is hybridized with at least two pairs of nucleic acids of one standard probe pair that are sufficiently homologous to the nucleic acid molecules present in a constant number;
(c) a hybridization reaction is carried out; Thereafter, the test hybrid and standard hybrid are separated if necessary, the label added to the detector probe is measured, and the number of nucleic acid molecules per fixed unit is compared with the number of test nucleic acids and the number of standard nucleic acids. A method for quantifying nucleic acid molecules by sandwich hybridization or solution hybridization, characterized in that the method is obtained by: 2. The assay method according to claim 1, wherein the test nucleic acid and the standard nucleic acid are deoxyribonucleic acids. 3. The assay method according to claim 1, wherein the test nucleic acid is ribonucleic acid and the standard nucleic acid is deoxyribonucleic acid. 4. The assay method according to claim 1, wherein the test nucleic acid and the standard nucleic acid are ribonucleic acids. 5. The assay method according to claim 1, wherein the test nucleic acid is deoxyribonucleic acid and the standard nucleic acid is ribonucleic acid. 6 Hind fragment of human c-Ki-ras gene
A recombinant plasmid consisting of a 1.1 kb Bgl-Bgl fragment, in which the Hind fragment is a plasmid.
The Bgl-Bgl cloned into pBR322
The fragment contains the restriction enzyme BamH of plasmid pBR322.
the detector probe of the standard probe pair, which is subcloned into the restriction enzyme site of
and the Hind fragment of the human c-Ki-ras gene.
A recombinant phage consisting of a 0.5kb Bgl-Hind fragment, and the Hind fragment is a plasmid.
The Bgl-
Capturing probes of the standard probe pair, in which the Hind fragment was subcloned between the BamH and Hind restriction enzyme sites of the M13mp10 and M13mp11 phage vectors, were isolated individually or together with the test probe pair. 4. A quantitative method according to claim 1, 2 or 3, in which the nucleic acid sample is contacted with the isolated nucleic acid sample when necessary. 7 EcoRI-KpnI (Hind) fragment from upstream of the promoter region of mouse metallotionin gene
A recombinant plasmid consisting of a 1.2 kb fragment subcloned between the restriction enzyme sites of the restriction enzymes EcoR and Hind of plasmid pAT153, the detector probe of the standard probe pair, and the promoter region of the metallothionin gene and its Kpn− from upstream region
The capturing probe of the standard probe pair is a recombinant phage consisting of the Bgl fragment 0.8kb, which is subcloned between the restriction enzyme sites of the restriction enzymes Kpn and BamH of the M13mp18 and M13mp19 phage vectors. , individually or together with test probe pairs, are contacted with an unseparated or, if necessary, separated, nucleic acid sample. 8 To determine the degree of amplification of the c-myc oncogene and/or the number of messenger RNA molecules corresponding to the gene,
A recombinant plasmid consisting of a Hind-Xba fragment of 3.7 kb, which is subcloned into the restriction enzyme site of the restriction enzyme Hind of plasmid pBR322, and a detector probe of the test probe pair and an Xba of the c-myc gene. −
The capturing probe of the test probe pair is a recombinant phage consisting of a 1.1 kb Pst fragment, which is subcloned between the restriction enzyme sites of the restriction enzymes Xba and Pat of the M13mp10 and M13mp11 phage vectors. ,
Claims 1, 2, 5, 6 or 7, wherein the nucleic acid sample, either individually or together with a standard probe pair, is contacted with an undivided or, if necessary, divided nucleic acid sample. quantitative method. 9. In order to measure the degree of amplification of the dihydrofolate reductase or DHFR gene and/or the number of messenger RNA molecules corresponding to the gene, a recombinant plasmid consisting of a 0.75 kb Hind-Bgl fragment encoding the DHFR gene of plasmid pMTVdhfr was used. The fragment was then digested with the restriction enzymes Hind and Hind of plasmid vector pAT153.
The detector probe of the test probe pair, which is subcloned between the restriction enzyme sites of BamH, and the detector probe of the plasmid pMTVdhfr.
