GB2187283A

GB2187283A - Quantification of nucleic acid molecules

Info

Publication number: GB2187283A
Application number: GB08704517A
Authority: GB
Inventors: Marjut Ranki; Hans Soderlund
Original assignee: Orion Yhtyma Oy
Current assignee: Orion Oyj
Priority date: 1986-02-27
Filing date: 1987-02-26
Publication date: 1987-09-03
Anticipated expiration: 2007-02-26
Also published as: DK99387A; AU603562B2; FI860836A; IE66904B1; FR2594849B1; GB8704517D0; IT1202581B; ES2061411A6; CA1287558C; FI76119C; IL81695A; IS3197A7; DD270383A5; IT8719501A0; NL8700483A; HUT43647A; FI76119B; HU201808B; BE1001168A4; IL81695A0

Description

GB 2 187 283A 1

SPECIFICATION

Quantification of nucleic acid molecules and the reagent kit used The invention relates to the quantification of certain nucleic acid molecules, particularly the 5 degree of amplification of genes and/or corresponding messenger RNA molecules using the sandwich or solution hybridization method, and the reagent kit used.

The number of -copies of individual genes in the genome is usually constant. In some instances there is only one gene per haploid genome and in others several. Under certain circumstances the number of copies may change. The amplification of certain genes has for example been 10 found to be associated with the development of cancer. It is also known that external factors such as pharmaceuticals and metals etc. cause certain genes to be amplified. For the develop ment of a disease, the faulty or enhanced expression level of a gene, such as an oncogene, i.e.

the quantity of messenger RNA in the cell, is of major importance. Increased numbers of some chromosomes is the cause of certain hereditary diseases or other disturbances, whereas some 15 hereditary diseases only require duplication of one recessive gene. In all such instances 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 given genes, is currently determined by digesting the extracted DNA to be studied by means of restriction enzymes and by separating the nucleotide fragments according to length by agarose gel electro- 20 phoresis. Subsequently the single-stranded DNA is transferred and affixed to a nitrocellulose filter, where hybridization takes place using the gene to be studied or part of the gene as a probe. The results are obtained by autoradiography (Southern, J. Mol. Biol. 98, 503-517, 1975). In each parallel analysis, the quantity of cellular DNA is the same.

The intensities of the hybridization bands, i.e. the signals are compared and the ratios between 25 the copy numbers of the genes under study in the test samples are deduced. The method only yields approximate results. Likewise, RNA is measured using Northern- blotting or dot-blotting methods. These methods are quantitatively very inaccurate (Thomas, Methods in Enzymol., 100, pp. 255-2667, 1983).

Known methods, such as the Southern and Northern blotting methods, are slow and difficult 30 to perform. Since they only yield approximate results their diagnostic value is doubtful in cases in which 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 US Patent No. 4,486,539 and in the British Patent Application No. GB 2 169 403 are quantitative (Virtanen et al., Lancet 1, pp. 35 381-383, 1983). In addition, the method of the present invention requires a standard nucleic acid, the copy number of which is constant and enables the determination of the number of relevant nucleic acid molecules per given unit such as a cell, nucleus, ribosome or chromosome.

The purpose of this invention is to produce an accurate and rapid quantitative method of nucleic acid molecule determination which is also faster and simpler to perform than those 40 currently used. It can be used for cancer and prenatal diagnostics, for detecting agents which cause gene amplification and for demonstrating the development of e.g. drug resistance as well as for the determination of the expression level of messenger RNA.

At least two determinations are required in the present invention. One determines the nucleic acid molecule, which may be present in several copies, the test nucleic acid. The other deter- 45 mines the constitutive nucleic acid molecule advantageously present in constant number, the standard nucleic acid. In the method according to the invention, a nucleic acid molecule denotes a certain 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 amplicone. 50 The determination of test and standard nucleic acids is done using an otherwise normal sandVvich hybridization method 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. The invention also relates to a reagent kit containing nucleic acid reagents consisting of at least one test probe pair and at least one standard probe pair. 55 The reagents, or probes, used in the method, are prepared, using recombinant-DNA tech niques, from nucleic acids sufficiently homologous to the test and standard nucleic acids. Suffici ently homologous nucleic acids can also be prepared synthetically and semisynthetically.

