NL8700483A - QUANTIFICATION OF NUCLEIC ACID MOLECULES AND REAGENT SET USED THEREIN. - Google Patents

Description

87302

ORION PHARMACEUTICAL

QUANTIFICATION OF NUCLEIC ACID MOLECULES AND REAGENT SET USED THEREOF

The invention relates to the quantification of certain nucleic acid molecules, more particularly the degree of amplification of genes and / or corresponding messenger RNA molecules using the sandwich or solution hybridization method, and the reagent set used.

The number of copies of the individual genes in the genome is usually 10 constant. In some cases there is only one gene per haploid genome and in others several, In some circumstances the number of copies may change. For example, the enhancement of certain genes has been found to be linked to the development of cancer. It is also known that external factors such as pharmaceutics and metals etc. can cause the amplification of certain genes. For the development of a disease, the erroneous or increased expression level of a gene, such as an oncogene, i.e. the amount of messenger RNA and a cell, is of great significance.

Increased numbers of single chromosomes are the cause of certain 20 hereditary diseases or other disturbances, while some hereditary diseases require only duplication of a 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 amplification of certain genes, is now determined by cutting the extracted DNA to be studied by means of restriction enzymes and by separating the nucleotide fragments to length by means of agarose gel electro-30 forese. The single-stranded DNA is then transferred and fixed to a nitrocellulose filter, hybridization taking place using the gene to be studied or part of the gene as a sniffing molecule.

8700483 -2- 87302 1 ï '

The results are obtained by autoradiography (Southern, J. Mol. BIOL. 98, p. 503-517, 1975). The amount of cellular DNA is the same with each parallel analysis.

The intensities of the hybridization bands, i.e. the signals, are compared and the ratios between the copy numbers of the genes to be studied in the test samples are determined.

The method provides approximate results only.

Analogously, RNA is measured by the Northern transfer or dot transfer method. These methods are quantitatively inaccurate (Thomas, methods in Enzymol., 100, 255-266, 1983).

Known methods such as the Southern and Northern transfer methods are slow and difficult to perform. Since they provide only 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.

•

The sandwich or solution hybridization methods described in U.S. Patent No. 4,486,539 and in British Patent Application No.

GB 2 169 403 are quantitative (Virtanen et al., Lancet JL, pp. 381-383, 1983). In addition, the method of the present invention requires a standard nucleic acid, the copy number of which is constant and allows the determination of the number of the relevant nucleic acid molecules per given unit, such as a cell, nucleus, ribosome or chromosome.

The object of the present invention is to produce an accurate and fast quantitative method for nucleic acid molecule determination, which can also be performed faster and simpler than currently used. It can be used for cancer and prenatal diagnostics, for detecting substances that cause gene amplification or for demonstrating the development of, for example, drug resistance, as well as for determining the expression level of messenger RNA.

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At least two determinations are required according to the present invention. One defines the nucleic acid molecule, which may be present in several copies, the test nucleic acid. The other determines the essential nucleic acid molecule which is advantageously present in a constant number, the standard nucleic acid. In the method of the invention, a nucleic acid molecule indicates a particular nucleotide sequence of 10-12 nucleotides or a gene containing several thousand nucleotides. It can also mean a messenger RNA or a nucleotide sequence significantly longer than a single gene, i.e. an amplicon.

The assay and standard nucleic acids are determined using an otherwise normal sandwich hybridization method, which is described, for example, in US Patent No. 4,486,539, or a solution hybridization method described in British Patent Application No. GB 2 169 403. invention also relates to a reagent set containing nucleic acid reagents consisting of at least one test sniff molecule pair and at least one standard sniff molecule pair.

20

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

The assay and standard nucleic acids can be isolated and identified directly from cells by various hybridization techniques. However, such assay and standard nucleic acids are also commercially available and from various gene banks.

Test and standard nucleic acids can be either DNA or RNA.

Snuff molecule pairs suitable for the sandwich or solution hybridization method are prepared from nucleic acids that are sufficiently homologous to the assay and standard nucleic acids 8700483-4-4-87302 by recombinant DNA techniques. The relevant nucleic acids are cut by of suitable restriction enzymes; at least two of the resulting restriction segments located relatively close to each other are donated via at least two suitable vectors. One of the fragments, the detector sniffing molecule, is labeled with a suitable detectable identifier and the other, the capturing sniffing molecule, is either fixed to a suitable carrier or a substance attached to it, which substance separates the resulting hybrid from the hybridization mixture. 10 through another substance, such as the complementary component of an affinity pair.

