DK174784B1 - Quantification of nucleic acid molecules and reagent kits thereto - Google Patents

Description

DK 174784 B1

The present invention relates to the quantification of certain nucleic acid molecules, in particular the degree of amplification of genes and / or corresponding messenger RNA molecules using the sandwich or solution hybridization method, and a reagent kit used therefor.

The number of copies of individual genes in the genome is usually constant. In some cases, there is only one gene per haploid genome and in others several. Under certain circumstances, the number of copies may change. For example, the increase of certain genes has been shown to be associated with the development of cancer. It is also known that external factors such as pharmaceuticals and metals etc. cause multiple genes to be multiplied. For the development of a disease, it is the wrong or increased expression level of a gene, eg an oncogene, ie. the amount of messenger RNA in the cell, of great importance. Increased numbers of some chromosomes are the cause of certain hereditary diseases and other disorders, while some hereditary diseases require only one replication of one recessive gene. In all such cases, it is important to determine the number of chromosomes or genes present.

The number of certain DNA molecules, for example the degree of increase of certain genes, is currently determined by digesting the extracted DNA to be examined by restriction enzymes and by separating the nucleotide fragments according to the length of agarose gel electrophoresis. Then, the single-stranded DNA is transferred and attached to a nitro cellulose filter where hybridization takes place using the gene or part of the gene as probe. The results are obtained by autoradiographing (Southern, J. Mol. Biol. 98, pp. 503-517, 1975). With each parallel analysis, the amount of cellular DNA is the same.

The intensities of the hybridization bands, i.e. the signals are compared and the ratios of the copy number of the genes under study in the test samples are deduced. The method only gives approximate results. Likewise, RNA is measured by Northern staining or dot staining methods. These methods are quantitatively inaccurate (Thomas, Methods in Enzymol., 100, p.

255-266, 1983).

Known methods such as the Southern and Northern staining methods are slow and difficult to implement. Since they only give approximate results, their diagnostic value is questionable in cases where it is important to know the number of certain nucleic acid molecules per cell. given unit such as a cell.

The sandwich or solution hybridization methods described in U.S. Patent No. 4,486,539 and in British Patent Application No. 2,169,403 are quantitative (Virtanen et al., Lancet, J, pp. 381-383, 1983). . In addition, the method of the present invention requires a standard nucleic acid whose copy number is constant and constitutes the determination of the number of 20 relevant nucleic acid molecules per given unit such as a cell, nucleus, ribosome or chromosome.

The object of the present invention is to provide an accurate and rapid quantitative method for determining nucleic acid molecules, a method which is also faster and simpler to implement than the one currently used. It can be used for cancer and prenatal diagnosis, for detecting agents that cause reformation and for detecting the development of, for example, drug resistance as well as for determining the level of expression of messenger RNA.

At least two provisions are needed in the present invention. One determines the nucleic acid molecule which may be present in a number of copies, the test nucleic acid. The second determines the constitutive nucleic acid molecule which is advantageously present in a constant number of standard nucleic acids. By the method of the invention is meant by a nucleic acid molecule a given nucleotide sequence of 10-12 nucleotides or a gene containing several thousand nucleotides. It can also mean a messenger RNA or nucleotide sequence that is significantly longer than a single gene, i.e. an ampiicon.

The determination of test and standard nucleic acid is carried out using the otherwise normal sandwich hybridization method described, for example, in U.S. Patent No. 4,486,539, or a solution hybridization method disclosed in published British patent application. No. 2,169,403. The invention also relates to a reagent set containing nucleic acid reagents consisting of at least one test probe pair and at least one standard probe pair.

The reagents or probes used in the method are prepared using recombinant DNA techniques from nucleic acids sufficiently homologous to the test and standard nucleic acids. Sufficiently homologous nucleic acids may also be prepared synthetically or semi-synthetically.

