IE870496L - Quantitative determination of nucleic acids. - Google Patents

Abstract

The invention is related to the quantification of certain nucleic acid molecules, particularly the degree of amplification of genes and/or corresponding messenger RNA molecules using the sandwich or solution hybridization method, and the reagent kit used. The determination is carried out by comparing the number of the test nucleic acid molecules potentially present in several copies in given unit to the number of chosen standard nucleic acid molecules advantageously present in a constant number per same unit. [CA1287558C]

Description

6 6 9 0 4 - 2 - The invention relates to the quantification of certain nucleic acid molecules, particularly the degree of amplification of genes and/or corresponding messenger RMA molecules using the sandwich or solution hybridization method e and the reagent kit 5 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 10 amplification of certain genes has for example been found to be associated with the development of cancer. It is also known that external factors such as pharmaceuticals and metals cause certain genes to be amplified. For the development of a disease,, the faulty or enhanced expression level of a gene, 15 such as an oncogenef i.e. the quantity of messenger RHA in the cellf is of major importance. Increased numbers of some chromosomes is the cause of certain hereditary diseases or other disturbances, whereas some hereditary diseases only require duplication of one recessive gene. In all such 20 instances it is important to determine the number of chromosomes or genes present.

The number of certain DMA molecules e for example the degree of amplification of given genes, is currently determined by digesting the extracted DMA to be studied by means of 25 restriction ensymes and by separating the nucleotide fragments according to length by agarose gel electrophoresis. Subsequently the single-stranded DMA is transferred and affixed to a nitrocellulose filter£, where hybridisation takes place using the gene to be studied or part of the gene as a - 3 ~ probe. The results are obtained by autoradiography (Southern, J, Mol. Biol 98, pp. 503-517, 1975). In each parallel analysis, the quantity of cellular DMA is the same.

The intensities of the hybridisation bands, i.e. the signals 5 are compared and the ratios between 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 10 Enzymol,, 100, pp. 255-266, 1983).

Known methods, such as the Southern and northern blotting methods, are slow and difficult 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 15 certain nucleic acid molecules per given unit, such as a cell.

The present invention now provides an accurate and rapid quantitative method of nucleic acid molecule determination which is also faster and simpler to perform than those currently used. It can be used for cancer and prenatal 20 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 REA.

In accordance with the present invention, there is provided a 25 quantitative method for the determination of nucleic acid molecules by sandwich solution hybridization, the method comprising determining the number of nucleic acid molecules per given unit by comparing the number of test nucleic acid molecules potentially present in several copies in the unit to 30 the number of chosen standard nucleic acid, molecules advantageously present in a constant number in the same unit, wherein the nucleic acids present in the sample (a) are rendered, if necessary, into a form whereby they can participate in the hybridization reaction, any nucleic acids potentially disturbing the hybridisation reaction being rendered, if necessary, into a form whereby they cannot interfere with the hybridization testand (b) are brought into contact £, either undivided or, when necessary dividedt, 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 e the detector probes of the test probe pair and standard probe pair being labelled with a detectable label and the capturing probes having been affixed to a suitable carrier or a substance having been affixed to the capturing probes which enables isolation of the resulting hybrids? and wherein (c) after hybridization? the test hybrid and standard hybrid are separated when necessary and the attached label is measured and the number of nucleic acid molecules per given unit is obtained by comparing the test and standard nucleic acid numbers.

The present invention provides also a reagent kit for use in such a method, the kit comprising 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 having been affixed to the capturing probesP enabling isolation of sandwich hybrids. „ 5 - In the present context, a nucleic acid molecule is considered to be of a size ranging from a sequence of 10 - 12 nucleotides to a gene containing several thousand nucleotides. It can also mean a messenger RNA or a nucleotide sequence 5 considerably longer than a single gene, i.e. an amplicone.

In the method of the invention, the determination of test and standard nucleic acids is carried out using an otherwise normal sandwich hybridisation described, for example, in U.S. Patent Mo. 4,485,539 or a solution hybridization described in 10 British Patent Application No. GB 2,169,403. Such sandwich and solution hybridization have the distinction of being quantitative (¥irtanen et al. , Lancet _1, pp. 381-383, 1983).