The test and standard sniff molecule pairs can be assembled into appropriate reagent sets, wherein the test and standard 15 sniff bright molecule pairs are either DNA or RNA, or the test sniff bright molecule pair is DNA and the standard sniff molecule pair RNA or vice versa. The pretreatment and further treatment of samples prior to the hybridization and the hybridization conditions should therefore be appropriate for the sniffing molecule pairs used in the assay.

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

It is possible to perform both DNA and RNA assays using the method of the invention. Deoxyribonucleic acids are denatured to obtain single strands if necessary. Single stranded messenger RNA molecules potentially interfering with the hybridization assay can be hydrolyzed, for example, by caustic boiling. The sample is not denatured 8700483 87302-5 with respect to ribonucleic acid assays since the double stranded deoxyribonucleic acid does not interfere with the RNA assay. It is, of course, possible to disrupt the DNA with deoxyribonuclease or to modify it either chemically or mechanically so that it cannot participate in the hybridization reaction. Therefore, in connection with the DNA and RNA assays, an appropriate method for further treatment of the sample should be selected, or alternatively, this further treatment may be omitted. The selection of a suitable method for further treatment naturally depends on the method used for the preliminary treatment of the nucleic acid sample. Numerous pretreatment and further treatment methods of nucleic acid samples have been described in the literature, allowing selection of the most suitable methods for each case.

Assays in which both the assay and standard nucleic acids, either DNA or RNA, can be performed using an undivided sample. Assays in which the test nucleic acids are DNA and the standard nucleic acids RNA or vice versa can be performed using a divided sample, since different methods are required for further treatment. The sample can of course be divided even if the test and standard nucleic acids are of the same nucleic acid type.

25

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

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In any case, regardless of whether the sample is divided or not, the hybridization can be allowed to proceed under the most advantageous conditions and time. Once the reaction (s) has taken place, the resulting test and standard hybrids are separated from the hybridization mixture (s) by the carrier and washed, or by means of an isolation reagent such as the complementary part of an affinity pair. The attached identifier to the test and standard hybrids is measured and the result compared to standard curves. In this way, the number of nucleic acid molecules to be studied can be determined per chosen unit.

The method according to the invention is of practical diagnostic value, in particular for the detection of some types of cancer.

In small cell lung carcinomas, the c-myc gene is often amplified and the level of expression inherited is significantly higher than in normal tissue. In cases of 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 substances or the development of drug resistance.

It is known that external selection pressure can result in an increased expression of a particular gene. In the treatment of cancer, cells develop resistance to a particular drug by enhancing the gene, the expression product of which 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 metallothionine under the influence of cadmium.

30

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

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The expression levels of an oncogene vary depending on the cell type, level of differentiation and stage of development of the cell.

For example, at some stage of fetal development, the c-myc oncogene is copied rapidly, while at another stage it is very slowly copied. The degree of amplification is often correlated with the expression level of the gene, although the latter may increase significantly without the former. In such cases, the role of an oncogene is best determined by measuring its level of expression rather than the number of copies. In some cases, the quantitative determination of the messenger RNA may be simpler and more convenient than the quantification of the gene product itself. As an example, c-myc oncogene, a labile protein that can be easily coagulated by heat, can be mentioned.

The method of the invention can also be used to identify numerical chromosomal abnormalities such as Down syndrome. In prenatal diagnosis, it is also possible to determine whether the fetus has abnormalities, i.e. homozygous for some recessive gene.

20

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

Example 1 25

Quantification of an amplified oncogene (a) Nucleic acid reagents and their preparation 30 STANDARD SNUFFLE MOLECULES ·

Cell standard nucleic acid. The c-Ki-rasI gene is present in all human cells. The snuff molecule pairs for sandwich hybridization were prepared by subcloning the Hindii fragment of the 35 c-Ki rasI gene, measuring 3.8 kb in length, of which the 8700483

tl W

873Q2-8 restriction map is described by Chang et al., PNAS 79, pp. 4848-52, 1982. The fragment is available e.g. donated in the pBR322 plasmid (ATCC 41032) and may e.g. are obtained from the ATCC culture collection.

5

Further treatment of the cell standard nucleic acid. The previously described pBR322 clone was treated with BglII and HindII restriction enzymes and the resulting fragments isolated from the agarose gel; closely spaced purified fragments were subcloned into two suitable vectors for the preparation of the detector and the scavenging molecules.

