LU86792A1 - QUANTIFICATION OF NUCLEIC ACID MOLECULES AND REAGENT KIT USED - Google Patents

Description

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

The number of copies of individual qenes in the genome is generally constant - In some cases, there is only one gene per haploid genome and several in others.

10 In certain circumstances, the number of copies may change. It has been found, for example, that the amplification of certain genes is associated with the development of cancer. It is also known that external factors, such as pharmaceuticals and metals, etc., cause the amplification of certain suppressors. The defective or increased level of expression of a gene, such as an oncogene, i.e. the amount of messaqer RNA in the cell is of major importance for the development of a disease. An increased number of certain chromosomes is the cause of certain hereditary diseases or other disorders, while certain hereditary diseases require only the duplication of a recessive gene. In any case, it is important to determine the number of genes or chromosomes present.

The number of certain DNA molecules, for example the degree of amplification of given qenes, is commonly determined by digestion of the extracted DNA to be studied with restriction enzymes and separation of the nucleotide fragments according to their length, by electrophoresis on agarose qel. The single stranded DNA is then transferred and fixed on a nitrocellulose filter, where hybridization takes place with the gene to be studied or part of the gene as a probe. The results are obtained by autoradiography (Southern, J. Mol. Biol. §, pp. 503-517, 1975). In each parallel analysis, the amount of cellular DNA is the same. The intensities of the 35 hybridization bands, that is to say the signals, are compared and 2 the ratios between the replication rates of the genes studied in the test samples are obtained by deduction.

The method provides only approximate results. Likewise, RNA is measured using Northern blotting or point blotting methods. These methods are very imprecise quantitatively (Thomas, Methods in Enzymol., 100, pp. 255-266, 1983).

Known methods, such as the Southern and Northern filter paper fixing methods, are slow and difficult to perform. Since they only provide 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. in solution, described in US Patent No. 4,486,539 and in British Patent Application No. GB 2,169,403 are quantitative (Virtanen et al., Lancet 1 ^, pp. 381-383, 1983). In addition, the method of the present invention requires a standard nucleic acid whose replication rate is constant and allows the determination of the number of corresponding nucleic acid molecules per given unit, such as a cell, a nucleus, a ribosome or a chromosome.

The object of the present invention is to produce a precise and rapid quantitative method for the determination of nucleic acid molecules, which is also faster and simpler to carry out than the methods commonly used. It can be used for cancer and prenatal diagnostics, to detect agents that cause amplification of genes and to demonstrate the development, for example, of drug resistance, as well as to determine the level of expression of d 'Messenger RNA.

At least two determinations must be made in the present invention. The nucleic acid molecule, which can be present in more than one copy, is determined, namely the test nucleic acid. Furthermore, the constituting nucleic acid molecule advantageously present in constant number is determined, the so-called "standard" standard nucleic acid.

In the method according to the present invention, a nucleic acid molecule represents a certain nucleotide sequence of 10 to 12 nucleotides or a gene containing several thousand nucleotides. It can also signify a messenger RNA or a nucleotide sequence much longer than a simple gene, that is to say an amplicone.

The determination of the test and standard nucleic acids is carried out according to an otherwise normal sandwich hybridization method, described for example in US Patent No. 4,486,539 or a solution hybridization method described in the Application for British Patent No. GB 2,169,403. The invention also relates to a reagent kit containing nucleic acid reagents, comprising at least one pair of test probes and at least one pair of standard probes.

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

Test and standard nucleic acids can be directly isolated from cells and identified by various hybridization techniques. These test and standard nucleic acids are, however, also available industrially and from various gene banks. Test and standard nucleic acids can be DNA or RNA.

Pairs of probes 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 corresponding nucleic acids are digested with suitable restriction enzymes; at least two of the resulting restriction fragments located relatively close to each other are cloned into at least two suitable vectors. One of the fragments, the detection probe, is labeled with a suitable detectable marker and the other, the capture probe, is attached to a suitable support or a substance is attached thereto, which substance allows the separation of the resulting hybrid from the hybridization mixture using another substance, such as the complementary component of an affinity pair.