The reagents, or probes, used in the method of the invention may be prepared using r e c omb i nan t - DMA techniques, from nucleic 15 acids sufficiently homologous to the test and standard nucleic acids. Sufficiently 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 hybridisation techniques. 20 Such test and standard nucleic acids are however also available commercially and from various gene banks. Test and standard nucleic acid may be either DMA or RNA.

Probe pairs suitable for the sandwich or solution hybridisation are prepared from nucleic acids sufficiently 25 homologous to the test and standard nucleic acids by recombinant-DBA techniques. The relevant nucleic acids are digested by suitable restriction enzymes; at least two of the resulting restriction fragments situated relatively close together are cloned to at least two suitable vectors. One of 30 the fragments, the detectors 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 pair.

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

It is possible to carry out both DM& and RNA determinations using the method of the invention. Deoxyribonucleic acids are denatured to obtain single strands if necessary. Single-stranded messenger RHA molecules potentially disturbing the hybridisation test can be hydrolyzea, for example by alkaline boiling. The sample is not denatured in connection with ribonucleic acid determinations since the double-stranded deoxyribonucleic acid does not interfere with RHA determination* It is of course possible to disrupt the DMA with deoxyribonuclease or alter it either chemically or mechanically so that it cannot participate in the hybridisation reaction. Therefore in connection with DMA and RNA determinations a suitable method for further treatment of the sample must be selected or, alternatively, this further treatment may be omitted. The choice of a suitable method for - 7 - 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 5 most suitable method to be chosen in each case* Determinations in which both the test and standard nucleic acids are either DMA or RNA can be performed using an undivided sample. Determinations in which the test nucleic acids are DMA and the standard nucleic acids RNA or vice versa 10 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 hybridisation test itself is performed by bringing the 15 undivided sample solution into contact 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 instancesp the quantity of test nucleic 20 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 f hybridization is allowed to take place in the most advantageous conditions and time in each case. Once the 25 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 30 and the result compared with standard curves. In this way the number of nucleic acid molecules to be studied can be determined per selected unit. - 8 - 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 5 tissue. In cases of neuroblastoma the M-myc gene is amplified.

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 10 that external pressure of selection can result in enhanced expression of a certain gene. In the treatment of cancer, cells develop resistance to a given drug by amplification of the gene, the expression product of which inactivates the drug. One such case is methotrexate which induces 15 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 phenotvpe and function of the cell. This can be 20 investigated by measuring the quantity of messenger RNA which correlates to the quantity of protein coded by it. The transcription product of an oncogene determines the way in which it will ultimately be expressed.

The expression levels of an oncogene vary depending on the 25 cell type, differentiation level and phase of development of the cell. For exa.mple, 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, 30 although the latter may 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 - 9 - determination of the messenger RMA 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. 10 The method of the invention and the nucleic acid reagents used in the method are described in greater detail below.

EX&HELE 1 .QsajitificatiOR of an amplified oncogene a.) Nucleic acid reagents and their preparation 15 STANDARD PROBES Cell stiaxidarri imcleic acid. The c-Ki-rasI gene is present in all human cells. The probe pairs for sandwich hybridization were prepared by subcloning the HindiII fragment of the c-Ki-rasl gene, measuring 3.8 kb in length, the restriction map of 20 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.

Farther treatment, of the cell standard r^acleic acid. The 25 pBE322 clone described above was treated with Bglll and HindiII restriction enzymes and the resulting fragments were isolated from the agarose gel % purified fragments located - 10 - close together were subcloned into two suitable vectors for preparation of the detector and capturing probes.

Standard detector -probe. A BglXI-Bglll fragment measuring about 1.1 kb in length was subcloned into the BamHI restriction enzyme site of the pBR322 plasmid and labelled by nick-translation with 125I-labelled dCTP.

Standard capturing probe. The Bgl 11-Hindi 11 fragment of about 0.5 kb was inserted into the Ml3 mplO and mpll phage vectors between the restriction sites of BamHI and HindiII restriction enzymes and affixed to a nitrocellulose filter (150 ng DNA/dia 1 cm).