Standard detector sniffing molecule. A BglII-BglII fragment, the measured length of which was about 1.1 kb, was subcloned into the BamHI restriction enzyme site of the pBR322 plasmid and labeled by 125 by means of a marker translation Inet dCTP.

Standard catching sniffing molecule. The approximately 0.5 kb BglII-HindII fragment was inserted into the M13 mplO and mplI phage vectors between the restriction sites of the BamHI and HindII restriction enzymes and fixed on a nitrocellulose filter (150 ng DNA / dia 1 cm).

TEST SNUFFLE MOLECULES 25

Test nucleic acid. A snuff molecule pair for the sandwich hybridization was prepared from a donated 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. pp, 6307-6311, 1983.

Further treatment of the test nucleic acid. The c-myc gene was treated with HindIII, Xbal and PstI restriction enzymes and the fragments isolated from the agarose gel, purified and subcloned into suitable vectors to prepare the detector and catcher sniffing molecules.

8700483 Μ -9- 87302

Tesfc detector sniffing molecule. The single stranded tails of the HindII-Xbal restriction fragment of the e-myc gene, the measured length of which was 3.7 kb, were double stranded by DNA polymerase. The Hind III linkers were inserted through T4 DNA ligase into the resulting straight cut DNA fragments; after phenol extraction, the DNA was treated with the HindII restriction enzyme. The DNA fragment was then donated into the pBR322 plasmid at the restriction site of the HindIII * restriction enzyme and labeled by marker translation with 125 I-labeled dCTP.

Test catcher sniffing molecule. The 1.1 kb Xbal-PstI fragment of the c-myc gene was donated in the M13 mplO and mpll phage cloning vectors between the restriction sites of the Xbal and PstI restriction enzymes and fixed to the nitrocellulose filter (150 ng DNA / slide 1) cm).

(b) Determination of the standard curve. The sample used for the determination of the standard curve consisted of an alkaline denatured pBR322 clone of the c-myc gene. The sandwich hybridization solution to which the aforementioned test sniffing molecules were added consisted of 4 x SSC, 1 x Denhardt solution, 200 µg / ml herring sperm DNA and 0.2% SDS.

The 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 identifier attached to the sandwich hybrids was then counted in the gamma counter.

30 - 8700433 »» 87302 -10-

Table 1

Sample tpm_ molecules / test c-myc filter 5 ___ 0 40 106 75 5 x 106 190 107 340 10 108 2200 (c) Determination of the number of genes 15 The samples contained 1) cells of a human placenta and 2)

Cola 320 cells, e.g. can be obtained from the ATCC culture collection (ATCC-CCC220). DNA was isolated from both samples, and the same amount of cell DNA, denatured by caustic boiling, was added to both tests. Alkaline denaturation hydrolysed any RNA present in the sample.

The test was performed by adding to each sample both the c-myc and the c-Ki-rasI filter and the two labeled reagents, allowing both standard and test DNA to be measured for each sample. Based on the c-Ki-ras I assays, each test was found to contain the same amount of DNA from which it can be deduced that the c-myc gene is present in Colo 320 cells in approximately 16-20 higher copy number than in the normal situation.

The results are shown in Table 2.

30

Table 2 ___

Sample c-Ki-rasI filter _c-myc filter _tpm * _tpm * _number

Human placental cells 486 340 107 35 Colo 320 cells_432_3205_1.6 x 10 ° 8700483 φ. + -11- 87502 * the resulting blank filter observation was subtracted from the observations.

EXAMPLE 2 5

Quantification of amplified gene

(a) Nucleic acid reagents and their preparation 10 STANDARD SNUGLE MOLECULES

Cell standard nucleic acid. The control nucleic acid was taken from the promoter region of the mouse matallothionin gene, i.e., the MT gene, and the DNA in immediate proximity in the reading sequence. 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. donated in the pBPV-MN.Tneo (432-12) vector (ATCC 37224) and may e.g. are obtained from the ATCC culture collection.

20

Further treatment of the cell standard nucleic acid. The MT gene described above was treated with Kpnl, BglII and EcoRI restriction enzymes for subcloning into the pAT153 plasmid. The Kpnl tail was converted into a HindlII tail with a bridge former.

