FI76119C - Quantitative determination of nucleic acid molecules and reagent packaging used in the process - Google Patents

Description

1 76119

Quantitative determination of nucleic acid molecules and reagent kit used in the method

The invention relates to the quantitative determination of certain nucleic acid molecules, in particular the degree of gene amplification and / or the corresponding messenger RNA molecules, by a layer or solution hybridization method and a reagent kit used in the method.

10 The copy number of individual genes in the genome is usually constant. Some genes have only one per haploid genome, while others have several. In special situations, the number of copies may change. Reproduction of some genes has been shown to be linked to the development of cancer. It has also been found that some of the external factors such as drugs, metals and the like.

as a result, the genes are amplified or amplified. Incorrect / overexpressed expression levels of a gene, e.g. an oncogene, i.e. the amount of messenger RNA in a cell, are relevant to the development of the disease. The presence of certain chromosomes in more than one body causes certain inherited diseases or other disorders. Other hereditary diseases occur only when the disease-causing gene occurs in duplicate, i.e., it is a recessive trait. In all these cases, it is important to indicate the number of chromosome 25 en / genes.

The number of specific DNA molecules, e.g., the degree of gene amplification, is currently measured by digesting the test DNA isolated from the cells with restriction enzymes, after which the 30 DNA fragments are separated by size on an agarose gel by electrophoresis. The DNA is then transferred to nitrocellulose paper, attached to it in single-stranded form, and finally hybridized using the gene of interest or a portion thereof as a probe. Results 35 are obtained by autoradiography (Southern, J. Mol. Biol. 98, pp. 503-517, 1975). In the assay, each parallel assay contains the same amount of cellular DNA. The intensities of the 2 761 19 lines to be hybridized are compared. Based on the differences in intensity, the ratios of the copy numbers of the studied genes in the studied samples are deduced. The study gives only approximate results. RNA is measured by 05 Northern blotting or dot blotting methods, respectively. These methods are highly inaccurate in quantitative terms (Thomas, Methods in Enzymol., 100, pp. 255-266, 1983).

Known methods such as Southern and Northern blotting-10 are slow and cumbersome to perform. Because they provide only approximate results, their diagnostic significance is questionable in cases where it is necessary to know the number of particular nucleic acid molecules per unit, e.g., per cell.

15

The layer and solution hybridization methods described in e.g. in Finnish patent 63,596 and in Finnish patent application 85,0004, are quantitative methods (Virtanen et al., Lancet, 1, pp. 381-383, 1983). The method according to the invention 20 also requires a standard nucleic acid, the number of molecules of which determines the number of nucleic acid molecules to be determined per specific unit, for example a cell, nucleus, ribosome or chromosome.

25

It is an object of the present invention to provide a rapid method for the accurate quantification of nucleic acid molecules suitable for cancer diagnosis, prenatal diagnosis and the detection of agents causing gene amplification and for the diagnosis of resistance to these agents, as well as for the rapid quantification of nucleic acid molecules.

At least two assays are required to perform the method of the invention. The second determines the 3,761 19 nucleic acid molecules to be detected, possibly in multiple copies, the so-called test nucleic acid. The second defines a constitutive, preferably a constant number of nucleic acid molecule, the so-called standard nucleic acid. In the method of the invention, a nucleic acid-05 molecule means a specific nucleic acid sequence.

A nucleic acid molecule can be a nucleic acid sequence of 10-12 nucleotides, but also a gene or messenger RNA containing several thousand nucleotides or a nucleic acid sequence of considerably longer than a single gene, the so-called amplicon.

10

The assay and standard nucleic acids are otherwise determined using the standard layer hybridization method described in e.g. in Finnish Patent 63,596 or the solution hybridization method described in Finnish Patent Application 850004. The invention also relates to a reagent kit containing the nucleic acid reagents used in the method, at least one pair of test probes and at least one pair of standard probes.