The pairs of test and standard probes can be assembled into suitable reagent kits, in which the pairs of test and standard probes are both DNA or RNA, or the pair of test probes is formed of DNA and the pair of standard 15 RNA probes, or vice versa. Pretreatment and further processing of samples before hybridization and hybridization conditions should therefore be suitable for the pairs of probes used in the test.

The method of the present invention is particularly suitable for determining the number of nucleic acid molecules directly from cellular homogens.

The method can obviously also be used for the determination of purified or pure nucleic acids. However, before carrying out the method of the invention, one must choose the most suitable pretreatment of the nucleic acid sample.

It is possible to carry out both DNA and RNA determinations according to the method of the invention. The deoxyribonucleic acids are denatured to obtain single strands if necessary. Single-stranded messenger RNA molecules potentially disrupting the hybridization test can be hydrolyzed, for example by alkaline boiling. The sample is not denatured in the case of ribonucleic acid determinations, since double-stranded deoxyribonucleic acid does not interfere with the determination of RNA. It is of course possible to break the DNA with a deoxyribonuclease or to modify it chemically or mechanically so that it cannot participate in the hybridization reaction. With regard to DNA and RNA determinations, a suitable method for further processing of the sample should therefore be chosen or, alternatively, such processing may be omitted. The choice of a suitable method for further processing obviously depends on the method used for the preliminary processing of the nucleic acid sample. Many methods of preprocessing and further processing of nucleic acid samples have been described in the literature, so that it is possible to choose the most suitable method in each case.

Determinations in which both the test and standard nucleic acids are either DNA or RNA can be made with a sample which is not divided. Determinations in which the test nucleic acids are DNA and the standard nucleic acids are RNA or vice versa, should be made with a divided sample, since different methods are required for additional treatment. The sample can obviously be divided, even if the test and standard nucleic acids are of the same type of nucleic acid.

The hybridization test itself is carried out by placing the undivided sample solution, simultaneously in contact with at least one pair of test probes, and one pair of standard probes. If the solution of sample 30 has been divided, it is brought into contact separately with at least one pair of test probes and one pair of standard probes. In these cases, the amount of test nucleic acid is determined in one reactor and the amount of standard nucleic acid in another.

Whether the sample is divided or not, hybridization is allowed to take place under the most advantageous conditions and during the time in each case. When the reaction (s) have taken place, the resulting test and standard hybrids are separated from the hybridization mixture (s) by the support and washed, or by an isolating agent such as the complementary member of a pair of affinity. The marker attached to the test and standard hybrids is measured and the result is compared to calibration curves. In this way, the number of nucleic acid molecules to be studied can be determined per chosen unit.

The method of the invention has 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 considerably higher than that observed in normal tissue. In neuroblastomas, the N-myc gene is amplified.

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

The level of expression of a gene is important from the point of view of the phenotype and the functioning of the cell. It can be studied by measuring the amount of messenger RNA which is related to the amount of protein encoded by it. The transcript of an oncogene determines how it will ultimately be expressed.

7

The expression levels of an oncogene vary according to the type of cell, the rate of differentiation and the development phase of the cell. For example, at some stage of fetal development, the c-myc oncogene is rapidly replicated, while at another stage this is very slow. The degree of amplification is often correlated with the level of expression of the gene, although the latter can increase markedly without the former increasing. In these cases, the role of an oncogene is best determined by measuring its level of expression rather than the number of replications. In some cases, quantitative determination of the messenger RNA may be simpler and more convenient than quantifying the gene itself. By way of example, mention may be made of the oncogene c-myc, a labile protein easily coagulated by heat.

The method of the invention can also be used to identify digital chromosomal abnormalities such as Down syndrome. In prenatal diagnostics, it is also possible to determine whether the fetus is abnormal, that is, homozygous for a certain recessive gene.

The method of the invention and the nucleic acid reagents used in the method are further described below.