25

Standard detector sniffing molecule. The EcoRI-Kpn1 (HindlII) fragment about 1.2 kb in length, located in the reading direction of the promoter region of the metallothionine gene, was donated to the pAT 153 plasmid between the restriction sites of the EcoRI and HindlII. restriction enzymes and labeled by 32 a marker translation with P-labeled nucleoside triphosphates.

Standard catcher sniffing molecule. The 0.8 kb Kpnl-BglII fragment containing the promoter region of the metallothionine gene and the region in the reading direction thereto was donated in the 9700483 -12- 87302 M13 mpl8 and M13 mpl9 phage vectors between the restriction sites of the Kpnl and BamHI restriction enzymes and fixed to the nitrocellulose filter.

5 TEST SNUFFLE MOLECULES-

Test nucleic acid. The sniff molecule pair for the sandwich hybridization assay was prepared using the commercially available pMTVdhfr plasmid (Bethesda Research Laboratories; 10 product No. 5369SS), the structure of which is described by Lee et al., Nature 294, pp. 228-232, 1981.

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

Test detector sniffing molecule. The HindII-BglII fragment, 0.75 kb in length and corresponding to the region encoding the DHFR gene of the pMTVdhfr plasmid, was inserted into the plasmid pAT153 vector between the restriction sites of the HindII and

BamHI restriction enzymes and labeled by a marker-32 translation with P-labeled nucleoside triphosphates.

Test catcher sniffing molecule. A 1.4 kb HindIII fragment, 25 * extracted from the MMTV gene region of the pMTVdhfr plasmid, was donated into the M13 mpl8 and M13 mpl9 phage vectors.

(b) Determination of the 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 except that a liquid scintillation counter was used for counting. The resulting standard curve is shown in Table 3.

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Table 3

Sample_rpm_ ~ molecules / test DFRH filter 5 ____; __ 0 17 106 45 3 x 106 79 10 107 210 (c) Determination of the number of genes 15 Cell lines transfected with different amounts of cDNA corresponding to the mRNA of the DHFR gene were grown on food culture plates and used as sample. The cells were lysed using sodium dodecyl sulfate and their DNA was sheared by squeezing it through a fine hypodermic needle of an infection syringe. A 250 x / 1 sample corresponding to about 10 4 cells was taken from homogenate and 50 IA 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 previously described sniffing molecules were added simultaneously and a so-called blank filter was added as background control. Hybridization, washing and mark counting were performed as in Example 1b except that a liquid scintillation counter was used for the counting. The results are shown in Table 4.

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Table 4

Cell MT Number of cells _OHFR__ rpm * in the sample rpm * Number of molecules Number 5 in the sample copies

Control cell (no DHFR cDNA) 182 1.05 x 106 21 <106 10 Line I 138 0.9 x 106 80 3 x 10³ 3

Line II 210 1.25 x 106 732 5 x 107 40 * rpm: the reading obtained from the blank filter has been subtracted 15

The MT gene is an internal identifier that measures the number of cells present in a sample. The results show that in this test 10 ^ cells gave an MT specific signal of 165 + 20 rpm. The DHFR 20 reagents measure the amount of DHFR cDNA. The number of cells was determined from the MT-specific signal. It was therefore possible to determine the number of DHFR cDNA copies in different cell lines as shown in Table 4.

25 EXAMPLE 3

Quantification of the messenger DNA

(a) Nucleic acid reagents and their preparation 30

Using the test sniffing molecules described in Example 2, it is also possible to measure the amount of mRNA 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 are approximately 1.0 kb in length. Of these, 8 7 0 4 S 3 87 302-15 are about 0.25 kb from the MMTV promoter region and the remainder from DHFR cDNA (Lee et al., Nature 294, pp. 228-232, 1981).

STANDARD SNUFFLE MOLECULES 5

The cell standard nucleic acid, the standard detector and the standard catcher molecule were as described in Example 2.

TEST SNUFFLE MOLECULES

10

The test nucleic acid, the test detector and the test catcher sniff molecule were as described in Example 2.

(b) Determination of the standard curve 15.

The sample used for the standard curve assay consisted of messenger RNA corresponding to the dihydrofolate reductase gene produced by in vitro transcription. The DNA required for transcription was prepared by subcloning 2Q of the 1.4 kb Hind III fragment of the MMTV promoter of the p MTVdhfr plasmid and the 0.75 kb HindII-BglII fragment (DHFR cDNA) located side by side in the pSP64 plasmid (Promega Biotec) between the restriction sites of the Hindii and BamHI restriction enzymes.