The reagents, probes, used in the method of the invention are prepared by recombinant DNA technology from nucleic acid sufficiently homologous to the test nucleic acid and sufficiently homologous to the standard nucleic acid. Sufficiently homologous nucleic acids for test and standard nucleic acids can also be prepared synthetically or semi-synthetically.

The required test and standard nucleic acids can be isolated directly from the cells and identified by a variety of hybridization methods. On the other hand, such test and standard nucleic acid and test probes for preparation are available both commercially and from various gene banks. Test and standard nucleic acids can be either DNA or RNA.

From nucleic acids sufficiently homologous to test and standard nucleic acids, probe pairs suitable for the layered or solution hybridization method are prepared by recombinant DNA technology, cleaving those nucleic acids into appropriate vectors with appropriate restriction enzymes, and transferring at least two of the nucleic acids thus prepared relatively close to each other. Another 05 fragments thus obtained, the so-called. Detector probe is labeled with any suitable label and address of a second, so-called. Capturing probe attached to a suitable support or attached to the material, which allows the separation of the hybrid formed from the hybridization solution by means of the second material 10, for example, the other party by means of an affinity pair.

The test and standard probe pairs can be assembled into suitable reagent kits so that both the test and standard probe pairs are each DNA or RNA, or so that the test probe pair is DNA and the standard probe pair is RNA, or vice versa. In this case, the sample pre- and post-treatments and hybridization conditions prior to the hybridization test must be selected to match the probe pairs used in the test.

The method of the invention is particularly well suited for determining the number of nucleic acid molecules directly from cell homogenates. Of course, the method can also be used to determine purified or pure nucleic acid. Before carrying out the method according to the invention, it is only necessary to select the most suitable pretreatment procedure for the nucleic acid sample.

Both DNA-30 and RNA assays can be performed with the method of the invention. The deoxyribonucleic acids are denatured to a single-stranded form, if necessary. Single-stranded messenger RNA molecules that potentially interfere with the hybridization assay can be hydrolyzed, e.g., by alkaline cooking. When determining ribo nucleic acids, the sample is not denatured.

In this case, the double-stranded deoxyribonucleic acid present does not interfere with the RNA assay. Of course, it is possible to destroy the DNA by DNAase treatment or by chemically or mechanically rendering the DNA in a form that is incapable of participating in the hybridization assay. Thus, when determining DNA or RNA, an appropriate sample further processing procedure must be selected or, alternatively, this further processing may be omitted. The choice of a suitable further processing procedure will, of course, depend on the nucleic acid sample pretreatment procedure. Numerous methods for pre- and post-processing nucleic acid samples have been described in the literature, so one skilled in the art will find a suitable procedure for each situation.

Assays in which the test and standard nucleic acids are both DNA or RNA can be performed on an undivided sample. Assays in which the test nucleic acids are DNA and the standard nucleic acids are RNA or vice versa must be performed on a split sample, as further processing procedures are different. Of course, the sample can be split if desired, even if the test and standard nucleic acids are of the same nucleic acid type.

20

The hybridization test itself is performed by simultaneously contacting the undivided sample solution with at least one pair of test probes and one pair of standard probes. If the sample solution is split, it is separately contacted with at least one pair of test probes and one pair of standard probes. In this case, the amount of test nucleic acid is determined in one reaction vessel and the amount of standard nucleic acid in the other.

Regardless of whether the sample is undivided or split, the hybridization reaction is allowed to proceed under preferred hybridization conditions and for a preferred time in each case. After the reaction or reactions have occurred, the resulting test and standard hybrids are separated from the hybridization solution or solution 35 via the carrier to enable the isolation of the substance and, for example, the other party by means of an affinity pair rinsed or separated from the hybridization. The label bound to the test and 6 7611 9 standard hybrids is measured and the result is compared to the standard curves. In this way, the number of nucleic acid molecules to be determined is determined per suitably selected unit.

05

The method according to the invention has practical diagnostic significance, especially in the detection of certain cancerous tumors. In lung microcellular carcinoma, the c-myc gene is often amplified and its expression level is also considerably higher than in normal tissue. In neuroblastoma cases, on the other hand, the amplified N-myc gene is found.

