NO175380B - Method for Quantitative Determination of Nucleic Acid Molecules and Reagent Packages for Use in Performing the Process - Google Patents

Description

The invention relates to a method for the quantitative determination 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. Another aspect of the invention relates to the reagent pack used in the method.

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 other cases there are several. Under certain circumstances, the number of copies may change. The amplification of certain genes is e.g. found to be associated with the development of cancer. It has also been shown that external factors such as pharmaceuticals and metals etc. cause amplification of certain genes. For the development of a disease, the incorrect or increased expression level of a gene such as an oncogene, i.e. the amount of messenger RNA in the cell, is of utmost importance. Increased numbers of some chromosomes are the cause of certain hereditary diseases or other disorders, while some hereditary diseases only require the duplication 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, e.g. the degree of amplification of given genes is currently determined by digesting the extracted DNA to be examined with restriction enzymes and by separating the nucleotide fragments according to length by agarose gel electrophoresis. The single-stranded DNA is then transferred and fixed to a nitrocellulose filter, where hybridization takes place using the gene to be investigated or part of this gene as a probe. The results are obtained by autoradiography (Southern, J. Mol. Biol. 98, pp. 503-517, 1975). In each parallel assay, the amount of cellular DNA is the same.

The intensities of the hybridization bands, i.e. the signals, are compared and the ratios between the copy numbers of the genes under investigation in the experimental samples are estimated. The procedure only gives approximate results. Similarly, RNA is measured using Northern blotting methods or dot blotting methods. These methods are quantitatively very inaccurate (Thomas, Methods in Enzymol-, 100. pp. 255-266, 1983).

Known methods, such as Southern and Northern blotting methods are slow and difficult to perform. As 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 given unit, such as a cell.

Sandwich or solution hybridization methods described in US-PS 4,486,539 and in British Patent Application No. GB 2,169,403 are quantitative (Virtanen et al., Lancet 1, pp. 381-383, 1983). Moreover, the method according to the invention requires a standard nucleic acid, whose copy number is constant and allows the determination of the number of relevant nucleic acid molecules per given unit, such as a cell, nucleus, ribosome or chromosome.

The purpose of the invention is to provide an accurate and rapid quantitative method for nucleic acid molecular determination which is also faster and easier to perform than the methods currently used. It can be used for the diagnosis of cancer and prenatal diagnosis, for the detection of agents that cause gene amplification and for demonstration of the development of e.g. drug resistance, as well as to the determination of the expression level of messenger RNA. The purpose of the invention is achieved by a method and a reagent pack for use in the method, characterized by the features that appear in the patent claims.

At least two provisions are necessary in the present invention. One determines the nucleic acid molecule, which may exist in multiple copies, the experimental nucleic acid. The second determines the basic nucleic acid molecule which is advantageously present in constant numbers, the standard nucleic acid. In the method according to the invention, a nucleic acid molecule indicates a specific nucleotide sequence of 10-12 nucleotides or a gene containing several thousand nucleotides. It can also mean a messenger RNA or a nucleotide sequence considerably longer than a single gene, i.e. an ampicon.

The determination of test and standard nucleic acids is carried out using an otherwise common sandwich hybridization method described e.g. in US-PS 4,486,539 or a solution hybridization method described in British patent application No. GB 2 169 403. The invention also relates to a reagent pack containing nucleic acid reagents consisting of at least one experimental 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 that are sufficiently homologous to the experimental and standard nucleic acids. Sufficiently homologous nucleic acids can also be produced synthetically and semisynthetically.

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

Probe pairs suitable for the sandwich or solution hybridization method are prepared from nucleic acids sufficiently homologous to the test and standard nucleic acids by recombinant DNA techniques. The relevant nucleic acids are digested by suitable restriction enzymes; at least two of the resulting restriction fragments which are arranged 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 label, and the other, the capture probe, is either attached to a suitable support or a substance is attached thereto, which substance allows separation of the resulting hybrid from the hybridization mixture by means of another substance, such as the complementary component of an affinity pair.

The test and standard probe pairs can be built together in suitable reagent packs, where the test and standard probe pairs are both DNA or RNA or the test probe pair is DNA and the standard probe pair is RNA, or vice versa. The prior and further treatment of the samples before the hybridization and the hybridization conditions should therefore follow the probe pairs used in the experiment.

The method according to the invention is particularly suitable for determining the number of nucleic acid molecules directly from cellular homogenates. The method can of course also be used for the determination of purified or pure nucleic acids. However, before the method according to the invention is carried out, the most suitable pretreatment of the nucleic acid sample should be selected.