The test and standard nucleic acids can be isolated directly from cells and identified by various hybridization techniques. However, such test and standard nucleic acids are also commercially available from various gene banks. The test and standard nucleic acids 25 can be either DNA or RNA.

Such pairs suitable for sandwich or solution hybridization methods are prepared from nucleic acids sufficiently homologous to the test and standard nucleic acids with recombinant DNA techniques. The relevant nucleic acids are degraded (digested) by appropriate restriction enzymes; at least two of the resultant restriction fragments, which are relatively close to each other, are cloned into at least two suitable vectors.

One of the fragments, the detector probe, is labeled with a suitable, detectable marker and the other, the capture probe, is either attached to a suitable carrier or a substance is attached thereto which allows separation of the resulting hybrid from the hybridization mixture by using an additional substance such as the complementary component of an affinity pair.

The test and standard probe pairs can be assembled into appropriate reagent sets where the test and standard probe pairs are both DNA or RNA, or where the test probe pair is DNA and standard probe paired RNA or vice versa. The pretreatment and further processing of the samples prior to the hybridization and hybridization conditions must then be 10 with these other pairs used in the test.

The process of the invention is particularly well suited for determining the number of nucleic acid molecules directly from cell homogenates. Of course, the process can also be used to determine purified or pure nucleic acids. However, before carrying out the method according to the invention, the most appropriate pretreatment of the nucleic acid sample should be chosen.

It is possible to perform both DNA and RNA assays by the method of the invention.

20 Deoxyribonucleic acids are denatured to obtain single strands if necessary. Single-stranded messenger RNA molecules that could potentially interfere with the hybridization test can be hydrolyzed, for example, by boiling with alkali. The sample is not denatured for ribonucleic acid assays since the double-stranded deoxyribonucleic acid does not interfere with the RNA assay. Of course, it is possible to break down DNA with deoxyribonuclease or change it either chemically or mechanically so that it cannot participate in the hybridization reaction. Therefore, in connection with DNA and RNA assays, an appropriate method for further processing of the sample must be selected or this additional treatment may be discharged. The choice of a suitable method for further processing, of course, depends on the method used for the preliminary treatment of the nucleic acid sample. Numerous methods for pre-processing and further processing of nucleic acid samples have been described in the literature, and from this, the most appropriate method can be selected in each case.

Determinations in which both the test and standard nucleic acids are either DNA or RNA can be made using an undivided sample. Determinations in which the test nucleic acids are DNA and the standard nucleic acids RNA or vice versa must be carried out by means of a split sample, as different methods of further processing are necessary. Of course, the sample can be split even if the test and standard nu-10 small acids are of the same nucleic acid type.

The hybridization test itself is performed by contacting the undivided sample solution simultaneously with at least one test probe pair and one standard probe pair. If the sample solution is divided, it is contacted separately with at least one test probe pair and one standard probe pair.

In such cases, the amount of test nucleic acid in one reaction vessel and the amount of standard nucleic acid in the other are determined.

Whether or not the sample is split, the hybridization is allowed to take place under the most advantageous conditions and times in each case. When the reaction or reactions have taken place, the resulting test and standard hybrids are separated from the hybridization mixture or mixtures by the carrier and washed, or by an insulating agent such as a complementary member of an affinity pair. The marker associated with the test and standard hybrids is measured and the result is compared with standard curves. In this way, one can determine the number of nucleic acid molecules under study per cell. selected device.

The method of the invention has practical diagnostic value, especially in detecting some types of cancer. In small cell lung carcinomas, the c-myc gene is often multiplied and its expression level is significantly higher than in normal tissue. In the case of neuroblastoma, the N-myc gene is multiplied.

6 DK 174784 B1

The method of the invention can also be used to detect the mutagenic or carcinogenic effects of certain agents or the development of drug resistance. It is known that external selection pressures may result in increased expression of a given gene. In the treatment of cancer, cells develop resistance to a given drug by multiplying the gene whose expression product inactivates the drug. One such case is methotrexate which induces the propagation of the dihydrofolate reductase (DHFR) gene. A further example is the propagation of the gene for metallothionine under the influence of cadmium.