The method according to the invention can also be used to demonstrate the mutagenic or carcinogenic effect of certain substances or the development of drug resistance. It is known that a certain external selection pressure leads to a more efficient expression of a particular gene than normal. In the treatment of anticancer drugs, the cells develop resistance to the drug. This is done by amplifying the gene whose product makes the drug ineffective. An example is the amplification of the dihydrofolate reductase (DHFR) gene by methotrexate. Another example is the amplification of a gene expressing metallothionein by, for example, cadmium.

25 The level of gene expression is relevant to cell phenotype and function. The level of expression can be examined by measuring the amount of messenger RNA, which in turn correlates with the amount of protein produced from it. The oncogene product ultimately determines how the effect of the oncogene occurs.

30

Oncogene expression levels vary depending on the cell type and its degree and stage of differentiation. For example, at a certain stage of fetal development, the c-myc oncogene is copied vigorously, in the second stage very little. The degree of gene amplification 35 often correlates with the level of gene expression, but the level of expression may be significantly elevated without gene amplification.

7 76119 In this case, the best information about the significance of the oncogene is obtained by measuring its expression level instead of the copy number. In some cases, quantification of messenger RNA may be more convenient / easier than quantification of the 05 gene product itself. An example is the product of the c-myc oncogene, which is a labile, easily heat precipitated protein.

The method according to the invention can also be used to detect disorders of chromosome number 10, such as Down's syndrome. In prenatal diagnosis, it is also possible to show whether a fetus is diseased, i.e., homozygous for a recessive gene.

The method of the invention and the nucleic acid reagents used in the method are described in more detail in the following examples.

Example 1 20

Quantitative determination of amplified oncogene by layer hybridization a) Nucleic acid reagents and their preparation 25

STANDARDIKOETTIMET

Solustandardinukleiinihappo. c-Ki-rasI is a human gene that is present in all cells. Probe pairs for layer hybridization were prepared by subcloning from the HindIII fragment of the 3.8 kb 31-length c-Ki-rasl gene, the restriction map of which is described in Chang et al., PNAS 79, p. 4848-52, 1982. The fragment is available e.g. cloned into plasmid pBR322, e.g., from ATCC strain collections (ATCC 41032).

36 Further processing of cellular standard nucleic acid. The pBR322 clone described above was treated with BglII and HindIII restriction enzymes and the resulting fragments were isolated from an agarose gel and 76119 purified contiguous fragments were subcloned into two suitable vectors to prepare a detector and fixative probe.

05 Standard Detector Probe. The approximately 1.1 kb BglII-BglII fragment was recloned into plasmid pBR322 at its BamHI restriction enzyme cleavage site and labeled by the nick-125 translation method using I-labeled dCTP.

10 Standard mounting probe. A BglII of about 0.5 kb in length

The HindIII fragment was transferred into phage vectors M13 mp10 and mp11 between the cleavage sites of these BamHI and HindIII restriction enzymes and attached to a nitrocellulose filter (150 ng DNA / 1 cm diameter).

15

TESTIKOETTIMET

Testinukleiinihappo. A probe pair for layer hybridization was prepared from a cloned c-myc gene available from, e.g., the ATCC strain collection (ATCC 41010), the restriction map of which is described in Watt et al., PNAS iM), p. 6307-6311, 1983.

Further processing of the test nucleic acid, the c-myc gene was treated with restriction enzymes HindIII, XbaI and PstI, and the fragments were isolated from an agarose gel and purified and subcloned into appropriate vectors to prepare a detector and capture probe.

The single-stranded tails of the 3.7 kb HindIII-XbaI restriction fragment of the test detector probe, the c-myc gene, were double-stranded by DNA polymerase. HindIII linkers were ligated to the resulting blunt-end DNA fragments using T4 DNA ligase, and after phenol extraction, the DNA was treated with HindIII restriction enzyme. The DNA fragment was then cloned into the HindIII restriction enzyme cleavage site of plasmid pBR322 and 9,716,115,125 were labeled by nick translation using I-labeled dCTP.