It is possible to carry out both DNA and RNA determinations using the method according to the invention. Deoxyribonucleic acids are denatured to obtain single strands, if necessary. Single-stranded messenger RNA molecules that potentially interfere with the hybridization experiment can be hydrolyzed, e.g. by alkaline boiling. The sample is not denatured in connection with ribonucleic acid determinations, as the double-stranded dioxyribonucleic acid does not interfere with the RNA determination. It is of course possible to break up DNA with deoxyribonuclease, or to change it chemically or mechanically so that it cannot take part in the hybridization reaction. Therefore, in connection with DNA and RNA determinations, a suitable method for further processing of the sample must be selected, or alternatively this further processing can be omitted. The choice of a suitable method for the further processing obviously depends on the method used in the pre-processing of the nucleic acid sample. A number of methods for pre-treatment and further treatment of nucleic acid samples have been described in the literature, which allows the most suitable method to be selected in each case.

Determinations where both the test and standard nucleic acids are either DNA or RNA can be performed using an undivided sample. Determinations where the experimental nucleic acids are DNA and the standard nucleic acids RNA or vice versa must be carried out using a split sample, as different methods for further processing are necessary. The sample can of course be split, even if the test and standard nucleic acids are of the same nucleic acid type.

The hybridization experiment itself is carried out by bringing the undivided sample solution into contact simultaneously with at least one experimental probe pair and one standard probe pair. If the sample solution has been divided, it is separately brought into contact with at least one experimental probe pair and a standard probe pair. In such cases, the amount of test nucleic acid is determined in one reaction container and the amount of standard nucleic acid in the other.

Regardless of whether the sample is split or not, the hybridization is allowed to take place under the most favorable conditions and time period in each case. Once the reaction or reactions have taken place, the resulting test and standard hybrids are separated from the hybridization mixture or mixtures by the support and washed, or by an isolation agent such as the complementary member of an affinity pair. The marker attached to the experimental and standard hybrids is measured and the result compared with standard curves. In this way, the number of nucleic acid molecules to be examined can be determined per selected unit.

The method according to the invention is of practical diagnostic value, 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 considerably higher than in normal tissue. In cases of neuroblastoma, the N-myc gene is amplified.

The method according to the invention can also be used to show the mutagenic or carcinogenic effects of certain agents or the development of drug resistance. It is known that external selection pressure can lead to increased expression of a particular gene. In the treatment of cancer, cells develop resistance to a given drug by amplification of the gene whose expression product inactivates the drug. One such case is methotrexate which induces amplification of the dihydrofolate reductase (DHFR) gene. A further example is the amplification of the gene for metallothionein under the influence of cadmium.

The expression level of a gene is important from the point of view of phenotype and cell function. This can be investigated by measuring the amount of messenger RNA, which correlates with the amount of protein that it codes for. The transcription product of an oncogene determines the way in which it will ultimately be expressed.

The expression levels of an oncogene vary depending on the cell type, differentiation level and developmental phase of the cell. E.g. at a certain stage of fetal development the c-myc oncogene is copied rapidly, while at another stage this is very slow. The degree of amplification often correlates with the expression level of the gene, although the latter may increase significantly without the former. In such cases, the role of the oncogene is best determined by measuring its expression level rather than its copy number. In some cases, quantitative determination of messenger RNA may be easier and more practical than quantification of the gene product itself. As an example, the c-myc oncogene can be mentioned, a labile protein that is easily coagulated by heat.

The method according to the invention can also be used for the identification of numerical chromosomal abnormalities, such as Down's syndrome. In prenatal diagnostics, it is also possible to determine whether the fetus is defective, i.e. homozygous for some recessive gene.

The method according to the invention and the nucleic acid reagents used in the method are described in more detail below.

Example 1

Quantification of an amplified oncogene

a) Nucleic acid reagents and their preparation

STANDARD PROBE

Cell standard nucleic acid

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 measuring 3.8 kb in length, the restriction map of which has been described by Chang et al., PNAS 79, pp 4848-52, 1982 The fragment is available e.g. cloned into the pBR322 plasmid (ATCC 41032) and can be obtained e.g. from

ATCC culture collection.

Further treatment of cell standard nucleic acid

The pBR322 clone described above was treated with BglII and HindIII restriction enzymes, and the resulting fragments were isolated from agarose gel; purified fragments that were arranged close to each other were subcloned into two suitable vectors for making the detector and capture probes.

