AU603562B2

AU603562B2 - Quantitative determination of nucleic acid molecules and the reagent kit used

Info

Publication number: AU603562B2
Application number: AU69275/87A
Authority: AU
Inventors: Marjut Ranki; Hans Soderlund
Original assignee: Orion Yhtyma Oy
Current assignee: SANGTEC MOLECULAR DIAGNOSTICS AB
Priority date: 1986-02-27
Filing date: 1987-02-26
Publication date: 1990-11-22
Anticipated expiration: 2007-02-26
Also published as: FI76119B; DK99387A; GB2187283B; NL8700483A; FI860836A0; IL81695A; IT8719501A0; LU86792A1; PT84368A; IL81695A0; ZA871401B; HUT43647A; DE3706285C2; NZ219421A; GB2187283A; IE66904B1; IE870496L; CH675593A5; NO870794D0; IT1202581B

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: This document contains tIh amendments made utnder Section 49 and is correct for printing.

rr>J[, .HfM MW Q C C C C C C C I C C C: C C C C 'S CS c APPLICANT'S REFERENCE: FI-860836 Name(s) of Applicant(s): Orion-Yhtyma Oy Address(es) of Applicant(s): Valtakatu 2, SF-53600 Lappeenranta

FINLAND.

Address for Service is: PHILLIPS ORMONDE and FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Complete Specification for the invention entitled: QUANTITATIVE DETERMINATION OF NUCLEIC ACID MOLECULES AND THE REAGENT KIT USED Our Ref 46379 POF Code: 46284/20383 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): 6003q/l r n r n QUANTIFICATION OF NUCLEIC ACID MOLECULES AND THE REAGENT KIT

USED

The invention relates 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 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 found to be associated with the development of cancer. It is also S i known that external factors such as pharmaceuticals and metals etc. cause certain genes to be amplified. For the S development 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 i numbers of some chromosomes is the cause of certain i hereditary diseases or other disturbances, whereas some 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 electrophoresis.

SSubsequently 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, pp. 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 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., I 100, pp. 255-266, 1983).

I Known methods, such as the Southern and Northern blotting methods, are slow and difficult to perform. Since they only I 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.

IThe sandwich or solution hybridization methods, described in SUS Patent No. 4,486,539 and in the British Patent Application No. GB 2 169 403 are quantitative (Virtanen et al., Lancet 1, S pp. 381-383, 1983). In addition, the method of the present S e invention requires a standard nucleic acid, the copy number of which is constant and enables the determination of the i number of relevant nucleic acid molecules per given unit such as a cell, nucleus, ribosome or chromosome.

I 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 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 y expression level of messenger RNA.

At leas t two adcteminai-et s---e-r-equ-^ed-i-n-t-he -tPe g invention. One determines the nuc-c r molecule, which may be present -e~a±copies, the test nucleic acid. The t-M stitutive nucleic acid molecul:e- J

T

2a According to the present invention there is provided a quantitive method for the determination of nucleic acid molecules by sandwich or 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 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 are rendered, 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, into a form whereby they i cannot interfere with the hybricTization test; and p a Qo o are brought into contact, either undivided or, when necessary divided, with at least one test probe pair S sufficiently homologous to the nucleic acid potentially present in several copies and with at least one chosen and FE suitable standard probe pair sufficiently homologous to the nucleic acid molecule advantageously present in a constant number, the detector probes of the test probe pair and standard probe pair being labelled with a detectable label Ft £C and 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 tt after hybridization, the test hybrid and standard hybrid are separated when necessary and the attached label S is measured and the number of nucleic acid molecules per given unit is obtained by comparing the test and standard nucleic acid numbers.

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 determies the constitutive nucleic acid molecule I rf 3 advantageously present in constant number, the standard ~nucleic acid. In the method according to the invention, a Snucleic acid molecule denotes a certain nucleotide sequence Sof 10 12 nucleotides or a gene containing several thousand i! nucleotides. It can also mean a messenger RNA or a Snucleotide sequence considerably longer than a single gene, i.e. an amplicone.

SThe determination of test and standard nucleic acids is done g using an otherwise normal sandwich 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 j A least one test probe pair and at least one standard probe pair.

S ta d The reagents, or probes, used in the method, are prepared, i using recombinant-DNA techniques, from nucleic acids sufficiently homologous to the test and standard nucleic C t V acids. Sufficiently homologous nucleic acids can also be prepared synthetically and semisynthetically.

z The test and standard nucleic acids may be isolated directly

S

I from cells and identified by various hybridization id techniques. Such test and standard nucleic acids are however also available 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 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 I 4 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 -i separation of the resulting hybrid from the hybridization i, 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 |i 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 i| probe pairs used in the test.