Test clamp Probe. The 1.1 kb XbaI-05 PstI fragment of the c-myc gene was cloned into M13 mp10 and mpII phage cloning vectors between the XbaI and PstI restriction enzyme cleavage sites and attached to a nitrocellulose filter (150 ng DNA / diameter 1 cm).

10 b) Determination of the standard curve

The alkaline-denatured clone pBR322 of the c-myc gene was used as a sample to determine the standard curve. As a layer hybridization solution, 4 x SSC, 1 x Denhardt's solution and 15,200 ug / ml herring sperm DNA and 0.2% SDS were used, to which the test probes described above were added. The hybridization reaction was performed at 65 ° C for 17-19 h. After the hybridization reaction, the filters were washed in a wash solution (0.1 x SSC in 0.2%: SDS) at 50 ° C. The label bound to the layer hybrids was then counted in a counter.

Table 1 25 Number of samples cpm_ Molecule / test c-myc filter 0 40 106 75 30 5x106 190 107 340 108 2200 10 761 1 9 c) Determination of gene number Samples are 1) cells from the human placenta; 2) Colo 320 cells available e.g. from ATCC 05 strain collections (ATCC-CCC220). DNA was isolated from both and the same amount of cellular DNA denatured with alkaline soup was placed in each assay. Alkaline saturation also hydrolyzes any RNA present in the sample.

The assay was performed by adding both c-myc and c-Ki-rasI filters and both labeled reagents to each sample. In this case, both standard and test DNA can be measured from one sample. Based on C-Ki-rasl assays, the assays contained the same amount of DNA. From this, it can be concluded that the c-myc gene has been amplified in Colo 320 cells by approximately 16-20-fold compared to the normal situation. The results are shown in Table 2.

Table 2 20 _Internal standard Test__ Sample with c-Ki-rasI- c-myc filter with filter cpm * cpm * number 25 --_- human placental cells 486 340 10

Colo 320 cells 432 3205 1,6.10® f 30 The number given by the zero filter has been deducted from the numbers.

Example 2 35 Quantitative Determination of Amplified Gene by Layer Hybridization a) Nucleic Acid Reagents and Their Preparation 11 76119

STANDARDIKOETTIMET

Solustandardinukleiinihappo. The promoter region of the mouse metallothionein gene and the 05 DNA region above that gene, the so-called MT gene, were used as the reference nucleic acid.

The structure of the MT gene is described in Pavlakis and Hamer, PNAS 80, p. 397-401, 1983. A reference nucleic acid fragment is available e.g. From the ATCC strain collection from the vector pBPV-MMTneo (342-12) (ATCC 37224).

10

Further processing of cellular standard nucleic acid. The MT gene described above was treated with KpnI, BglII and EcoRI restriction enzymes for further cloning into plasmid pAT153. The Kpnl end was converted to a HindIII end using a linker.

15

Standardidetektorikoetin. The approximately 1.2 kb EcoRI-KpnI (HindIII) fragment above the metallothionein gene was cloned into plasmid pAT153 between the EcoRI and HindIII restriction enzyme cleavage sites and labeled by the nick-trans-20 lation method using P-labeled phosphates.

Standard capturing probe. The 0.8 kb KpnI-BglII fragment formed by the promoter region of the metallothionein gene and the region above it was cloned into the M13 mp18 and M13 mp19 phage vectors between the KpnI and BamHI restriction enzyme cleavage sites and attached to a nitrocellulose filter.

30 TEST PROBES

Testinukleiinihappo. A probe pair for the layer hybridization assay was prepared from the commercially available plasmid pMTVdhfr (Bethesda Research Laboratories, Product No. 5369SS), the structure of which is described in Lee et al., Nature 294, p. 228-232, 1981.