Example 1 - 25 Quantification of an amplified oncogene, a) Nucleic acid reagents and their preparation.

Standard probes:

Standard cell nucleic acid: The c-Ki-rasI gene is present in all human cells. Probe pairs for sandwich hybridization were prepared by subcloning the Hind III fragment of the c-Ki-rasI gene, which is 3.8 kb in length and whose restriction map has been described by Chang et al., PNAS 79, pp. 4848-52, 1982. The fragment is available, for example cloned into the plasmid pBR 322 (ATCC 35 41032) and can be obtained for example from the coloration of ATCC cultures.

-Additional processing of standard cell nucleic acid.

The pBR 322 clone described above was treated with the restriction enzymes BglII and Hind III and the resulting fragments were isolated from the agarose gel; purified fragments located in a close vicinity, were subcloned into two vectors suitable for the preparation of detection and capture probes.

10 - Standard detection probe. A BglII-BglII fragment

measuring approximately 1.1 kb in length was subcloned into the site of the restriction enzyme BaraHI of the plasmid pBR 322 and labeled 125 by mck-translation with dCTP labeled with 1 '.

- Standard capture probe. The Bglli ~ Hindlll fragment of about -15 kb, about 0.5 kb, was inserted into the vectors constituted by the phages Ml3 mplO and mpll between the restriction sites of the restriction enzymes BamHI and Hindm and fixed to a nitrocellulose filter ( 150 ng DNA / dia 1 cm).

Test probes.

20 - Test nucleic acid. A pair of probes for sandwich hybridization was prepared from a cloned c-myc gene which can be obtained for example from the collection of ATCC cultures (ATCC 41010). The gene restriction map has been described by Watt et al., PNAS 130, pp. 6307-6311, 1983). 25 - Additional processing of the test nucleic acid.

The c-myc gene was treated with the restriction enzymes Hindm / Xbal and PstI and the fragments isolated from the agarose gel, purified and subcloned into suitable vectors, to prepare the detection and capture probes.

- Test detection probe: the single-stranded tails of the HindHI-Xbal restriction fragment of the c-myc gene, measuring 3.7 kb in length, were made double-stranded by DNA polymerase. Hindlll linkers have been inserted by T4-DNA ligase into DNA fragments with sticky-ended ends; after phenol extraction, the DNA was treated with the Hindill restriction enzyme. The DNA fragment was then cloned into the plasmid pBR 322 at the restriction site of the restriction enzyme Hindill and labeled by nick-translation with dCTP labeled with 1 '.

5 - Test capture probe: the 1.1 kb Xbal-PstI fragment of the c-myc gene was cloned into the cloning vectors constituted by the phages Ml3 mplO and mpll between the restriction sites of the restriction enzymes Xbal and PstI and attached to a nitrocellulose filter (150 ng DNA / dia. 1 cm).

B) Determination of the calibration curve.

The sample used for the determination of the standard curve consisted of a clone pBR 322 denatured by an alkali, of the gene c-myc. The sandwich-hybridization solution to which the above test probes were added, consisted of 4 x SSC, 1 x Denhardt solution, 200 µg / ml herring sperm DNA and 0.2% from SDS. Hybridization took place at 65 ° C for 17-19 hours, then the filters were washed in the washing solution (0.1 x SSC, 0.2% SDS) at 50 ° C. The label attached to the sandwich hybrids was then counted in a gamma counter.

Table I

Sample cpm_ molecules / test 'filter c-myc 25 0 40 106 75 5 x 106 190 107 340 30 108 2200 c) Determination of the number of genes

The samples included 1) cells from a human placenta and 2) colo 320 cells, which can be obtained, for example, from the collection of 10 ATCC cultures (ATCC-CCC220). The DNA was isolated from the two samples and the same amount of cell DNA, denatured by an alkali at the boil, was added to the tests. Alkaline denaturation hydrolyzed all of the RNA present in the sample.