The level of expression of a gene is significant from the standpoint of the phenotype and function of the cell.

This can be investigated by measuring the amount of messenger RNA correlated with the amount of protein seeded by it. The transcription product of an oncogene determines the way in which it will be finally expressed.

20 The levels of expression from oncogen vary in dependence on the cell type, the level of differentiation, and the developmental phase of the cell. For example, at a certain stage of fetal development, the c-myc oncogene is copied rapidly, while the development at another stage is very slow. The degree of multiplication or amplification is often correlated with the level of expression of the gene, although the latter can be significantly increased without the former. In such cases, the role of an oncogene is best determined by measuring its expression level rather than the number of 30 copies. In some cases, quantitative determination of messenger RNA may be simpler and easier to handle than quantification of the gene product cell. An example is the c-myc oncogene, a labile protein that is easily coagulated by heat.

The method of the invention may also be used to identify numerical chromosomal abnormalities such as Down syndrome. In prenatal diagnosis, it is also possible to determine if the fetus is defective, ie. homozygous for some recessive gene.

The process of the invention and the nucleic acid reagents used in the process will be described in more detail below.

Example 1 1 o Quantification of a Multiplied (Amplified) Oncogene a) Nucleic Acid Reagents_02_ Prepare Li n2_der; a.f

STANDARD WITHOUT

Standardnukleinsyre-T cell is intended. The c-Ki-rasI gene is present in all human cells. The probe pairs for sandwich hybridization were prepared by subcloning the HindIII fragment of the c-Ki rasI gene, whose length is 3.8 kb, and whose restriction map is described by Chang et al., In PNAS 79_, p. 4852, 1982. The fragment is available, for example, cloned into the pBR322 plasmid (ATCC 41032) and can be acquired, for example, from the ATCC culture collection.

Further treatment of standard cell nucleic acid. The above-described pBR322 clone was treated with BglII and HindIII restriction enzymes and the resulting fragments were isolated from the agarose gel; purified fragments located close to each other were subcloned into two suitable vectors to produce the detector and the capture probes.

30 Standard detector probe. A BglII-BglII fragment having a length of approx. 1.1 kb was subcloned into the BamHI restriction enzyme site on the pBR322 plasmid and labeled by 1 25 incision translation with I-labeled dCTP.

35 Standard Prisoner Probe. The BglII-HindIII fragment of ca. 0.5 kb was inserted into M13 mp10 and mpl1 phage vectors between the restriction sites of the BamHI and HindIII restriction enzymes and attached to a nitrocellulose filter (150 ng DNA / dia 1 cm).

8

DK 174784 W

TEST ONDER

5

The test nucleic acid. A sandwich pair for sandwich hybridization prepared from a cloned c-myc gene which can be obtained, for example, from the ATCC culture collection (ATCC 41010). The gene restriction map is described by Watt et al., PNAS 80, pages 6307-6311, 1983.

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

Test-probe detector. The single-stranded ends of the HindIII-XbaI restriction fragment of the c-myc gene, with a length of 3.7 kb, were double-stranded by DNA polymerase. The HindIII couplers were inserted by T4 DNA ligase into the resultant but-terminated DNA fragments; after phenol extraction, DNA was treated with the HindIII restriction enzyme. The DNA fragment was then cloned into the pBR322 plasmid at the restriction site of the HindIII restriction enzyme and labeled by incision 125 translation with I-labeled dCTP.

Test-capturing probe. The 1.1 kb x-bale PstI fragment of the c-myc gene was cloned into M13 mp1Q and mp11 phage cloning vectors between the restriction sites of XbaI and the PstI restriction enzymes and attached to the nitrocellulose filter (150 ng DNA / dia 1 cm).