I The method of the present invention is particularly suitable Iq for determining the number of nucleic acid molecules directly from cellular homogenates. The method may of course also be "r "used for the determination of purified or pure nucleic acids.

t:u However, before carrying out the method of the invention, the I 'most suitable pretreatment of the nucleic acid sample should 1|i be selected.

v (e It is possible to carry out both DNA and RNA determinations I using the method of the invention. Deoxyribonucleic acids ji ,c are denatured to obtain single strands if necessary.

SSingle-stranded messenger RNA molecules potentially C C disturbing the hybridization test can be hydrolyzed, for ii 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 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 treatment of the sample must be selected or, alternatively, this further treatment may be omitted. The choice of a suitable method d for the further treatment is of course dependent on the Smethod 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 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.

tt Z The hybridization test itself is performed by bringing the 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 o standard probe pair. In such instances, the quantity of test i 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 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 can be determined per selected unit.

I' 6 The method of the invention is of practical diagnostic value, particularly in the detection of some types of cancer. In !i 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.

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 given drug by amplification of the gene, the expression product of which inactivates the drug. One such case is methotrexate which induces amplification of the gene for dihydrofolate reductase (DHFR). A further example is amplification of the gene for I metallothionine under the influence of cadmium.

I The expression level of a gene is important from the point of I view of the phenotype and function of the cell. This can be rIi investigated by measuring the quantity of messenger RNA which correlates to the quantity of protein coded by it. The I transcription product of an oncogene determines the way in I which it will ultimately be expressed.

c tc SThe expression levels of an oncogene vary depending on the Scell type, differentiation level and phase of development of the cell. For example, at a certain stage of fetal development, the c-inyc 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 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 i than quantification of the gene product itself. As an example the c-myc oncogene, a labile protein readily coagulated by heat, can be mentioned.

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.

EXAMPLE 1 Quantification of an amplified oncogene a) Nucleic acid reagents and their preparation STANDARD PROBES Cell standard nucleic acid. The c-Ki-rasl gene is present in all human cells. The probe pairs for sandwich hybridization were prepared by subcloning the HindIII fragment of the c-Ki-rasI gene, measuring 3.8 kb in length, the restriction map of which has been described by Chang et al., PNAS 79, pp. 4848-52, 1982. The fragment is available e.g. cloned into the pBR322 plasmid (ATCC 41032) and can be obtained e.g.

from the ATCC culture collection.

Further treatment of the cell standard nucleic acid. The pBR322 clone described above was treated with BglII and HindIII 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.

8 Standard detector probe. A BglII-BglII 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 1 2 5 I-labelled dCTP.

Standard capturing probe. The BglII-HindIII fragment of about 0.5 kb was inserted into the M13 mplO and mpll phage fvectors between the restriction sites of the BamHI and i HindIII restriction enzymes and affixed to a nitrocellulose i filter (150 ng DNA/dia 1 cm).

*I TEST PROBES STest nucleic acid. A probe pair for sandwich hybridization was prepared from a cloned c-myc gene which can be obtained 1 e for example, from the ATCC culture collection (ATCC 41010).

e The restriction map of gene has been described by Watt et {i e( al., PNAS 80, pp. 6307-6311, 1983.

j C -Further treatment of the test nucleic arid. The c-myc gene i was treated with HindIII, Xbal and PstI restriction enzymes i e tand the fragments isolated from the agarose gel, purified and i subcloned into suitable vectors in order to prepare the Sdetector and capturing probes.

i Test detector probe. The single-stranded tails of the HindIII-XbaI restriction fragment of the c-myc gene, d measuring 3.7 kb in length, were rendered double-stranded by i i DNA polymerase. The HindIII linkers were inserted by T4-DNA-ligase into the resulting blunt-end DNA fragments; after phenol extraction the DNA was treated with the HindIII restriction enzyme. The DNA fragment was subsequently cloned into the pBR322 plasmid at the restriction site of the HindIII restriction enzyme and labelled by nick-translation with 125 I-labelled dCTP.

Test capturing probe. The 1.1 kb XbaI-PstI fragment of the 9 Sc-myc gene was cloned into the M13 mplO and mpll phage i cloning vectors between the restriction sites of the Xbal and PstI restriction enzymes and affixed to the nitrocellulose l filter (150 ng DNA/dia 1 cm).