Standard detector probe

A Bglll-Bglll fragment measuring approx. 1.1 kb was subcloned into the BamHI restriction enzyme site of the pBR322 plasmid and labeled by nick translation with <125>I-labeled dCTP.

Standard capture probe

The Bglll-Hindlll fragment of approx. 0.5 kb was inserted into M13 mplO and mpll 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).

EXPERIMENTAL PROBE

Experimental nucleic acid

A probe pair for sandwich hybridization was prepared from a cloned c-myc gene which can be obtained e.g. from the ATCC culture collection (ATCC 41010). The restriction map for the gene has been described by Watt et al., PNAS 80, pp. 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 isolated from the agarose gel, purified and subcloned into appropriate vectors to prepare the detector and capture probes.

Experimental detector probe

The single-stranded tails of the HindIII-XbaI restriction fragment of the c-myc gene, measuring 3.7 kb in length, were made double-stranded by DNA polymerase. The HindIII linkers were inserted by T4 DNA ligase into the resulting blunt-ended 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 nick translation with 125 I-labeled dCTP.

Experimental capture probe

The 1.1 kb XbaI-Pstl fragment of the c-myc gene was cloned into the M13 mplO and mpll phage cloning vectors between the restriction sites of the XbaI and Pstl restriction enzymes and attached to the nitrocellulose filter (150 ng DNA/dia 1 cm) .

b) Determination of the standard curve

The samples used to determine the standard curve consisted of

of an alkaline-denatured pBR322 clone of the c-myc gene. The sandwich hybridization solution, to which the above test probes were added, consisted of 4 x SSC, 1 x Denhardt's solution, 200 µg/ml herring sperm DNA and 0.2 percent SDS. Hybridization took place at 65°C for 17-19 hours, after which the filters were washed in the washing solution (0.1 x SSC 0.2 percent SDS) at 50°C. The marker attached to the sandwich hybrids was then counted in the gamma counter.

c) Determination of the number of genes

The samples included 1) cells from a human placenta and 2)

Colo 3 20 cells which can be obtained e.g. from the ATCC culture collection (ATCC-CCC220). DNA was isolated from both samples, and the same amount of cell DNA denatured by alkaline boiling was added to both experiments. Alkaline denaturation hydrolyzed any RNA present in the sample.

The experiment was carried out by adding both the c-myc and c-Ki-rasI filters and the two labeled reagents to each sample, which allowed measurement of both standard and test DNA for each sample. On the basis of the c-Ki-rasI determinations, each sample was found to contain the same amount of DNA, and it can therefore be concluded that the c-myc gene in Colo 320 cells is present in a 16-20 times higher copy number than in the normal situation. The results are shown in table 2.

Example 2

Quantification of amplified gene

a) Nucleic acid reagents and their preparation

STANDARD PROBE

Standard cell nucleic acid

The reference nucleic acid was taken from the promoter region of the mouse metallothionein gene, i.e. the MT gene, and the DNA immediately upstream of it. The structure of the MT gene has been described by Pavlakis and Hamer, PNAS 80_, pp. 397-401, 1983. The reference nucleic acid fragment is available e.g. cloned into the pBPV-MMTneo (342-12) vector (ATCC 37224) and can be obtained e.g. from the ATCC culture collection.

Further processing of cell standard nucleic acid

The MT gene described above was treated with KpnI, BglII and EcoRI restriction enzymes for subcloning into the pAT153 plasmid. The KpnI tail was converted to a HindIII tail with a linker.

Standard detection probe

The EcoRI-KpnI-(HindIII) fragment measuring ca. 1.2 kb and was located upstream of the promoter region of the metallothionein gene was cloned into the pAT153 plasmid between the restriction sites of the EcoRI and HindIII restriction enzymes and labeled by nick translation with <32>P-labeled nucleoside triphosphates.

Standard capture probe

The KpnI-Bglll fragment of 0.8 kb comprising the promoter region of the metallothionein gene and the region upstream thereof was cloned into M13 mpl8 and M13 mpl9 phage vectors between the restriction sites of the KpnI and BamHI restriction enzymes and attached to the nitrocellulose filter.

EXPERIMENTAL PROBE

Experimental nucleic acid

The probe pair for the sandwich hybridization experiment was prepared using the commercially available pMTVdhfr plasmid (Betfiesda Research Laboratories, product no. 53 69SS), the structure of which is described by Lee et al., Nature 294. pp. 228-232, 1981.