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 8_0, pp» 5307-6311, 1983.

Fmrthar treatment of the test rascleic acid- The c~myc gene was treated with Hindi 11JCbal and PstI 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 KindIII~XbaI restriction fragment of the c-myc gene, measuring 3.7 kb in length, were rendered double-stranded by DNA polymerase. The HindiII linkers were inserted by T4-DMA-ligase into the resulting blunt-end DNA fragments; after phenol extraction the DMA was treated with the HindiII restriction ensyme. The DMA fragment was subsequently cloned into the pBR322 plasmid at the restriction site of the HindiII - 11 restriction enzyme and labelled by nick-translation with 1251-labelled dCTP.

Test caot^rinq probe. The 1.1 kb Xbal-Pstl fragment of the c-myc gene was cloned into the Ml3 mplO and mpll phage cloning 5 vectors between the restriction sites of the Xbai and PstI restriction enzymes and affixed to the nitrocellulose filter (150 ng DMA/dia 1 cm) . b) netezminafcion of the standard rarve.

The sample used for determination of the standard curve 10 consisted of an alkaline-denatured pBR322 clone of the c-myc gensThe sandwich hybridization solution to which the above test probes were added consisted of 4 x SSC, 1 x Denhardt solution, 200 jig/ml herring sperm DMA and 0.2 % SDS. Hybridization took place at 65°C for 1? - 19 hours,, whereafter 15 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.

Table 1 20 Sample crma aolscTales/t-est. G-sye-filt.ar 0 40 10s 75 25 5 x 1KJ6 107 340 l®8 220© 12 c) Determination of the mamlber of oreiaes „ The samples comprises 1) cells from a human placenta and 2) Colo 320 cells, which can be obtained e.g. from the ATCC culture collection (ATCC-CCC220). DNA was isolated from both 5 samples, and the same quantity of cell DRA, denatured by alkaline boiling, was added to both tests. Alkaline denaturation hydro ly zed. any RNA present in the sample.

The test was performed by adding to each sample both the c-myc and c-Ki-rasI filters and the two labelled reagents, enabling 10 both the standard and test DNA to be measured for each sample. On the basis of c-Ki-rasI determinations, each test was found to contain the same quantity of DMA 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 15 are shown in Table 2.

Table 2 Sample c-Ki~rasI filter c-Mtvc filter cpai* iMjjfflber 20 Human placental cells Colo 320 cells 486 432 340 3205 107 1.6 x 108 25 *the reading obtained from the blank filter has been subtracted from the readings. - 13 - EZBMPLE 2 Quantification of amplified, gene a) Mmcleic acid referents and their preparation STANDARD PROBES 5 Cell standard nucleic acid. The control nucleic acid was taken from the promoter area of the metallothionine gene in the mouses, i.e. the MT gene, and the DMA immediately upstream of it. The structure of the MT gens has been described by Pavlakis and Earner, PHAS 80. pp. 397-401, 1983. The reference 10 nucleic acid fragments 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.

Farther treataaent of cell standard macleic acid. The MT gene described above was treated with Kpnl,, Bglll and ScoRI 15 restriction enzymes for subcloning into the pAT153 plasmid. The Kpnl tail was converted into a Hindi 11 tail with a linker.

Standard detector probe. The EcoRI-Kpnl-(HindiII) fragment measuring about 1.2 kb and located upstream of the promoter area of metallothionine gene was cloned to the pAT153 plasmid 20 between the restriction sites of the EcoRI and HindiII restriction enzymes and labelled by nick-translation with 3 2p-labelled nucleoside triphosphates.

Standard c&ofarina probe. The 0.8 kb Kpnl-Bglll fragment comprising the promoter area of the metallothionine gene and 25 the area upstream of it was cloned, into the Ml3 mpl8 and Ml3 mp19 phage vectors between the restriction sites of the Kpnl and BamHI restriction enzymes and affixed to the nitrocellulose filter. - 14 - TEST PROBES Test: raacle5.c acid. The probe pair for the sandwich hybridization test was prepared using the commercially available pMTVdhfr plasmid. (Bethesda Research Laboratories, 5 product Ho. 5369SS), the structure of which is described by Lee et al., Nature 294, pp. 228-232, 1981.