The RNA sample was stored in 0.2% SDS aqueous solution.

25

The sandwich hybridization test was performed as described in Examples 1b and 2b but the denaturation was omitted.

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Table 5

Sample_rpm

Molecules / test DHFR filter 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 example shown in Example 2 described cell lines.

The cells were lysed using sodium dadecyl sulfate and their DNA lightly sheared by pressing through a fine hypodermic syringe needle. A 250 µl sample of the homogenate was taken corresponding to approximately 5 x 10 4 cells. The homogenate was then added to the sandwich hybridization test without denaturation. The sandwich hybridization took place as given in Examples 2c and 1b, with the exception that only the DHFR sniffing molecules were added to the hybridization solution. In a parallel sample of 250 µl homogenate, the cell number was determined using the MT sniffing molecule as described in Example 2c.

The results are shown in Table 6.

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Table 6

Cell MT Cell number in _ DHFR__ rpm * the sample rpm * Number of molecules Number per 5 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 10 * rpm: the reading obtained from the blank filter has been subtracted.

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

EXAMPLE 4

Quantification of amplified gene by solution hybridization 20 (a) Nucleic acid reagents and their preparation STANDARD SNUFF MOLECULES The cell standard nucleic acid, standard detector and standard catcher molecule were as described in Example 2.

The 1.2 kb EcoRI-Kpnl (HindIII) fragment in pAT153 was labeled by 125 by marker translation with I-labeled deoxycytidine.

The 0.8 kb Kpnl-BglII fragments in M13 mpl8 and M13 mpl9 were

30 TM

modified with biotin using the Photoprobe reagent (Vector Laboratories, CA, USA, product No SP-1000); 8700433.

87302 -18-

TEST SNUFFLE MOLECULES

The test nucleic acid, the test detector and the test capture snuff molecule 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 HindII fragments in

M13 mpl8 and M13 mpl9 were biotinylated using TM

from Photoprobe as before.

10 (b) Determination of the standard curve

A cell standard curve was prepared using a known amount of cells, the hybridization signal of which was measured using the standard sniffing molecules recognizing the MT gene. A test nucleic acid standard curve was prepared with the pMTVdhfr plasmid and the test sniffing molecules recognizing this plasmid. Hybridizations are performed in 200 L of a solution consisting of 0.6 M NaCl, 20 mM

phosphate buffer, pH 7.5, ImM EDTA, 4% polyethylene glycol (PEG 6000) for 1.5 hours at 70 ° C. After the reaction, 50 l of streptavidin agarose (Bethesda Research Laboratories, Maryland, USA, product No. 5943SA), and 1 M NaCl, 10 mM sodium phosphate, pH 7.5, 1 mM EDTA were added to a final volume of 500 l. The hybrids were collected on the streptavidin agarose at 37 ° C for 15 min.

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 (Syvanen et al., Nucleic Acids Res. 14, 5037-5048, 1966).

30 The results are shown in Tables 7 and 8.

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Table 7

Sample_ rpm_ _ cells / test MT sniffing molecules 5 _ ^ _____ 0.8 x IQ6 162 1.6 x 106 216 3 x 106 298 10

Table 8

Sample_rpm_ molecules / test DHFR sniffing molecules 15 __: _; ___ IQ6 148 5 x 106 .394 5 x 107 2240 20 (c) Determination of the number of genes

Samples of the cell lines described in Example 2 were treated analogously, except that the volume per sample corresponding to about 2 x 10 cells was 125 liters. The cell number and the number of test nucleic acid molecules were determined in separate tubes by adding the cell sample, the appropriate detector and catcher sniffing molecules, and the hybridization mixture components to a final volume of 200 l. Control assays without cell standard or test DNA were included. Hybridization, hybridization, washing and measuring 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.

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Table 9

Cell _MT _; _ DHFR_ rpm * Number of cells in rpm * Number of molecules Number of the sample in the sample copies

Control cell 253 2.3 x 10 ^ 73 <10 ^

Line I 210 1.5 x 106 233 3.8 x 106 3

Line II 237 2.1 x 106 3059 8.8 x 107 42 * rpm: values of the controls without cell standard or test nucleic acid have been subtracted.