The test and standard nucleic acids may be isolated directly from cells and identified by various hybridization techniques. Such test and standard nucleic acids are however also available 60 commercially and from various gene banks. Test and standard nucleic acids may 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 65 2 GB2187283A 2 of the resulting restriction fragments situated relatively close together are cloned to at least two suitable vectors. One of the fragments, the detector probe, is labelled with a suitable detectable label and the other, the capturing probe, is either affixed to a suitable carrier or an substance is affixed to it, which substance enables separation of the resulting hybrid from the hybridization mixture by means of another substance, such as the complementary- component of an affinity 5 pair.

The test and standard probe pairs can be assembled into suitable reagent kits wherein the test and standard probe pairs are both DNA or RNA, or the test probe pair is DNA and the standard probe pair RNA or vice versa. The pre- and further treatment of samples prior to hybridization and the hybridization conditions should therefore comply with the probe pairs used 10 in the test.

The method of the present invention is particularly suitable for determining the number of nucleic acid molecules directly from cellular homogenates. The method may of course also be used for the determination of purified or pure nucleic acids. However, before carrying out the method of the invention, the most suitable pretreatment of the nucleic acid sample should be 15 selected.

It is possible to carry out both DNA and RNA determinations using the method of the invention. Deoxyribonucleic acids are denaturated to obtain single strands if necessary. Singlestranded messenger RNA molecules potentially disturbing the hybridization test can be hydro- lyzed, for example by alkaline boiling. The sample is not denatured in connection with ribonucleic 20 acid determinations since the double- stranded deoxyribonucleic acid does not interfere with RNA determination. It is of course possible to disrupt the DNA with deoxyribonuclease or alter it either chemically or mechanically so that it cannot participate in the hybridization reaction. Therefore in connection with DNA and RNA determinations a suitable method for further treat- ment of the sample must be selected or, alternatively, this further treatment may be omitted. 25 The choice of a suitable method for the further treatment is of course dependent on the method used for the preliminary treatment of the nucleic acid sample. Numerous methods of pre- and further treatment of nucleic acid samples have been described in the literature, enabling the most suitable method to be chosen in each case.

Determinations in which both the test and standard nucleic acids are either DNA or RNA can 30 be performed using an undivided sample. Determinations in which the test nucleic acids are DNA and the standard nucleic acids RNA or vice versa must be performed using a divided sample, as different methods for further treatment are necessary. The sample may of course be divided even if the test and standard nucleic acids are of the same nucleic acid type.

The hybridization test itself is performed by bringing the undivided sample solution into contact 35 simultaneously with at least one test probe pair and one standard probe pair. If the sample solution has been divided it is brought separately into contact with at least one test probe pair and one standard probe pair. In such instances, the quantity of test nucleic acid is determined in one reaction vessel and the quantity of the standard nucleic acid in the other.

Regardless of whether the sample is divided or not, hybridization is allowed to take place in 40 the most advantageous conditions and time in each case. Once the reaction(s) has/have taken place, the resulting test and standard hybrids are separated from the hybridization mixture(s) by the carrier and washed, or by an isolation agent such as the complementary member of an affinity pair. The label attached to the test and standard hybrids is measured and the result compared with standard curves. In this way the number of nucleic acid molecules to be studied 45 can be determined per selected unit.