35 b) §§§ ^ Ξίϋ! ££ ^ §® _? ^ § £ §ηά3Γά1ςηΓνθη

The sample used to determine the standard curve consisted of an alkali-denatured pBR322 clone of the c- 9 DK 174784 B1 myc gene. The sandwich hybridization solution to which the above test probe was added consisted of 4 x SSC, 1 x Denhardt solution, 200 µg / ml herring sperm DNA and 0.2% SDS. Hybridization took place at 65 ° C for 17-19 hours, after which the filters were washed in the wash solution (0.1 x SSC 0.2% SDS) at 50 ° C. The marker associated with the sandwich hybrids was then counted in a gamma counter.

Table 1 10 '

Sample, c-myc_ molecules / test c-myc filter 0 40 106 75 15 5 x 106 190 107 340 108 2200 C) Determination of number of genes 20

The samples included 1) cells from a human placenta and 2) Colo 320 cells obtainable, for example, from the ATCC culture collection (ATCC-CCC220). DNA was isolated from both samples and the same amount of cell DNA, denatured by boiling in alkali, was added to both samples. Denaturation with alkali hydrolyzed all the RNA present in the sample.

The test was selected by adding to each of the samples both the c-myc and c-Ki-rasI filters and the two labeled 30 reagents, which made it possible to measure both the standard and the test DNA for each sample. On the basis of c-Ki-rasI assays, each test was found to contain the same amount of DNA, and it can be deduced from this that the c-myc gene in Colo 320 cells is present in about 35 to 20-20 higher copies than in the normal situation. The results are shown in Table 2.

Table 2 10 DK 174784 B1

Sample c-Ki-rasI filter c-myc filter cpm * cpm * number - - 5 Human placental cells 486 340 10

Colo 320 cells 432 3205 1.6 x 10® * The reading obtained from the blank filter is subtracted from the readings.

Example 2

Quantification of Multiplied Gene a) Nucleic Acid Reagents_and_production_of

STANDARD WITHOUT

1 5

Standard cell nucleic acid. The control nucleic acid was taken from the promoter region of the mouse metallothionine gene, i. The MT gene, and the DNA immediately above it. The structure of the MT gene is described by Pav-lakis and Hamer, PNAS 8JD, pages 397-401, 1983. The reference nucleic acid fragment is available, for example, cloned into the pBPV-MMTneo (432-12) vector (ATCC 37224) and can eg from the ATCC culture collection.

Further treatment of cell standard nucleic acid.

2 5

The MT gene described above was treated with KpnI, BglII and EcorRI restriction enzymes for subcloning into pAT153 plasmid. The KpnI end was converted to a HindIII end with a coupler.

Standard Detector evil. The EcoRI-KpnI (HindIII) fragment having a size of ca. 1.2 kb and located above the (upstream of) promoter region of the metallothionine gene was cloned to the pATI53 plasmid between the restriction sites of the Eco RI and HindIII restriction enzymes 32 and labeled by incision translation with P-labeled nucleoside triphosphates.

11 DK 174784 B1

Standard capturing probe. The 0.8 kb KpnI-BgIII fragment comprising the promoter region of the metallothionine gene and the region above it was cloned into M1 3 mp18 and M13 mp19 phage vectors between the restriction sites of 5 KpnI and BamHI restriction enzymes and attached to the nitrocellulose filter.

TEST ONDER

10 Test nucleic acid. The probe pairs for the sandwich hybridization test were prepared using the commercially available pMTVdhfr plasmid (Bethesda Research Laboratories, Product No. 5369SS), whose structure is described by Lee et al., In Nature 294, pages 228-232, 1981.

15

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

20

Test-probe detector. The HindIII-BglII fragment, measuring 0.75 kb and corresponding to the region encoding the DHFR gene in the pMTVdhf plasmid, was inserted into the plasmid pAT153 vector between the restriction sites of HindIII 25 and the BamHI restriction enzymes and labeled by section 32 translation with P-labeled nucleoside trip, etc.