This is a recombinant phage consisting of a 1.4kb Hind fragment of the MMTV gene region, and this fragment is M13mp18.
and restriction enzymes of M13mp19 phage vector
Capturing probes of the test probe pair, which have been subcloned between the Hind restriction enzyme sites, are contacted with the nucleic acid sample, either individually or together with the standard probe pair, unsplit or, when necessary, split. A quantitative method according to claim 1, 2, 5, 6 or 7. 10 Containing at least one test probe pair and at least one standard probe pair, the detector probes of both the test probe pair and the standard probe pair are labeled with an appropriate label, and the test probe pair and the standard probe pair are labeled with an appropriate label. Quantitation of a nucleic acid molecule, characterized in that both of the capturing probes of the pair are immobilized on a suitable carrier, or a substance that allows the isolation of Sand-Germany hybrids is immobilized on the capturing probes. reagent for. 11 Degree of amplification and/or c-myc oncogene
or messenger RNA corresponding to the gene
The detector probe of the test probe pair used to measure the number of molecules is Hind- of the c-myc gene.
This is a recombinant plasmid consisting of a 3.7 kb Xba restriction enzyme fragment, and this fragment is subcloned into the restriction enzyme site of the restriction enzyme Hind of plasmid pBR322, and the capturing probe of the test probe pair is the Xba restriction enzyme fragment of the c-myc gene. −Pst
It is a recombinant phage consisting of a 1.1 kb fragment,
The fragment was subcloned between the Xba and Pat restriction enzyme sites of the M13mp10 and M13mp11 phage vectors, and the detector probe of the standard probe pair was human c-Ki-ras.
Bgl-Bgl fragment of Hind fragment of gene
The Hind fragment is 1.1kb and the plasmid
It has been cloned into pBR322, and the Bgl−Bgl
The fragment contains the restriction enzyme BamH of plasmid pBR322.
The capturing probe of the standard probe pair is subcloned into the restriction enzyme site of the c-Ki-ras gene.
A recombinant phage consisting of a Hind fragment of 0.5 kb, the Hind fragment being subcloned into the plasmid pBR322 of the c-Ki-ras gene,
The Bgl-Hind fragment has M13mp10 and
Restriction enzyme BamH of M13mp11 phage vector
11. The reagent according to claim 10, which is subcloned between the restriction enzyme sites of and Hind. 12 Dihydrofolate reductase or DHFR
The detector probe of the test probe pair used to measure the degree of gene amplification and/or the number of messenger RNA molecules corresponding to the gene is a recombinant plasmid consisting of a 0.75 kb Hind-Bgl fragment encoding the DHFR gene of plasmid pMTVdhfr, The fragment is a plasmid vector
It is subcloned into the restriction enzyme sites of Hind and BamH of pAT153, and the capturing probe of the test probe pair is subcloned into the Hind and BamH restriction enzyme sites of plasmid pMTVdhfr.
It is a recombinant phage consisting of a 1.4 kb fragment,
The fragment was subcloned into the restriction enzyme site of the restriction enzyme Hind of the M13mp18 and M13mp19 phage vectors, and the detector probe of the standard probe pair was subcloned into the restriction enzyme site of the restriction enzyme Hind of the M13mp18 and M13mp19 phage vectors, and the detector probe of the standard probe pair was subcloned into the restriction enzyme site of the M13mp18 and M13mp19 phage vectors.
It is a recombinant plasmid consisting of a 1.2 kb fragment,
This fragment contains the restriction enzyme EcoR of plasmid pAT153.
and Hind restriction enzyme sites, and the capturing probe of the standard probe pair is a recombinant phage consisting of a 0.8 kb Kpn-Bgl fragment of the metallothionine gene formed by the promoter region and its upstream region. Yes, the fragment is M13mp18 and
11. The reagent according to claim 10, which is subcloned between the restriction enzyme sites of the restriction enzymes Kpn and BamH of the M13mp19 phage vector.