Further treatment of rest nucleic acid. The pMTVdhfr plasmid containing cDEA of the dihydrofolate reductase (DHFR) gene was treated with HindiII and Bglll restriction enzymes. 10 Test, defector probe. The Hindlll-Bglll fragmentt 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 HindiXI and BamHI restriction enzymes and labelled by nick-translation with 15 32P-labelled nucleoside triphosphates. fast capturing probe. A HindiII fragment measuring 1.4 kb taken from the MMTV gene area of the pMTVdhfr plasmid was cloned into the Ml3 mpl8 and M13 mpl9 phage vectors. b} Determination of the standard cuarve 20 The sample used for the test was purified DNA from the pMTVdhfr plasmid. The test itself was carried out as described in Example lb except that a liquid scintillation counter was used for counting. The resulting standard curve is shown in Table 3. - 15 - Table 3 Saarejie ssolecmiles /test DHFR filter 0 17 10s 45 3 x 10* 19 107 210 c) ParterTeiiinafcicm of the number of oemes.

Cell lines (derived from the mouse fibroblast cell MIH 3T3 and available from the ATCC culture collection under the number CRL 1658) which has been trans fee ted with different quantities 15 of cDNA corresponding to the mRHA 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 DMA was sheared by squeezing through a fine hypodermic needle from a syringe. A 250 (J.1 sample corresponding to about 106 cells 20 was taken from the homogenate and 50 p.1 NaOH added. The sample was boiled and neutralised with acetic acid and the hybridisation 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. 25 Hybridisation, washing and label counting were done as in Example lb except that a liquid scintillation counter was used for counting. The results are shown in Table 4 . - 16 - Table 4- Call MT cam® f**o. a's aslkt in ths ssraiate cpm® DHFR E>*a. ai ETsol-Jffislsa is) ma ttstnpSs Ma. of caprfis Control csil (SSa DHFB-eD;>2A) 182 1.03 x 10s 21 < icf - Lins 1 138 0.S x 1(f eo 3ii 1(f 3 Lto II 210 •\m k 1G® 732 5x 10t 40 * cpm s the reading subtracted. given by the blank filter has been The MT gene is an internal marker which measures the number of 15 cells present in a sample. The results show that in this test 106 cells gave an MT-specific signal of 165 + 20 cpm. The DHFR reagents measure the quantity of DHFR-cDMA. The number of cells was deduced from the MT-specif ic signal. It was thus possible to determine the number of DHFR-cDMA copies in 20 different cell lines as shown in Table 4.

EXAHPX& 3.

Quantification of saessenger RNA a) Bfaicleic acid reacemts aad their preparation Using the test probes described in Example 2 it is also 25 possible to measure the quantity of mRMA 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 about 1.0 kb in length. Of this, about 0.25 kb are derived from the HMTV promoter area and the 30 rest from DHFR-cDNA (Lee et al.# Nature 294, pp. 228-232, 1981) • - 17 - 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) DetermdLfflat:iosi of time standard curve The sample used for standard curve determination consisted of messenger RNA corresponding to the dihydrofolate reductase 10 gene produced by in vitro transcription. The DMA needed for transcription was prepared by subcloning the 1.4 kb HindiII fragment of the MMTV promoter of the pMTVdhfr plasmid and the 0,75 kb HindIII-Bg111 fragment (DHFR-cDHA) next to each other into the p3P64 plasmid (Promega Biotec) between the 15 restriction sites of the HindiII and BamHI restriction enzymes. The sample RNA was stored in 0.2 % SDS aqueous solution.