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-myc gene. The sandwich hybridization solution to which the above test probes were added consisted of 4 x SSC, 1 x Denhardt solution, 200 pg/ml 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 0 C. The label attached to the sandwich I hybrids was then counted in the gamma counter.

*j Table 1 Sample cpm c molecules/test c-myc-filter i cl i ti i i C i tet i "cile i z cc Ti c; I: r. I r r i i E i i 0 106 5 x 106 190 107 340 108 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 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

I

denaturation hydrolyzed 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 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 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 U UC CP i LU U U U U: Ue U LU U C LU CU U: UU C

LU

U U U ULL U EL UI U UE LU C C U C C C Sample c-Ki-rasI filter c-myc filter cpm* cpm* number Human placental cells 486 340 Colo 320 cells 432 3205 1.6 x 108 *the reading obtained from the blank filter has been subtracted from the readings.

EXAMPLE 2 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 I

'II

11 the mouse, i.e. the MT gene, and the DNA immediately upstream of it. The structure of the MT gene has been described by Pavlakis and Hamer, PNAS 80, pp. 397-401, 1983. The reference nucleic acid fragment is available e.g, cloned into the pBPV-MMTeo(342-12) 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 with KpnI, BglII and EcoRI restriction enzymes for subcloning into the pATl53 plasmid.

The KpnI tail was converted into a HindIII tail with a linker.

Standard detector probe. The EcoRI-KpnI-(HindIII) 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 'oRI and HindIII restriction enzymes and labelled by 32 nick-translation with P-labelled nucleoside triphosphates.

Standard capturing probe. The 0.8 kb KpnI-BglII fragment c comprising the promoter area of the metallothionine gene and the area upstream of it was cloned into the M13 mpl8 and M13 mpl9 phage vectors between the restriction sites of the KpnI and BamHI restriction enzymes and affixed to the °nitrocellulose filter.

S 'TEST PROBES I i Test nucleic acid. The probe pair for the sandwich hybridization test was prepared using the commercially available pMTVdhfr 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 dihydrofolate reductase (DHFR) gene

I

fl 12 was treated with HindIII and BglII restriction enzymes.

Test detector probe. The HindIII-BglII 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 HindIII and BamHI restriction enzymes and labelled by nicktranslation with 32 P-labelled nucleoside triphosphates.

Test capturing probe. A HindIII fragment measuring 1.4 kb taken from the MMTV gene area of the pMTVdhfr plasmid was cloned 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 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.

Table 3 i i;a 1

P

i I e :li;

S£

CIC

Sample molecules/test cpm DHFR filter 0 17 106 3 x 106 79 7 210 c) Determination of the number of genes The cell lines mentioned are derived from the mouse fibroblast cell NIH 3T3, which cell line is available from the ATCC culture collection and the number of which is CRL 1658.

-12 a- Cell lines which had been transfected with different quantities of cDNA corresponding to the mRNA of the DHFR gene I~ CC

~C

CCC C

CC

C CC

CC

C C

C

CCC

CC

C

C~.

C

C CC

CC

13 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 pj sample corresponding to about cells was taken from the homogenate and 50 pl NaOH added.

The sample was boiled and neutralized with acetic acid and the hybridization mixture. The total volume was 0.5 ml. All the probes described above were added simultaneously and a so-called blank filter was added as a background control.

Hybridization, 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.

Table 4 ct 91 c eC C CC EC r C C

CCC

c C 1i Cell MT No.of cells DHFR cpm* in the sample cpm* No. of molecules No. of in the sample copies Control cell (No DHFR-cDNA) 182 1.05 x 106 21 106 Line I 138 0.9 x 10 6 80 3 x 106 3 Line II 210 1.25 x 106 732 5 x 107 cpm: the reading given by the blank filter has been subtracted 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 nT-specific signal of 165 20 cpm. The DHFR reagents measure the quantity of DHFR-cDNA. The number of cells was deduced from the MT-specific signal. It was thus possible to determine the number of DHFR-cDNA copies in different cell lines as shown in Table 4.

14 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 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 about 1.0 kb in length. Of this, about 0.25 kb are derived from the MMTV Spromoter area and the rest from DHFR-cDNA (Lee et al., Nature 294, pp. 228-232, 1981).

C C t c 9 S• STANDARD PROBES I V C r C C The cell standard nucleic acid, standard detector and Sstandard capturing probe were as described in Example 2.