Test-capturing probe. A 1.4 kb HindIII fragment taken from the MMTV genome region of the pMTVdhfr plasmid was cloned into the M13 mp18 and M13 mp19 phage vectors.

b) §®§ void_of_standard curve

The sample used for the test was purified DNA from the pMTVdhfr plasmid. The test itself was performed as described in Example 1b, with the exception that a liquid scintillation counter was used for the count. The resulting standard curve is shown in Table 3.

12 DK 174784 B1

Table 3 5 Sample_ cmp_ molecules / test DHRF filter 0 17 1 06 45 3 x 106 79 10 7 υ 1θ '210

Cell lines derived from the mouse fibroblast cell NIH 3T3, which cell line can be acquired from the ATCC culture collection and whose number is CRL 1658, which had been transfected with different amounts of cDNA corresponding to mRNA in the DHFR gene, were seeded on cell culture plates and used as the sample. The cells were lysed by sodium dodecyl sulfate and their DNA was excised by pressing from a syringe through a fine injection needle. A 250 μΐ sample corresponding to 10 ° cells was taken from the homogenate and 50 μΐ NaOH was added. The sample was boiled and neutralized with acetic acid and the hybridization mixture. The total volume was 0.5 ml. All the 25 probes described above were added at the same time and a so-called blank filter was added as a background check. Hybridization, washing, and marker counting were performed as in Example 1b, with the exception that a liquid scintillation counter was used for the counting. The results are shown in Table 4.

35

Table 4 13 DK 174784 B1

Cell MT Number of cell _DHFR

cpm * smiles in sample cpm * number of mole number 5 friend cool in samples _ven_

Control cell (no DHFR, cDNA) 182 1.05 x 10 21 <10 ° 1Q Line I 138 0.9 x 106 80 3 x 106 3

Line II 210 1, 25 x 106 732 5 x 107_40 * cpm: the reading given by the blank filter is deducted.

The MT gene is an internal marker that measures the number of cells present in a sample. The results show that 10 cells in this test gave an MT-specific signal of 165 + 20 cpm. The DHFR reagents measure the amount of DHFR cDNA. The number of cells was deduced by the MT-20 specific signal. Thus, it was possible to determine the number of DHFR cDNA copies in different cell lines as shown in Table 4.

Example 3 25

Quantification of messenger RNA

a) Nucleic Acid Reagents and Preparation

Using the test probes described in Example 2, it is also possible to measure the amount of mRNA 30 derived from DHFR cDNA. The structure of the pMTVdhfr plasmid is such that transcription of the DHFR gene begins at the MMTV promoter. The resulting messengers have a length of approx. 1.0 kb. Of this, approx. 0.25 kb from the MMTV promoter region and the rest of the DHFR cDNA (Lee et al., Nature 2 £ 4, pages 228-232, 1981.

14 DK 174784 B1

STANDARD PROBES

Cell standard nucleic acid, standard detector and standard capture probe were as described in Example 2.

5

TEST PROBES

The test nucleic acid, test detector, and test capture probe were as described in Example 2.

10 b) Determination of the standard curve

The sample used to determine the standard curve consisted of messenger RNA corresponding to the dihydrofolate reductase gene produced by transcription in vitro. The DNA required for transcription was prepared by subcloning the 1.4 kb HindIII fragment of the MMTV promoter from the pMTVdhfr plasmid and the 0.75 kb HindIII-BglII fragment (DHFR cDNA) side by side into the pSP64 plasmid (Progema Biotec) between the restriction sites of HindIII and BanHI restriction enzymes. The sample of RNA was stored in 0.2% SDS aqueous solution.

The sandwich hybridization experiment was performed as described in Examples Ib and 2b, but denaturation was omitted.

Table 5

Sample cmp 30 molecules / test DHFR filter 0 20 5 x 106 65 107 130 5 x 107 390 J D β 10B 653 15 DK 174784 B1 c) Determination_of_the_number_of_messenger RNA_riol

The number of messenger RNA molecules corresponding to the DHFR gene was determined from the cell lines described in Example 2.