The sandwich hybridisation test was carried out as described in Examples lb and 2b but denaturation was omitted. - 18 - Table 5 Sample molecules/test DHFR filter ClOffii 5 10 20 65 130 390 653 c) DetemffldLHiafcion of the number of messenger HMA molecules The number of. messenger RNA molecules corresponding to the DHFR gene was determined from the cell lines described in 15 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 jal sample of the homogenate was taken corresponding to about 5 x 106 cells. The 20 homogenate was then added to the sandwich hybridisation test without denaturation. Sandwich hybridization took place as described in Examples 2c and lb, except that only the DHFR probes were added to the hybridisation solution. In a parallel sample of 250 [il of homogenate, the cell number was 25 determined using the MT probe as described in Example 2c.

The results are shown in Table 6. - 19 - Table 6 ©ail IMF GeI BtwiDcir ispsffi* fe iBiaes saKwie! 5 Lime U 396 SJS x id* Um HI 400 *2 x *? CHRR cfxn* No. of iraateeste Me. pwsr fe stsei smpto eel 1485 3WB x «? 10B 4a@e 2 x id9 see 10 *cpm: The reading given by the blank filter has been subtracted.

The results showed that cell line I produced per cell about 100 messenger RNA molecules from the DHFR genes and cell line II produced about 500 messenger RHA molecules from the DHFR 15 genes.

EXAMPLE 4.

Quantification of amplified gene by solis.ti.ori hybridization. a) Ktecleic acid reagents and their preiparaticiis STANDARD PROBES 20 The cell standard nucleic acidP standard detector and standard capturing probe were as described in Example 2. The 1.2 kb EcoRI-KpnI-(HindT.II) fragment in pAT153 was labelled by nick-translation with 125I~labelled deoxycytidine. The 0.8 kb Kpnl-Bglll fragments in Ml3 mpl8 and HI3 mpl9 were modified with 25 biotin using the Photoprobe™ reagent (Vector Laboratories, CA, USA, product Mo SP-1000). - 20 - TEST PROBES The test nucleic acid, test detector and test capturing probe were as described in Example 2. The 0.75 kb HindlXI-Bglll fragment in pAT153 was labelled with 125I-labelled 5 deoxycytidine. The 1.4 kb HindiII fragments in Ml3 mpl8 and Ml3 mpl9 were biotinylated using Photoprobe™ as above. b) roefceriBii-ittatiOffli of the standard cu.r-i.res A cell standard curve was prepared using a known amount of cells, from which the hybridisation signal was measured using 10 the standard probes recognising the MT-gene. A test nucleic acid standard curve was prepared with the pMTVdhfr plasmid and the test probes recognising this plasmid. Hybridisations were carried out in 200 jaI of a solution consisting of 0.5 M NaCl, 20 mM phosphate buffer, pH 7.5, 1 mM EDTA, 4% polyethylene 15 glycol (PEG 6000) for 1.5 hours at 70°C- After the reaction 50 111 of streptavidin-agarose (Betheseda Research Laboratories, Maryland, USA, product Mo. 5942SA), and 1 M NaCl, 10 mM sodium phosphate, pH 7.5, 1 mM EDTA was added to a final volume of 500 fil. The hybrids were collected on the streptavidin-20 agarose at 3/°C for 15 min. The agarose was washed once for 5 min. with the buffered 1 M MaCL 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 25 Res. 14., 5037-5048, 1986). The results are shown in table 7 and 8. - 21 - Table 7 Saaole com cells/test MT probes 0,8 x 10s 162 1,6 x 106 216 3 x 10s 298 10 Table 8 .Sample cam molecules/-test DHFE probes 15 10s 148 5 x 10s 394 5 x 107 2240 Determination of the number of creiaes. 20 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 10s cells was 125 jaI. The determinations of number of cells and number of test nucleic acid molecules were carried out in separate vials by adding 25 the cell sample, the appropriate detector and capturing probes j, and the components of the hybridisation mixture to a final volume of 200 {a1. Control assays without cell standard or test DMA were included. Hybridisation^ collection of hybrids t, washing and measurement t#as done as described in - 22 - 10 Example 4b. The results were read from standard curves prepared in parallel as described in Example 4b. The results are shown in Table 9.