Claims (13)

A quantitative method for the determination of nucleic acid 5 molecules by a sandwich or solution hybridization method, characterized in that the number of determined nucleic acid molecules per given unit is determined by comparing the number of the test nucleic acid molecules potentially present in different copies in unit IQ relative to the number of selected standard nucleic acid molecules favorably present in a constant number per the same unit. Determination method according to Claim 1, characterized in 15. if the nucleic acids present in the sample (a) are converted, if necessary, into a form in which they can participate in the hybridization reaction, (b) all nucleic acids that potentially can interfere with the hybridization reaction, if necessary, are converted into a form in which they cannot interfere with the hybridization test, (c) are contacted, either undivided or shared if necessary, with at least one test sniff molecule pair sufficiently homologous to the nucleic acid potentially present in several copies and with at least one selected and suitable standard sniffing molecule pair sufficiently homologous to the nucleic acid molecule advantageously present in a constant number; the detector sniffing molecules of said test sniffing molecule pair and said standard sniffing molecule pair are labeled with a suitable, detectable identifier, and the catcher sniffing molecules are fixed to a suitable support or where a substance is attached to said catcher sniffing molecules, thereby isolating allows the resulting hybrids; (d) after the hybridization reaction or reactions have occurred, the test hybrid and the standard hybrid are separated 8700483-87302-22 if necessary, and said attached identifier is measured; wherein the number of nucleic acid molecules per given unit is obtained by comparison of the assay and standard nucleic acid numbers. 5 The method according to Claims 1 and 2, characterized in that the test and standard nucleic acids are deoxyribonucleic acids. The method according to Claims 1 and 2, characterized in that the test nucleic acid is ribonucleic acid and the standard nucleic acid is deoxyribonucleic acid. The method according to Claims 1 and 2, characterized in that the test and standard nucleic acids are ribonucleic acids. The method according to Claims 1 and 2, characterized in that the test nucleic acid is deoxyribonucleic acid and the standard nucleic acid is ribonucleic acid. 20 7. The method according to Claims 1, 2, 3 and 4, characterized in that the detector sniffing molecule of the standard sniffing molecule pair - a recombinant plasmid containing a 1.1 kb BglII-BglII fragment of the Hindii fragment of the human c-Ki -rasI gene, wherein said HindIII fragment is donated into the pBR322 plasmid and said BglII-BglII fragment is subcloned at the restriction site of the BamHI restriction enzyme of the pBR322 plasmid - and the capture snuff molecules - recombinant phages containing 0.5 kb Contain BglII-HindlII fragment of the HindlII fragment of the human 'c-Ki-rasI gene, said HindlII fragment being donated in the pBR322 plasmid and said BglII-HindlII fragment being subcloned into the M13 mplO and M13 mpll phage vectors between the restriction sites of the BamHI and HindlII 35 restriction enzymes - either individually or together with the test 8700483 87302-23-. sniffing molecule pair are brought into contact with an undivided or if necessary divided nucleic acid sample. 8. The method according to Claims 1,2,3 and 4, characterized in that the detector sniffing molecule of the standard sniffing molecule pair - a recombinant plasmid comprising a 1.2 kb EcoRI-KpnI (HindIII} fragment, determined by the reading order, from the promoter region of the mouse metallothionine gene, which fragment is subcloned into the pAT153 plasmid between the restriction sites of the EcoRI and Hindii restriction enzymes - and the catcher snuff molecules - recombinant phages containing a 0.8 kb Kpnl-BglII fragment from the promoter region of the metallothionine gene and its sequenced region, which fragment is subcloned into the M13 mpl8 and M13 mpl9 phage vectors between the restriction sites of the Kpnl and BamHI restriction enzymes - either individually or together with the test sniff molecule pair, are brought into contact with an undivided or if necessary divided nucleic acid sample. The 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, detector sniffing molecule 25 of the test sniffing molecule pair - recombinant plasmid comprising a 3.7 kb HindII-Xbal fragment of the c-myc gene, whereby cleaved fragment is subcloned into the pBR322 plasmid at the restriction site of the Hindii restriction enzyme - and the catcher snuff molecules - recombinant phages comprising a 1.1 kb Xbal-PstI fragment of the c-myc gene, which fragment is subcloned into the M13 mplO and M13 mpl1 vectors between the restriction sites of the Xbal and PstI restriction enzymes - either individually or together with the standard sniffing molecule pair, were