12 761 1 9

Further processing of test nucleic acid. The pMTVdhfr plasmid containing the dihydrofolate reductase gene (DHFR) cDNA was treated with HindIII and BglII restriction enzymes.

05 Test Detector Probe. The 0.75 kb HindIII-BglII fragment corresponding to the DHFR gene coding region of the pMTVdhfr plasmid was transferred into the plasmid vector pAT153 with HindIII and

Between the cleavage sites of BamHI restriction enzymes and labeled using the nick translation method, P-labeled nucleoside triphosphates.

Test clamp Probe. The MMTV gene region of plasmid pMTVdhfr was cloned as a 1.4 kb HindIII fragment into the phage vectors M13 mp18 and M13 mp19.

15 b) Determination of the standard curve.

Purified pMTVdhfr plasmid DNA was used as a sample in the assay. The test itself was performed as described in Example 20 Ib except that the calculation was performed using a liquid scintillation counter. The resulting standard curve is shown in Table 3.

Table 3 25 Number of samples cpm_molecular / test DHFR filter 30 0 17 106 45 3x106 79 107 210 13 761 1 9 c) Determination of gene number

Cell lines into which different amounts of cDNA corresponding to the messenger RNA of the DHFR gene had been artificially introduced were cultured in cell-05 culture dishes and used as a sample. The cells were detached using sodium dodecyl sulfate and their DNA was somewhat digested by inserting a syringe through a narrow needle. 250 of the homogenate, corresponding to about 10 cells, was taken and 50 NaOH was added, the sample was boiled and neutralized with acetic acid and hybridization solution.

The total volume was 0.5 ml. All the probes described above were added simultaneously to this solution. In addition, the so-called zero filter for background control. Hybridization, washing and counting were performed as in Example Ib 15 except that counting was performed using a liquid scintillation counter. The results are shown in Table 4.

Table 4 20 _Internal standard_Test_

Cell MT Number of cells _PH FR_ cpm in sample cpm number of copies of molecules number / cells _in_sample 25 Control cell (no DHFR cDNA) 182 l, 05x106 21 <106 line I 138 0.9 x106 80 3x106 3 line II 210 l, 25x10 7 732 5xl07 40 30 _ * cpm: The number given by the zero filter has been subtracted from the numbers · The MT gene serves as an internal marker in the assay that measures the number of 35 cells. Our results showed that 10 cells in this experiment gave a 165 + 20 cpm MT-specific signal. DHFR reagents measure the amount of DHFR cDNA. Based on the 14 76119 signal number of the MT gene, the number of cells was known. Thus, the copy number of DHFR cDNA from different cell lines was measurable as shown in Table 4.

05 Example 3 Quantitative determination of messenger RNA by multi-hybridization 10 a) Nucleic acid reagents and their preparation

The amount of messenger RNA produced from DHFR cDNA can also be measured using the test probes described in Example 2. The pMTVdhfr plasmid is constructed such that transcription of the DHFR gene begins at the MMTV promoter. The transmitters produced are about 1.0 kb in length. Of this, about 0.25 kb is from the MMTV promoter region and the rest is from DHFR cDNA (Lee et al., Nature 294, pp. 228-232, 1981).

20 STANDARD PROBES

The cell standard nucleic acid, standard detector and standard detector probe were the same as in Example 2.

25 TEST PROBES

The test nucleic acid, test detector and test fixative probe were the same as in Example 2.

30 b) Determination of the standard curve

To determine the standard curve, messenger RNA corresponding to the dihydrofolate reductase gene produced by in vitro transcription was used as a sample. The DNA required for transcription 35 was prepared by subcloning the 1.4 kb HindIII fragment from the MMTV promoter and the 0.75 kb HindIII-BglII fragment (DHFR cDNA) of plasmid pMTVdhfr sequentially into plasmid pSP64 (Promega 15 76119).

Biotec) between the cleavage sites of the HindIII and BamHI restriction enzymes. Sample RNA was stored in 0.2% aqueous SDS.