Table 3 Cell cgm* Mo of cells in the sample DHFR cam* No. of No. of molecules copies in the sample Control 253 cell 2.3 x 10s 73 < I05 Lin© I 210 1.5 x 10* 233 3.8 as 10s 3 Lime II 237 2.1 x 3059 ,8 x 107 42 15 * cpas values from control assays without cell standard or test nucleic acid have bees subtracted.

Claims (14)

- 23 - eukiMS

1. A quantitative method for the determination of nucleic acid molecules by sandwich or solution hybridization, the method comprising determining the number of nucleic acid. 5 molecules per given unit by comparing the number of 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 in the same unit, wherein the nucleic 10 acids present in the sample (a) are renderedf if necessary, into a form whereby they can participate in the hybridization reaction, any nucleic acids potentially disturbing the hybridization reaction being rendered, if necessary, 15 into a form whereby they cannot interfere with the hybridisation test^ and (b) are brought into contact, either undivided or, when necessary divided, with at least one test probe pair sufficiently homologous to the nucleic acid 20 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 numbert, the detector probes of the test probe pair and standard 25 probe pair being labelled with a detectable label and the capturing probes having been affixed to a suitable carrier or a substance having been affixed to the capturing probes which enables isolation of the resulting hybrids? and wherein 30 (c) after hybridisation, the test hybrid and standard hybrid are separated when necessary and the attached label is measured and the number of nucleic acid molecules per given unit is obtained by comparing the test and standard nucleic acid numbers.

2. A method according to claim 1, wherein the test and standard nucleic acids are deoxyribonucleic acids.

3. A method according to claim lf, wherein the test nucleic acid is ribonucleic acid and the standard nucleic acid is deoxyribonucleic acid.

4. A method according to claim 1t wherein the test and standard nucleic acids are ribonucleic acids.

5. A method according to claim wherein the test nucleic acid is deoxyribonucleic acid and the standard nucleic acid is ribonucleic acid.

6. A method according to any one of claims 1 to 3, wherein the detector probe of the standard probe pair is a recombinant plasmid. comprising a 1.1 kb Bglll-Bglll fragment of the Hindlll fragment of the human c-Ki-rasI gene? the HindiII fragment being cloned into the pBR322 plasmid and the Bglll-Bglll fragment being subcloned into the restriction site of the BamHI restriction enzyme of the pBR322 plasmid, and the capturing probes are recombinant phages comprising a 0.5 kb Bglll-HindiII fragment of the Hindlll fragment of the human c-Ki-rasI gene, the Hindlll fragment being cloned into the pBR322 plasmid and the Bglll-Hindlll fragment being subcloned into the Ml3 mplO and Ml3 mpll phage vectors between the restriction sites of the BamHI and Hindlll restriction ensymes and wherein the probes are brought, either individually or together with the test probe pair, into contact with an undivided or when necessary divided nucleic acid sample. - 25 -

7. A method according to any one of claims 1 to 3, wherein the detector probe of the standard probe pair is a recombinant plasmid comprising a 1.2 kb EcoRI-Kpnl(Hindlll) 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 Hindlll restriction enzymes, and the capturing probes are recombinant phages comprising a 0.8 kb KpnI-Bglll fragment from the promoter area of the metallothionine gene and the area upstream of it, the fragment having been subcloned into the El3 sap 18 and Ml3 mpl9 phage vectors between the restriction sites of the Kpnl and BamHI restriction enzymes, and wherein the probes are brought, either individually or together with the test probe pair, into contact with an undivided or when necessary divided nucleic acid sample.

8. A method according to claim S or claim 7, wherein in order to determine the degree of amplification of the c-myc oncogene and/or the number of messenger RNA molecules corresponding to that gene, the detector probe of the test probe pair is a recombinant plasmid comprising a 3.7 kb Hindlll-Xbal fragment of the c-myc gene, the fragment being subcloned into the pBR322 plasmid at the restriction site of the Hindlll restriction enzyme, and the capturing probes are recombinant phages comprising a 1.1 kb Xbal-PstI fragment of the c-myc gene, the fragment having been subcloned into the Ml 3 mplO and Ml 3 mpll vectors between the restriction sites of the Xbal and Pstl restriction enzymes, and wherein the probes are brought, either individually or together with the standard probe pair, into contact with an undivided or when necessary divided nucleic acid sample.