The method of the invention is of practical diagnostic value, particularly in the detection of some types of cancer. In small cell lung carcinoma, the c-myc gene is often amplified and its level of expression considerably higher than in normal tissue. In cases of neuroblastoma the N myc gene is amplified. 50 The method of the present invention can also be used for demonstrating the mutagenic or carcinogenic effects of certain agents or the development of drug resistance. It is known that external pressure of selection can result in enhanced expression of a certain gene. In the treatment of cancer, cells develop resistance to a griven drug by amplification of the gene, the expression product of which inactivates the drug. One such case is methotrexate which induces 55 amplification of the gene for dihydrofolate reductase (DHFR). A further example is amplification of the gene for metallothionine under the influence of cadmium.

The expression level of a gene is important from the point of view of the phenotype and function of the cell. This can be investigated by measuring the quantity of messenger RNA which correlates to the quantity of protein coded by it. The transcription product of an onco- 60 gene 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 phase of development of the cell. For example, at a certain stage of fetal development, the c myc oncogene is copied rapidly, whereas at another stage this is very slow. The degree of amplification often correlates with the level of expression of the gene, although the latter may 65 3 GB 2 187 283A 3 significantly increase without the former. In such instances the role of an oncogene is best determined by measuring its level of expression rather than the number of copies. In some instances, quantitative determination of the messenger RNA may be simpler and handier than quantification of the gene product itself. As an example the c-myc oncogene, a labile protein readily coagulated by heat, can be mentioned. 5 The method of the invention can also be used for identifying 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 of the invention and the nucleic acid reagents used in the method are described in greater detail below. 10 EXAMPLE 1 a) Nucleic acid reagents and their preparation STANDARD PROBES Cell standard nucleic acid. The c-Ki-rasi gene is present in all human cells. The probe pairs for 15 sandwich hybridization were prepared by subcloning the Hindill 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 the ATCC culture collection.

Further treatment of the cell standard nucleic acid. The pBR322 clone described above was 20 treated with Bgill and Hindill restriction enzymes and the resulting fragments were isolated from the agarose gel; purified fragments located close together were subcloned into two suitable vectors for preparation of the detector and capturing probes.

Standard detector probe. A Bgill-Bgill fragment measuring about 1. 1 kb in length was sub cloned into the BamHI restriction enzyme site of the pBR322 plasmid and labelled by nick- 25 translation with 1251-labelled dCTP.

Standard capturing probe. The Bgill-Hindill fragment of about 0.5 kb was inserted into the M13 mp10 and mpl 1 phage fvectors between the restriction sites of the BamHI and Hindill restriction enzymes and affixed to a nitrocellulose filter (150 ng DNA/dia 1 em).

30 TEST PROBES Test nucleic acid. A probe pair for sandwich hybridization was prepared from a cloned c-myc gene which can be obtained for example, from the ATCC culture collection (ATCC 41010). The restriction map of 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 Hindill, Xbal and 35 Pstl restriction enzymes and the fragments isolated from the agarose gel, purified and subcloned into suitable vectors in order to prepare the detector and capturing probes.

Test detector probe. The single-stranded tails of the Hindll]-Xbal restriction fragment of the c myc gene, measuring 3.7 kb in length, were rendered double-stranded by DNA polymerase. The Hindill linkers were inserted by T4-DNA-ligase into the resulting blunt- end DNA fragments; after 40 phenol extraction the DNA was treated with the Hindill restriction enzyme. The DNA fragment was subsequently cloned into the pBR322 plasmid at the restriction site of the Hindill restriction enzyme and labelled by nick-translation with 1251-labelled dCTP.

Test capturing probe. The 1.1 kb Xbal-Pstl fragment of the c-myc gene was cloned into the M 13 mp 10 and mp 11 phage cloning vectors between the restriction sites of the Xbal and Pstl 45 restriction enzymes and affixed to the nitrocellulose filter (150 ng DNA/dia 1 em).

(b) Determination of the standard curve The sample used for determination of the standard curve consisted of an alkaline-denatured pBR322 clone of the c-mye gene. The sandwich hybridization solution to which the above test 50 probes were added consisted of 4 x SSC, 1 x Denhardt solution, 200 jig/mi herring sperm DNA and 0.2% SDS. Hybridization took place at 65'C for 17-19 hours, whereafter the filters were washed in the wash solution (0. 1 x SSC 0.2% SDS) at 50'C. The label attached to the sandwich hybrids was then counted in the gamma counter.