The test was carried out by adding to each sample both the c-myc and c-Ki-rasI filters and the two labeled reagents, which made it possible to measure both the test and standard DNA for each sample. Based on the 10 c-Ki-rasI determinations, it was found that each assay contained the same amount of DNA and it was possible to deduce that the c-myc gene in colo 320 cells is present at a replication rate of 16 to 20 times higher than that observed in the normal situation. The results are shown in Table II.

Table II

C-Ki-rasI filter sample c-myc filter cpm * cpm * number 20

Human placental cells 7 486 340 10 '

Colo cells 320 432 3205 1.6 x 10 ^ the reading obtained for the white filter was subtracted 25 from the readings.

Example 2

Quantification of amplified discomfort, a) Nucleic acid reagents and their preparation.

30 Standard probes.

- Standard cell nucleic acid: the control nucleic acid was taken from the promoter region of the metallothionine gene in mice, that is to say the MT gene with DNA immediately upstream thereof. The structure of the MT gene has been described by Pavlakis and Hamer, PNAS 8 £, pp. 397-401, 1983.

he

The reference nucleic acid fragment is available, for example, cloned into the vector pBPV-MMneo (442-12) (ATCC 37224) and can be obtained, for example, from the collection of ATCC cultures.

5 - Additional treatment of the standard cell nucleic acid ”The MT gene described above was treated with the restriction enzymes Kpnl, Bglll and EcoRI for the subclonaqe in the plasmid pAT153. The Kpnl tail was converted to a Hindm tail with a linker, 10 - Standard detection probe. The EcoRI-Kpnl-Olindlil fragment) measuring approximately 1.2 kb and located upstream of the promoter region of the metallothionine gene, was cloned into the plasmid pAT! 53 between the restriction sites of the restriction enzymes EcoRI and HindiJT, and labeled é pure nick-translation with nucleoside triphosphates labeled with. 32 p, - Standard capture probe, The 0.8 kb Kpni-Bglil fragment comprising the promoter region of the metallothionine gene and the region upstream thereof, has been cloned into the vectors 20 constituted by the phages M13 mpl8 and Ml3 mpl9 between the restriction sites of the restriction enzymes Kpnl and BamIII and attached to a nitrocellulose filter.

Test probes.

- Test nucleic acid, The pair of probes for the sandwich hybridization teu 25 was prepared with the plasmid pMTVdhfr commercially available (Bethesda Research Laboratories, product w ° 5369SS) whose structure is described by Lee et al., Nature 294, pp. 228-232, 1981,

Additional processing of the test nucleic acid.

The plasmid pMTVdhfr containing the qene cDNA for dihydro-folate reductase (DHFR) was treated with the restriction enzymes HindIII and BglII.

- Test detection probe. The Hindu 1- Bglll fragment measuring 0.75 kb and corresponding to the region coding for the DHFR gene of the plasmid pMTVdhfr was inserted into the vector

U

constituted by the plasmid ρΛΤΐ53 between the restriction sites

restriction enzymes HindTII and BanüII

and labeled by nick-translation with nucleoside-32 triphosphates labeled with p.

5 - Test capture probe. A Hindin fragment measuring 1.4 kb originating from the region of the MMTV qne of the plasmid pMTV-dhfr, was cloned into the vectors constituted by the phages M13 mpl8 and M13 mpl9, 10 b) Determination of the calibration curve The sample used for the test was purified DNA originating from the plasmid pMTVdhfr. The test itself was carried out according to the procedure of Example lbf except that a liquid scintillation counter was used for counting. The resulting standard curve is shown in Table lii.

Table III

Sample_ cpm_

molecules / DHFR filter test

20 ““ '0 17 106 45 3 x 106 79 107 210 25 "'" ".......

c) Determination of the number of genes

Cell lines originating from NIH 3Γ3 mouse fibroblast cell (ATCC culture collection, CRL 1658), which were transfected with different quantities of cDNA corresponding to the AHNni of the DHFR gene, were cultured on culture plates. cell and used as a sample. The cells were lysed with sodium dodecylsulfate and their DNA was sheared by compression through a thin hypodermic needle from a serine. A sample of 250 μl corresponding to approximately 10® cells was taken from the homogenate and 1350 μl of NaOH were added. The sample was heated to boiling 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 control. Hybridization, washing and counting of the marker were carried out according to the procedure of Example 1b, except that a liquid scintillation counter was used for counting.