Further processing of experimental nucleic acid

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

Experimental detection probe

The HindIII-BglII fragment measuring 0.75 kb and corresponding to the region coding for the DHFR gene of the pMTVdhfr plasmid was inserted into the plasmid pAT153 vector between the restriction sites of the HindIII and BamHI restriction enzymes and tagged by nick translation with <32 >P-labeled nucleoside triphosphates.

Experimental capture probe

A HindiII fragment measuring 1.4 kb taken from the MMTV gene region of the pMTVdhfr plasmid was cloned into the M13 mpl8 and M13 mpl9 phage vectors.

b) Determination of the standard curve

The sample used in the experiment was purified DNA from the pMTVdhfr plasmid. The experiment itself was carried out as described in example 1b, with the exception that a liquid scintillation counter was used for counting. The resulting standard curve is shown in Table 3.

c) Determination of the number of genes

Cell lines obtained from mouse fibroblast cell NIH 3T3,

which cell line is available from the ATCC culture collection and which has the number CRL 1658 and which had been transfected with different amounts of cDNA corresponding to mRNA of the DHFR gene was grown on cell culture plates and used as the sample. The cells were lysed using sodium dodecyl sulfate and their DNA was broken up using shear forces as they were pushed through a fine hypodermic needle from an injection syringe. A 250 pl sample corresponding to approx. 10<6> cells were taken from the homogenate and 50 µl NaOH added. The sample was boiled and neutralized with acetic acid and the hybridization mixture. The total volume was 0.5 ml. All the probes described above were added simultaneously, and a so-called blank filter was added as a background comparison sample. Hybridization, washing and marker counting were performed as in

example lb, except that a liquid scintillation counter was used for counting. The results are shown in table 4.

The MT gene is an internal marker that measures the number of cells present in a sample. The results show that in this experiment IO<6> cells gave an MT-specific signal of 165 + 20 cpm. The DHFR reagents measure the amount of DHFR cDNA. The number of cells was deduced from the MT-specific signal. It was thus possible to determine the number of DHFR cDNA copies in different cell lines as shown in Table 4.

Example 3

Quantification of messenger RNA

a) Nucleic acid reagents and their preparation

When using the experimental probes described in example 2, it is also

possible to measure the amount of mRNA obtained 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 are approx. 1.0 kb long. Of this, 0.25 kb is obtained from the MMTV promoter region and the rest from the DHFR cDNA (Lee et al., Nature 294, pp. 228-232, 1981).

STANDARD PROBE

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

EXPERIMENTAL PROBE

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

b) Determination of the standard curve

The sample used for standard curve determination consisted of

messenger RNA corresponding to the dihydrofolate reductase gene produced by in vitro transcription. The DNA required for transcription was prepared by subcloning the 1.4 kb HindIII fragment of the MMTV promoter of the pMTVdhfr plasmid and the 0.75 kb HindIII-BglII fragment (DHFR cDNA) side by side into pSP64- plasmid (Promega Biotec) between the restriction sites of the HindIII and BamHI restriction enzymes. Sample RNA was stored in 0.2 percent SDS aqueous solution.

The sandwich hybridization experiment was performed as described in examples 1b and 2b, but the denaturation was omitted.

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 as described in Example 2.

The cells were lysed using sodium dodecyl sulfate, and their DNA was subjected to light shearing by pressing through a fine hypodermic needle from a hypodermic syringe. A 250 µl sample of the homogenate was taken, corresponding to approx. 5 x IO<6> cells. The homogenate was then added to the sandwich hybridization experiment without denaturation. Sandwich hybridization took place as described in Examples 2c and 1b, with the exception that only the DHFR probes were added to the hybridization solution. In a parallel sample of 250 µl homogenate, the cell count was determined using the MT probe as described in example 2c.

The results are shown in table 6.

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

Example 4

Quantification of amplified gene by resolution hybridization

a) Nucleic acid reagents and their preparation

STANDARD PROBE

Cell standard nucleic acid, standard detector and standard capture probe were as described in Example 2. The 1.2 kb EcoRI-Kpnl-(HindIII) fragment in pAT153 was labeled by nick translation with <125>I-labeled deoxycytidine. The 0.8 kb Kpnl-Bglll fragments in M13 mpl8 and M13 mpl9 were modified with biotin using the Photoprobe™ reagent (Vector Laboratories, CA, USA, Product No. SP-1000).