4 GB2187283A 4 Table 1

Sample opm molecules/test c-mye-filter 5 0 40 106 75 X 106 190 107 340 10 108 2200 c) Determination of the number of genes The samples comprised 1) cells from a human placenta and 2) Colo 320 cells, which can be 15 obtained e.g. from the ATCC culture collection (ATCC-CCC220). DNA was isolated from both samples, and the same quantity of cell DNA, denatured by alkaline boiling, was added to both tests. Alkaline denaturation hydrolyzed any RNA present in the sample.

The test was performed by adding to each sample both the c-myc and c-Kirasi filters and the two labelled reagents, enabling both the standard and test DNA to be measured for each 20 sample. On the basis of c-Ki-rasi determinations, each test was found to contain the same quantity of DNA and it can be deduced that the c-myc gene in Colo 320 cells is present in about 16-20 higher copy number than in the normal situation. The results are shown in Table 2.

Table 2 25

Sample c-Ki-rasi filter c-myc filter epm cpm number Human placental cells 486 340 107 30 Colo 320 cells 432 3205 1.6 x 1011 the reading obtained from the blank filter has been subtracted from the readings.

EXAMPLE 2 35

Quantification of amplified gene a) Nucleic acid reagents and their preparation STANDARD PROBES Cell standard nucleic acid. The control nucleic acid was taken from the promoter area of the metallothionine gene in the mouse, i.e. the MT gene, and the DNA immediately upstream of it. 40 The structure of the MT gene has been described by Paviakis and Hamer, PNAS 80, pp.

397-401, 1983. The reference nucleic acid fragment is available e.g. cloned into the pBPV-MMTneo(432-12) vector (ATCC 37224) and can be obtained, for example, from the ATCC culture collection.

Further treatment of cell standard nucleic acid. The MT gene described above was treated 45 with Kpni, Bgill and EcoRI restriction enzymes for subcloning into the pAT153 plasmid. The Kpni tail was converted into a Hindill tail with a linker.

Standard detector probe. The EcoR]-Kpni-(Hindill) fragment measuring about 1.2 kb and located upstream of the promoter area of metallothionine gene was cloned to the pAT153 plasmid between the restriction sites of the EcoRI and HindIll restriction enzymes and labelled by nick- 50 translation with 31P-labelled nucleoside triphosphates.

Standard capturing probe. The 0.8 kb Kpni-Bgill fragment comprising the promoter area of the metallothionine gene and the area upstream of it was cloned into the M13 mp18 and M13 mp19 phage vectors between the restriction sites of the Kpni nitrocellulose filter.

55 TEST PROBES Test nucleic acid. The probe pair for the sandwich hybridization test was prepared using the commercially available pIVITVdW plasmid (Bethesda Research Laboratories, 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 dihydro- 60 folate reductase (DHFR) gene was treated with Hindill and Bgill restriction enzymes.

Test detector probe. The Hindill-Bgill fragment, measuring 0.75 kb and corresponding to the area coding for the DHFR gene of the pMTVdhfr plasmid, was inserted into the plasmid pAT153 vector between the restriction sites of the Hindill and BarnHI restriction enzymes and labelled by nick-translation with 32P-labelled nucleoside triphosphates. 65 GB 2 187 283A 5 Test capturing probe. A Hindill fragment measuring 1.4 kb taken from the MIVITV gene area of the pMTVdhfr plasmid was cloned into the M13 mp18 and M13 mp19 phage vectors.

b) Determination of the standard curve The sample used for the test was purified DNA from the pMTVdhfr plasmid. The test itself 5 was carried out as described in Example 1b except that a liquid scintillation counter was used for counting. The resulting standard curve is shown in Table 3.