The cells were analyzed by sodium dodecyl sulfate and their DNA was weakly cut by pressing a syringe through a fine injection needle. A sample of 250 μΐ of the homogenate was taken, corresponding to approx. 5x10 5 cells. The homogenate was then added to the 10 sandwich hybridization test without denaturation. Sandwich hybridization took place as described in Examples 2c and 1b, with the exception that only DHFR probes were added to the hybridization solution. In a 250 µΐ parallel homogenize sample, the cell number was determined using the 15 MT probe as described in Example 2c.

The results are shown in Table 6.

Table 6 20 Cell MT Cell count _DHFR_ cpm * in sample cpm * number of mole number.

cools in the sample cell

Line I 380 3.5 x 106 1465 3.45 x 108 100

Line II 430 4.2 x 106 4800 2 x 109 500 25 ---- * cmp: The reading obtained with the blank filter is deducted.

The results showed that cell line I per cell produced approx. 100 messenger RNA molecules from the DHFR genes and cell line II produced approx. 500 messenger RNA molecules from the DHFR genes.

35 16 DK 174784 B1

Example 4

Quantification of Amplified Gene by Solution Hybridization_ (a) Nucleic Acid Reagents_and_ Preparation of

5 STANDARD WITHOUT

The cell standard nucleic acid, standard detector and standard capture probe were as described in Example 2.

The 1.2 kb Eco RI-KpnI (HindIII) fragment in pAT153 was 125 labeled by cut-in translation with I-labeled 10 deoxycytidine. The 0.8 kb Kpnl-Bglll fragments in M13 mp18 and M13 mpl9 were modified with biotin using the Photoprobe reagent (Vector Laboratories, CA, USA, product no. SP-1000).

15 TESTS WITHOUT

The test nucleic acid, test detector and test capture probe were as described in Example 2. The 0.75 kb HindIII-BglII fragment in pAT153 was labeled with I-labeled deoxycytidine. The 1.4 kb HindIII fragments in M13 20 mp18 and M1 3 mp19 were biotinylated by "Photo-probe" as above.

b) §§§ mood_of_standard curves

A cell standard curve was prepared using a known amount of cells from which the hybridization signal was measured using the standard probes recognizing the MT gene. A test nucleic acid standard curve was prepared by the pMTVdhfr plasmid and the test probes recognizing this plasmid. Hybridizations were performed in 200 µl of a solution consisting of 0.6 M NaCl, 20 mM phosphate buffer, pH 7.5, 1 mM EDTA, 4% polyethylene glycol (PEG 6000) for 1.5 hours at 70 ° C. . After the reaction, 50 μΐ of streptavidin agarose (Betheseda Research Laboratories, Maryland, USA, product no. 5942SA) and 1 M NaCl, 10 mM sodium phosphate, pH 7.5, 1 mM EDTA were added to a final volume of 500 μΐ. The hybrids were collected on the strep-tavidine agarose at 37 ° C for 15 minutes. The agarose was washed once for 5 minutes with the buffered 1 M NaCl solution at 37 ° C and twice for 2 minutes with 15 mM NaCl, 1.5 µM sodium citrate at 55 ° C. The amount of bound hydrides 5 was determined by measuring the agarose in a gamma counter (Syvånen et al., Nucleic Acids Res. J_4, 5037-5048, 1986). The results are shown in Tables 7 and 8.

Table 7

Sample_ cpm_ cells / test MT probes 0.8 x 106 162 1.6 x 106 216 15 3 x 106 298

Table 8 20 Sample_ cpm_ molecules / test DHFR probes 106 148 5 x 106 394 5 x 107 2240 25 c) Determination_of_number of genes

Samples of the cell lines described in Example 2 were treated in a similar manner, however, so that the sample corresponding to 2 x 10 6 cells was 125 μΐ.