The results are given in Table IV.

10 Table IV

Cell No. of cell- - -r Iules in cpm * No. of molecules No. of cpm sample- in sample αο ^ 1 s

Ion 15

Control cell (no DHFR-, cDNA) 182 1.05x10 21 <I0b

Line I 138 0.9 xlO ^ 80 3 x 10 ^ 3

Line II 210 l, 25xl06 732 5 x 107 40 20 - * cpm: the reading obtained with the white filter has been subtracted.

The MT gene is an internal marker that measures the number of cells present in a sample. The results show that in this test, 10 ^ cells gave a specific MT signal of 165 + 20 cpm. DHFR reagents measure the amount of DHFR-cDNA. The number of cells was deduced from the specific signal of MT. It was therefore possible to determine the number of copies of DHFR-cDNA in different cell lines, as shown in Table 30 IV.

Example 3

Quantification of messenger RNA, a) Nucleic acid reagents and their preparation.

It is also possible to measure the amount of mRNA derived from the DHFR cDNA with the test probes described in Example 2. The structure of the plasmid pMTVdhfr is such that the transcription of the DHFR gene begins at the MMTV promoter.

The resulting messengers are approximately 1.0 kb in length, of which approximately 0.25 kb is derived from the 5 MMTV promoter region and the remainder of DHFR cDNA (Lee et al., Nature 294 pp. 228-232 , nineteen eighty one).

Standard probes.

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

Test probes.

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

B) Determination of the calibration curve.

The sample used for the determination of the standard curve consisted of messenger RNA corresponding to the gene for dihydrofolate dreamuctase produced by in vitro transcription. The DNA required for transcription was prepared by subcloning the 1.4 kb HindiII fragment of the MMTV promoter from the plasmid pMTVdhfr and the 0.75 kb HindiII Bglil fragment (neighboring DHFR cDNA) into the plasmid pSP64 (Promega Biotec) between the restriction sites of the restriction enzymes HindIII and BamHI. The sample RNA was stored in 0.2% aqueous SDS solution.

The sandwich-hybridization test was carried out as described in Examples 1b and 2b, but the denaturation was omitted.

30 - 35 _ 15

Table V

Cpm_ sample

molecules / DHFR filter test

5 - 0 20 5 x 106 65 107 130 5 x 107 390 10 108 653 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.

The cells were lysed with sodium dodecyl sulfate and their DNA was sheared slightly by compression through a thin hypodermic needle from a syringe. A 250 µl sample of the homogenization product was taken, corresponding to approximately 5,108 cells. The homoaenate was then added to the sandwich-hybridization test without denaturation. The sandwich hybridization took place according to the procedure of Examples 2c and 1b, except that the only DHFR probes were added to the hybridization solution. The number of cells in a parallel 250 µl homogenate sample was determined with the MT probe as described in Example 2c. The results are provided in Table VI.

Table VI

30----

Number of „

Iules cell in 1 '-. ^ -îs = ü- r sample cnm,,., - cells in I by ____ sample j cell

Line I 380 3.5 x 106 1465 3.45 x 108 100 35 Line II 430 4.2 x 106 4800 2 x 109 500 ie • ft

Cpm: the reading obtained with the white filter has been subcontracted.

The results showed that cell line I produced approximately 100 mRNA molecules per cell from DHFR genes and that cell line II produced approximately 500 mRNA molecules from DHFR genes.

Example 4

Quantification of genes amplified by hybridization of solution 10 a) Nucleic acid reagents and their preparation Standard probes.