Table 3

10 Sample cpm molecules/test DHFR filter 0 17 106 45 15 3x 106 79 101 210 c) Determination of the number of genes 20 Cell lines (derived from the mouse fibroblast cell N1H 3T3 and available from the ATCC culture collection under the number CRL 1658) which has been transfected with different quantities of cDNA corresponding to the mRNA of the DHFR gene were cultured on cell culture plates and used as the sample. The cells were lysed using sodium dodecyl sulphate and their DNA was sheared by squeezing through a fine hypodermic needle from a syringe. A 250 111 sample 25 corresponding to about 106 cells was taken from the homogenate and 50 pI NaOH added. The sample was boiled and neutralized with acetic acid and the hybridization mixture. The total volume was 0.5 mi. All the probes described above were added simultaneously and a so-called blank filter was added as a background control. Hybridization, washing and label counting were done as in Example 1b except that a liquid scintillation counter was used for counting. The 30 results are shown in Table 4.

Table 4 cell MT No.of cells DHFR 35 cpm in the sample cpm No. of molecules No. of in the sample copies 40 Control cell (No DHFR-cDNA) 182 1.05 X 106 21 < 106 Line 1 138 0.9 X 106 80 3 x 106 3 45 Line 11 210 1.25 x 106 732 5 x.10 7 40 cpm: the reading given by the blank filter has been subtracted 50 The MT gene is an internal marker which measures the number of cells present in a sample.

The results show that in this test 106 cells gave an MT-specific signal of 165+20 epm. The DHFR reagents measure the quantity of DI-Iffi-cDNA. The number of cells was deduced from the MT-specific signal. It was thus possible to determine the number of DI- Iffi-cDNA copies in different cell lines as shown in Table 4. 55 EXAMPLE 3

Quantification of messenger RNA a) Nucleic acid reagents and their preparation Using the test probes described in Example 2 it is also possible to measure the quantity of 60 mRNA derived from DI-Iffi-cDNA. The structure of the pMTVdhfr plasmid is such that transcrip tion of the DHFR gene begins at the MIVITV promoter. The resulting messengers are about 1.0 kb in length. Of this, about 0.25 kb are derived from the MMTV promoter area and the rest from DI-Iffi-cDNA (Lee et al., Nature 294, pp. 228-232, 1981).

6 GB 2 187 283A 6 STANDARD PROBES The cell standard nucleic acid, standard detector and standard capturing probe were as described in Example 2.

TEST PROBES 5 The test nucleic acid, test detector and test capturing 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 needed for 10 transcription was prepared by subcloning the 1.4.kb Hindill fragment of the MMTV promoter of the pMTVdhfr plasmid and the 0,75 kb Kindill-Bgill fragment (DHFR-cDNA) next to each other into the pSP64 plasmid (Promega Biotec) between the restriction sites of the Hindill and BamHI restriction enzymes. The sample RNA was stored in 0.2% SDS aqueous solution.

The sandwich hybridization test was carried out as described in Examples 1b and 2b but 15 denaturation was omitted.

Table 5

Sample cpm 20 molecules/test DHFR filter 0 20 X 106 65 107 130 25 X 107 390 1011 653 c) Determination of the number of messenger RNA molecules 30 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 using sodium dodecyl sulphate and their DNA was sheared slightly by squeezing through a fine hypodermic needle from a syringe. A 250 111 sample of the homogenate was taken corresponding to about 5 X 106 cells. The homogenate was then added to the 35 sandwich hybridization test without denaturation. Sandwich hybridization took place as described in Examples 2c and lb, except that only the DHFR probes were added to the hybridization solution. In a parallel sample of 250 111 of homogenate, the cell number was determined using the MT probe as described in Example 2c.

The results are shown in Table 6. 40 Table 6

45 Cell MT Cell number DHFR cpm in the sample cpm No of molecules No. per in the sample cell 50 Line 1 380 3.5 x 10 6 1465 3.45 x 10 8 100 Line 11 430 4.2 x 10 6 4800 2 x 10 9 500 55 cmp: The reading given by the blank filter has been subtracted.