The determinations of the number of cells and the number of test nucleic acid molecules were carried out in separate ampoules by the addition of the cell sample, the detector and capture probes, and the components of the hybridization mixture to a final volume of 200 μΐ. Control determinations were also included without cell standard or test DNA. Hybridization, collection of hybrids, washing and measurement were performed as described in Example 4b. The results are read from standard curves prepared in parallel as described in Example 4b. The results are shown in Table 9.

Table 9

Cell _J4T_ DHFR______ cpm * number of cell cpm * number of mole number of clays in sample cool in sample copies 10--

Control cell 253 2.3 x 106 73 <105

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 15 * cpm: Values from control assays without cellular or test nucleic acid are deducted.

20 25 30 35

Claims (12)

A method of quantitatively determining nucleic acid molecules by a sandwich or solution hybridization method, characterized in that the number of nucleic acid molecules given per biological unit found in the investigated organism is determined by comparing the number of test nucleic acid molecules potentially present in multiple copies within the unit with the number of selected standard nucleic acid molecules advantageously present in constant numbers per the same unit, by contacting the nucleic acids in the sample, either undivided or, if necessary, with at least one test probe pair sufficiently homologous to the nucleic acid determined and at least one selected and appropriate standard probe pair sufficiently homologous to the nucleic acid molecule advantageously present in a constant number, which probe pair consists of a double probe and a capture probe, the detector probes 20 of said test probe pair and said standard probe pair being labeled with an appropriate detectable marker and the captive probes being attached to a suitable carrier or a substance being attached to said capture probes, allowing isolation of the resulting hybrids, upon which the test hybrid and the standard hybrid after the hybridization reaction or reactions have taken place, if necessary, are separated and said associated marker is measured, the number of nucleic acid molecules per given unit is found by comparing the number of 30 test nucleic acids and standard nucleic acids. DK 174784 B1 2. A process according to claim 1, characterized in that the test and standard nucleic acids are deoxyribonucleic acids. The method of claim 1? known gene in that the test nucleic acid is a ribonucleic acid and the standard nucleic acid a deoxyribonucleic acid. The method of claim 1, wherein 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 Qg 3, characterized in that the detector probe in the standard probe pair - a recombinant plasmid comprising a 1.1 kb BglII-BglII fragment of the HindIII fragment of the human c-K The Rasl gene, which HindIII fragment is cloned into the pBR322 plasmid and said BglII-BgIII fragment is subcloned into the restriction site of the BamHI restriction enzyme in the pBR322 plasmid - and the capture probes - recombinant phage comprising a 0.5 kb BglII HindIII fragment of the human c-Ki rasI gene, which HindIII fragment is cloned into the pBR322 plasmid and which BglII-HindIII fragment is subcloned into the M1 3 mp10 and M13 mp11 phage vectors between the restriction sites of the BamHI and HindIII restriction enzymes - either individually or together with the test probe pair is contacted with an undivided or, if necessary, shared nucleic acid sample. 7. 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 Eco RI-KpnI (HindIII) fragment from the above promoter region of the mouse metallothionine gene, which fragment is subcloned into the pAT153 plasmid between the restriction sites of EcoRI-35 and the HindIII restriction enzymes - and the captive probes - recombinant phage comprising a 0.8 kb KpnI-BgIII fragment from the promoter region of the metallothionine gene and the region DK 174784 01 above, which fragment are subcloned into the M13 mp18 and M13 mp19 phage vectors between the restriction sites of the KpnI and BamHI restriction enzymes - either individually or together with the test probe pair being contacted with a undivided or, if necessary, shared nucleic acid sample. Method according to claims 1, 2, 5, 6 ° 8 Λ characterized in that to determine the degree of multiplication 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 