The layer hybridization test itself was performed as shown in Examples 05 lb and 2b except that no denaturation was performed.

Table 5 10 _ Number of samples cpm_ molecules / test DHFR filter 0 20 15 5x106 65 107 130 5x107 390 108 653 20 c) Determination of the number of messenger RNA molecules The number of messenger RNA molecules corresponding to the DHFR gene was determined from the cell lines described in Example 2.

25

The cells were detached using sodium dodecyl sulfate and their DNA was somewhat digested by inserting a syringe through a narrow needle. 250 μl was taken from the homogenate, corresponding to about 5x10 8 cells. The homogenate was added without denaturation to the layer hybridization assay. The layer hybridization assay was performed as described in Examples 2c and Ib except that only DHFR probes were added to the hybridization solution. The cell number of the sample from the parallel 250 homogenate was determined using an MT probe 35 as described in Example 2c.

The results are shown in Table 6.

16 761 1 9

Table 6 _Internal standard_Test__

Cell MT Number of cells _DHFR_ 05 cpm * in sample cpm * molecular number number in the sample per cell line I 380 3.5xl06 1465 3.45xl08 100 10 line II 430 4.2xl06 4800 2xl09 500 * cpm: The numbers given by the zero filter have been deducted from the numbers.

Our results showed that about 100 messenger RNA molecules corresponding to the DHFR gene were produced in cell line I and about 500 messenger RNA molecules corresponding to the DHFR gene per cell in cell line II.

20 Example 4

Quantitative determination of the amplified gene by solution hybridization

25 a) Nucleic acid reagents and their preparation STANDARD PROBES

The cellular standard nucleic acid, standard detector and standard attachment probe were the same as in Example 2. The 1.2 kb EcoRI-KpnI (HindIII) fragment 125 cloned into plasmid pAT153 was labeled by nick translation using purple-labeled deoxycytidine. The 0.8 kb Kpnl-BglII fragments cloned into phages M13 mpl8 and M13 mpl9 were modified

TM

35 with biotin using Photoprobe reagent (Vector Laboratories, CA, USA, product No. SP-1000).

17 761 1 9

TESTIKOETTIMET

The test nucleic acid, test detector, and test clamp probe were the same as in Example 2. The 0.75 kb HindIII-BglII fragment cloned into plasmid pAT153 was labeled with 125 L of labeled deoxycytidine. Phages M13 mpl8 and

The 1.4 kb HindIII fragments cloned from M13 mp19 were biotinylated by TM

Using Photoprobe reagent as above.

10 b) Determination of standard curves

A cell standard curve was prepared using a known number of cells whose hybridization signal was measured using standard probes recognizing the MT gene. A post-standard curve of test nucleic acid-15 was prepared using plasmid pMTVdhfr and test probes recognizing this plasmid.

Hybridizations were performed in 200 μΐ of a solution having the following composition: 0.6 M NaCl, 20 mM phosphate buffer, pH 7.5, 1 mM EDTA, 4% polyethylene glycol (PEG 6000).

The hybridization time was 1.5 hours and the temperature was 70 ° C. After the hybridization reaction, 50 μΐ streptavidin-agarose (Bethesda Research Laboratories, Maryland, USA, Product No. 5942SA) was added to a solution of 1 M NaCl, 10 mM sodium phosphate, pH 7.5, 1 mM EDTA to give a final volume of 500 μΐ. Hybrids were collected on streptavidin-agarose at 37 ° C for 15 minutes. The agarose was washed once for five minutes with buffered 1 M NaCl solution at 37 ° C and twice for two minutes in a solution of 15 mM NaCl and 1.5 mM sodium citrate at 55 ° C. The number of hybrids was measured from agarose with a gamma counter (Syvänen et al., Nucleic Acids Res. 14, 5037-5048, 1986). The results are shown in Tables 7 and 8.