60 The results showed that cell line 1 produced per cell about 100 messenger RNA molecules from the DHFR genes and cell line 11 produced about 500 messenger RNA molecules from the DHFR genes.

EXAMPLE 4 65

7 GB 2 187 283A 7 Quantification of amplified gene by solution hybridization a) Nucleic acid reagents and their preparation STANDARD PROBES The cell standard nucleic acid, standard detector and standard capturing probe were as described in Example 2. The 1.2 kb EcoRl-Kpni-(Hindill) fragment in pAT153 was labelled by 5 nick-translation with 1251-labelled deoxycytidine. The 0.8 kb Kpnl-Bgill fragments in M13 mp18 and M13 mp19 were modified with biotin using the Photoprobe Tm reagent (Vector Laboratories, CA, USA, product No SP-1000).

TEST PROBES 10 The test nucleic acid, test detector and test capturing probe were as described in Example 2. The 0.75 kb Hindill-Bgiii fragment in pAT153 was labelled with 1251-labelled deoxycytidene. The 1.4 kb Hindill fragments in M13 mp18 and M13 mp19 were biotinylated using Photoprobe W as above.

15 b) Determination of the standard curves A cell standard curve was prepared using a known amount of cells, from which the hybridization signal was measured using the standard probes recognizing the MT-gene. A test nucleic acid standard curve was prepared with the pMTVdW plasmic! and the test probes recognizing this plasmid. Hybridizations were carried out in 200 ul of a solution consisting of 0.6 M NaCI, 20 mM phosphate buffer, pH 7.5, 1 mM EDTA, 4% polyethylene glycol (PEG 6000) for 1.5 hours at 7WC. After the reaction 50 lim of streptavid i n-aga rose (Betheseda Research Labora tories, Maryland, USA, product No. 5942SA), and 1 M NaCI, 10 mM sodium phosphate, pH 7.5, 1 mM EDTA was added to a final volume of 500 Id. The hybrids were collected on the streptavidin-aga rose at 37'C for 15 min. The agarose was washed once for 5 min. with the 25 buffered 1 M NaCI solution at 37'C and twice for 2 min. with 15 mM NaCI, 1.5 mM sodium citrate at WC. The amount of bound hybrids was determined by measuring the agarose in a gamma counter. (Syvinen et al., Nucleic Acids Res. 14, 5037-5048, 1986). The results are shown in table 7 and 8.

30 Table 7

Sample cpm cells/test MT probes 35 0,8 X 106 162 1,6 x 106 216 3 x 106 298 40 Table 8

Sample cpm molecules/test DHFR probes 45 106 148 X 106 394 5x 107 2240 50 c) Determination of the number of genes Samples of the cell lines described in Example 2 were treated in a similar way, except that the volume per sample corresponding to approximately 2 x 106 cells was 125 11. The determinations of number of cells and number of test nucleic acid molecules were carried out in separate vials by adding the cell sample, the appropriate detector and capturing probes, and the components 55 of the hybridization mixture to a final volume of 200 jil. Control assays without cell standard or test DNA were included. Hybridization, collection of hybrids, washing and measurement was done 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.

8 GB2187283A 8 Table 9

5 Cell MT DE1FR cpm No of cells cpm No. Of No. of 10 in the sample molecules copies in the sample 15 Control cell 253 2.3 x 10 6 73 < 10 5 Line 1 210 1.5 X 10 6 233 3.8 x 10 6 3 20 Line 11 237 2.1 X 10 6 3059 8.8 X 10 7 42 25 cpm: values from control assays without cell standard or test nucleic acid have been subtracted.