C TEST PROBES c tC The test nucleic acid, test detector and test capturing probe I C were as described in Example 2.

Sb) 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 transcription was prepared by subcloning the 1.4 kb HindIII fragment of the MMTV promoter of the pMTVdhfr plasmid and the 0,75 kb HindIII-BglII fragment (DHFR-cDNA) next to each other into the pSP64 plasmid (Promega Biotec) between the restriction sites of the HindIII and BamHI restriction enzymes. The sample RNA was stored in 0.2 SDS aqueous solution.

-2I I I id I:i !1 i i-

I

1 i!i ;1 ;1t 13

I

jj r i

L

ii ::s The sandwich hybridization test was carried out as described in Examples Ib and 2b but denaturation was omitted.

Table Sample molecules/test cpm DHFR filter 0 5 x 10 6 107 5 x 107 108 130 390 653 cgr C

C

C r t-C S c) Determination of the number of messenger RNA molecules The number of messenger RNA molecules corresponding to the DHFR gene was determined from the cell lines described in *c Example 2.

S 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 pl sample of the homogenate was taken corresponding to about 5 x 106 cells.

The homogenate was then added to the 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 pl of homogenate, the cell number was determined using the MT probe as described in Example 2c.

The results are shown in Table 6.

i

I

i Table 6 -Cell MT cpm* Cell number in the sample

DHFR

cpm* No of molecules No. per in the sample cell t S S S C C S C St C S S 5555 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 *cmp: 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 RNA molecules from the DHFR genes.

EXAMPLE 4- 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 EcoRI-KpnI-(HindIII) fragment in pAT153 was labelled by nick-translation with 125 I-labelled deoxycytidine. The 0.8 kb KpnI-BglII fragments in M13 mpl8 and M13 mpl9 were modified with biotin using the Photoprobe TM reagent (Vector Laboratories, CA, USA, product No SP-1000).

55 C: c c St 17 TEST PROBES The test nucleic acid, test detector and test capturing probe i were as described in Example 2. The 0.75 kb HindII-BglII fragment in pAT153 was labelled with 1 25 I-labelled deoxycytidine. The 1.4 kb HindIII fragments in M13 mpl8 and M13 mpl9 were biotinylated using Photoprobe as above.

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 S .F the standad probes recognizing the MT-gene. A test nucleic i c acid standard curve was prepared with the pMTVdhfr plasmid i and the test probes recognizing this plasmid. Hybridizations were carried out in 200 pl of a solution consisting of 0.6 M NaCi, 20 mM phosphate buffer, pH 7.5, 1 mM EDTA, 4 i polyethylene glycol (PEG 6000) for 1.5 hours at 70°C. After the reaction 50 pl of streptavidin-agarose (Betheseda Research Laboratories, Maryland, USA, product No. 5942SA), and 1 M NaCi, 10 mM sodium phosphate, pH 7.5, 1 mM EDTA was i added to a final volume of 500 pi. The hybrids were collected on the streptavidin-agarose at 37 C for 15 min. The agarose Sc I 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 I t determined by measuring the agarose in a gamma counter.

I (Syvanen et al., Nucleic Acids Res, 14, 5037-5048, 1986).

The results are shown in table 7 and 8.

Ii "I r i 18 Table 7 Sample cpm cells/test MT probes 0,8 x 10 6 162 1,6 x 106 216 3 x 106 298 Table 8 Sample cpm molecules/test DHFR probes 106 148 5 x 106 394 5 x 10 7 2240 <i1 t r C C: g

CC

C S ct C C C~ C C 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 approximatedly 2 x 10 6 cells was 125 pl.

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 of the hybridization mixture to a final volume of 200 p1. 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.

19 Table 9 Cell MT DHFR cpm No of cells cpm* No. of No. of in the sample molecules copies in the sample Control cell 253 2.3 x 10o 6 73 10 5 Line 1 210 1.5 x 10 6 233 3.8 x 106 3 Line 11 237 2.1 x 10 6 3059 8.8 x 10 42 cpm: values from control assays without cell standard or test nucleic acid have been subtracted.