contacted with an undivided or if necessary divided nucleic acid sample. §700483 .w -24- 87302 The 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 DHFR gene and / or the number of messenger RNA molecules corresponding to this gene the detector sniffing molecule of the test sniffing molecule pair - a recombinant plasmid comprising a 0.75 kb HindIII-BglII fragment encoding the DHFR gene of the pMTVdhfr plasmid, which fragment is subcloned into the pAT153 plasmid vector between the * restriction sites of the HindlII and BamHI restriction enzymes -10 and the catcher snuff molecules - recombinant phages containing a 1.4 kb HindII fragment from the MMTV gene region of the pMTVdhfr plasmid, which fragment is subcloned into the M13 mpl8 and M13 mpl9 phage vectors at the restriction sites of the HindII restriction enzyme - contacted individually or together with the standard 15 sniff molecule pair with an undivided or if necessary divided nucleic acid sample. 11. A reagent set for the quantitative determination of nucleic acid molecules, characterized in that the set contains at least one test sniff molecule pair and at least one standard sniff molecule pair, the detector sniff molecules of both the test sniff molecule pair and the standard sniff molecule pair. with a suitable identification means and the catcher sniffing molecules are fixed on a suitable support or wherein a substance is attached to said catcher sniffing molecules, which enables isolation of the sandwich hybrids. A reagent set according to Claim 11, characterized in that the detector sniffing molecule of the test sniffing molecule pair becomes corresponding to this gene for determining the degree of amplification of the c-myc oncogene and / or the number of messenger RNA molecules. used, a recombinant plasmid containing a 3.7 kb HindIII-Xbal restriction fragment of 35 c-myc gene, which fragment is subcloned into the pBR322 S70 0 483 '-Y-f -25- 87302 plasmid at the restriction site of the Hindii restriction enzyme, and the capture snuff molecules are recombinant phages, comprising a 1.1 kb Xbal-PstI fragment of the c-myc gene, which is subcloned into the M13 mplO and M13 mpll phage 5 vectors between the restriction sites of the Xbal and PstI restriction enzymes, and the detector sniffing molecule of the standard sniffing molecule pair is a 1.1 kb BglII-BglII fragment of the HindII fragment of the human c-Ki rasI gene, wherein said HindII fragment has been donated d is in the pBr322 ig plasmid and said BglII-BglII fragment is subcloned into the pBR322 plasmid at the restriction site of the BamHI restriction enzyme, and the capture snuff molecules are recombinant phages comprising a 0.5 kb BglII-HindII fragment of the HindII fragment of the c-Ki-rasI gene, wherein said HindiI fragment is subcloned into the pBR322 plasmid of the c-Ki-rasI gene, and said BglII-HindlII fragment is subcloned into the M13 mplO and M13 mpll phage vectors between the restriction sites of the BamHI and HindlII restriction enzymes. The reagent set according to Claim 11, characterized in that the detector sniffing molecule of the test sniffing molecule pair is used to determine the degree of amplification of the dihydrofolate reductase or DHFR gene and / or the number of messenger DNA molecules corresponding to this gene 25 is a recombinant plasmid comprising a 0.75 kb HindlII-BglII fragment encoding the DHFR gene of the pMTVdhfr plasmid, which fragment is subcloned into the pAT153 plasmid vector between the restriction sites of the HindlII andBamHI restriction enzymes, and wherein the catcher sniffing molecules are recombinant phages comprising a 1.4 kb HindII fragment from the MMTV gene region of the pMTVdhfr plasmid, which fragment is subcloned into the M13 mpl8 and M13 mpl9 phage vectors at the restriction site of the HindII restriction enzyme, and wherein the detector sniff molecule of the standard sniffing molecule pair 35 is a recombinant plasmid containing a 1.2 kb EcoRI-Kpnl S7Q 0 483 -i V -26- 87302 fragment located in the reading sequence from the promoter region of the mouse metallothionine gene, which fragment is subcloned into the pAT153 plasmid between the restriction sites of the EcoRI and HindII restriction enzymes, and where the catcher is sniffing. 5 molecules are recombinant phages, comprising a 0.8 kn Kpnl-BglII fragment of the metallothionine gene formed by the promoter region and the region in the reading direction located below, which fragment is subcloned into the M13 mpl8 and M13 mpl9 phage vectors between the restriction sites of the 10 Kpnl and BamHI restriction enzymes. §700483 * f 87302. ABSTRACT The invention relates to the quantification of certain 5 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 to the reagent set which is used for this. The assay is made by comparing the number of test nucleic acid molecules potentially present in different copies in given unit to the number of selected standard nucleic acid molecules favorably present in a constant number per same unit. 8700433