9. A method according to claim 6 or claim 7, wherein 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 that gene, the detector probe of the test probe pair is a recombinant plasmid comprising a 0.75 kb Hindlll-Bglll fragment coding for the DHFR gene of the pMTVdhfr plasmid, the fragment having been subcloned into the pAT153 plasmid vector between the restriction sites of the Hindlll and BamHI restriction enzymes, and the capturing probes are recombinant phages comprising a 1.4 kb Hindlll fragment of the MMTV gene area of the pMTVdhfr plasmid, the fragment having been subcloned into the Ml3 mpl8 and Ml3 mpl9 phage vectors at the restriction site of the Hindlll restriction enzyme and wherein the probes are brought, either individually or together with the standard probe pair, into contact with an undivided or when necessary divided nucleic acid sample.

10. A reagent kit for the quantitative determination of nucleic acid molecules by the method of claim 1, the kit comprising 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 having been affixed to the capturing probes, enabling isolation of sandwich hybrids.

11. A reagent kit according to claim 10, wherein 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 that gene is a recombinant plasmid comprising a 3.7 kb HindIII-XbaI restriction fragment of the c-myc gene, the fragment having been subcloned into - 27 - the pBR322 plasmid at the restriction! site of the Hindlll restriction enzyme, the capturing probes are recombinant phages comprising a 1.1 kb XhaJ-PstI fragment of the c-myc gene, the fragment having been subcloned into the 5 Ml 3 rap 10 and Ml 3 mpll phage vectors between the restriction sites of the Xbal and PstI restriction enzymes t, the detector probe of the standard probe pair is a 1.1 kb Bglll-Bglll fragment of the Hindlll fragment of the human c-Ki-rasI genef the Hindlll fragment having 10 been cloned into the pBR322 plasmid and the Bglll-Bglll 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 Bglll-HindXII fragment of the Hindlll fragment of 15 the c-Ki-rasI gene, the Hindlll fragment having been subcloned into the pBR322 plasmid of the c-Ki-rasl gene, and the Bglll-HindiII fragment having been subcloned into the Ml3 mplO and Ml3 mpll phage vectors between the restriction sites of the BamHI and Hindlll restriction 20 enzymes.

12. A reagent kit according to claim 10, wherein the detector probe of the test probe pair used for determination of the degree of amplification of the dihydrofolate reductase of DHFR gene and/or the number of messenger RNA 25 molecules corresponding to that gene is a recombinant plasmid comprising a 0*75 kb HinduI-Bglll fragment coding for the DEFR gene of the pMTVdhfr plasmidf the fragment having been subcloned into the pATl53 plasmid. vector between the restriction sites of the Hindlll and 30 BamHI restriction enzymes„ the capturing probes are recombinant phages comprising a 1.4 kb Hindlll fragment of the MMTV gene area of the pMTVdhfr plasmid t the fragment having been subcloned into the Ml3 rap 18 and Ml3 mpl9 phage vectors at the restriction site of the Hindlll 35 restriction enzyme, the detector probe of the standard.;- 28 -;probe pair is a recombinant plasmid comprising a 1.2 kb EcoRI-Kpnl fragment from upstream of the promoter area of the mouse metallothionine gene, the fragment having been subcloned into the pAT153 plasmid between the restriction sites of the EcoRI and Hindlll restriction enzymes, and the capturing probes are recombinant phages comprising a 0.8 kb Kpnl-Bglll fragment of the metallothionine gene formed by the promoter area and the area upstream of it, the fragment having been subcloned into the HI 3 mplS and Ml3 mpl9 phage vectors between the restriction sites of the Kpnl and BamHI restriction enzymes.;

13. A method according to claim 1, substantially as hereinbefore described.;

14. A reagent kit according to claim 10, substantially as hereinbefore described.;Dated this 26th day of February, 1987;CRUICKSHANK & CO.;Agents for the Applicant;1 Holies Street;Dublin 2;*.%WfTO\{gi3^Ssf