Table 7 76119 18 Number of samples cpm_ 05 cells / test MT filter 0.8 x 106162 0.6 x 106 216 3 x 106 298 10

Table 8 15__ Number of samples cpm_ molecule / test DHFR filter ΰβ 20 5 x 106 394 5 x 107 2240 c) Determination of the number of genes 25

The cell line samples described in Example 2 were treated in the same manner as in Example 2. is described in the example, except that the volume was 125 μΐ per sample, corresponding to about 2 x 10® cells. The number of cells and test nucleic acid molecules was determined in different dishes by adding a cell sample, the respective detector and probe probes, and the components of the hybridization mixture so that the final volume was 200 μΐ. At the same time, control probes without cellular standard and test nucleic acid were added. Hybridization, hybrid collection, washing 35, and measurement were performed as described in Example 4b. The results were read from standard curves prepared as described in Example 4b. The results are shown in Table 9.

19 761 1 9

Table 9 _Internal standard_Test_

Cell MT_ DHFR_ 05 cpm * Cell number cpm * Mol. Num. The number of copies in the sample

Control cell 253 2.3 x 10 ^ 73 <10 ^ 10 Line I 210 1.5 x 106 233 3.8 x 106 3

Line II 237 2.1 x 106 3059 8.8 x 107 42 * cpm: The figure given by the zero filter has been deducted from the figures

Claims (16)