The standard cell nucleic acid, the standard detection probe and the standard capture probe were the same as in Example 2. The 1.2 kb fragment EcoRT- Kpnl- (HindIII) in the plasmid pAT153 a was marked by nick-translation with deoxycytidine labeled with ^^ I. The 0.8 kb Kpnl-BglII fragments in the ranges Ml3 mpl8 and M13 mpl9 were modified by biotin using the

TM

Photoprobe reagent (Vector Laboratories, CA, USA, product 20 SP-1000).

Test probes.

The test nucleic acid, the test detection probe and the test capture probe are the same as · * in Example 2. The 0.75 kb HindIII-IIIIII fragment in plasmid pAT153 was labeled with deoxycyt.i di 125 labeled 1 'I, The 1.4 kb HindIII fragments in the ranges M13 mpl8 and M13 mp! 9 were biotynilized using

TM

the Photoprobe reagent as above, b) Determination of the calibration curves A cell calibration curve was prepared using a known amount of cells, from which the hybridization signal was measured using the probes standards recognizing the MT gene. A calibration curve for the test nucleic acid was prepared with the plasmid pMTVdhfr and the test probes recognizing this 17 * plasmid. The hybridizations were carried out in 200 μΐ of a solution comprising 0.6 M NaCl, 20 mM phosphate buffer, pH 7.5, 1 mM d1 EDTA and 4% polyethylene glycol (JPEG 6000) laying one and a half hours at 70 ° C. After 5 the reaction, 50 μΐ agarose-streptavidin (Bethesoda

Research Laboratories, Maryland, USA, product # 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 agarose-10 streptavidin at 37UC for 15 minutes. The agarose was washed once for 5 minutes with the solution buffered with 1 M NaCl at 37 ° C and twice for 2 minutes with 15 mM NaCl, 1.5 mM sodium citrate at 55 ^ 0. The amount of bound hybrids was determined by measuring agarose in a gamma counter (Syvânen et al., Nucloie Acids Rus. 14, 5037-5048, 1986). The results are shown in Tables VII and VIII.

TABLE VII

2 0 Sample cpm

cells / MV probe test

0.8 x 106 162 1.6 x 106 216 25 3 x 106 298

TABLE VIII

Epm sample

30 molecules / DHFR probe test

106 148 5 x 1Û6 394 5 x 107 2240 35 ------------------- 18 c) Determination of the number of genes

Samples of the cell lines described in Example 2 were treated in a similar manner, except that the volume per corresponding sample - approximately 5 to 2 × 10 ^ cells was 125 μΐ- Determinations of the number of cells and the number of molecules test nucleic acid were made in flask-rents by adding the cell sample, appropriate detection and capture probes, and the mc-10 hybridization compound compounds to a final volume 200 μΐ. Control tests without cell calibration or test DNA were included. Hybridization, collection of hybrids, washing and measurement were performed as described in Example 4b. The results were read from the calibration curves in parallel with what is described in Example 4b. The results are shown in Table IX.

TABLE IX

20 cell MT_ DHFR_____ L 1 1 cpm No of cel 1 u— cpm No of mole- No, the in the cells in samples I sample copies

Control cell 253 2.3 x 10 ^ 73 <10 ^ 25 Line I 210 1.5 x 106 233 3.8 x 10ft 3

Line II 237 2.1 x 106 3059 8.8 x 107 42 cpm The values of the control tests without calibration of test cell or nucleic acid were deduced.

Claims (13)