EXPERIMENTAL PROBE

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 <125>I-labeled deoxycytidine. The 1.4 kb HindiII fragments in M13 mpl8 and M13 mpl9 were biotinylated using Photoprobe™ as indicated above.

b) Determination of the standard curves

A cell standard curve was prepared using a known quantity

cells, from which the hybridization signal was measured using the standard probes that recognized the MT gene. An experimental nucleic acid standard curve was prepared with the pMTVdhfr plasmid and the experimental probes that recognized 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 percent polyethylene glycol (PEG 6000) for 1.5 hours at 70°C. After the reaction, 50 µl of streptavidin-agarose (Bethesda Research Laboratories, Maryland, USA, product no. 5942SA), and 1 M NaCl, 10 mM sodium phosphate, pH 7.5, 1 mM EDTA were added to the final volume of 500 µl. The hybrids were collected on the streptavidin-agarose at 37°C for 15 minutes. The agarose was washed once for 5 min with the buffered 1 M NaCl solution at 37°C and twice for 2 min with 15 mM NaCl, 1.5 mM sodium citrate at 55°C. The amount of bound hybrids was determined by measuring the agarose in a gamma counter. (Syvanenen et al., Nucleic Acids Res. 14, 5037-5048, 1986). The results are shown in tables 7 and 8.

c) Determination of the number of genes

Samples of the cell lines described in Example 2 were treated on

similar way, except that the volume per sample corresponding to approx. 2 x IO<6> cells were 125 fil. The determination of the number of cells and the number of test nucleic acid molecules was carried out in separate vials by adding the cell sample, the appropriate detector and capture probes and the components of the hybridization mixture to a final volume of 200 μl. Control samples without cell standard or experimental DNA were included. Hybridization, collection of hybrids, washing and measurement were performed as described in Example 4b. The results were read from standard curves prepared in parallel as described in example 4b. The results are shown in table 9.

Claims (12)