Claims

CLAIMS 30

1. A quantitative method for determination of nucleic acid molecules by a sandwich or solution hybridization method, characterized in that the number of given nucleic acid molecules per given unit is determined by comparing the number of the test nucleic acid molecules potentially present in several copies in the unit to the number of chosen standard nucleic acid molecules advantageously present in a constant number per same unit. 35

2. The method according to claim 1 characterized in that the nucleic acids present in the sample a) are rendered, if necessary, into a form whereby they can participate in the hybridization reaction, b) any nucleic acids potentially disturbing the hybridization reaction are rendered, if necessary, 40 into a form whereby they cannot interfere with the hybridization test, c) are brought into contact, either undivided or, when necessary divided, with at least one test probe pair sufficiently homologous to the nucleic acid potentially present in several copies and with at least one chosen and suitable standard probe pair sufficiently homologous to the nucleic acid molecule advantageously present in a constant number; the detector probes of the said test 45 probe pair and said standard probe pair are labelled with a suitable, detectable label and the capturing probes have been affixed to a suitable carrier or a substance has been affixed to the said capturing probes which enables isolation of the resulting hybrids; d) after the hybridization reaction or reactions have taken place the test hybrid and standard hybrid are separated when necessary and the said attached label measured; the number of 50 nucleic acid molecules per given unit is obtained by comparing the test and standard nucleic acid numbers.

3. The method according to Claims 1 and 2, characterized in that the test and the standard nucleic acids are deoxyribonucleic acids.

4. The method according to claims 1 and 2,. characterized in that the test nucleic acid is 55 ribonucleic acid and the standard nucleic acid is deoxyribonucleic acid.

5. The method according to Claims 1 and 2, characterized in that the test and standard nucleic acids are ribonucleic acids.

6. 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. 60

7. The method according to Claims 1, 2, 3 and 4, characterized in that the detector probe of the standard probe pair-a recombinant plasmid comprising a 1. 1 kb Bgill- Bgill fragment of the Hindill fragment of the human c-Ki-rasi gene, said Hindill fragment being cloned into the pBR322 plasmid and said 13g111-139111 fragment being subcloned into the restriction site of the BamHI restriction enzyme of the pBR322 plasmid-and the capturing probes- recombinant phages 65 9 GB2187283A 9 comprising a 0.5 kb Bgill-Hindill fragment of the Hindill fragment of the human c-Ki-rasi gene, said Hindill fragment being cloned into the pBR322 plasmid and said Bgill- Hindill fragment being subcloned into the M 13 mp 10 and M 13 mp 11 phage vectors between the restriction sites of the BarnHI and Hindill restriction enzymes-are brought, either individually or together with the test probe pair, into contact with an undivided or when necessary divided nucleic acid sample. 5

8. The method according to Claims 1, 2, 3 and 4 characterized in that the detector probe of the standard probe pair-a recombinant plasmid comprising a 1.2 kb EcoRl- Kpni(Hindill) fragment from upstream of the promoter area of the mouse metallothionine gene, which fragment has been subcloned into the pAT153 plasmid between the restriction sites of the EcoRI and Hindill restriction enzymes-and the capturing probes-recombinant phages comprising a 0.8 kb Kpn]- 10 Bgill fragment from the promoter area of the metallothionine gene and the area upstream of it, which fragment has been subcloned into the M13 mp18 and M13 mp19 phage vectors between the restriction sites of the KpM and BamHI restriction enzymes-are brought, either individually or together with the test probe pair, into contact with an undivided or when necessary divided nucleic acid sample. 15

9. The method according to Claims 1, 2, 3, 6, 7 and 8, characterized in that in order to determine 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 HindliMbal fragment of the c-myc gene, said fragment being subcloned into the pBR322 plasmid at the restriction site of the Hindill restriction enzy- 20 me-and the capturing probes-recombinant phages comprising a 1. 1 kb Xbal- Pstl fragment of the c-myc gene, which fragment has been subcloned into the M 13 mp