Ie Er 49

Claims

4.*44 1 i. A quantitative method for the determination of nucleic acid molecules by sandwich or solution hybridization, the method comprising determining the 5 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 the number of chosen standard nuxcleic acid molecules advantageously present in a constant number in the same unit, wherein the nucleic acids present in the sample are rendered, 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, into a form whereby they cannot interfere with the hybridization test; and 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 25 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 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 L, I 21 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 1, wherein the test nucleic acid is ribonucleic acid and the standard Snucleic acid is deoxyribonucleic acid. 4. A method according to claim 1, wherein the test and standard nucleic acids are ribonucleic acids. 10 5. A method according to claim 1, wherein the t 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 BglII-BglII fragment of the HindIII fragment of the human c-Ki-rasI gene, the HindIII fragment being cloned into the pBR322 plasmid and the BglII-BglII fragment being subcloned into the restriction site of S 20 the BamHI restriction enzyme of the pBR322 plasmid, and the capturing probes are recombinant phages comprising a 0.5 kb BglII-HindIII fragment of the I HindIII fragment of the human c-Ki-rasI gene, the HindIII fragment being cloned into the pBR322 plasmid and the BglII-HindIII fragment being subcloned into the M13 mplO and M13 mpll phage vectors between the restriction sites of the BamHI and HindIII 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 r:! 22 divided nucleic acid sample.
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-KpnI(HindIII) 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 HindIII restriction enzymes, and the capturing probes are recombinant phages comprising a 0.8 kb KpnI-BglII fragment from the promoter area of the metallothionine gene and the area upstream of it, the fragment having been subcloned into the M13 mpl8 and M13 mpl9 phage vectors between the restriction sites of the KpnI and 15 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 6 or claim 7, 20 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 HindIII-XbaI fragment of the c-myc gene, the fragment being subcloned into the pBR322 plasmid at the restriction site of the HindIII restriction enzyme, and the capturing probes are recombinant phages comprising a 1.1 kb XbaI-PstI fragment of the c-myc gene, the fragment having been subcloned into the M13 mplO and M13 mpll vectors between the restriction sites of the XbaI and PstI restriction enzymes, and wherein the probes are brought, either individually or -together 23 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 ^i 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 Itest probe pair is a recombinant plasmid comprising a 0.75 kb HindIII-BglII 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 HindIII and BamHI restriction enzymes, and the capturing probes are recombinant phages comprising a 1.4 kb HindIII fragment cf the MMTV gene area of the pMTVdhfr plasmid, the fragment having been subcloned into the M13 mpl8 and M13 mpl9 phage vectors at the restriction site of the HindIII 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. 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 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. A, Ty^\ 24
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 the pBR322 plasmid at the restriction site of the HindIII restriction enzyme, the capturing probes are recombinant phages comprising a 1.1 kb XbaI-PstI fragment of the c-myc gene, the fragment having been subcloned into the M13 mplO and M13 mpll phage vectors between the restriction sites of the XbaI and PstI restriction enzymes, the detector probe of the standard probe pair is a 1.1 kb BglII-BglII fragment'of the HindIII fragment of the human c-Ki- rasI gene, the HindIII fragment having been cloned into the pBR322 plasmid and the BglII-BglII 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 BglII-HindIII fragment of the HindIII fragment of the c-Ki-rasI gene, the HindIII fragment having been subcloned into the pBR,22 plasmid of the c-Ki-rasI gene, and the BglII-HindIII fragment having been subcloned into the M13 mplO and M13 mpll phage vectors between the restriction sites of the BamHI and HindIII restriction 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 or DHFR gene and/or the number of messenger RNA molecules corresponding to that gene is a recombinant plasmid comprising a 0.75 kb HindIII- BglII 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 HindIII and BamHI restriction enzymes, the capturing probes are recombinant phages comprising a 1.4 kb HindIII fragment of the MMTV gene area of the pMTVdhfr plasmid, the fragment having been subcloned into the M13 mpl8 and M13 mpl9 phage vectors at the restriction site of the HindIII restriction enzyme, the detector probe of the standard probe pair is a recombinant plasmid comprising a 1.2 kb EcoRI-KpnI fragment 'from upstream of the promoter area of the mouse metallothionine gene, the fragment having been Ssubcloned into the pAT153 plasmid between the restriction sites of the EcoRI and HindIII restriction enzymes, and the capturing probes are recombinant phages comprising a 0.8 kb KpnI-BglII fragment of the metallothionine gene formed by the promoter area and the area upstream of it, the fragment having been subcloned into the M13 mpl8 and M13 mpl9 phage vectors between the restriction sites of the KpnI and BamHI restriction enzymes.
13. A method according to claim 1, substantially as hereinbefore described.
14. A reagent kit according to claim substantially as hereinbefore described. DATED: 6 April, 1990 PHILLIPS ORMONDE FITZPATRICK Attorneys for: ORION-YHTYMA OY