19 1, Quantitative method for the determination of nucleic acid molecules by a d1 hybridât.Lon-sandwich or solution method, characterized in that the number of 5 nucleic acid molecules given per given unit is. determined by comparison of the number of test nucleic acid molecules potentially present in several copies in the unit, with the number of standard nucleic acid molecules chosen advantageously present in constant number in the same unit. 2. Method according to claim 1, characterized in that the nucleic acids present in the sample: a) are put, if necessary, in a form in which i is can participate in the hybridization reaction, * b) any nucleic acids possibly disrupting the hybridization reaction are put if necessary in a form in which they cannot interfere with the hybridization test; C) are brought into contact, either undivided or after compulsory division, with at least one pair of test probes sufficiently homologous to the nucleic acid possibly present, in several copies and with at least one pair of standard probes selected and suitable sufficiently homologous to the nucleic acid molecule advantageously present in constant number; the detection probes of this pair of test probes and of this pair of standard probes are labeled with a suitable detectable marker and the capture probes have been fixed to a suitable support where a substance has been fixed to the capture probes, which allows the isolation of the resulting hybrids \ d) after completion of the hybridization reaction (s), the test hybrid and the standard hybrid are separated when necessary and the attached label is measured; the number of nucleic acid molecules per given unit is obtained by comparison of the numbers of test and standard nucleic acid molecules. 3. Method according to claims 1 and 2, characterized in that the standard and test nucleic acids 5 are deoxyribonucleic acids. 4. Method according to claims 1 and 2, characterized in that the test nucleic acid is a ribonucleic acid and the standard nucleic acid is a deoxyribonucleic acid. 10 5. Method according to claims 1 and 2, charac terized in that the test and standard nucleic acids are ribonucleic acids. 6. Method according to claims 1 and 2, characterized in that the test nucleic acid is a deoxyribonucleic acid and the standard nucleic acid is a ribonucleic acid. 7. Method according to claims 1, 2, 3 and 4, characterized in that the detection probe of the pair of standard probes - a recombinant plasmid comprising a 1.2 kb BglII -Bglll fragment of the HindIII fragment of the human gene c-Ki-rasI, this HindIII fragment being cloned in the plasmid pBR322 and this fragment Bglll -Bglll being subcloned in the restriction site of the restriction enzyme BamHI of the plasmid pBR322 - and the capture probes - of the phages recombinants comprising a 0.5 kb BglII-HindIII fragment of the HindIII fragment of the human c-Ki-rasi gene, this HindIII fragment being cloned in the plasmid pBR322 and the BglII-HindIII fragment being subcloned in the vectors constituted by the phages M13 mplO and M13 mpll between the restriction sites of the restriction enzymes BamHI and HindIII - are brought into contact, either individually or with the pair of test probes, with an undivided or divided nucleic acid sample when necessary . 8. Method according to claims 1, 2, 3 and 35 4, characterized in that the detection probe of the pair 21 of standard probes - a recombinant plasmid comprising an EcoRI-Kpnl fragment (HindIII of 1.2 kb upstream of the promoter region of the mouse metallothionin gene, which fragment was subcloned into the plasmid pAT153 between the restriction sites of the restriction enzymes EcoRI and HindIII - and the capture probes - of the recombinant phages comprising a KpnI-BglII fragment 0.8 kb from the promoter region of the metallothionine gene and the region upstream thereof, which fragment has been subcloned into the vectors constituted by the phages M13 mpl8 and M13 mp19 between the restriction sites of the enzymes restriction Kpnl and BamHl - are contacted, either individually or with the pair of test probes, with an undivided or divided nucleic acid sample, if necessary. 9. Method according to claims 1, 2, 3, 6, 7 and 8, characterized in that to determine the degree of amplification of the c-myc oncogene and / or the number of messenger RNA molecules corresponding to this gene, the probe of the test probe pair - a recombinant plasmid comprising a HindIII-Xbal 3.7 kb fragment of the c-myc gene, this fragment being subcloned in the plasmid pBR322 at the restriction site of the restriction enzyme HindIII - and the capture probes - of the recombinant phages comprising a 1.1 kb Xbal-PstI fragment of the c-myc gene, which fragment was subcloned into the vectors M13 mplO and M13 mpll between the restriction sites of the restriction enzymes Xbal and PstI - are contacted, either individually or with the pair of standard probes, with an undivided or divided nucleic acid sample, if necessary. 