1. 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 that exists in the examined organism, is determined by comparing the number of experimental nucleic acid molecules, which potentially exist in several copies in the unit, with the number of selected standard nucleic acid molecules that advantageously exist in a constant number per same unit, in that the undivided, or if necessary divided, nucleic acid sample is brought into contact with at least one preliminary probe pair that is sufficiently homologous with the nucleic acid being determined and with at least one selected and suitable standard probe pair that is sufficiently homologous with the nucleic acid molecule, which is advantageously present in a constant number, which probe pair consists of a detector probe and a capture probe, the detector probes of said experimental probe pair and said standard probe pair being marked with a suitable detectable marker and the capture probes having been attached to a suitable carrier or a substance having been attached to the said capture probes, allowing isolation of the resulting hybrids, and after the hybridization reaction or reactions have taken place, the probe hybrid and the standard hybrid are separated, if necessary, and said attached marker is measured, the number of nucleic acid molecules per given biological unit that exists in the examined organism, is obtained by comparing the number of experimental and standard nucleic acids. 2. Method as stated in claim 1, characterized in that the test and standard nucleic acids are deoxyribonucleic acids. 3. Method as stated in claim 1, characterized in that the test nucleic acid is ribonucleic acid and the standard nucleic acid is deoxyribonucleic acid. 4. Method as stated in claim 1, characterized in that the test and standard nucleic acids are ribonucleic acids. 5. Method as stated in claim 1, characterized in that the test nucleic acid is deoxyribonucleic acid and the standard nucleic acid is ribonucleic acid. 6. Method as stated in claims 1, 2 and 3, characterized in that the detector probe in the standard probe pair - a recombinant plasmid comprising a 1.1 kb Bglll-Bglll fragment of the Hindlll fragment of the human c-Ki-rasI gene, wherein said HindIII fragment is cloned into the pBR322 plasmid and said BglII-BglII fragment is subcloned into the restriction site for the BamHI restriction enzyme in the pBR322 plasmid - and the capture probes - recombinant phages comprising a 0.5 kb BglII-HindIII- fragment of the HindIII fragment of the human c-Ki-rasI gene, said HindIII fragment being cloned into the pBR322 plasmid and said BglII-HindIII fragment being subcloned into the M13 mplO and M13 mpll phage vectors between the restriction sites for the BamHI and HindiII restriction enzymes - are brought, either individually or together with the experimental probe pair, into contact with an undivided or, if necessary, divided nucleic acid sample. 7. Method as stated in 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-Kpnl(Hindlll) fragment from the upper side of the promoter region of the mouse metallothionein gene, which fragment is has been subcloned into the pAT153 plasmid between the restriction sites for the restriction enzymes EcoRI and HindIII - and the capture probes - recombinant phages comprising a 0.8 kb KpnI-BglII fragment from the promoter region of the metallothionein gene and the region upstream of it, which fragment has been subcloned into M13 The mpl8 and M13 mpl9 phage vectors between the restriction sites for the KpnI and BamHI restriction enzymes - are brought, either individually or together with the experimental probe pair, into contact with an undivided or, when necessary, divided nucleic acid sample. 8. Method as stated in claims 1, 2, 5, 6 and 7, characterized in that for determining the degree of amplification 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, said fragment having been subcloned into the pBR322 plasmid at the restriction site for the HindIII restriction enzyme - and the capture probes - recombinant phages comprising a 1.1 kb XbaI-Pstl fragment of the c-myc gene, which fragment has been subcloned into the M13 mplO and M13 mpll phage vectors between the restriction sites of the XbaI and Pstl restriction enzymes - is brought, either separately or together with the standard probe pair, in contact with an undivided or, if necessary, a divided nucleic acid sample. 9. Method as stated in claims 1, 2, 3, 6 and 7, characterized in that for determining the degree of amplification of the dihydrofolate reductase or DHFR gene and/or the number of messenger RNA molecules corresponding to this gene, the detector probe of the experimental 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 pAT153 plasmid vector between the restriction sites of the HindIII and BamHI restriction enzymes - and the capture probes - recombinant phages comprising a 1.4 kb Hindi 11 fragment of the MMTV gene region of the pMTVdhf r plasmid, which fragment has been subcloned into the M13 mpl8 and M13 mpl9 phage vectors at the restriction site of the HindIII restriction enzyme - are brought, either separately or together with the standard probe pair, in contact with an undivided or, if necessary, a divided nucleic acid sample. 10. Reagent package for the quantitative determination of nucleic acid molecules, by a method according to claim 1, characterized in that the package contains at least one experimental probe pair and at least one standard probe pair, the detector probes of both the experimental probe pair and the standard probe pair being marked with a suitable marker, and the capture probes have been attached to a suitable carrier or a suitable substance has been attached to the capture probes, which allows isolation of sandwich hybrids. 11. Reagent package as stated in claim 10, characterized in that the detector probe in the experimental probe pair used for determining the degree of amplification of the c-myc oncogene, 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 HindIII restriction enzyme, and the capture probes are recombinant phages comprising a 1.1 kb XbaI Pstl fragment of the c-myc gene, which fragment has been subcloned into the M13 mplO and M13 mpll phage vectors between the restriction sites of the XbaI and Pstl restriction enzymes, and the detector probe in the standard probe pair is 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 into the pBR322 plasmid and said Bglll-Bglll fragment has been subcloned into the pBR322 plasmid at the restriction site t for the BamHI restriction enzyme, and the capture probes are recombinant phages comprising a 0.5 kb BglII-HindII fragment of the HindIII fragment of the c-Ki-rasI gene, said HindIII fragment having been subcloned into the pBR322 plasmid for the c-Ki-rasI gene, and the aforementioned Bglll-Hindlll fragment has been subcloned into the M13 mplO and M13 mpll phage vectors between the restriction sites for the BamHI and Hindllll restriction enzymes. 12. Reagent package as stated in claim 10, characterized in that the detector probe in the test probe pair used to determine the degree of amplification of the dihydrofolate reductase or DHFR gene and/or the number of messenger RNA molecules corresponding to this gene is 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 pAT153 plasmid vector between the restriction sites of the HindIII and BamHI restriction enzymes, and the capture probes are recombinant phages comprising a 1.4 kb HindIII fragment of the MMTV gene region of the pMTVdhfr plasmid, which fragment has been subcloned into the M13 mpl8 and M13 mpl9 phage vectors at the restriction site of the HindIII restriction enzyme, and the detector probe of the standard probe pair is a recombinant plasmid comprising a 1.2 kb EcoRI-Kpnl fragment from upstream of the promoter region of the mouse metallothionein gene, which fragment has been subcloned into pAT153-pla forged between the restriction sites of the EcoRI and HindIII restriction enzymes, and the capture probes are recombinant phages comprising a 0.8 kb KpnI-BglII fragment of the metallothionein gene formed by the promoter region and the region upstream of it, which fragment has been subcloned into M13 the mpl8 and M13 mpl9 phage vectors between the restriction sites of the KpnI and BamHI restriction enzymes.