from the c-myc gene, which fragment is subcloned into the pBR322 plasmid at the restriction site of the HindIII restriction enzyme - and the capture probes - recombinant phage comprising a 1.1 kb XbaI -PstI fragment of the c-myc gene, which fragment is subcloned into the M1 3 mp10 and M13 mpl1 vectors between the restriction sites of the XbaI and PstI restriction enzymes - in contact, either individually or together with the standard probe pair, with an undivided or if necessary divided nu-20 small acid sample. Method according to claims 1, 2, 3, 6 and 7, characterized in that to determine the degree of multiplication of the dihydrofolate reductase or 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 in the pMTVdhfr plasmid, which fragment is subcloned into the pAT153 plasmid vector between the restriction sites for HindIII and BamHI restriction enzymes - and the capture probes - recombinant phages comprising a 1.4 kb HindIII fragment in the MMTV genome region of the pMTVdhfr plasmid, which fragment is subcloned into the M13 mpl8 and M1 3 mp19 phage vectors at the restriction site of the HindIII restriction enzyme - in contact, either individually or together with the standard probe pair, with either a undivided or, if necessary, divided nucleic acid sample. DK 174784 B1 Reagent kit for quantitative determination of nucleic acid molecules according to the method of claim 1, characterized in that the kit contains 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 being labeled with an appropriate marker and: the capture probes are attached to a suitable carrier, or; a substance is attached to said captive probes, which allows the isolation of sandwich hybrids. Reagent set according to claim 10, characterized in that the detector probe in the test probe pair used to determine the degree of multiplication of the c-myc oncogene and / or the number of messenger RNA molecules corresponding to this gene is a recombinant plasmid comprising 3.7 kb HindIII-XbaI restriction fragment from the c-myc gene, which fragment is subcloned into the pBR322 plasmid at the restriction site of the HindIII restriction enzyme, and that the capture probes are recombinant phages comprising a 1.1 kb XbaI-PstI fragment of c. the myc gene, which fragment is subcloned into the M13 mp10 and M13 mp11 phage vectors between the restriction sites of the XbaI and PstI restriction enzymes, the detector probe of the standard probe pair being a 1.1 kb BgIII-BgIII fragment of the HindIII fragment of the human c Ki-rasI gene, which HindIII fragment is cloned into the pBR322 plasmid and which BglII-BgIII fragment is subcloned into the pBR322 plasmid at the restriction site of the BamHI restriction enzyme, and the capture probes are recombinant phages comprising a 0.5 kb BglII-HindIII fragment of the HindIII fragment of the c-Ki-rasI gene, which HindIII fragment is subcloned into the pBR322 plasmid of the c-Ki-rasI gene and which BglII-HindIII fragment is subcloned into the M13 mp10 and M13 mp11 phage vectors between the restriction sites of the BamHI and HindIII restriction enzymes. Reagent set according to claim 10. characterized in that the detector probe in the test probe pair used to determine the degree of multiplication of the dihydrofolate reductase or DHFR gene and / or the number of messenger RNA molecules corresponding to this gene is a recombinant DK 174784 B1 plasmid comprising a 0.75 kb HindIII-BgIII fragment encoding the DHFR gene in the pMTVdhfr plasmid, which fragment is subcloned into the pAT153 plasmid vector between the restriction sites of the HindIII and BamHI restriction enzymes and that the captive probes are recombinant. a 1.4 kb HindIII fragment in the MMTV genome region of the pMTVdhfr plasmid, which fragment is subcloned into the M13 mp18 and M13 mp19 phage vectors at the restriction site of the HindIII restriction enzyme, the detector probe of the standard probe pair being a recombinant plasmid comprising a 1.2 kb Eco RI-KpnI fragment from the above promoter region of the mouse metal lothionine gene, which is subcloned into the pAT153 plasmid flour are the restriction sites for the Eco RI and HindIII restriction enzymes, while the capture regions are recombinant phages comprising a 0.8 kb KpnI-BgIII fragment of the metallothionine gene formed by the promoter region and the region above it, which is subcloned into M13 mp18 and M13 mp19 the phage vectors between the restriction sites of the KpnI and BamHI restriction enzymes.