30 10. Method according to claims 1,2, 3, 6, 7 and 8, characterized in that to determine the degree of amplification of the dihydrofolate reductase or of the DHFR gene and / or the number of messenger RNA molecules corresponding to this gene, the detection probe of the pair of test probes - a recombinant plas-35 mide comprising a 0.75 kb HindIII-BglII fragment coding for the DHFR gene of the plasmid pMTVdhfr, which fragment has been subcloned in the vector constituted by the plasmid pAT153 between the restriction sites of the restriction enzymes HindiII and BamHI - and the capture probes - of the recombinant phages comprising a HindIII fragment of .5 1.4 kb from the region of the MMTV gene of the plasmid pMTVdhfr , which fragment was subcloned into the vectors constituted by the phaaes M13 mpl8 and M13 mpl9 at the restriction site of the restriction enzyme HindIII - are brought into contact, either individually or with the pair of standard probes, with 10 a naked acid sample undivided or divided clique if necessary. 11. Kit of reagents for the quantitative determination of nucleic acid molecules, characterized in that the kit contains at least one pair of test probes and at least one pair of standard probes, the detection probes of both the pair of test probes and the pair of standard probes being labeled with a suitable marker and the capture probes having been fixed to a suitable support or a substance having been fixed to the capture probes, allowing the isolation of the sandwich hybrids. 12. Reagent kit according to claim 11, characterized in that the detection probe of the pair of test probes used for the determination of the degree of amplification of the c-myc oncogene and / or of the number of mole- 25 messenger RNA cules corresponding to this gene is a recombinant plasmid comprising a HindIII Xbal restriction fragment of 3.7 kb from the c-mvc gene, which fragment was subcloned into the plasmid pBR322 at the restriction site of l restriction enzyme HindIII, and the capture probes are recombinant phages comprising a 1.1 kb Xbal-PstI fragment of the c-myc gene, which fragment was subcloned into the vectors constituted by the phages M13 mplO and M13 mpll between the restriction sites of the restriction enzymes Xbal and PstI, and the detection probe of the pair of standard probes is a 1.1 kb BglII-BglII fragment of the HindIII fragment of 23 / (./ human gene c -Ki-rasI, this HindIII fragment having been cloned into the plasmid pBR32 2 and the BglII-BglII fragment having been subcloned into the plasmid pBR322 at the restriction site of the restriction enzyme BamHI, 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, this HindIII fragment having been subcloned into the plasmid pBR322 of the c-Ki-rasI gene, and this BglII-HindIII fragment having been subcloned into the vectors constituted by the phages M13. mplO and Ml3 mpll between the restriction sites of the restriction enzymes BamHI and HindlII. 13. Reagent kit according to claim 11, characterized in that the detection probe of the pair of test probes used for the determination of the degree of amplification of the dihydrofolate reductase or of the DHFR gene and / or of the number of messenger RNA molecules corresponding to this gene, is a recombinant plasmid comprising a 0.75 kb HindIII-BglII fragment coding for the DHFR gene of the plasmid pMTVdhfr, which fragment was subcloned into the vector constituted by the plasmid DAT153 between the restriction sites of the restriction enzymes HindIII and BamHI, and the capture probes are recombinant phages comprising a HindIII fragment of 1.4 kb from the region of the MMTV gene of the plasmid pMTVdhfr, which fragment has been subcloned in the 25 vectors constituted by phages M13 mpl8 and M13 mpl9 at the restriction site of the restriction enzyme HindIII, and the detection probe for the pair of standard probes is a recombinant plasmid comprising a fragment 1.2 kb EcoRI-Kpnl from the region upstream of the promo-30 region of the mouse metallothionin gene, which fragment was subcloned into the plasmid pAT153 between the restriction sites of the EcoRI restriction enzymes and HindIII, and the capture probes are recombinant phages comprising a 0.8 kb KpnI-BglII fragment of the metallothionin gene 35 formed by the promoter region and the region upstream thereof, which fragment has been subcloned into the vectors constituted by the phages M13 mpl8 and M13 mpl9 between the restriction sites of the restriction enzymes Kpnl and BamHI.