10 and M 13 mp 11 vectors between the restriction sites of the Xbal and Pstl restriction enzymes-are brought, either individually or together with the standard probe pair, into contact with an undivided or when necessary divided nucleic acid sample. 25 10. The method according to Claims 1, 2, 3, 6, 7 and 8, characterized in that in order 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 probe of the test probe pair-a recombinant plasmid comprising a 0.75 kb Hindill-Bgill fragment coding for the DI-IFR gene of the pMTVdhfr plasmid, which fragment has been subcloned into the pAT153 30 plasmid vector between the restriction sites of the Hindill and BamH[ restriction enzymes-and the capturing probes-recombinant phages comprising a 1.4 kb Hindill fragment of the MIVITV gene area of the pMTVdhfr plasmid, which fragment has been subcloned into the M13 mp18 and M13 mp19 phage vectors at the restriction site of the Hindill restriction enzyme-are brought, either individually or together with the standard probe pair, into contact with an 35 undivided or when necessary divided nucleic acid sample.

11. A reagent kit for the quantitative determination of nucleic acid molecules characterized in that the kit contains at least one test probe pair and at least one standard probe pair, the detector probes of both the test probe pair and the standard probe pair being labelled with a suitable label and the capturing probes having been affixed to a suitable carrier or a substance 40 having been affixed to the said capturing probes, enabling isolation of sandwich hybrids.

12. The reagent kit according to Claim 11, characterized in that the detector probe of the test probe pair used for the determination of 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 HindllMbal restriction fragment of the c-myc gene, which fragment 45 has been subcloned into the pBR322 plasmid at the restriction site of the Hindill restriction enzyme, and the capturing probes are recombinant phages comprising a 1.1 kb Xbai-Pstl frag ment of the c-myc gene, which fragment has been subcloned into the M13 mp1O and M13 mpl 1 phage vectors between the restriction sites of the Xbal and Pstl restriction enzymes, and the detector probe of the standard probe pair is a 1.1 kb Bgill-Bgill fragment of the Hindill 50 fragment of the human c-Ki-rasi gene, said Hindill fragment having been cloned into the pBR322 plasmid and said Bgill-Bgill fragment having been subcloned into the pBR322 plasmid at the restriction site of the BamHI restriction enzyme, and the capturing probes are recombinant phages comprising a 0.5 kb 139111-Hindill fragment of the Hindill fragment of the c-Ki-rasi gene, said Hindill fragment having been subcloned into the pBR322 plasmid of the c-Ki-rasi gene, and 55 said Bgill-Hindill fragment having been subcloned into the M 13 mp 10 and M 13 mp 11 phage vectors between the restriction sites of the BamHI and Hindill restriction enzymes.

The reagent kit according to Claim 11, characterized in that the detector probe of the test probe pair used for determination of 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 60 recombinant plasmid comprising a 0.75 kb Hindill-Bgill fragment coding for the DHFR gene of the pIVITVdW plasmid, which fragment has been subcloned into the pAT153 plasmid vector be tween the restriction sites of the Hindill and BamHI restriction enzymes, and the capturing probes are recombinant phages comprising a 1.4 kb Hindill fragment of the MMTV gene area of the pIVITVdW plasmid, which fragment has been subcloned into the M13 mp18 and M13 mp19 65 GB2187283A 10 phage vectors at the restriction site of the Hindill restriction enzyme, and the detector probe of the standard probe pair is a recombinant plasmid comprising a 1.2 kb EcoRl-Kpril fragment from upstream of the promoter area of the mouse metallothionine gene, which fragment has been subcloned into the pAT153 plasmid between the restriction sites of the EcoRI and Hindll restriction enzymes, and the capturing probes are recombinant phages comprising a 0.8 kb Kpn]- 5 B9111 fragment of the metallothionine gene formed by the promoter area and the area upstream of it, which fragment has been subcloned into the M13 mp18 and M13 mp19 phage vectors between the restriction sites of the Kpn] and BamHI restriction enzymes.

Printed for Her Majesty's Stationery Office by Burgess & Son (Abingdon) Ltd, Dd 8991685, 1987. Published at The Patent Office, 25 Southampton Buildings, London, WC2A 1 AY, from which copies may be obtained.