Method for Quantitative Determination of Nucleic Acid Molecules by a Sandwich or Solution Hybridization Method, characterized in that the number of certain nucleic acid molecules per given biological unit existing in the investigated organism is determined by comparison of the number of test nucleic acid molecules present in potential multiple copies in the unit, and the number of selected standard nucleic acid molecules, which are advantageously present in a constant number per unit, by either dividing or, if necessary, the shared nucleic acid sample, into contact with at least one test probe pair which is sufficient degree is homologous to the nucleic acid as determined and with at least one selected and suitable standard probe pair sufficiently homologous to the nucleic acid molecule advantageously present in a constant number, the detector probes of said test probe pair and said standard probe pair being labeled with a suitable one. marker and phase the test probes have been fixed to a suitable carrier or to said recipient probes a sustenance has been fixed which allows isolation of the resulting hybrids, and after the hybridization reaction (s) has taken place, the test hybrid and the standard hybrid, if necessary, are separated and said attached marker is measured, whereby the number of nucleic acid molecules per given biological unit existing in the organism examined is obtained by comparing the number of test and standard nucleic acids. 30 Process according to claim 1, characterized in that the test and standard nucleic acids are deoxyribonucleic acids. Method according to claim 1, characterized in that the test nucleic acid is ribonucleic acid and the standard nucleic acid is deoxyribonucleic acid. 26 7 61 1 9 Method according to claim 1, characterized in that the test and standard nucleic acids are ribonucleic acids. Process according to claim 1, characterized in that the test nucleic acid is deoxyribonucleic acid and the standard nucleic acid is ribonucleic acid. Method according to claims 1, 2 and 3, characterized in that the detector probe of the standard probe pair - a recombinant plasmid comprising a 1.1 kb BglII-BglII fragment of the HindIII fragment of the human c-Ki-rasi gene, wherein said HindIII fragment has been cloned of the pBR322 plasmid and said BglII-BglII fragment have been subcloned into the restriction site of the restriction enzyme BamHI of the pBR322 plasmid -15 and the capturing probes - recombinant phages comprising a 0.5 kb BglII-HindIII fragment of the HindIII fragment of the human c-Ki gene wherein said Hind fragment has been cloned into the pBR322 plasmid and said BglII-HindIII fragment has been subcloned into the phage vectors M13 mp10 and M13 mp11 between the restriction sites of the restriction enzymes BamHI and HindIII - contacted, either individually or together with the test probe, or it is necessary split nucleic acid sample. 25 A method according to claims 1, 2 and 3, characterized in that the detector probe of the standard probe pair - a recombinant plasmid comprising a 1.2 kb EcoRl-KpnI (HindIII) fragment upstream from the promoter area of the mouse metallothionic gene, which fragment has been subcloned into the pATL53 plasmid between the restriction sites of the restriction enzymes Eco RI and HindIII - and the capturing probes - recombinant phages comprising a 0.8 kb KpnI-BglII fragment from the promoter area of the metallothionein gene and the upstream region thereof, which has been subcloned into the phage vectors 35 M13 mpl8 and M13 The restriction enzymes KpnI and BamHI - are contacted, either individually or together with the test probe pair, with an undivided, or if sh is necessary, divided nucleic acid sample. 05 Method according to claims 1, 2, 5, 6 and 7, characterized in that in order to determine the amplification rate of the c-myc oncogene and / or the number of messenger RNA molecules corresponding to this gene, the detector probe of the test probe pair - a recombinant plasmid comprising a 3.7 kb HindIII-XbaI fragment of the c-myc gene, which fragment has been subcloned into the pBR322 plasmid at the restriction site of the restriction enzyme HindIII - and the capturing probes - recombinant phage comprising a 1.1 kb XbaI-PstI fragment of c-myc The gene, which fragment has been subcloned into the phage vectors M13 mp10 and M13 mp11 between the restriction sites of the restriction enzymes XbaI and PstI, is contacted, either individually or together with the standard probe pair, with an undivided, or if necessary, a shared, nucleic acid sample. Method according to claims 1, 2, 3, 6 and 7, characterized in that, in order to determine the degree of amplification of the dihydrofolate reductase or the DHFR gene and / or the number of messenger RNA molecules corresponding to this gene, the detector probe of the test probe pair, a recombinant plasmid comprising a 0.75 kb HindIII-BglII fragment encoding the DHFR gene of the pMTVdhfr plasmid, which fragment has been subcloned into the pAT1532 plasmid vector between the restriction sites of the restriction enzymes HindIII and BamHI kb HindiII fragments of the MMTV gene area of the pMTVdhfr plasmid, which fragment has been subcloned into the phage vectors M13 mpl8 and M13 mpl9 at the restriction site of the restriction enzyme HindIII - are contacted, either individually or together with the standard probe pair, or if necessary split nucleic acid sample. 28 761 1 9 Reagent kit for quantitative determination of nucleic acid molecules by a method according to claim 1, characterized in that said kit contains at least one test probe pair and at least one standard probe pair, wherein the detector probes of both the test probe pair and the standard probe pair are labeled with a suitable marker, wherein the scaffold probes have been fixed to a suitable carrier and a substance has been fixed to said scaffold probes, enabling isolation of sandwich hybrids. 10 Reagent kit according to claim 10, characterized in that the detector probe of the test probe pair used to determine the degree of amplification of the c-myconogen and / or the number of messenger RNA molecules corresponding to this gene is a recombinant plasmid comprising a 3.7 kb HindIII-XbaI. restriction fragment of the c-myc gene, which fragment has been subcloned into the pBR322 plasmid at the restriction site of the restriction enzyme Hindin, and the capturing probes are recombinant phages comprising a 1.1 kb XbaI-PstI fragment of the c-myc gene, which fragment has been subcloned into the phage vectors M13 mp10 and M13 mp11 between the restriction sites of the restriction enzymes XbaI and PstI, and the detector probe of the standard probe pair is a 1.1 kb BgIII-BgIII fragment of the HindIII fragment of the human c-Ki-rasI gene, which HindIII fragment has been cloned into the pBR322 plasmid BglII-BglII fragments have been subcloned into the pBR322 plasmid at the restriction site of the restriction enzyme BamHI, and The genes are recombinant phages comprising a 0.5 kb BglII-HindIII fragment of the HindIII fragment of the c-Ki-rasI gene, said HindIII fragment being subcloned into the pBR322 plasmid of the c-Ki-rasI gene, and said BglII-HindIII fragment having been subcloned in the phage vectors M13 mp10 and M13 mp11 between the restriction sites of the restriction enzymes BamHI and HindIII.